Location Transcriptional unit Functional Expressed ICR Protein RNA b

Human (Mouse) Human (Mouse) component Human Mouse allele methylation Name or description Description

1p36 (4 E2) TP73 (Trp73) I NR M Tumour related protein1p31 DIRAS3 PD NO P Ras homolog2p12 (6 C3) LRRTM1 (Lrrtm1) I NR leucine rich repeat transmembrane2p15 (11 A3) COMMD1 (Commd1) NI I M Copper metabolism gene Murr1

(Zrsr1) NO I P M U2 small nuclear RNP auxiliary factor4q22.1 (6 C1) NAP1L5 (Nap1l5) I I P M Nucleosome assembly protein6p11 (1 B) PRIM2 (Prim2) I NR M Primase, polypeptide 26q24 (10 A1) HYMAI (Hymai) I PD P M misc RNA

PLAGL1 (Plagl1) I I P Zinc finger protein6q25 (17 A1) IGF2R (Igf2r) PI? I M Insulin-like growth factor receptor 2

(Air) NO I P M Igf2r ASSLC22A2 (Slc22a2) PI? I M Organic cation transporterSLC22A3 (Slc22a3) PI? I M Organic cation transporter

7p12 (11 A1) DDC (Ddc) Exon1a transcript NR I P Dopa decarboxylaseGRB10 (Grb10) I I M(P)c

M Growth factor receptor-bound protein

7q21 (6 A1) CALCR (Calcr) PD I M Calcitonin receptor

7q21.3 (6 A1) SGCE (Sgce) I I P Sarcoglycan, epsilonPEG10 (Peg10) I I P M Retroviral gag pol homologuePPP1R9A (Ppp1r9a) I I M Protein phosphatase inhibitorPON1 (Pon1) PD NI P Paraoxonase 1PON3 (Pon3) NR PD M Paraoxonase 3PON2 (Pon2) NR PD M Paraoxonase 2ASB4 (Asb4) NR I M Ankyrin repeat and SOCS box

7q32.2 (6 A3) CPA4 (Cpa4) I NR M CarboxypeptidaseMEST (Mest) I I P M Alpha/beta hydrolase fold familyMIRN335 (Mirn335) NR I microRNAMESTIT1 I NO P MEST ASCOPG2IT1 (Copgas2) I I P COPG2 ASCOPG2 (Copg2) CD I P(M)d Coatomer protein complex subunit KLF14 (Klf14) I I M Krüppel-like factor 14

8p23 (8 A1.1) DLGAP2 I ND P Membrane associated guanylate kinase

8q24.3 (15 D3) KCNK9 (Kcnk9) I I M Potassium channel(Peg13) NO I P M misc RNA

10p14 (2 A1) SFMBT2 (Sfmbt2) NR I P M Scm-like with 4 mbt domains

10q22 (10 B4) CTNNA3 (Ctnna3) PD NR M Catenin, alpha 3

10q26.11 (7 F3) INPP5F_V2 (Inpp5f_v2) V2 isoform only I I P M misc RNA (retro)

11p15 (7 F5) H19 (H19) I I M P miRNA hostmiR-625 I I M miRNA

IGF2 (Igf2) I I P Insulin-like growth factor 2IGF2AS (Igf2as) I I P IGF2 AS INS (Ins2) I I P InsulinTH (Th) NR I M ? Tyrosine hydroxylaseASCL2 (Ascl2) CD I M HLH transcription factorTSPAN32 (Tspan32) NI I M Tetraspanin 32CD81 (Cd81) NI I M Transmembrane 4 superfamily TSSC4 (Tssc4) NI I M Tumor suppressing candidateKCNQ1 (Kcnq1) I I M Voltage-gated potassium channelKCNQ1OT1 (Kcnq1ot1) I I P M KCNQ1 ASKCNQ1DN I NO M BWRT proteinCDKN1C (Cdkn1c) I I M Cyclin-dependent kinase inhibitor(Msuit1, AF313042) NO I M misc RNASLC22A18AS PD NO M SLC22A18AS putative proteinSLC22A18 (Slc22a18) I I M Organic cation transporterPHLDA2 (Phlda2) I I M Pleckstrin homology-like domainNAP1L4 (Nap1l4) NR I M Nucleosome assembly protein(Tnfrsf23) NO I M TNF receptor superfamily

Imprinting status

OSBPL5 (Osbpl5) I I M Oxysterol binding protein-like 5ZNF215 PD NO M Zinc finger protein

11p15.4 (7 E3) AMPD3 (Ampd3) NI I M AMP deaminase (isoform E)

11p13 (2 E) WT1-Alt transcript (Wt1) I NR P Zinc finger proteinWT1AS (Wt1as) PD NI P WT1 AS

11q13.4 (7 F5) DHCR7 (Dhcr7) NI I M 7-dehydrocholesterol reductase11q23 (9 A5) SDHD (Sdhd) CD NR P Succinate dehydrogenase, subunit12q13 (15 F1) SLC38A4 (Slc38a4) NR I P Amino acid transporter12q21 (10 C3) DCN (Dcn) NI PD M Proteoglycan13q14 (14 D2) HTR2A (Htr2a) NI/CD I M Serotonin receptor

14q32 (12 F1) BEGAIN [BEGAIN - sheep] NR NR P brain-enriched guanylate kinase-associated (imprinted in sheep)DLK1 (Dlk1) I I P Delta-like 1 homolog DLK1 downstream transcripts NR I P misc RNA(Mico1) NR I M Circadian oscillating (Mico1os) NR I M Circadian oscillating MEG3 (Meg3) I I M P misc RNAmiR-337 NR I M miRNARTL1 (Rtl1) NR I P Retrotransposon-like 1Anti-PEG11 (anti-Rtl1) anti-Rtl1 NR I M Rtl1-AS

miR-431 NR I M miRNAmiR-433 NR PD M miRNAmiR-127 NR I M miRNAmiR-434 NR PD M miRNAmiR-432 NR PD M miRNAmiR-136 NR I M miRNA

MEG8 (Rian) MEG8 (Rian) NR I M snoRNA hostmiR-370 NR I M miRNA(MBII-78) NO I M snoRNA (MBII-19) NO I M snoRNA 14q(0) NR I M snoRNA 14q(I) (MBII-48) NR I M snoRNA(MBII-49) NO I M snoRNA (MBII-426) NO I M snoRNA 14q(II) (MBII-343) NR I M snoRNA[RBII-36-rat] NO NO ? snoRNA 86 copies

(Mirg) (Mirg) NR I M miRNA hostmiR-411 NR I M miRNAmiR-380 NR I M miRNAmiR-376b NR I M miRNAmiR-376 NR I M miRNAmiR-134 NR I M miRNAmiR-154 NR I M miRNAmiR-410 NR I M miRNA

DIO3 (Dio3) NR I P Deiodinase, iodothyronine type III

Imprinted transcriptional units in humans and mice. Modified from http://igc.otago.ac.nz/home.html.

X-inactive specific transcriptXIST antisense

Imprinted transcript variant1

15q11-q12 (7C-B5) (Peg12) NO I P Gsk-3-binding protein familyMKRN3 (Mkrn3) I I P Makorin, ring finger proteinZNF127AS (Zfp127as) NR I P MKRN3 ASMAGEL2 (Magel2) I I P MAGE-like proteinNDN (Ndn) I I P Necdin, neuronal growth suppressor(AK014392) NR PD P Ndn ASBM117114 NO I P EST(Pec2) NR I P LINE-rich intergenic(BB077283) NO I P EST(Pec3) NR I P LINE-rich intergenic(Nccr) NR I P ?miRNA host PWRN1 I ?NO NK misc RNAC15ORF2 I NO NK 1156 aa intron-less gene in primates onlySNURF-SNRPN SNURF (Snurf) I I P SNRPN upstream reading frame

SNRPN (Snrpn) I I P M Small nuclear ribonucleoproteinSNORD107 (MBII-436) I I P snoRNASNORD64 (MBII-13) I I P snoRNA SNORD108 I NO P snoRNASNORD109A I NO P snoRNASNORD116@ I I P snoRNA clusterSNORD115@ I I P snoRNA clusterSNORD109B I NO P snoRNAUBE3A-AS I I P UBE3A AS

UBE3A (Ube3a) I I M Ubiquitin protein ligaseATP10A (Atp10a) I CD M ATPase, Class VGABRB3 (Gabrb3) CD NI P Gamma-aminobutyric acid receptorGABRA5 (Gabra5) CD NI P Gamma-aminobutyric acid receptorGABRG3 (Gabrg3) CD NI P Gamma-aminobutyric acid receptor

15q21 (2 E5) GATM (Gatm) NI I M Glycine amidinotransferase

15q24 (9 E3.1) MIRN184 (Mirn184) NR I P microRNA(AS4) NR I P misc RNA(4930524O08Rik, A19) NO I P misc RNARASGRF1 (Rasgrf1) NR I P P Guanine nucleotide exchange factor

16p13 (16 A1) ZNF597 (Zfp597) I NR M Zinc finger protein18q11 (18A2-B2) IMPACT (Impact) NI I P Imprinted and ancient18q21.1 TCEB3C I NO M Transcription elongation factor19q13.41 ZNF331 PD NO M Zinc finger protein

19q13.43 (7A2-B1) ZIM2 (Zim2) I I P(M)d Zinc-finger protein(Zim1) NO I M Zinc-finger proteinPEG3 (Peg3) I I P M Zinc-finger proteinITUP1/MIMT1 (Usp29) I NO PUSP29 (Usp29)e NR I P Ubiquitin-specific proteaseZIM3 (Zim3) NR I M Zinc-finger protein (human) No ORF (mouse)ZNF264 (Zfp264) NR I P Zinc-finger protein (human) No ORF (mouse)

20q11.21 (2 H1) MCTS2 (Mcts2) I I P M RNA binding proteinHM13 (H13) NR I M Signal peptide peptidase

20q11.23 (2 H1) NNAT (Nnat) I I P M Neuronatin20q13 (2 H3) L3MBTL (L3mbtl) I NI P DMR Polycomb group

20q13 (2 E1-H3) GNAS (Gnas) NESP55 I I M Neuroendocrine secretory protein 55GNASXL I I P M Large isoform of GS-a

(F7) NO PD M Hypothetical protein (Mm.125770)Exon-1A I I P M misc RNAGS-alpha I I M Stimulatory G-protein, alpha subunit

SANG (Nespas) I I P GNAS AS

(X) All genes affected by X inactivation NI I M

(X A7) (Xlr3b) NO I M X linked lymphocyte regulated(Xlr4b) NO I M X linked lymphocyte regulated(Xlr4c) NO I M X linked lymphocyte regulated

Xq13 (X D) XIST (Xist) NI I PTSIX (Tsix) NI I M

Abbreviations. AS, antisense transcript; miRNA, microRNA; misc RNA, RNA of unknown functionCD, conflicting data; I, reported to be imprinted; ICR, Imprint control region; ND, not detected; NI, reported to be not imprinted; NO, no orthologue known; NR, no reports of imprinting status; M, maternal; P, paternal; PD, provisional data; PI, polymorphic imprinting

bNoncoding RNAs onlycImprinting is isoform dependent.dZIM2 and COPG2 are reported to be oppositely imprinted in human and mouse.e Mouse Usp29 appears to split into two genes in human and cow (ie MIMT1 and Usp29).

Location Transcriptional unit Functional Expressed ICR Protein RNA b

Human (Mouse) Human (Mouse) component Human Mouse allele methylation Name or description DescriptionImprinting status

All genes affected by X inactivation

157

APPENDIX 2: THE EVOLTION OF IMPRINTING:

CHROMOSOMAL MAPPING OF ORTHOLOGUES

OF MAMMALIAN IMPRINTED DOMAINS IN

MONOTREME AND MARSUPIAL MAMMALS

The following appendix is a publication from mid-2007 which examines the evolution

of genomic imprinting and X inactivation through localisation of orthologues of

imprinted genes in marsupials and monotremes.

Edwards CA, Rens W, Clarke O, Mungall AJ, Hore TA, et al. (2007) The

evolution of imprinting: chromosomal mapping of orthologues of mammalian

imprinted domains in monotreme and marsupial mammals. BMC Evol Biol 7:

157.

Although my final contribution to this publication was relatively minor, the time taken

generating my results consumed a reasonable proportion of my early PhD project. I

isolated three imprinted gene orthologues in platypus (UBE3A, GNAS and DLK1) and

determined their localisation on the challenging platypus chromosomes. My results

were later independently confirmed by co-authors.

As discussed in Chapter 6, this publication significantly contributed to our

understanding of the evolution of genomic imprinting and imprinted loci. Specifically, it

disproved the hypothesis that imprinted genes evolved mono-allelic gene expression at

one chromosomal region (such as the sex chromosomes) and then transferred this to

other genomic regions upon rearrangement.

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BioMed Central

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BMC Evolutionary Biology

Open AccessResearch articleThe evolution of imprinting: chromosomal mapping of orthologues of mammalian imprinted domains in monotreme and marsupial mammalsCarol A Edwards†1, Willem Rens†2, Oliver Clarke2, Andrew J Mungall3, Timothy Hore4, Jennifer A Marshall Graves4, Ian Dunham3, Anne C Ferguson-Smith*1 and Malcolm A Ferguson-Smith2

Address: 1Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK, 2Cambridge Resource Centre for Comparative Genomics, Department of Veterinary Medicine, University of Cambridge, Cambridge CB3 OES, UK, 3Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK and 4Research School of Biological Sciences, The Australian National University, Canberra, Australia

Email: Carol A Edwards - cae28@cam.ac.uk; Willem Rens - wr203@mole.bio.cam.ac.uk; Oliver Clarke - oc226@cam.ac.uk; Andrew J Mungall - ajm@sanger.ac.uk; Timothy Hore - TIMOTHY.HORE@ANU.EDU.AU; Jennifer A Marshall Graves - graves@rsbs.anu.edu.au; Ian Dunham - id1@sanger.ac.uk; Anne C Ferguson-Smith* - afsmith@mole.bio.cam.ac.uk; Malcolm A Ferguson-Smith - maf12@cam.ac.uk* Corresponding author †Equal contributors

AbstractBackground: The evolution of genomic imprinting, the parental-origin specific expression ofgenes, is the subject of much debate. There are several theories to account for how the mechanismevolved including the hypothesis that it was driven by the evolution of X-inactivation, or that itarose from an ancestrally imprinted chromosome.

Results: Here we demonstrate that mammalian orthologues of imprinted genes are dispersedamongst autosomes in both monotreme and marsupial karyotypes.

Conclusion: These data, along with the similar distribution seen in birds, suggest that imprintedgenes were not located on an ancestrally imprinted chromosome or associated with a sexchromosome. Our results suggest imprinting evolution was a stepwise, adaptive process, with eachgene/cluster independently becoming imprinted as the need arose.

BackgroundGenomic imprinting is an epigenetic phenomenon thathas been well-characterised in eutherian mammals.Imprinted genes are expressed from one of the two paren-tally inherited chromosome homologues and repressedon the other. The mechanism of parental-origin specificgene expression is associated with heritable differentialmodifications to the DNA and chromatin that are pro-grammed during gametogenesis [1]. Since the discovery of

imprinting in placental mammals over 20 years ago therehas been much speculation about how the mechanismhas evolved. Despite this, the range of mammalian speciestested for imprinting is limited and very few non-mam-malian vertebrates have been experimentally assessed.Mammals that diverged early from the lineage of euthe-rian mammals are ideally suited for investigating imprint-ing evolution by comparing epigenetic mechanismswithin mammalian species. Such comparative analysis

Published: 6 September 2007

BMC Evolutionary Biology 2007, 7:157 doi:10.1186/1471-2148-7-157

Received: 13 March 2007Accepted: 6 September 2007

This article is available from: http://www.biomedcentral.com/1471-2148/7/157

© 2007 Edwards et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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has wider implications for our understanding of the evo-lution of the epigenetic control of genome function. Todate, based on investigations of eutherian imprintedorthologues, imprinting has been demonstrated at someloci in marsupials (both Macropus eugenii {tammar wal-laby} and Monodelphis domestica {grey short-tailed opos-sum} which diverged from each other approximately 70million years ago) but not in monotremes (platypus andechidna) [2-5]. This suggests that, if imprinting arose onlyonce in mammals, it evolved somewhere between thedivergence of monotremes (prototherians) from therianmammals around 166 million years ago (MYA) [6] andthe divergence of marsupials (metatherians) from euthe-rian mammals approximately 147 MYA.

The egg-laying monotreme is an important link betweenbirds and viviparous mammals, and is therefore of inter-est for studies on the evolution of imprinting. In addition,the platypus has been shown to possess 10 sex chromo-somes, 5 Xs and 5 Ys [7,8]. In male meiosis these 10 chro-mosomes form a multivalent chain consisting ofalternating X and Y chromosomes [7]. The 5Y and 5Xchromosomes segregate alternately from a translocationchain to form male (5Y) and female (5X) determiningsperm. Dosage compensation mechanisms have not beenelucidated in monotremes. Parallels have been drawnbetween epigenetic mechanisms associated with genomicimprinting and X chromosome dosage compensation infemale eutherian mammals. Hence determining the pres-ence, organisation and location of imprinted orthologuesin the monotreme can provide a useful framework forcomparative mechanistic and evolutionary studies.

Recently, different views on the evolution of imprintingmechanisms have been expressed. Two views are based onthe similarities between X chromosome inactivation(XCI) and autosomal genomic imprinting that have longbeen noted [9]. Since both have a number of features incommon, such as the association with non-coding andanti-sense RNA and some related patterns of histone mod-ifications, it has been suggested that X-inactivation wasthe 'driving force' behind the evolution of imprinting[10]. This idea has grown from the finding that, in marsu-pials, XCI is an imprinted event with the paternal X beingpreferentially inactivated in all tissues [11,12]. In Musmusculus (mouse) and Bos taurus (cow), imprinted XCI isan early event confined to extra-embryonic tissues [13,14]and occurring prior to the reprogramming of the X in theepiblast which leads to random XCI in embryonic deriva-tives [15,16]. Once inactivation was fixed on the X chro-mosome in ancestral mammals, it has been suggested thatthese mechanisms were adopted by autosomes to estab-lish genomic imprinting[10]. An alternative to the 'drivingforce' hypothesis is the view that imprinting and X-inacti-vation co-evolved when the placenta emerged [17]. In this

perspective, the evolution of placentation exerted selectivepressure to imprint growth-related genes present on boththe X and the autosomes. The basis of this model is thesuggestion that genes imprinted in the placenta utilise anon-coding RNA mechanism that parallels the function ofthe Xist non-coding RNA essential for X inactivation inplacental mammals. Most recently, data have emergedproving that marsupial imprinted X-inactivation and plat-ypus sex chromosome dosage compensation occur via amechanism that is independent of the XIST-mediatedmechanism occurring in mouse and man [18,19]. Thisfinding is not consistent with either of the two proposedmodels linking X inactivation to autosomal imprinting.

Another theory postulates that imprinted domainsevolved through chromosomal duplication and thatimprinted genes were originally located on one (or a few)ancestral pre-imprinted chromosome region(s) and thendispersed in mammalian genomes through recombina-tion or transposition events [20]. Duplication of a set ofgenes may have led to random monoallelic expression asa means of dosage compensation and, subsequently,imprinting (parental-origin specific gene activity/repres-sion) following divergence of the paralogues. If imprintedgenes were found to be located on one or two platypusautosomes this would constitute some evidence for thishypothesis. Alternatively, given the large number of plat-ypus sex chromosomes that may have epigenetically regu-lated dosage compensation mechanisms, it is possiblethat autosomal imprinted domains might have arisenthrough translocation of sex chromosome-linked genesonto autosomes carrying with them vestiges of the regula-tory sequences required for parental origin specific sexchromosome dosage compensation. It is relevant to notehowever, that the platypus sex chromosome system bearsno relationship to the XY system in viviparous mammals(Rens et al. submitted for publication).

In order to understand the emergence of imprinting afterthe divergence of monotremes from the mammalian line-age we have isolated platypus (Ornithorhynchus anatinus)and tammar wallaby (Macropus eugenii) bacterial artificialchromosome (BAC) clones that contain orthologues ofmouse and human imprinted domains and investigatedtheir localisation on tammar wallaby and platypus chro-mosomes. We have determined the chromosomal loca-tion of 8 imprinted gene orthologues in the platypus,representing 7 different clusters of imprinted genes in themouse or human (the IGF2 imprinted domain, IGF2R, theDLK1/DIO3 imprinted domain, GRB10, the GNAS com-plex, a gene from the Prader-Willi/Angelman Syndromecomplex and SLC38A4). In addition 8 imprinted geneorthologues were mapped in the tammar wallaby – aninth was mapped previously. Three of these genes belongto the Beckwith-Wiedemann Syndrome (BWS) ortholo-

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gous region and two to the DLK1/DIO3 region. The genesinvestigated here represent the best-characterisedimprinted domains known in the mammalian genomeand can be considered in the context of the informationavailable on their imprinting status. Our analysis contrib-utes to the identification of regions of syntenic homologyacross a range of vertebrates including chicken and theprototherian, metatherian and eutherian mammals.

ResultsIdentification of platypus and tammar wallaby BACs containing imprinting orthologuesEach of the imprinted genes described in this report havebeen mapped by fluorescent in situ hybridisation (FISH)of BAC clones to metaphase chromosomes of platypusand wallaby cells in culture to determine their regionalposition and in some instances, to confirm retention ofclustering across the cluster (Figures 1 & 2).

The orthologues of both the insulin like growth factor 2(IGF2) and one of its receptors (M6P/IGF2R), have previ-ously been characterised in the platypus [Gen-bank:AY552324 and Genbank:AF151172] [3,21]. IGF2 isa paternally expressed imprinted gene in both eutherianand marsupial mammals but has been shown not to beimprinted in birds and monotremes [2,4,22]. In mouseand human it forms part of a large imprinted cluster thatcan be divided into two imprinted subdomains – one con-taining the IGF2 and H19 genes, and the other containingCDKN1C and several genes showing tissue-specificimprinting in the mouse placenta including CD81. Thesetwo contiguous subdomains map to chromosome11p15.5 in humans (BWS critical region) and mouse dis-tal chromosome 7. A fragment of IGF2 was amplifiedfrom platypus DNA using primers from the highly con-served second coding exon C2 in platypus. This was usedas a probe to screen platypus and wallaby BAC libraries.M6P/IGF2R is a large gene consisting of 48 exons whichencodes a protein of 2482 amino acid residues in mouse.It is expressed from the maternally inherited chromosomein mice [23] and has also been shown to be imprinted inthe opossum Didelphis virginiana[3]. This gene is bialleli-cally expressed in monotremes and also lacks IGF2 bind-ing properties in these species [3]. To screen the wallabyBAC library, a probe was designed to Macropus rufogriseus(red-necked wallaby) IGF2R mRNA [Genbank:AF339159].

The other genes/regions chosen for this study had not pre-viously been characterised in monotremes or marsupials.The CD81 gene encodes a member of the transmembrane4 superfamily which is preferentially expressed from thematernal allele in mouse placentas [24]. CD81 is approx-imately 240 kb downstream of IGF2 in human. A probe ofthe entire human CD81 coding sequence was used toscreen the wallaby BACs and 5 positives were found. DIO3

is an intronless gene that codes for type III iodothyroninedeiodinase (D3), a 278 amino acid selenoprotein inhuman. It is a predominantly paternally expressed genewhich is part of the DLK1/DIO3 cluster which is found at14q32 in humans and distal chromosome 12 in mice.DLK1 is a Delta-like protein member of the Notch familyof signalling molecules and is found in all vertebrates.Despite this DLK1 is not as conserved as the otherimprinted genes in this study so in order to produceprobes to screen the libraries, the trace archives fromNCBI were searched with DLK1 sequences from other spe-cies. By searching the Monodelphis domestica tracearchive with human DLK1 [Genbank:NM_003836],TI_395847291 was identified and a probe designed tothe most conserved regions between the two sequenceswas used to screen the wallaby library. Chicken DLK1

FISH mapping on platypus metaphase chromosomes of BACs containing orthologues of imprinted genesFigure 1FISH mapping on platypus metaphase chromosomes of BACs containing orthologues of imprinted genes. (A) DIO3, (B) DLK1, (C) IGF2R, (D) SLC38A4, (E) IGF2, (F) GRB10, (G) GNAS (and platypus 8 paint in green) and (H) UBE3A. Scale bar is 10 !m.

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sequence [Genbank:XM_421369] identified the platypustrace file TI_752207707 to which a probe was designed toscreen the platypus library. The growth factor receptor-bound protein 10 gene (GRB10) is expressed from thepaternally inherited chromosome in both mouse andhuman brain. In other organs, it is maternally expressed inmouse and biallelically expressed in the human. Itappears to be a solitary imprinted gene which is locatedon human 7p12 and mouse proximal 11. The GNAS com-plex is located on human 20q13.3 and mouse distal 2.This is a complex domain with a number of differentiallyimprinted, alternatively spliced transcripts. The guaninenucleotide binding protein, alpha stimulating gene(GNAS) is highly conserved in vertebrates. The Prader-Willi/Angelman Syndrome cluster is located at human15q11–13 and mouse central chromosome 7. This is a

large cluster that spans 4 Mb in human and includes theubiquitin protein ligase E3A gene (UBE3A) that isexpressed from the maternally inherited chromosome.This gene has previously been assigned to wallaby chro-mosome 5 [25,26]. Finally, solute carrier family 38, mem-ber 4 (SLC38A4 also called ATA3) is located on human12q13 and mouse distal chromosome 15. It is found in agene cluster with two other solute carriers of which it isthe only imprinted one, being repressed on the maternallyinherited chromosome. Probes were designed to highlyconserved regions in each of these genes and used toscreen platypus and tammar wallaby BAC libraries.

Further information on all probes used for library screens,the sequences they were designed against and the numberof BACs identified can be found in [see Additional file 1].

FISH mapping of Platypus BACsThe platypus karyotype (2 n = 52) consists of 21 auto-somes and 10 sex chromosomes (5X's and 5Y's in maleand 5 X-pairs in female). One positive BAC for each genewas chosen for FISH analysis. The BACs were labelled withbiotin using a standard nick translation protocol andlocalised on platypus chromosomes by FISH on maleplatypus metaphase preparations. Fig 1a shows the local-ization of DIO3 to a site distal to the centromere of platy-pus chromosome 1. DLK1 maps close to DIO3 in platypus(Fig 1b) IGF2R and SLC38A4 both localise to platypuschromosome 2, IGF2R to a position close to the centro-mere of chromosome 2, and SLC38A4 to distal 2q (Fig 1cand 1d). IGF2 maps to distal platypus chromosome 3p(Fig 1e). GRB10 is positioned near the centromere of plat-ypus 4 (Fig 1f). Fig 1g shows GNAS on platypus chromo-some 8 as confirmed by FISH using a chromosome 8specific paint. A fainter signal was also observed on platy-pus X5. UBE3A is found on platypus chromosome 18 (Fig1h). All gene locations are shown on the platypus G-banded karyotype (Fig 3).

FISH mapping of Tammar Wallaby BACsThe tammar wallaby karyotype (2 n = 16) consists of 7autosomes and the two sex chromosomes. The tiny Ychromosome is not shown in Figure 3. The genes werelocalised on male tammar wallaby metaphase chromo-somes using FISH with labelled BAC DNA (as above).DIO3 and DLK1 (Fig 2a) were mapped to tammar wallabychromosome 1q about one third distal from the centro-mere. GNAS also was mapped to chromosome 1q butconsiderably more distal from the centromere (Fig 2b).IGF2, CD81, and MRLP23 were mapped to the samecytogenetic region on tammar wallaby chromosome 2p(Fig 2c, 2d and 2g). GRB10 localised to tammar wallaby3p (Fig 2f).IGF2R was mapped to 2q, half way down thatarm (Fig 2e). SLC38A4 was mapped to tammar wallaby

FISH mapping on tammar metaphase chromosomes of BACs containing orthologues of imprinted genesFigure 2FISH mapping on tammar metaphase chromosomes of BACs containing orthologues of imprinted genes. (A) DIO3 (green) and DLK1 (red), (B) GNAS, (C) IGF2, (D) CD81, (E) IGF2R, (F) GRB10, (G) MRPL23, and (H) SLC38A4 (red) with chromosome 3 in green. Scale bar is 10 !m.

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chromosome 3p, as confirmed by chromosome paintingwith a chromosome 3 specific paint (Fig. 2h).

Conservation of syntenyDlk1 and Dio3 encompass a 1 MB region in the mouse. Inorder to ascertain whether synteny is conserved within theDLK1/DIO3 domain, DLK1 containing BACs were identi-fied in both species. One BAC from each species was usedfor FISH analysis. DIO3 (Fig 1a) and DLK1 (Fig 1b)mapped to a site on the long arm 1/3 the arm length fromthe centromere of platypus chromosome 1. In tammarwallaby DLK1 and DIO3 also mapped to the same loca-tion as shown by FISH analysis with the probes labelled intwo different colours (Fig 2a).

Lambda clones containing IGF2 have previously beenmapped to tammar wallaby chromosome 2p [27]. Inorder to confirm this location and see if synteny was con-served in this species, BACs containing 2 other genes fromthis region were isolated. CD81 is preferentially expressedfrom the maternally inherited allele in mouse placentas.MRPL23 is located 175 kb upstream of IGF2 in humansand it encodes the mitochondrial ribosomal protein L23.This gene does not appear to be imprinted in mammals.Hence the genes selected here fall into three differentfunctional and regulatory categories which may not haveconserved ancestral linkage. For example the two differentimprinted subdomains might be separated from eachother and/or the unrelated mitochondrial protein. One

Location of orthologues of mammalian imprinted genes on the karyotypes of platypus (A) and tammar wallaby (B) in redFigure 3Location of orthologues of mammalian imprinted genes on the karyotypes of platypus (A) and tammar wallaby (B) in red. Gene names in black are those previously mapped genes from other studies, (reviewed in [25, 44]). The position of the orthol-ogous genes in human are shown on the left.

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positive BAC for each of these genes was used for FISHwhich showed that IGF2,CD81 and MRPL23 do indeedmap together on tammar wallaby chromosome 2p (Fig2c, 2d and 2g).

The location of imprinted orthologues in the chicken byin-silico methods has been recently published [28]. Wehave also performed an in-silico analysis to identify thechromosomal locations of the imprinted genes in theopossum using the UCSC genome browser[29,30]. Theresults of this analysis and the FISH mapping are summa-rised in Table 1.

In silico identification of orthologues and chromosomal locations of genes contiguous with imprinted genesThe transcripts of the human imprinted genes werealigned by BLAST to find orthologues within the EnsemblPlatypus Ornithorhynchus anatinus database release 5. Theplatypus contigs in the database contain several predictedgenes, which were then identified by blasting to findalignments with the NCBI human genome database.Orthologues of these genes were subsequently localised inchicken and opossum by BLAST alignment in Ensembl.The results are shown in Table 1 and Table 2.

DLK1 is located on platypus 1q in ultracontig378 whichcontains 49 predicted genes, most of them with ortho-logues on human 14q, chicken 5, and opossum 1. Thethree genes that have orthologues elsewhere might bemistakes in the ultracontig assembly. Genes that arepresent on either side of DIO3 on human chromosome14q are mapped in the same platypus ultracontig378. Onopossum chromosome 1 DLK1 and DIO3 are 1.6 Mbapart according to Ensembl-opossum. The platypusultracontig378 does not correspond to a continuousregion in opossum. The predicted genes betweenKIAA1622 and GSC are not identified in opossum but

instead are replaced by regions homologous to regionsother then human 14q13.2 and chicken 5.

GNAS is located on platypus 8p in contig16 together with31 other genes (4 unidentified) all of which have ortho-logues on human 20q13, chicken 20, and opossum 1.Only one gene (Fam38A) is located on human 16q andchicken 11 and is probably a mistake in this contig assem-bly. GRB10 was found on platypus 4p in contig107, whichcontains 13 other genes. All of the genes have orthologueson chicken 2. Three of them have orthologues on human7q36.1 and the other eight are on human 7p12 (4 genesare unidentified). An inversion in the eutherian lineageseparated these genes from each other. In the marsupialMonodelphis domestica these two gene clusters are not syn-tenic but are localized on different chromosomes (chro-mosome 8, 6 and 3 respectively, Ensembl Opossumrelease 4). IGFR2 and SLC38A4 are found in small contigswith a limited number of genes.

UBE3A is located on platypus 18p in contig121 togetherwith 15 other genes (3 unidentified). All of the genes haveorthologues on chicken 1. However, UBE3A is localizedon human 15q. Two other genes are on human 2q and theremaining genes in this contig are on human Xp21.2 orXp11.4. As these genes are syntenic in platypus andchicken, this contig represents the ancestral configuration.Before the marsupial-eutherian split, one fission sepa-rated the human Xp region from the human 15q andhuman 2q regions; the latter two regions are still togetherin opossum. A subsequent fission in the eutherian lineageseparated the human 15q and human 2q regions. Unfor-tunately, IGF2, CD81, MRPL23 and SNRPB are not yet rec-ognised in the Ensembl Platypus Ornithorhynchus anatinusdatabase 5.

This approach identified conserved synteny at the major-ity of extended loci examined. We identified one large

Table 1: Summary of chromosomal locations of genes studied in human, mouse, wallaby, opossum, platypus and chicken

Gene Human location Mouse location Wallaby location

Opossum location

Platypus location

Chicken location

DIO3 14q Distal 12 1q 1 1q 5DLK1 14q Distal 12 1q 1 1q 5GNAS 20q Distal 2 1q 1 8p 20GRB10 7p Proximal 11 3p 6 4p 2IGF2 11p Distal 7 2p 5q [35] 3p 5CD81 11p Distal 7 2p Unplaced - 5

MRPL23 11p Distal 7 2p Unplaced - 5IGF2R 6q Proximal 17 2q 2 Centric 2 3

SLC38A4 12q Distal 15 3q 8 2q 1UBE3A 15q Central 7 5 [23] 7 18p 1SNRPB 20p 2 1q [23] 1 - 20

Chicken and opossum locations are taken from the UCSC genome browser except for opossum IGF2 [35]

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Table 2: In silico identification of orthologues and chromosomal locations of genes contiguous with imprinted genes.

Platypus Contig Predicted gene Human orthologue Human location Chicken assignment Opossum assignment

1q Ultracontig378 Ox_plat42681 LGMN 14q32.12 5 1Ox_plat497848 High E-value - - -Nov499641/Nov5433 GOLGA5 14q32.12 5 1Nov5434 CHGA 14q32.12 5 1Ox_plat52244 ITPK1 14q32.12 5 1Ox_plat7773 MOAP1 14q32.13 5 -Ox_plat1058 C14orf130 14q32.13 5 1Nov0444/Ox_plat88666 BTBD7 14q32.13 5 1Ox-plat454053/Nov11241/Ox_plat35285 KIAA1409 14q32.13 5 1Ox_plat548259 High E-value - - -Nov11242/Ox_plat367667 ASB2 14q32.13 5 1Nov11243 High E-value 14q32.13 - -Nov11244/Ox_plat4560 OTUB2 14q32.13 5 1Nov11245 KIAA1622 14q32.13 5 1Ox_plat116474 KIAA1622 14q32.13 5 1 insertionNov11246/Ox_plat42680 High E-value - - - insertionNov12040/Ox_plat411980 SERPINA11 14q32.13 5 - insertionNov12038/Ox_plat468381/Nov12037 High E-value - - - insertionNov12036 GSC 14q32.13 5 1 insertionNov12034/Ox_plat475598 DICER1 14q32.13 5 1Nov12033 CLMN 14q32.13 5 -Nov9154 C14orf49 14q32.13 5 1Ox_plat409095 High E-value - - -Nov65921/Nov6591 BDKRB2 14q32.2 5 1Nov65905/Ox_plat42182/Nov6589;6588 C14orf103 14q32.2 5 1Nov6588 C14orf129 14q32.2 5 1Nov13812/Ox_plat6298 PAPOLA 14q32.2 5 -Nov12141/Ox_plat6576 VRK1 14q32.2 5 1Nov14770 BCL11B 14q32.2 5 1Nov14773 KRT19 17q21.2 27 2Nov14774/Nov3153/Ox_plat403179 SETD3 14q32.2 5 1Nov2410 KIAA1822 14q32.2 5 1Nov7692 CYP46A1 14q32.2 5 -Nov7691 LOC91461 14q32.2 5 -Nov4045/Ox_plat43367 EML1 14q32.2 5 1Nov12424/Ox_plat123106 DEGS2 14q32.2 5 1Nov5723/Ox_plat43391 YY1 14q32.2 5 1Nov5724 SLC25A29 14q32.2 5 1Nov5726 C14orf68 14q32.2 5 1Nov5727/Ox_plat494845 WARS 14q32.2 5 1Nov5730 High E-value - - -

* Nov5731 DLK1 14q32.2 5 1Nov5732/Ox_plat43477 DNAH1 3p21.1 12 6Nov5733/Ox_plat522828 DYNC1H1 14q32.32 5 1Nov5734/Ox_plat6302 HSP90AA1 14q32.32 3 -Nov5735/Ox_plat461283 WDR20 14q32.32 5 1Nov5736/Ox_plat549051/Ox_plat5738/Ox_plat456837

RAGE 14q32.32 5 1

Nov5740/Ox_plat526165 KIAA0329 14q32.32 5 1Nov5741 ANKRD9 14q32.32 5 1Nov9609 KIAA1446 14q32.2 5 -

8p Contig16 Ox_plat6649 CYP24A 20q13.2 20 1Ox_plat6759 PFDN 20q13.2 20 -Ox_plat44306 DOK 20q13.2 20 -Ox_plat1864 CBLN4 20q13.31 20 1Ox_plat373291 High E-value - - -Ox_plat6760 CSTF1 20q13.31 20 1Ox_plat485472 C20orf32 20q13.31 20 1Ox_plat509072 C20orf43 20q13.31 20 1Ox_plat21038/Ox_plat49662 High E-value - - -Ox_plat50570 BMP7 20q13.31 20 1Ox_plat6664 SPO11 20q13.32 20 1Ox_plat6762 RAE1 20q13.32 20 1Ox_plat501210 RBM30 20q13.32 20 -Ox_plat21169 CTCFL 20q13.32 20 1Ox_plat364767 PCK1 20q13.32 20 1Ox_plat50577 TMEPAI 20q13.32 20 1Nov 9262 TMEPAI 20q13.32 20 1Ox_plat21326 C20orf80 20q13.32 20 -Ox_plat21104 RAB22A 20q13.32 20 1Ox_plat21317 C20orf80 20q13.32 20 -Ox_plat69044 PPP4R1L 20q13.32 20 1

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inversion, and potential errors in the platypus contigassembly. Finally, we determined that the UBE3A regionon platypus chromosome 18 and chicken chromosome 1represent an ancestral configuration of 15 genes whichduring eutherian evolution has undergone fission placingseveral of them on two regions on the human X chromo-some.

DiscussionStudies that consider the chromosomal relationshipsbetween autosomal imprinting and dosage compensationmechanisms in the range of mammals that includemonotremes, marsupials, mouse and man are likely toprovide insights into the evolution of the mechanismsinvolved. In a wider context, this will aid in understandingthe evolution of epigenetic controls regulating genomefunction.

Monotremes, due to their early offshoot from the othermammalian species, are an ideal class for various kinds ofgenetic, cytogenetic and epigenetic research. Whereasmost male mammals have an XY complement and femalebirds have a ZW complement, the male platypus has five

X- and five Y chromosomes. Furthermore, X5 carries theDMRT1 orthologue present on the avian Z and thought tobe sex determining. Platypus X1 was previously thought toshow homology with the human X (see for example ref29), but this is not confirmed by the draft platypussequence (Ensembl release 44) that instead shows homol-ogy to chicken chromosome 3, 11, 12, 13, and Z andhuman chromosome 2 and 5 (Rens et al submitted). The-rian X-linked genes mapped to date are predominantlylocalised to platypus chromosome 6 [31]. The results indi-cate that the monotreme sex chromosome system is unre-lated to the XY sex chromosome system of othermammals which must have arisen after the divergence ofmonotremes 166 MYA. This intriguing system combinedwith an apparent absence of genomic imprinting makes itimportant to localize imprinted genes on platypus chro-mosomes in order to consider the evolution of epigeneti-cally regulated dosage compensation systems. Theselocalizations also serve to define regions of syntenichomology between vertebrates including monotremesand eutherian mammals. In addition, the placement ofsuch genes on the cytogenetic map will contribute to

Ox_plat50567 VAPB 20q13.32 20 1Ox_plat499610 STX16 20q13.32 20 -Nov9272 Fam38A 16q24.3 11 1Ox_plat499488 C20orf45 20q13.32 20 1

* Nov9275 GNAS 20q13.32 20 1Ox_plat6777 TUBB1 20q13.32 - 1Ox_plat21080 High E-value 20q13.32 - -Nov9282 C20orf174 20q13.32 20 1Ox_plat4456 PHACTR3 20q13.33 20 1Ox_plat454388 CDH26 20q13.33 - 1

4p Contig107 Ox_plat400863 CUL1 7q36.1 2 8Ox_plat10731 EZH2 7q36.1 2 3Nov0280 PDIA4 7q36.1 2 8Nov0281 High E-value - - -Ox_plat49897 COBL 7p12.1 2 6

* Ox_plat451754/Nov0283 GRB10 7p12.1 2 6Nov0284 DDC 7p12.2 2 6Nov0285 FIGNL1 7p12.2 - 6Ox_plat403769/Nov0286 IKZF1 7p12.2 2 6Nov0287 ZPBP 7p12.2 2 6Nov0288 High E-value - - -Ox_plat4844817/Ox_plat467096 High E-value - - -

2 Contig1301 Nov7295 SLC22A2 6q25.3 3 2* Q9N1T1 IGF2R 6q25.3 3 2

2q Contig538 Nov6345/Nov6346/Ox_plat486528 SLC38A1 12q13.11 1 8* Nov6348 SLC38A4 12q13.11 1 8

18p Contig121 Ox_plat15397 UBE3A 15q11.2 1 7Ox_plat498667 MGC26733 2q11.2 1 -Ox_plat3315 TMEM131 2q11.2 1 7Ox_plat59493 TMEM47 Xp21.2 1 4Nov7105/Nov7106/Ox_plat390388 High E-value - 1Ox_plat85743 CXorf22 Xp21.2 1 4Ox_plat472396 PRRG1 Xp21.2 1 4Ox_plat1731 XK Xp11.4 1 4Nov7111 CYBB Xp11.4 1 4Ox_plat85753 DYNLT3 Xp11.4 1 4Ox_plat514733 SYTL5 Xp11.4 1 4Ox_plat7251 SRPX Xp11.4 1 4Ox_plat375200 RPGR Xp11.4 1 4Ox_plat1440 OTC Xp11.4 1 4

Table 2: In silico identification of orthologues and chromosomal locations of genes contiguous with imprinted genes. (Continued)

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anchoring the platypus genomic sequence currently beinggenerated.

Here we mapped the chromosomal location of imprintedgenes in the platypus and tammar wallaby. Eightimprinted gene orthologues (representing six differentimprinted mouse/human clusters) localized to 6 differentautosomes in the platypus as shown in Fig. 1a–h. In tam-mar wallaby eight imprinted gene orthologues (threebelonging to the BWS region) representing the same siximprinted domains were mapped to 5 of the 7 differentautosomes. First, the results will be discussed in relationto other genes mapped in platypus and tammar wallaby.Second, the imprinted gene orthologue localization willbe discussed in relation to imprinting evolution.

Comparative gene mappingGene mapping is one of the tools used to define regionsthat are conserved between different species. The localiza-tion of orthologues of imprinted genes (red) on platypuschromosomes is presented in Figure 3a with genesmapped previously in black [31-34]. Gene mapping dataare still limited in platypus, hence mapping the ortho-logues of imprinted genes will anchor contigs to specificchromosomes and aid in constructing a platypus-humanhomology map.

The localization of orthologues of imprinted regions intammar wallaby is presented in Fig 3b. We show that IGF2is located at the telomere of tammar chromosome 2. Arecent paper placed the M. domestica orthologue of IGF2on 5q [35] a region that was previously shown to beequivalent to 6p in tammar [36]. This discrepancy mightbe due to the poor resolution of chromosome paints atthe telomeres and suggests that there may be a smallregion at the tip of M. domestica 5q which is homologousto 2p in the tammar wallaby. It is interesting that GNASand SNRPB are close on tammar wallaby 1p (our resultsand Rapkins et al[26]), which is part of a region that isconserved in a large set of marsupial species[37]. GNAS islocated on human distal 20p and SNRPB on distal 20q.Human chromosome 20 is a chromosome that is con-served in all eutherian mammals, the mapping of GNASand SNRPB indicates that it is conserved in marsupials aswell. The four homologous regions mapped in this reportadd to the complexity of the rearrangements that haveoccurred during chromosome evolution between humanand tammar wallaby. For instance, tammar wallaby chro-mosome 1 has regions homologous to human 5, 7, 9, 10,14, 16, 20 and X (our results and Alsop et al.[25])

Imprinted gene orthologue localizationThe overall conclusion made from the mapping data isthat the orthologues of these imprinted genes are notfound on sex chromosomes in either species. Although

the mechanism of dosage compensation remains to bedetermined in platypus, the lack of imprinted orthologueson sex chromosomes does not favour the idea thatimprinted genes arose as duplications from the X.

This, and the absence of imprinting in the platypus todate, suggests that monotreme X chromosome dosagecompensation preceded genomic imprinting which sub-sequently adopted the same mechanism, or that sex chro-mosomes dosage compensation in monotremes is anunrelated event. The latter is more likely since monotremesex chromosomes share no homology with the human X(Rens et al submitted). The position of orthologues ofimprinted genes provides no insight regarding thehypothesis of co-evolution of X-inactivation and imprint-ing in mammals being associated with placentation [17].

The results show that the selected imprinted gene clustersare scattered among autosomes in the platypus and tam-mar wallaby karyotypes; the clusters do not grouptogether in either species. Data from comparison of thedistribution of the imprinted gene orthologues in platy-pus and tammar wallaby with their locations in thehuman karyotype reflects the high number of rearrange-ments that occurred in the lineages of either themonotremes or placental mammals. The position ofgenes on the prototherian ancestor will be more relevantto evaluating the imprinting duplication hypothesis andcomparing it with data generated here. However, the pro-totherian ancestral karyotype remains to be determinedand will be assisted by the establishment of a genomewide comparison between monotremes/marsupials andan outgroup species.

The SNRPN gene in the PWS/AS cluster arose from a tan-dem duplication of the SNRPB gene so its syntenic rela-tionship with imprinted GNAS is of interest. The SNRPBduplication had already occurred when the marsupialsdiverged from the eutherian line as SNRPB and SNRPNare tandemly arranged in both tammar and opossum. Insilico analysis of this region in the chicken shows that thereis only one copy of SNRPB and that it is only 166.9 kbaway from GNAS on chromosome 20 implying that thesegenes were close in the ancestral mammalian karyotype.

In-silico analysis reveals that SNRPB and GNAS are 36.6Mb apart in the opossum and 54.5 Mb apart in human.Therefore although these two genes are located on thesame chromosome they have become separated by one ormore inversions. Furthermore, in opossum, tammar, plat-ypus, chicken and zebrafish, the PWS/AS genes SNRPNand UBE3A are on separate chromosomes and areexpressed biallelically in tammar [26]. Together thesefindings suggest that imprinted regulation was acquiredafter the loss of close synteny with GNAS and a major rear-

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rangement that united SNRPN and UBE3A. However, italso remains theoretically possible that the SNRPN andUBE3A genes lost imprinting in the macropodid lineageand their imprinting state is ancestral for therians.

ConclusionThe combined data of chicken, marsupial and platypusgene position suggest that imprinted gene orthologueshave existed on separate chromosomes since beforeimprinting evolved. This makes the hypothesis, that therewas a single or small number of ancestrally imprintedchromosomes, unlikely. The observation that someimprinted domains in mouse and human are notimprinted in marsupials, suggests that imprinting was astep wise process during evolution beginning after theevolution of viviparity and continuing convergently in themarsupial and eutherian lineages. Thus the evolution ofimprinting has most likely been a long process with eachcluster independently evolving or indeed losing, itsimprinting mechanisms as the need arose. This suggestsan element of adaptation in the process of imprinting evo-lution.

MethodsAmplification and sequence analysisThe published coding sequences of the genes of interestwere obtained from as many species as possible from Ent-rez Gene at the NCBI webpage [38] [Additional File 1].Sequences were then aligned to each other using the Clus-talW program [39,40] and PCR Primers designed to theregions of greatest homology within the same exon.

Platypus genomic DNA (gDNA) was extracted from pri-mary fibroblasts using standard protocols [41]. IGF2,DIO3 and SLC38A4 were amplified in a 15 !l reactioncontaining 1! NEB buffer [42], 500 !M dNTPs, 2.5 !g BSA(Sigma), 0.067% v/v "-mercaptoethanol, 0.6 U Taqpolymerase (Applied Biosystems), 0.75 !M of each primerand 50 ng gDNA. GNAS, GRB10 and IGF2R were ampli-fied in a 25 !l reaction containing 1! PCR Buffer (Bio-line), 1.5 mM MgCl, 250 !M dNTPs, 1.5 U Taqpolymerase (Bioline), 0.3 !M of each primer and 50 nggDNA. PCR cycling was, 94°C for 5 min, 35 cycles at 94°Cfor 30 sec, annealing temperature (specific for each primersee table 1) for 30 sec, 72°C for 30 sec and 5 min at 72°C.UBE3A was amplified in a 25 !l reaction containing 1!PCR Buffer (KOD Hot Start, Novagen), 300 !M dNTPs, 1mM MgSO4, 0.5 U Hot Start KOD polymerase, 0.6 !M ofeach primer and 50 ng gDNA. PCR cycling was 94°C for 2min, 31 cycles of 94°C for 15 sec, 60°C for 30 sec, 68°Cfor 30 sec, then 5 minute at 68°C.

The PCR products were separated by electrophoresis andthe appropriately sized fragments were excised andcleaned (Qiaquick Gel Extraction Kit; Qiagen). These frag-

ments were cloned into pCR® 2.1-TOPO® (Invitrogen)using the manufacturers protocol. DNA from the plas-mids was prepared using GeneElute™ Plasmid MiniprepKit (Sigma) then sequenced to confirm its identity.

BAC IsolationThe OA_Bb Platypus BAC library (Clemson UniversityGenomics Institute, South Carolina, USA) and the ME_KBa Tammar wallaby BAC library (Arizona GenomicsInstitute, USA) were screened with [#-32P] dCTP (Amer-sham Pharmacia Biotech) labelled PCR products. Label-ling was performed under the following conditions 94°Cfor 5 minutes, 25 cycles of 93°C for 30 sec, 50°C for 30sec 72°C for 30 sec and 1 cycle of 72°C for 5 min. Probeswere denatured at 99°C for 5 min and snap chilled beforehybridisation. The library membranes were hybridisedand washed at low stringency (55°C). They were thenexposed to X-ray film at -70°C overnight. BACs werestreaked to single colony and tested by PCR with theiridentifying primers to ensure they contained the correctgene.

Preparation of BAC ProbesBAC DNA was isolated using the protocol described at theWellcome Trust Sanger Institute methods website [42].The DNA probes were labelled by nick translation withBiotin-16-dUTP using a standard protocol.

Localization of DNA probesChromosome specific DNA was prepared from flow-sorted platypus chromosomes and fluorescence in situhybridization was performed according to protocolsdescribed previously [7,43]. The labelled DNA probes(and chromosome paints for chromosome identification)were hybridized to male platypus and wallaby chromo-some preparations and detected with Cy3-avidin.

Image analysisImages were captured using the Leica QFISH software(Leica Microsystems) and a cooled CCD camera (Photo-metrics Sensys) mounted on a Leica DMRXA microscopeequipped with an automated filter wheel, DAPI, FITC, andCy3 specific filter sets and a 63!, 1.3 NA objective or100!, 1.4 NA objective.

AbbreviationsMYA – Million Years Ago

XCI – X Chromosome Inactivation

BAC – Bacterial Artificial Chromosome

FISH – Fluorescent In-situ Hybridisation

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Authors' contributionsCAE and AM conducted BAC library screening and probecharacterisation, WR and OC conducted FISH experi-ments, and TH conducted gene characterisation. CAE andWR carried out in silico analysis. CAE, WR, AFS drafted themanuscript, ID, MAFS and JMG contributed reagents andprovided input to the manuscript, AFS and MAFS con-ceived, designed and coordinated the study.

Additional material

AcknowledgementsCAE is funded by an MRC studentship. AFS is an associate member of the FP6 Epigenome Network of Excellence. We are grateful to the other mem-bers of the SAVOIR consortium, including Wolf Reik and Gavin Kelsey for helpful discussions during the course of this work. The research was sup-ported by a grant from the Wellcome Trust to the Cambridge Resource Centre for Comparative Genomics.

References1. Da Rocha ST, Ferguson-Smith AC: Genomic imprinting. Curr Biol

2004, 14:R646-9.2. Killian JK, Nolan CM, Stewart N, Munday BL, Andersen NA, Nicol S,

Jirtle RL: Monotreme IGF2 expression and ancestral origin ofgenomic imprinting. J Exp Zool 2001, 291:205-212.

3. Killian JK, Byrd JC, Jirtle JV, Munday BL, Stoskopf MK, MacDonald RG,Jirtle RL: M6P/IGF2R imprinting evolution in mammals. MolCell 2000, 5:707-716.

4. O'Neill MJ, Ingram RS, Vrana PB, Tilghman SM: Allelic expressionof IGF2 in marsupials and birds. Dev Genes Evol 2000, 210:18-20.

5. Suzuki S, Renfree MB, Pask AJ, Shaw G, Kobayashi S, Kohda T,Kaneko-Ishino T, Ishino F: Genomic imprinting of IGF2,p57(KIP2) and PEG1/MEST in a marsupial, the tammar wal-laby. Mech Dev 2005, 122:213-222.

6. Bininda-Emonds OR, Cardillo M, Jones KE, MacPhee RD, Beck RM,Grenyer R, Price SA, Vos RA, Gittleman JL, Purvis A: The delayedrise of present-day mammals. Nature 2007, 446:507-512.

7. Rens W, Grutzner F, O'Brien P C, Fairclough H, Graves JA, Ferguson-Smith MA: Resolution and evolution of the duck-billed platy-pus karyotype with an X1Y1X2Y2X3Y3X4Y4X5Y5 male sexchromosome constitution. Proc Natl Acad Sci U S A 2004,101:16257-16261.

8. Grutzner F, Rens W, Tsend-Ayush E, El-Mogharbel N, O'Brien PC,Jones RC, Ferguson-Smith MA, Marshall Graves JA: In the platypusa meiotic chain of ten sex chromosomes shares genes withthe bird Z and mammal X chromosomes. Nature 2004,432:913-917.

9. Cattanach BM, Beechey CV: Autosomal and X-chromosomeimprinting. Dev Suppl 1990:63-72.

10. Lee JT: Molecular links between X-inactivation and auto-somal imprinting: X-inactivation as a driving force for theevolution of imprinting? Curr Biol 2003, 13:R242-54.

11. Sharman GB: Late DNA replication in the paternally derivedX chromosome of female kangaroos. Nature 1971,230:231-232.

12. Richardson BJ, Czuppon AB, Sharman GB: Inheritance of glucose-6-phosphate dehydrogenase variation in kangaroos. Nat NewBiol 1971, 230:154-155.

13. Takagi N, Sasaki M: Preferential inactivation of the paternallyderived X chromosome in the extraembryonic membranesof the mouse. Nature 1975, 256:640-642.

14. Xue F, Tian XC, Du F, Kubota C, Taneja M, Dinnyes A, Dai Y, LevineH, Pereira LV, Yang X: Aberrant patterns of X chromosomeinactivation in bovine clones. Nat Genet 2002, 31:216-220.

15. Okamoto I, Otte AP, Allis CD, Reinberg D, Heard E: Epigeneticdynamics of imprinted X inactivation during early mousedevelopment. Science 2004, 303:644-649.

16. Mak W, Nesterova TB, de Napoles M, Appanah R, Yamanaka S, OtteAP, Brockdorff N: Reactivation of the paternal X chromosomein early mouse embryos. Science 2004, 303:666-669.

17. Reik W, Lewis A: Co-evolution of X-chromosome inactivationand imprinting in mammals. Nat Rev Genet 2005, 6:403-410.

18. Duret L, Chureau C, Samain S, Weissenbach J, Avner P: The XistRNA gene evolved in eutherians by pseudogenization of aprotein-coding gene. Science 2006, 312:1653-1655.

19. Hore T, Koina E, Wakefield M, Graves JAM: XIST is absent fromthe X chromosome, and its flanking region is disrupted innon-placental mammals. Chromosome Res 2007.

20. Walter J, Paulsen M: The potential role of gene duplications inthe evolution of imprinting mechanisms. Hum Mol Genet 2003,12 Spec No 2:R215-20.

21. Weidman JR, Murphy SK, Nolan CM, Dietrich FS, Jirtle RL: Phyloge-netic footprint analysis of IGF2 in extant mammals. GenomeRes 2004, 14:1726-1732.

22. Nolan CM, Killian JK, Petitte JN, Jirtle RL: Imprint status of M6P/IGF2R and IGF2 in chickens. Dev Genes Evol 2001, 211:179-183.

23. Barlow DP, Stoger R, Herrmann BG, Saito K, Schweifer N: Themouse insulin-like growth factor type-2 receptor isimprinted and closely linked to the Tme locus. Nature 1991,349:84-87.

24. Lewis A, Mitsuya K, Umlauf D, Smith P, Dean W, Walter J, Higgins M,Feil R, Reik W: Imprinting on distal chromosome 7 in the pla-centa involves repressive histone methylation independentof DNA methylation. Nat Genet 2004, 36:1291-1295.

25. Alsop AE, Miethke P, Rofe R, Koina E, Sankovic N, Deakin JE, HainesH, Rapkins RW, Marshall Graves JA: Characterizing the chromo-somes of the Australian model marsupial Macropus eugenii(tammar wallaby). Chromosome Res 2005, 13:627-636.

26. Rapkins RW, Hore T, Smithwick M, Ager E, Pask AJ, Renfree MB,Kohn M, Hameister H, Nicholls RD, Deakin JE, Graves JA: Recentassembly of an imprinted domain from non-imprinted com-ponents. PLoS Genet 2006, 2:e182.

27. Toder R, Wilcox SA, Smithwick M, Graves JA: The human/mouseimprinted genes IGF2, H19, SNRPN and ZNF127 map totwo conserved autosomal clusters in a marsupial. Chromo-some Res 1996, 4:295-300.

28. Dunzinger U, Nanda I, Schmid M, Haaf T, Zechner U: Chickenorthologues of mammalian imprinted genes are clustered onmacrochromosomes and replicate asynchronously. TrendsGenet 2005, 21:488-492.

29. UCSC Genome Browser [http://genome.ucsc.edu/]30. Karolchik D, Baertsch R, Diekhans M, Furey TS, Hinrichs A, Lu YT,

Roskin KM, Schwartz M, Sugnet CW, Thomas DJ, Weber RJ, HausslerD, Kent WJ: The UCSC Genome Browser Database. NucleicAcids Res 2003, 31:51-54.

31. Waters PD, Delbridge ML, Deakin JE, El-Mogharbel N, Kirby PJ, Car-valho-Silva DR, Graves JA: Autosomal location of genes fromthe conserved mammalian X in the platypus (Ornithorhyn-chus anatinus): implications for mammalian sex chromo-some evolution. Chromosome Res 2005, 13:401-410.

32. Wallis MC, Delbridge ML, Pask AJ, Alsop AE, Grutzner F, O'Brien PC,Rens W, Ferguson-Smith MA, Graves JA: Mapping platypus SOXgenes; autosomal location of SOX9 excludes it from sexdetermining role. Cytogenet Genome Res 2007, 116:232-234.

33. Delbridge ML, Wallis MC, Kirby PJ, Alsop AE, Grutzner F, Graves JA:Assignment of SOX1 to platypus chromosome 20q by fluo-rescence in situ hybridization. Cytogenet Genome Res 2006,112:342L.

34. Kirby PJ, Waters PD, Delbridge M, Svartman M, Stewart AN, NagaiK, Graves JA: Cloning and mapping of platypus SOX2 andSOX14: insights into SOX group B evolution. CytogenetGenome Res 2002, 98:96-100.

Additional file 1Probes used for BAC library screening, shows the number of BACs identi-fied by each probe.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2148-7-157-S1.doc]

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35. Lawton BR, Obergfell C, O'Neill RJ, O'Neill MJ: Physical mappingof the IGF2 locus in the South American opossum Monodel-phis domestica. Cytogenet Genome Res 2007, 116:130-131.

36. Rens W, O'Brien PC, Yang F, Solanky N, Perelman P, GraphodatskyAS, Ferguson MW, Svartman M, De Leo AA, Graves JA, Ferguson-Smith MA: Karyotype relationships between distantly relatedmarsupials from South America and Australia. ChromosomeRes 2001, 9:301-308.

37. Rens W, O'Brien PC, Fairclough H, Harman L, Graves JA, Ferguson-Smith MA: Reversal and convergence in marsupial chromo-some evolution. Cytogenet Genome Res 2003, 102:282-290.

38. NCBI Entrez Gene [http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene]

39. ClustalW [http://www.ebi.ac.uk/clustalw/index.html]40. Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving

the sensitivity of progressive multiple sequence alignmentthrough sequence weighting, position-specific gap penaltiesand weight matrix choice. Nucleic Acids Res 1994, 22:4673-4680.

41. Sambrook J, MacCallum P, Russell D: Molecular Cloning: A Labo-ratory Manual. Third edition. , CSHL Press; 2001.

42. Wellcome Trust Sanger Institute - Human Genome Meth-ods [http://www.sanger.ac.uk/HGP/methods/]

43. Rens W, O'Brien PC, Yang F, Graves JA, Ferguson-Smith MA: Kary-otype relationships between four distantly related marsupi-als revealed by reciprocal chromosome painting. ChromosomeRes 1999, 7:461-474.

44. Grutzner F, Deakin J, Rens W, El-Mogharbel N, Marshall Graves JA:The monotreme genome: a patchwork of reptile, mammaland unique features? Comp Biochem Physiol A Mol Integr Physiol2003, 136:867-881.

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APPENDIX 3. THE STATUS OF DOSAGE

COMPENSATION IN THE MULTIPLE X

CHROMOSOMES OF THE PLATYPUS

The following appendix is a publication from mid-2008 that reports for the first time

that female platypus do undergo dosage compensation of genes borne on the five X

chromosomes, and that this involves an incomplete form of random X inactivation.

Deakin JE, Hore TA, Koina EK, Graves JAM (2008) The status of dosage

compensation in the multiple X chromosomes of the platypus. PLoS Genet

4(7):e1000140.

My contribution to this publication consisted of the identification of single nucleotide

polymorphisms from multiple X chromosomes of the platypus individual (‘Glennie’)

that had its entire genome sequenced (Warren et al., 2008). These polymorphisms were

critical for RT-PCR experiments performed by Janine Deakin to show that the X

inactivation observed on platypus sex chromosomes is random (ie. no allelic bias at the

tissue level) as opposed to imprinted or non-random.

173

The Status of Dosage Compensation in the Multiple XChromosomes of the PlatypusJanine E. Deakin*, Timothy A. Hore, Edda Koina, Jennifer A. Marshall Graves

Research School of Biological Sciences, The Australian National University, Canberra, Australia

Abstract

Dosage compensation has been thought to be a ubiquitous property of sex chromosomes that are represented differentlyin males and females. The expression of most X-borne genes is equalized between XX females and XY males in therianmammals (marsupials and ‘‘placentals’’) by inactivating one X chromosome in female somatic cells. However, compensationseems not to be strictly required to equalize the expression of most Z-borne genes between ZZ male and ZW female birds.Whether dosage compensation operates in the third mammal lineage, the egg-laying monotremes, is of considerableinterest, since the platypus has a complex sex chromosome system in which five X and five Y chromosomes shareconsiderable genetic homology with the chicken ZW sex chromosome pair, but not with therian XY chromosomes. Theassignment of genes to four platypus X chromosomes allowed us to examine X dosage compensation in this uniquespecies. Quantitative PCR showed a range of compensation, but SNP analysis of several X-borne genes showed that bothalleles are transcribed in a heterozygous female. Transcription of 14 BACs representing 19 X-borne genes was examined byRNA-FISH in female and male fibroblasts. An autosomal control gene was expressed from both alleles in nearly all nuclei,and four pseudoautosomal BACs were usually expressed from both alleles in male as well as female nuclei, showing thattheir Y loci are active. However, nine X-specific BACs were usually transcribed from only one allele. This suggests that whilesome genes on the platypus X are not dosage compensated, other genes do show some form of compensation viastochastic transcriptional inhibition, perhaps representing an ancestral system that evolved to be more tightly controlled inplacental mammals such as human and mouse.

Citation: Deakin JE, Hore TA, Koina E, Graves JAM (2008) The Status of Dosage Compensation in the Multiple X Chromosomes of the Platypus. PLoS Genet 4(7):e1000140. doi:10.1371/journal.pgen.1000140

Editor: Jeannie T. Lee, Massachusetts General Hospital, Howard Hughes Medical Institute, United States of America

Received March 31, 2008; Accepted June 24, 2008; Published July 25, 2008

Copyright: ! 2008 Deakin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by a block grant from The Research School of Biological Sciences at Australian National University.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: Janine.Deakin@anu.edu.au

Introduction

Monotremes are unique mammals that exhibit a mix of reptilianand mammalian features, as they lay eggs, yet have fur andproduce milk for their young. Represented only by the fabledplatypus and four species of echidna, they are distantly related tohumans and other eutherian (‘placental’) mammals, havingdiverged from therian mammals (eutherians and marsupials) 166million years ago (MYA) [1].Monotreme genomes also show a curious mixture of reptilian and

mammalian characteristics. They have a smaller genome thantherian mammals [2], and their karyotype comprises a few largechromosomes, and many small ones, somewhat reminiscent ofchicken macro and microchromosomes. Most curious of all is the sexchromosome system of monotremes. Although monotremes, likeother mammals, subscribe to an XY system of male heterogamety,they have multiple X and Y chromosomes [3] which form amultivalent translocation chain during meiosis [4]. Platypus(Ornithorhynchus anatinus) have ten sex chromosomes; males have fiveX chromosomes (X1X2X3X4X5) and five Y chromosomes(Y1Y2Y3Y4Y5), and females five pairs of X chromosomes [5].During male meiosis, X and Y chromosomes pair within terminalpseudoautosomal regions [6], forming a chain of alternating X and Ychromosomes (numbered by their order in the chainX1–Y1–X2–Y2–X3–Y3–X4–Y4–X5–Y5) which segregate into five X-bearing (female-determining) and five Y-bearing (male-determining) sperm [7].

The sex chromosomes of therian mammals are remarkablyconserved. The X chromosomes of all placental mammals havevirtually identical gene contents, and the marsupial X chromo-some shares two thirds of the human X, defining it as the ancientX conserved region [8]. The largest platypus X was also thoughtto share this ancient region [9]. However, comparisons of the genecontents of platypus, human and marsupial sex chromosomesreveal that the ancient region of the therian X is entirelyhomologous to platypus chromosome 6 [6]. Instead, platypus Xchromosomes share considerable homology with the chicken Zchromosome, including DMRT1, a dosage-sensitive gene that is acandidate for bird sex determination [6,10].The monotreme sex chromosome complex is proposed to have

evolved by repeated autosome translocation onto an original bird-like ZW pair [5,11]. The possession of a chain of nine sexchromosomes by the echidna, seven of which are shared withplatypus [12], means that the chain is at least 30 M years old. Howa ZW system of female heterogamety was transformed into an XYsystem of male heterogamety has been vigorously debated [13].Mammalian Y chromosomes are much smaller and more

variable than their X chromosome partners, but share homologywithin pseudoautosomal regions, and also between coding geneson the X and Y. This supports the theory that heteromorphic sexchromosomes evolved from a pair of homologous autosomes in amammal ancestor after one member of the pair acquired a sexdetermining locus, which lead to suppression of recombination

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and ultimately resulted in differentiation between members of thepair (reviewed in [14,15]). A similar scenario is proposed for theevolution of the bird Z and W from an ancient autosomal pair [16].Comparative gene mapping between the mammal X and bird Z[17,18] shows that they arose from different autosomal pairs.Although they are non-homologous, the XY of therians and ZW

of birds do possess similar general properties. The bird Z, like themammal X, is highly conserved between species [18], whereas theWis degraded to different extents in different bird groups. Also, the birdZ and the mammal X are large chromosomes carrying many genes,and are well conserved between species, whereas the heterogameticchromosome (W and Y) is small, heterochromatic and varies greatlyin size and gene content. The X and Z chromosomes both appear tohave sex-biased gene content. For example, the human Xchromosome is enriched with genes involved in brain function, sexand reproduction [19–21], and in male (but not female) specificgenes [22], and the chicken Z is enriched with genes involved inmale(but not female) reproduction [23].Despite these similarities between the mammal XY and the bird

ZW sex chromosome systems, the extent to which genes on the Xand Z are dosage compensated is remarkably different. Xchromosome inactivation overcomes differences in gene dosagebetween XX females and XY males in therian mammals. Insomatic cells of female humans and mice, genes on one X becomegenetically inactive [24] and transcriptionally silenced [25] early inembryogenesis, a state that is somatically heritable. In marsupials,too, genes on one X chromosome are inactivated [26].X inactivation mechanisms in eutherians and marsupials differ

in a number of important aspects. In somatic cells of eutherians,inactivation is random between maternally and paternally derivedX chromosomes, whereas in marsupials only the paternal X issilenced. X inactivation in eutherians is more stable and completethan in marsupials [26], although it was recently discovered thatbetween 5% [27] and 15% [28] of genes on the human X escapeinactivation, mostly on the region added recently to the X in theeutherian lineage [28].At the molecular level, eutherian X inactivation results from a

complex process controlled by a master locus (the X inactivationcentre XIC), which includes the non-coding XIST gene [29,30]. An

array of epigenetic mechanisms, including binding with varianthistones [31], histone modifications [32,33] and differential DNAmethylation [34,35], contribute to the transcriptional silencing of theX-borne genes. An accumulation of LINE1 elements may provide‘‘booster stations’’ for the propagation of silencing signal along thechromosome [36]. The molecular mechanism of X inactivationseems to be much simpler in marsupials. The region homologous tothe XIC in eutherians is disrupted in marsupials and monotremesand no evidence of XIST has been found in the regions thatjuxtapose flanking markers [37–39]. XIST may have evolved ineutherians from relics of an ancient protein-coding gene [40].Molecular mechanisms shared between marsupial and eutherianinactivation so far have been limited to late replication [41] andhistone underacetylation of the inactive X [42]; DNA methylationdoes not seem to be involved in marsupial X inactivation [43].It was suggested that marsupial X inactivation might represent an

ancestral form of paternally imprinted X inactivation [26,44], andthis hypothesis is supported by imprinted inactivation in mouseextra-embryonic tissues [45], which, like marsupial X inactivation, isless stable and incomplete, and does not involve DNA methylation[46]. However, unlike marsupials, this imprinted X inactivation inmice requires Xist [47,48]. The XIC, along with an accumulation ofLINE1 elements on the X, may control random inactivation ineutherians and its absence correlates to the absence of XIST andLINE1 accumulation on the marsupial X [49].The dosage difference for Z-borne genes between ZZ male and

ZW female birds is equally as extreme as for the mammal X. Yetbirds do not appear to achieve dosage compensation by silencingone Z chromosome in males, since both alleles can bedemonstrated to be active by RNA-FISH and SNP analysis[50,51]. Quantitative PCR showed that nine of ten Z-borne geneshave a male-female ratio close to 1:1 [52], but in microarrays, 40zebrafinch and 964 chicken Z-borne genes showed a range of maleto female ratios from 2:1 (,10% of genes) to 1:1 (,10% of genes),with a mode in the middle [53]. In chicken embryos, the meanmale to female ratio is 1.4–1.6 for Z-linked genes, consistent withan absence of complete dosage compensation [54]. Thisincomplete dosage compensation suggests that differences in genedosage may be critical for only a few genes on the bird Zcompared to the mammal X.The molecular mechanisms behind bird dosage compensation

are yet to be elucidated. Differences in male to female ratiosbetween Z linked genes suggest that at least some are regulated atthe transcriptional level. A region on the short arm of the Zchromosome containing over 200 copies of a 2.2 kb repetitivesequence called MHM (male hypermethylated), is hypermethy-lated on the Z chromosomes in male embryos, but hypomethy-lated on the Z in females [55]. MHM is transcribed only in femalesand accumulates as non-coding RNA near the DMRT1 locus inthe nucleus. A higher proportion of genes subject to dosagecompensation are clustered in this MHM region [56]. Thissuggests that dosage compensation in birds is via upregulation ofgene expression in females, controlled by MHM [57].The platypus presents a fascinating system in which to study

dosage compensation. The need for such a system would appear tobe acute, since the five X chromosomes of the complex account for15% of the haploid genome, and are mostly unpaired by the five Ychromosomes, which together account for only 6%, and are atleast half heterochromatic. Thus 12% of the genome is subject to1:2 dosage differences. The homology of the platypus sexchromosomes with the bird Z, and lack of homology with themammal X, raises questions of whether dosage compensation isincomplete and bird-like, or related to the mammal X inactivationsystem–or is completely different from both.

Author Summary

Dosage compensation equalizes the expression of genesfound on sex chromosomes so that they are equallyexpressed in females and males. In placental and marsupialmammals, this is accomplished by silencing one of the twoX chromosomes in female cells. In birds, dosage compen-sation seems not to be strictly required to balance theexpression of most genes on the Z chromosome betweenZZ males and ZW females. Whether dosage compensationexists in the third group of mammals, the egg-layingmonotremes, is of considerable interest, particularly sincethe platypus has five different X and five different Ychromosomes. As part of the platypus genome project,genes have now been assigned to four of the five Xchromosomes. We have shown that there is someevidence for dosage compensation, but it is variablebetween genes. Most interesting are our results showingthat there is a difference in the probability of expressionfor X-specific genes, with about 50% of female cells havingtwo active copies of an X gene while the remainder haveonly one. This means that, although the platypus has thevariable compensation characteristic of birds, it also hassome level of inactivation, which is characteristic of dosagecompensation in other mammals.

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There are almost no studies of dosage compensation inmonotremes, and none using any molecular techniques. Earlystudies of replication timing of platypus X1 found no asynchronousreplication of the unpaired region of this chromosome [58]. Thissuggests that if the platypus does compensate for gene dosage, it isunlikely to do so by X inactivation. Determining whether theplatypus X chromosomes are dosage compensated has previouslybeen difficult in the absence of knowledge of the genes on platypusX chromosomes.The assignment of genes to four of the five X chromosomes as

part of the platypus genome project now presents an opportunityto investigate dosage compensation in this species. We used threedifferent approaches to determine activity of genes located on fourof the five platypus X chromosomes, and present evidence ofsignificant transcriptional silencing of platypus X-borne genes.

Results

We used quantitative real-time RT-PCR, SNPs (SingleNucleotide Polymorphisms) and RNA fluorescence in situ hybrid-ization (RNA-FISH) to examine dosage compensation in theplatypus. First we gained an overall assessment of the level ofdosage compensation by comparing the amounts of transcriptfrom X-specific, autosomal and pseudoautosomal genes in malesand females using quantitative real-time RT-PCR. We thenidentified SNPs within the sequence of X-borne genes todetermine if they are expressed from both alleles, or only one, aswould be expected from imprinted X inactivation. Finally, we usedRNA-FISH to examine the probability of transcription from thetwo alleles in female and male cells.

Determination of Male:Female Expression Ratios by qRT-PCRWe determined male to female gene expression ratios for two

autosomal genes and 19 genes on platypus X1, X2, X3 and X5, 10of which are X-specific and nine pseudoautosomal (shared withthe Y chromosomes adjacent in the meiotic translocation chain).Genes chosen were from BAC (Bacterial Artificial Chromosome)clones mapped to platypus X chromosomes as part of the genomeproject [6], as this localization indicated directly whether geneswere X-specific or pseudoautosomal. BAC-end sequences frommapped BACs were aligned to the genome to reveal the genomicsequence contained within each BAC. Genes within BACs wereidentified using the platypus genome Ensembl database (http://www.ensembl.org/Ornithorhynchus_anatinus/index.html) (Oana5.0).The presence of these genes within the BACs was confirmedby PCR and sequencing, and expression of these genes infibroblasts was determined (Table 1). We used RNA isolatedfrom independently derived primary fibroblast cell linesrepresenting 16 different individuals (eight males and eightfemales). Expression of these genes was normalized to theexpression levels of the housekeeping gene ACTB, an autosomalgene located on platypus chromosome 2.Male to female ratios were calculated for the normalized data

for each gene. The ratio was near 1 for both autosomal controlgenes (G6PD and HPRT1) on platypus chromosome 6. We alsomeasured expression levels for nine pseudoautosomal genes withcopies on X and Y. The expression ratios of seven genes were high(0.86–1.49), indicating that the Y-borne, as well as the X-borne,alleles are active. However, two pseudoautosomal genes (CDX1and GMDS) had ratios of about 0.5, suggesting that the Y locus isnot active.For five of the ten X-specific genes, ratios were high (0.81–0.99),

as would be expected if genes were largely or fully compensated.

However, for three X-specific genes, the ratio was near 0.5, whichwould be expected if the genes were not compensated between XYmales and XX females. Two genes had intermediate ratios (,0.7),suggesting partial dosage compensation (Table 2). Statistical testsof the null hypothesis that there is no difference in expression levelsbetween males and females, were compromised by the highvariability between individuals, which resulted in p-valuessupporting the null hypothesis (p=0.05) for all X-specific genes.This variation could not be attributed to particular cell linesconsistently showing higher or lower expression for the differentgenes tested (see Figure S1). The trend towards a higher level ofexpression in females than in males for X-specific genes suggeststhat different genes may be incompletely compensated to differentextents.

SNP Identification and ExpressionWe used a bioinformatics approach to identify SNPs in genes on

four of the five platypus X chromosomes (details in Materials andMethods). We searched the Ensembl database for exonic sequencefrom predicted genes on platypus chromosomes X1, X2, X3 andX5 and compared these to platypus whole genome traces. Withinthese alignments we searched for single nucleotide mismatchesappearing more than once at the same site. Possible SNPs were

Table 1. Genes contained within BACs mapped to Xchromosomes as part of genome sequencing project.

BAC Chromosome Gene Expression

636L7 X1/Y1 CRIM1 +

286H10 X1/Y1 CAMK2A +

SLC6A7 +

CDX1 +

EN022941 +

4D21 X1 Ox_plat_124086# +

271I19 X2/Y2 JARID2 +

DTNBP1 +

650K19 X2/Y2 GMDS +

158M16 X3 APC +

165F5 X3/Y2 IRX1 +

830M18 X5 EN149971 +

OaBb_24M14 X5 DMRT2 +

DMRT3* 2

DMRT1* 2

54B19 X5 FBXO10 +

22O3 X5 SHB +

752F12 X5 SEMA6A +

271G4 X5 SLC1A1 +

236A5 X5 ZNF474 +

LOX +

Expression detected in fibroblasts is indicated (+ expressed in fibroblasts; 2indicates no detectable expression in fibroblasts). Ensembl gene identifiers havebeen provided for genes not named in the Ensembl gene build (Jan. 2007).Unless otherwise stated, BAC clones are from the CHORI-236 female platypusBAC library.#Identifier assigned by the Oxford Functional Genomics group gene build.*Expression data from [10]1These gene names have been abbreviated from the Ensembl gene builddesignations ENSOANG00000002294, and ENSOANG00000004997.doi:10.1371/journal.pgen.1000140.t001

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found in the platypus genome sequence within 57 genes onplatypus chromosomes X1 (29), X2 (6), X3 (6) and X5 (16). Wevalidated a subset of these SNPs by sequencing PCR productsderived from genomic DNA isolated from the same female animal(‘‘Glennie’’) used for the genome sequencing project and testedexpression of these genes in fibroblast RNA isolated from this sameindividual. Of ten genes tested, seven were found to be expressedin fibroblasts (ss76901227–ss76901236) (Table 3).BAC clones for these seven potentially X-specific SNP-

containing genes were isolated, by using sequence up to 100 kbeither side of the gene to search the platypus trace archive forBAC-end sequences. We confirmed that BACs contained thegene(s) of interest by PCR and direct sequencing. BACs weremapped by DNA-FISH to male metaphase chromosomes toconfirm their location on an X and determine whether they haveY homologues (data not shown). Three genes with validated SNPson X1 were found to be pseudoautosomal, and based on genomeassembly co-ordinates, all other unvalidated X1 SNPs arepredicted to likewise fall within the pseudoautosomal region.Similarly, the SNP on X2 was shown to have a homologue on Y2

by FISH. However, the three X5 genes containing SNPs are X-specific.Sequencing of X-specific SNPs revealed that all genes were

biallelically expressed (Figure 1), as were the pseudoautosomalSNPs (data not shown). Allele specific real-time PCR was used todetermine if alleles were expressed to the same extent for the

pseudoautosomal gene GMDS and the X specific genes. Nosignificant difference from a 1:1 ratio was observed, implying theabsence of imprinting (Table 4 and Figure S2). Biallelic expressionwith equivalent expression from alternate alleles for the three X-specific genes eliminates the possibility that genes on platypus X5

are subjected to complete paternal inactivation (as is observed inmarsupials), and directed our approaches to examining theprobability of transcription from the two loci by RNA-FISH.

RNA-FISH Detection of Primary TranscriptsRNA-FISH detects the sites of primary transcription in

interphase cells by hybridization with large intronic sequencesthat are spliced from cytoplasmic mRNA. Thus large genomicprobes were required for the genes of interest.BAC clones mapped to platypus X chromosomes as part of the

genome project and found to contain genes expressed in fibroblast,were used for RNA-FISH experiments (Table 1). These includedthe four clones discussed above (one from X2 and three from X5).We also included BAC OaBb_24M14 (GenBank AccessionNo. AC152941) containing DMRT2, which had been fullysequenced previously and whose expression had been confirmedin fibroblast cell lines [10]. A BAC containing the HPRT1 genelocated on chromosome 6, OaBb_405M2 (GenBank AccessionNo. AC148426), was used as an autosomal control. HPRT1 wasdetected in the platypus fibroblast EST library sequenced as partof the genome project (GenBank Accession No. EG341684). The14 BACs together contained 19 genes; two pseudoautosomalBACs contained four and two genes respectively and one X-specific BAC contained two genes (Table S1).Transcription of the 14 BACs described above was initially

examined by RNA-FISH in female and male fibroblasts (Figure 2).As a control, RNA-FISH was followed by DNA-FISH to ensurethat RNA signals were located near one (X-specific genes in males)or both of the alleles (X-specific genes in females, autosomal andpseudoautosomal genes). Only those cells with two DNA-FISHsignals per nucleus (or one signal for X-specific genes in males)were included in analysis. Data from the male RNA-FISHexperiments was used to determine the efficiency of detection foreach gene which was then used to extrapolate the expectedpercent of nuclei with biallelic expression in females, which isexpected if there is no X inactivation (Table 5 - refer to Table S2for complete RNA-FISH dataset).

Table 2. Male:female ratio for expression of platypus X genesin fibroblast cells normalized to the autosomal ACTBhousekeeping gene.

Gene Chromosome Male:Female Ratio p-value

Autosomal

G6PD 6 1.12 0.21

HPRT1 6 0.97 0.96

Pseudoautosomal

CRIM1 X1/Y1 1.15 0.50

CAMK2A X1/Y1 1.39 0.11

CDX1 X1/Y1 0.50 0.01

EN02294 X1/Y1 1.48 0.57

SLC6A7 X1/Y1 1.49 0.06

DTNBP1 X2/Y2 0.86 0.47

JARID2 X2/Y2 0.93 0.88

GMDS X2/Y2 0.48 0.05

IRX1 X3/Y2 0.98 0.85

X-specific

Ox_plat_124086 X1 0.91 0.45

APC X3 0.85 0.76

SHB X5 0.81 0.43

LOX X5 0.94 0.88

EN14997 X5 0.71 0.18

FBXO10 X5 0.73 0.32

SLC1A1 X5 0.36 0.07

ZNF474 X5 0.99 0.69

DMRT2 X5 0.49 0.10

SEMA6A X5 0.55 0.14

doi:10.1371/journal.pgen.1000140.t002

Table 3. Genes with SNPs, identified from the genomesequence and validated by PCR and sequencing.

Gene Chromosome SNP Expressed in Fibroblasts

CCNG1 X1/Y1 C/T +

GABRB2 X1/Y1 C/A +

SYNPO X1/Y1 C/T +

GMDS X2/Y2 C/T +

ADAMTS16 X3 C/T 2

FRMPD1 X5 C/T 2

ACO1 X5 G/T 2

FBXO10 X5 A/C +

EN14997 X5 G/T +

SHB X5 A/G +

+ indicates expression detected in fibroblasts; 2 indicates no detectableexpression in fibroblasts.doi:10.1371/journal.pgen.1000140.t003

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HPRT1, an autosomal control gene located on chromosome 6,was expressed from both alleles in 96–97% of nuclei (Figure 3A).Genes within four pseudoautosomal BACs on X1, X2 (includingGMDS) and X3 were also expressed from both alleles in mostfemale nuclei (77–84%), as well as in most male nuclei (62–92%),showing that the Y, as well as the X, alleles are active (Figure 3B).Two pseudoautosomal BACs used for RNA-FISH contain morethan one gene, so it remains possible that not all genes within theseBACs have an active Y copy. We obtained quite different resultsfrom the BAC containing CRIM1, a X1-Y1 pseudoautosomal genewhich was expressed from only one allele in most male (81%) andfemale cells (71%) (Figure 3C). Except for this locus, we concludethat for the pseudoautosomal loci we tested, both X alleles areactive in females, and both X and Y alleles are active in males.We then tested transcription from nine X-specific BACs on

platypus X1, X3 and X5. Transcription from both alleles wasobserved on average in only 45% of nuclei (Figure 3D). Differentgenes showed a range of transcription of both alleles, from 20%(SEMA6A) to 53% (Ox_plat_124086). These X-specific genes weretherefore expressed very differently from the autosomal andpseudoautosomal genes, and significantly different to that expectedfor biallelic expression, indicating some level of transcriptionalinactivation for these genes.Two colour RNA FISH was performed with genes FBXO10 and

SHB, located within 500 kb of each other. Co-location of the twoRNA signals showed the same X in all of the 51% of cellsexpressing from only one allele. (Figure 4). A few cells (12%)displayed biallelic expression from SHB with monoallelic expres-sion of FBXO10, and in 37% of nuclei, both genes were expressedfrom both alleles. As a control, this experiment was performed onmale nuclei showing that RNA-FISH signals co-located in allnuclei in which genes were expressed. This experiment was carriedout only for two genes lying close together, as results from genessituated further apart (and hence with a gap between signalsexpressed from the same chromosome) would make results fromcells expressing only one of each gene, difficult to interpret.RNA-FISH results were validated for a subset of genes (HPRT1,

CRIM1, GMDS, SEMA6A and DMRT2) on four other independentlyderived primary fibroblast cell lines from different individuals (onemale and three females). Results for each cell line are shown in TableS3. As observed (Figure 2), the autosomal gene HPRT was expressedfrom both alleles in most nuclei (88% male and 83–90% female), aswas the pseudoautosomal gene GMDS (86%, 85–90%). Thepseudoautosomal gene CRIM1, as before, was expressed from bothX chromosomes in only 24–56% of female nuclei and X and Y inonly 24% of male nuclei. As observed (Figure 2), both X-specificgenes (DMRT2 and SEMA6A) were expressed from the single X in99% of male nuclei, and both X chromosomes in half of femalenuclei (45–60% and 38–43% respectively). Although there was somevariation between individuals, overall results were similar between allsix cell lines tested in this study. Statistical analysis revealed that onlythe two X-specific genes had a significant difference between themales and females for the number of nuclei expressing only one allele(p=0.0006 and 0.0008 respectively).

Discussion

The very large proportion of the genome (,12%) that is X-specific in the platypus, and the homology of the multiple platypusX chromosomes to the chicken Z but not the therian Xchromosome, makes them a most interesting species for exploringthe origins of dosage compensation in mammals.We therefore tested the transcription of genes on platypus X1, X2,

X3 and X5 in order to search for evidence of random X inactivation

Figure 1. Biallelic expression of three X-specific genes. SNPs(marked by boxes) were identified in the genome sequence demon-strated by sequencing fibroblast cDNA from the sequenced animal(‘‘Glennie’’).doi:10.1371/journal.pgen.1000140.g001

Table 4. Relative allele expression determined by allele-specific real-time RT-PCR.

Gene Allele A Allele B

FBXO10 (A/C) 0.47 0.53

EN14997 (G/T) 0.51 0.49

SHB (A/G) 0.50 0.50

GMDS (C/T) 0.52 0.48

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(as in eutherian mammals), paternally imprinted X inactivation (as inmarsupials), or incomplete and variable dosage compensation (as inchickens). Random inactivation would be manifested as dosageequality between males and females, expression from both SNPvariants overall but only a single allele per nucleus detected by RNA-FISH. Paternal inactivation would be manifest by dosage equality,but expression of only one SNP variant, and only one allele pernucleus would be detected by RNA-FISH. Bird-like incompletedosage compensation would be manifest as a wide range of dosagerelationships between males and females, expression of both SNPvariants and expression from both alleles in each nucleus.Our results are not strictly consistent with any of the above

predictions. Quantitative RT-PCR showed male:female expres-sion ratios near 0.5 or 1.0 for different genes, although both SNPalleles were expressed for all genes at an equal level in aheterozygote. Our examination of transcription of X-specificplatypus genes by RNA-FISH revealed that about half of femalecells expressed only one allele. The RNA-FISH results showed aclear difference between the transcription of X-specific locicompared with pseudoautosomal and autosomal loci.These data imply that genes from platypus X-specific regions

show some form of compensation via transcriptional inhibition, as

for mammals, but this is incomplete and variable between genes.Our demonstration that genes were expressed equally from bothalleles suggests that paternal inactivation and imprinted partialexpression is unlikely. Our demonstration that both alleles areexpressed in about half the nuclei rules out complete Xinactivation (random or imprinted), as is also seen for manypartially escaping genes in eutherians and marsupials.The variability in overall expression between different X-borne

genes resembles the range of expression of genes on the bird Z inmales and females that indicates a more relaxed, or more variable,dosage compensation system [53]. Biallelic expression of Z-bornegenes was also found by examining expression of different alleles oftwo genes from fibroblast cultures established from single cells[51]. These results taken together suggest that bird dosagecompensation is partial and differs between genes on the Z.Thus dosage compensation of X-borne genes occurs to some

extent in the platypus, and has features of both bird-like andmammal-like sex chromosome dosage compensation.

Is Partial Inactivation Ancestral?Together, our findings have parallels in observations of some

genes on the marsupial X and the mouse X in extra-embyronic

Figure 2. Summary of RNA-FISH results in platypus cells. Frequency of cells in which transcription of no (yellow), one (red) or two (blue) allelesis detected by RNA-FISH in male and female interphase nuclei. Autosomal control, pseudoautosomal and X-specific genes are grouped, with a distinctdifference observed between the X-specific genes and the autosomal and pseudoautosomal genes.doi:10.1371/journal.pgen.1000140.g002

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tissues, whose paternal alleles are partially inactive, or ‘‘escaper’’genes on the recently added region of the human and mouse X,which are partially expressed from the inactive X.The observations of partial inactivation in all three major

mammalian lineages suggests that partial inactivation observedhere in platypus represents a basic form of mammalian Xinactivation, which has come under tighter control during therianevolution, ultimately resulting in the highly stable and complexform of inactivation typical of most eutherian X-borne genes.Partial inactivation has been documented for two marsupial

genes (out of a total of five) in some tissues. PGK1 isozyme variantsshowed strong expression from the maternal allele and weakerexpression from the paternal allele in cells from heterozygousfemale kangaroos, even in single clones [59], and G6PD fromhybrid marsupials showed a heteropolymer band, diagnostic ofexpression from both alleles in a single cell [60]. Differencesbetween species, tissues and even between genes make it difficult togeneralize about the nature of marsupial X inactivation, and theseexperiments could not distinguish whether partial expression fromthe paternal X is due to low expression from paternal Xchromosomes in every cell, or to a mixture of two X-active andone X-active cells. RNA-FISH was used to show that the tammarwallaby X-borne gene SLC16A2 was expressed from only oneallele in most fibroblast cells [61].The partial silencing displayed for platypus X-specific genes also

has some parallels to genes on the human X that escapeinactivation. X inactivation in humans was initially thought to

involve all genes on the X chromosome, but in recent years it wasfound that 5% to as many as 15% of human genes escapeinactivation in lymphoblastoid [27] and fibroblast cell lines [28]respectively. Remarkably, transcription of some of these genes infibroblasts varies between individuals, as seems to be the case forplatypus. Partial expression of genes on the inactive X has alsobeen observed in other eutherians, including the mouse, cow andmole [62,63]. Typically, these escaper genes are fully expressed

Table 5. Expected vs observed frequency of nuclei withbiallelic expression in females.

Efficiency (p) Female Biallelic Frequency

Expected%

Observed%

P-value

Autosomal

HPRT1 0.98 96 97 0.96

Pseudoautosomal

CRIM1 0.60 36 26 ,0.01

CAMK2A, SLC6A7, CDX1,EN02294

0.84 71 77 0.16

JARID2, DNTBP1 0.96 92 80 ,0.01

GMDS 0.92 85 84 0.70

IRX1 0.77 59 83 ,0.01

X-specific

Ox_plat_124086 0.98 96 53 ,0.01

APC 0.97 94 52 ,0.01

SEMA6A 0.99 98 20 ,0.01

ZNF474, LOX 0.99 98 43 ,0.01

DMRT2 0.98 96 51 ,0.01

SHB 0.90 81 49 ,0.01

FBXO10 0.99 98 52 ,0.01

SLC1A1 0.99 98 39 ,0.01

EN14997 0.99 98 46 ,0.01

Efficiency (p) of RNA-FISH hybridisation was determined from the resultsobtained in male fibroblasts and extrapolated to determine the expectedfrequency of nuclei with two signals, one signal and no signal per cell using theformula p2+2pq+q2 = 1, where p2 is the number of nuclei with two signals, 2pqrepresents nuclei with one signal and q2 is the number with no signal. P-valueswere determined by a x2 test with 2 degrees of freedom.doi:10.1371/journal.pgen.1000140.t005

Figure 3. Co-localization of transcripts (RNA - green) and theircorresponding gene loci (DNA - red). (A) The autosomal controlHPRT1 is expressed from both loci in both sexes since two signals aredetected for both RNA and DNA-FISH in both males and females. (B)Pseudoautosomal BAC 286H10 is expressed from both X chromosomes infemales and the X and Y in males, since two signals are detected for bothRNA and DNA-FISH in males and females. (C) Pseudoautosomal CRIM1located on X1 is expressed from only one X in females and only one of theX and Y alleles in males, since two DNA signals but only one RNA signal isdetected in both males and females. (D) X-specific SEMA6A located on X5is expressed from only one of the two X chromosomes in females, as wellas from the single X in males, showing one RNA and DNA signal in malesbut two DNA signals and only one RNA signal in females.doi:10.1371/journal.pgen.1000140.g003

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from the active X and partially expressed from the inactive X[28,64].We propose that partial inactivation was the mechanism for

compensating differences in gene dosage in an ancestral mammal.

Partial X Inactivation and the Probability of TranscriptionTo date, it has been difficult to differentiate between the

alternative hypotheses that partial inactivation is due to a loweredrate of transcription in all cells, or from a lowered probability ofexpression per cell in the population. Ohlsson et al [65] arguedthat genes transcribed at a low level show a low probability oftranscription in the cell population, rather than a uniformly lowtranscription level. They propose that genomic imprinting and Xchromosome inactivation evolved by regulating, not the activity ofeach locus, but the probability that it is expressed, and making thisparent specific [65].This radical hypothesis is supported by our RNA-FISH data,

which show that platypus genes differ in the frequency of nuclei inwhich one or both alleles are transcribed, giving an overall partialdosage compensation that differs from gene to gene. The datafrom the bird Z is equivocal; the variability between genes isthought to reflect differences in the rate of transcription, but couldequally well reflect differences in the probability that a locus istranscribed. RNA-FISH of five chicken genes shows that most aretranscribed from both alleles in most cells [50]; however, the lowefficiency of signal detection (about a quarter of nuclei had nosignals), and the different tissues used makes this hard to interpret.Efficient RNA-FISH on the chicken Z genes for which we havedata in platypus would test the hypothesis that partial inactivationof the Z in male birds operates by altering the probability oftranscription, rather than uniformly downregulating transcription.Our finding that two genes located 500 kb apart are expressed

from the same chromosome implies that the stochastic expressionof X-specific genes is coordinated in cis. Furthermore, a recentstudy has shown that this type of probabilistic expression iswidespread on human autosomes, with their data suggesting thatas many as 1000 human genes are subject to stochastic monoallelicexpression [66]. Around 80% of these genes also showed some

level of biallelic expression. Unlike the hypothesis put forward byOhlsson et al [64], this type of expression is not limited to thosewith low levels of expressions.Is partial expression in therian mammals explained by stochastic

expression? Data on partial expression of genes on the paternal Xin marsupials are equivocal; the partial expression of the maternalPGK1 allele in clones, and the fainter paternal isozymeheteropolymer band for G6PD are explained equally well by bothhypotheses. The few data that would distinguish these hypothesesfor escapers on the inactive human X do not conclusivelyeliminate either hypothesis. Assays of the partially expressedhuman X-borne gene CHM (REP1) in single cells showed thatCHM was expressed from the inactive X in most (70%) but not allcells from one cell line, and in only seven out of ten hybrid celllines carrying an inactive X [67]. More recently, a study on dosagecompensation in human lymphoblastoid cell lines found that genesescaping X inactivation were not subject to the higher levels ofvariation found for fibroblast cell lines, suggesting that theexpression of the escaper genes is not stochastic but subject totight regulation [27]. RNA-FISH performed on both fibroblastsand lymphoblastoid cells for these escaper genes would conclu-sively rule out stochastic expression.It is important to note the difference in the number of genes in

human which escape inactivation between fibroblast cell lines,where 15% of genes are said to escape inactivation [28] andlymphoblastoid cell lines where only 5% of genes escape [27].Similarly in marsupials, differences have been found in theinactivation status of genes between tissues [26]. Our study hasonly used fibroblast cell lines due to the difficultly in obtainingtissue samples in large enough sample sizes, as the platypus is listedas a ‘‘vulnerable’’ species. A comparison of results for other tissuesmay show different results.Several human X-borne genes that escape from inactivation

have a widely expressed Y homologue, and some others havehomology to a Y-borne pseudogene that represents a recentlyinactivated partner on the Y. The Y homologue of an X/Y pairoften has a lower level of expression than its partner on the X(reviewed in [68]), similar to the lower level of expression exhibited

Figure 4. Two-colour RNA-FISH of neighbouring genes FBXO10 (red) and SHB (green). (A) Male nucleus expresses both genes from thesingle X. (B) Female nucleus expresses both genes from the same single X chromosome. (C) Female DNA-FISH showing that loci are located together.(D) Diagram depicting the region and the location of BACs used for RNA-FISH. Grey boxes indicate genes located between these two BACs.doi:10.1371/journal.pgen.1000140.g004

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by alleles on the inactive X in females. However, the presence of aY homologue does not necessarily negate the need for dosagecompensation, as some Y alleles have evidently taken on functionsdifferent from those of their X homologue. Nearly all escapergenes are part of the region added to the eutherian X chromosomeand only recently recruited to the inactivation system, suggestingthat their partial escape from X inactivation correlates withprogressive assimilation of genes into the X inactivation systemsonce the Y paralogue has degenerated.

Pseudoautosomal Genes and InactivationIn eutherian mammals, small terminal regions of the X and Y

are homologous, and pair and recombine at male meiosis. Thesepseudoautosomal regions (PARs) are relics of the X added regionthat have not yet degraded [15]. Genes within the PAR have noneed of dosage compensation.There are two PARs on the human X. PAR1 on the short arm

represents a relic of ancient XY homology, and contains genes thatare expressed from the Y, and not inactivated on the X [69]. Thesmaller PAR2 was added very recently to the long arm of the Yfrom the long arm of the X, but two genes in the region (SYBL1and SPRY3) are subject to inactivation, not only on the inactive X,but also on the Y [70].We observed that seven of the nine platypus genes from the

pseudoautosomal regions displayed as much or more expressionfrom males than females, as assessed by quantitative RT-PCR,suggesting that they are expressed from Y as well as the X alleles.RNA-FISH of these genes showed that both alleles were expressedin most cells in females (two X alleles) and males (X and Y alleles).Two of these BACs contained multiple genes, so detection ofpredominantly two signals per cell does not necessarily mean thatall genes are active on both chromosomes; however, expressionanalysis of transcripts from each of these BACs confirms that mostof these genes (3/4 in BAC 286H10 and 2/2 BAC 271I19) haveactive Y homologues. Two pseudoautosomal genes CDX1 andGMDS had male:female expression ratios near 0.5 but an almostequal probability of expression, suggesting that either both allelesare downregulated in males, or alternatively, the Y allele sequencehas sufficiently diverged from that of the X homologue, leaving itunable to be amplified by our primers.A fifth platypus pseudoautosomal gene showed a completely

different expression pattern. CRIM1 (cysteine rich transmembraneBMP regulator 1), located on platypus X1-Y1, had equivalentexpression in males and females, but was usually expressed fromonly one allele in both males (81% of nuclei) and females (69%).There are two possible explanations. Firstly, the Y homologue mayhave evolved a new male-specific function like many genes on thehuman Y [15], and be testis specific, so silencing of one X infemales evolved to equalize expression of the X homologue.Alternatively, inactivation of both X and Y could be equivalent tothe silencing of PAR2 genes on the long arm of the human X.SYBL1 and SPRY3 undergo silencing on both the X and Y, theproduct of their evolutionary history as a block transposed fromthe X (where it was subject to inactivation) to the Y, where it wasdosage compensated to match the X [70].Thus for most pseudoautosomal genes there is no need for

dosage compensation on the X because the Y allele is active, andno dosage compensation is observed.

Is More Tightly Controlled Dosage Compensation Linkedto Gene Function?The chromosome-wide X inactivation in mouse and human has

given rise to the expectation that dosage compensation for geneson sex chromosomes is critical for life. However, this does not

seem to be the case in birds. Dosage compensation for the 964genes on the bird Z chromosome extends over a range fromcomplete compensation (,10% genes) to no compensation (,10%genes) with most falling between these extremes [53]. This suggestseither that the necessity for strict dosage compensation has beenover-emphasized, or that genes on the bird Z chromosome aremuch more tolerant of dosage differences than genes on thetherian X [71].By no means are all genes dosage sensitive [71]. For instance,

many protein products, such as enzymes, are controlled atdifferent levels in the cell, so transcriptional control is not essential.For some genes, a dosage difference may even be essential forfunction; for instance, a 2:1 dosage of DMRT1 has been suggestedto define male versus female development in birds [72].One gene that does not display equal expression between males

and females and may even be hypertranscribed in females of bothplatypus and zebrafinch is SEMA6A, a gene on platypus X5 andthe avian Z. From our data, platypus SEMA6A appears not besubject to dosage compensation by real-time RT-PCR, yet RNA-FISH results show that it predominantly has only one allele activeper cell. In zebrafinch liver, SEMA6A is expressed more than two-fold more in females with just one copy than males with two copies[53]. Although these results were obtained from different cell typesin the different species, it is intriguing that in both cases there issome evidence of hypertranscription in females.It is therefore likely that only a minority of genes on the

mammalian X really need to be dosage compensated. Thedifference in the level of control of sex chromosome activity maytherefore be a side-effect of the mechanism used for dosagecompensation. Eutherian mammals subscribe to a whole-Xmechanism in which inactivation spreads along the X. The birdZ, however, seems to have a piecemeal dosage compensationsystem in which different genes appear to show different levels ofcompensation, and compensated genes are clustered [56].The alternative is that the genes on the bird Z and therian X

evolved under different selective pressures. We know that the genecontent of these chromosomes is different, having originated fromtwo different pairs of autosomes, and we also know that the genecontent of sex chromosomes is biased toward sex-specific expression.The human X is enriched for genes involved in brain function, andsex and (particularly male) reproduction [19–22]. The chicken Zchromosome gene content is male-biased yet noticeably deficient infemale-biased genes [23]. Commenting on the finding that dosagecompensation in birds is much less tightly controlled than in therianmammals, Graves and Disteche [71] suggested that expressiondifferences in Z-borne genes between males and females may havebeen selected for to control sex-specific characters. Since platypus sexchromosomes show considerable homology to the bird Z, thefunctions of platypus X-borne genes are likely to be equivalent tothose on the chicken Z.Perhaps, then, partial and variable silencing in the platypus

dosage compensates some essential genes, leaves some genesuncompensated where dosage differences are essential for sex-specific function, and partially compensates most genes inproportion to their dosage-sensitivity, as is evidently the case forbirds.

ConclusionsWe found that genes on the multiple platypus X chromosomes

show partial and variable dosage compensation. This is very similarto the partial and variable dosage relationships of genes on thechicken Z chromosome, with which the platypus X chromosomesshare considerable homology. However, unlike birds, platypusdosage compensation involves transcription from only one of the

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two alleles in a proportion of cells and is coordinated at least on aregional level. Transcriptional inhibition is a property shared by Xchromosome inactivation in therian mammals. Thus, platypusdosage compensation has features shared with dosage compensationof the bird Z and the mammal X.

Materials and Methods

Identification of Expressed Genes within BACs Mappingto the X ChromosomesBAC-end sequences from CHORI-236 BAC clones (http://

bacpac.chori.org), mapped to platypus X chromosomes as part ofthe genome project, were aligned against the genome sequence.Genes within the genomic region contained between the BAC-endsequences were identified by using the Ensembl database (http://www.ensembl.org/Ornithorhynchus_anatinus/index.html). Anadditional four BACs were chosen because they span genes withSNPs that were potentially X-specific. These BACs were identifiedby searching the platypus sequence trace archives containingBAC-end sequence data (http://www.ncbi.nlm.nih.gov/Traces)with genomic sequence from 100 kb up and downstream of thegene of interest.PCR was performed on the BACs to confirm that the genes

predicted to be contained within the BAC were present. The PCRcycling conditions for all primers were as follows: an initialdenaturing step of 94uC for 2 min, 30 cycles of 94uC for 30 sec,annealing for 30 sec at the appropriate temperature (Table S4),72uC for 1 min and a final extension at 72uC for 10 min.To determine whether genes within BACs were expressed in

fibroblasts, total RNA was extracted from female and malefibroblast cell lines using Gene Elute Mammalian Total RNAMiniprep extraction kit (Sigma). RNA was treated with DNA-free(Ambion) to remove any contaminating DNA and Superscript III(Invitrogen) was used to generate cDNA using random hexamersas primers for first strand synthesis. To ensure there was nogenomic DNA contamination in the cDNA sample, a RT-negativecontrol was made by excluding the Superscript III enzyme fromthe first strand synthesis reaction and was used as a negativecontrol in all RT-PCR experiments. Where possible, primers weredesigned to span introns. Primers, annealing temperatures andproduct sizes are listed in Table S4. PCR was carried out using thesame cycling conditions described above. Each set of primers wastested on female and male RT-positive and RT-negative samplesas well as genomic DNA. PCR products were gel purified using aQIAquick Gel Extraction kit (Qiagen) and directly sequenced byAGRF (Brisbane).

DNA-FISH on Metaphase ChromosomesFor the four BACs not previously mapped, 1 mg of DNA from

these BACs was labeled by nick translation with digoxigenin –11-dUTP (Roche Diagnostics), Spectrum-Orange or Spectrum-Green(Vysis). Unincorporated nucleotides were removed from Spec-trum-Orange and Spectrum-Green labeled probes using Probe-Quant G50 micro columns (GE Healthcare). Probes wereprecipitated with 1 mg platypus C0t1 DNA and hybridized tomale and/or female platypus metaphase chromosomes andfluorescent signals for digoxigenin labeled probes were detectedusing the protocol described by Alsop et al [73]. A Zeiss Axioplan2epifluorescence microscope was used to visualize fluorescentsignals. Images for DAPI-stained metaphase chromosomes andfluorescent signals were captured on a SPOT RT MonochromeCCD (charge-coupled device) camera (Diagnostic InstrumentsInc., Sterling Heights) and merged using IP Lab imaging software(Scanalytics Inc., Fairfax, VA, USA).

Quantitative Real-Time RT-PCRTotal RNA was extracted from eight different male and eight

different female fibroblast (toe web) cell lines (at passage 6 to 8) torepresent a total of 16 individuals. First-strand cDNA wassynthesized by oligo (dT) priming using Superscript III (Invitro-gen). Primers for each gene were designed using the Plexorprogram (Promega) (Table S4). PCR reactions were carried outusing Quantitect SYBR Green PCR kit (Qiagen) according to themanufacturer’s instructions. Amplifications were performed anddetected in a Rotorgene 3000 cycler (Corbett Research). Todetermine the detection range, linearity and real-time PCRamplification efficiency for each primer pair, standard curveswere calculated over a 10-fold serial dilution of fibroblast cDNA. Aseries of two-fold serial dilutions were also carried out to confirmthe ability of the PCR conditions to detect this level of difference inexpression. All dilutions and samples were run in triplicate.Cycling conditions consisted of an initial hold cycle of 95uC for15 min, 40 cycles of 94uC for 15 sec, annealing at the appropriatetemperature listed in Table S4 for 15 sec and extension at 72uCfor 20 sec for data acquisition. Melting curves were constructedfrom 45uC–95uC to confirm the purity of the PCR products anddirect sequencing of products was performed to confirm theiridentity. Relative expression of each gene was determined bynormalization to ACTB expression using the formula where theratio of ACTB to target = (1+ERef)

CtRef/(1+ETarget)CtTarget [74].

Statistical significance was assessed, for the null hypothesis thatthere was no difference between male and female expression levels,using an unrelated samples 2-tailed t test with unequal variance.

Bioinformatic Prediction of Expressed Single NucleotidePolymorphisms (SNPs) in PlatypusExonic sequence from predicted genes on platypus chromo-

somes X1, X2, X3 and X5 were extracted from the Ensembl 46database, using the Biomart tool (http://www.ensembl.org/biomart/martview). These sequences were compared to theplatypus whole genome shotgun sequence traces (‘‘Ornithorhynchusanatinus WGS’’) deposited on the trace archive at NCBI (http://www.ncbi.nlm.nih.gov/Traces), using MegaBLAST [75]. Poten-tial single-nucleotide polymorphisms (SNPs) were discovered bymanually searching within the BLAST output for single nucleotidemismatches occurring in approximately 50% of target traces. Thechromatogram files containing a potential SNP were extractedfrom the trace archive and assembled using SequencherTM 4.7(Gene Codes Corporation, Michigan). This assembled sequence(including surrounding intronic sequence) was tested for unique-ness within the platypus genome using BLAT [76] on the UCSCtest browser (http://genome-test.cse.ucsc.edu).

Allele Specific Real-Time PCRTo validate identified SNPs and test expression in fibroblasts,

DNA was extracted from the ‘‘Glennie’’ fibroblast cell line using theDneasy Blood and Tissue kit (Qiagen) and RNA was extracted asdescribed above. First strand synthesis was performed on RNA usingthe Supercript III First-Strand Synthesis System for RT-PCR kit(Invitrogen) according to manufacturer’s instructions. PCR and RT-PCR was carried out using the primers listed in Table S4.To quantify the expression level of SNPs for three X-specific

SNPs and one pseudoautosomal gene, allele-specific real-timePCR was carried out. Allele specific primers were designed withthe 39end base of either the forward or reverse primercorresponding to the specific allele (refer to Table S5 for primersequences and corresponding annealing temperatures). Thedifferent alleles were amplified in separate tubes. Real-time PCR

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was performed using Quantitect SYBR Green PCR kit (Qiagen)with amplifications performed and detected in a Rotorgene 3000cycler (Corbett Research). Cycling conditions are the same forthose described in the quantitative PCR section with all samplesrun in triplicate. Genomic DNA for ‘‘Glennie’’ was included as acontrol since the allele frequency ratio should be 1:1, permittingallele-specific amplification bias to be detected and corrected.Known homozygous cDNA samples and pooled homozygoussamples with varying ratios of each allele (0.2, 0.4, 0.6, 0.8) wereincluded to ensure the technique was sensitive enough to detectsmall differences. Allele relative expression levels were calculatedusing the formula: frequency of allele A= 1/(2EDCt+1) [77], whereDCt= (AcDNA2BcDNA)2(AgDNA2BgDNA) and converted to a ratioof allele A to allele B. PCR products were sequenced to confirmthe identity of products.

RNA/DNA-FISH on Interphase NucleiMale and female fibroblast cells (from toe web) were cultured on

gelatin-coated coverslips in AminoMax C100 medium (Invitrogen)at 30uC in an atmosphere of 5% CO2. Cells on coverslips werewashed with PBS, permeabilized for 7 minutes on ice using CSKbuffer plus Triton X (100 mM NaCl, 300mM sucrose, 3 mMMgCl2, 10 mM PIPES pH 6.8, 2 mm Vanadyl RibonucleosideComplex (VRC), 0.5% Triton X) and fixed in 3% paraformal-dehyde for 10 minutes. Coverslips were dehydrated via a series ofethanol washes (70%, 80%, 95%, 100%), air-dried and denatured.Probes were labeled as described in the DNA-FISH on metaphasechromosomes section. Hybridization buffer (46SSC, 40% dextransulphate, 2 mg/ml BSA, 10 mM VRC) was added to each probe.Probes were denatured at 75uC for 7 min and allowed topreanneal for 20 min. 10 ml of probe was added to each coverslipand hybridized overnight in a humid chamber at 37uC. Coverslipswere washed in 0.46SSC with 0.3% Tween 20 at 60uC for2 minutes followed by a wash in 26SSC with 0.1% Tween 20 for1 min at room temperature. Coverslips were fixed in 3%paraformaldehyde for 10 minutes, treated with 0.1 mg/ml RNasefor 1 hour at 37uC and subjected to DNA-FISH following thesame hybridization protocol described for DNA-FISH onmetaphase chromosomes. Nuclei were viewed under a fluores-cence microscope in several different focal planes, with 100 nucleiexamined for each probe for both males and females.Efficiency (p) of RNA-FISH hybridisation was determined from

the results obtained in male fibroblasts and extrapolated todetermine the expected frequency of nuclei with two signals, onesignal and no signal per cell using the formula p2+2pq+q2=1, wherep2 is the number of nuclei with two signals, 2pq (q=12p) representsnuclei with one signal and q2 is the number with no signal. P-valueswere determined by a x2 test with two degrees of freedom.Inconsistencies between RNA-FISH results in previous exper-

iments examining transcription have been attributed to theinability to detect weak signals, which could be overcome by,not only using a combination of RNA and DNA-FISH, but also byamplifying the RNA-FISH signal [78]. In order to ensure that thedifferences between autosomal, pseudoautosomal and X-specific

genes were not due to the inability of the technique to detect bothtranscripts, an experiment where BACs containing SEMA6A andCRIM1 were labeled with either Spectrum Green or SpectrumOrange (Vysis) or with biotin-16-dUTP (Roche Diagnostics) wasperformed. Biotin-labeled probes were detected with avidin-FITC(Vector Laboratories Inc.), with FITC signals amplified byadditional layers of biotinylated anti-avidin (Vector) and avidin-FITC. No differences between direct labeling and biotin labelingfollowed by amplification were detected.

Supporting Information

Figure S1 Real-time results for X-specific genes. Each point is adifferent cell line (shown in the same order in each graph). Malecell lines are shown in blue, female cell lines in red. Expression hasbeen normalised to ACTB.Found at: doi:10.1371/journal.pgen.1000140.s001 (0.15 MB PDF)

Figure S2 Allele-specific real-time RT-PCR results for EN14997.Standards for each allele are shown in red or green and ‘‘Glennie’’cDNA in pink. cDNA from homozygous individual for the oppositeallele in each case is in dark grey, showing that the primers do notamplify both alleles. No template control is light grey.Found at: doi:10.1371/journal.pgen.1000140.s002 (0.09 MB PDF)

Table S1 Ensembl Identifiers, genome co-ordinates and corre-sponding location in human and chicken for genes found withinBACs used for RNA FISH.Found at: doi:10.1371/journal.pgen.1000140.s003 (0.04 MBDOC)

Table S2 RNA-FISH dataset.Found at: doi:10.1371/journal.pgen.1000140.s004 (0.03 MBDOC)

Table S3 RNA-FISH results for three additional female and onemale cell lines.Found at: doi:10.1371/journal.pgen.1000140.s005 (0.03 MBDOC)

Table S4 List of primers used for SNP validation (SNP),confirmation of expression in fibroblasts (Expression), BACconfirmation (BAC) and qRT-PCR.Found at: doi:10.1371/journal.pgen.1000140.s006 (0.06 MBDOC)

Table S5 Primers used for allele-specific real-time PCR.Found at: doi:10.1371/journal.pgen.1000140.s007 (0.03 MBDOC)

Acknowledgments

We thank Colin L. Kremitzki for providing BAC clones.

Author Contributions

Conceived and designed the experiments: JED JAMG. Performed theexperiments: JED TAH. Analyzed the data: JED TAH EK JAMG. Wrotethe paper: JED JAMG.

References

1. Bininda-Emonds OR, Cardillo M, Jones KE, MacPhee RD, Beck RM, et al.(2007) The delayed rise of present-day mammals. Nature 446: 507–512.

2. WarrenWC, Hillier LW, Graves JAM, Birney E, Ponting CP, et al. (2008) Genomeanalysis of the platypus reveals unique signatures of evolution. Nature 453: 175–183.

3. Bick YA, Sharman GB (1975) The chromosomes of the platypus (Ornithorhynchus:Monotremata). Cytobios 14: 17–28.

4. Murtagh CE (1977) A unique cytogenetic system in monotremes. Chromosoma65: 37–57.

5. Rens W, Grutzner F, O’Brien PC, Fairclough H, Graves JAM, et al. (2004)Resolution and evolution of the duck-billed platypus karyotype with an

X1Y1X2Y2X3Y3X4Y4X5Y5 male sex chromosome constitution. Proc NatlAcad Sci U S A 101: 16257–16261.

6. Veyrunes F, Waters PD, Miethke P, Rens W, McMillan D, et al. (2008) Bird-likesex chromosomes of platypus imply recent origin of mammal sex chromosomes.Genome Res 18: 965–973.

7. Grutzner F, Rens W, Tsend-Ayush E, El-Mogharbel N, O’Brien PC, et al.(2004) In the platypus a meiotic chain of ten sex chromosomes shares genes withthe bird Z and mammal X chromosomes. Nature 432: 913–917.

8. Graves JAM (1995) The origin and function of the mammalian Y chromosomeand Y-borne genes–an evolving understanding. Bioessays 17: 311–320.

Dosage Compensation in Platypus

PLoS Genetics | www.plosgenetics.org 11 July 2008 | Volume 4 | Issue 7 | e1000140

184

9. Watson JM, Spencer JA, Riggs AD, Graves JAM (1990) The X chromosome ofmonotremes shares a highly conserved region with the eutherian and marsupialX chromosomes despite the absence of X chromosome inactivation. Proc NatlAcad Sci U S A 87: 7125–7129.

10. El-Mogharbel N, Wakefield M, Deakin JE, Tsend-Ayush E, Grutzner F, et al.(2007) DMRT gene cluster analysis in the platypus: new insights into genomicorganization and regulatory regions. Genomics 89: 10–21.

11. Gruetzner F, Ashley T, Graves JAM (2006) How did the platypus get its sexchromosome chain? A comparison of meiotic multiples and sex chromosomes inplants and animals. Chromosoma 115: 75–88.

12. Rens W, O’Brien PC, Grutzner F, Clarke O, Graphodatskaya D, et al. (2007)The multiple sex chromosomes of platypus and echidna are not completelyidentical and several share homology with the avian Z. Genome Biol 8: R243.

13. Ezaz T, Stiglec R, Veyrunes F, Graves JAM (2006) Relationships betweenvertebrate ZW and XY sex chromosome systems. Curr Biol 16: R736–743.

14. Charlesworth D, Charlesworth B, Marais G (2005) Steps in the evolution ofheteromorphic sex chromosomes. Heredity 95: 118–128.

15. Graves JAM (2006) Sex chromosome specialization and degeneration inmammals. Cell 124: 901–914.

16. Fridolfsson AK, Cheng H, Copeland NG, Jenkins NA, Liu HC, et al. (1998)Evolution of the avian sex chromosomes from an ancestral pair of autosomes.Proc Natl Acad Sci U S A 95: 8147–8152.

17. Nanda I, Zend-Ajusch E, Shan Z, Grutzner F, Schartl M, et al. (2000)Conserved synteny between the chicken Z sex chromosome and humanchromosome 9 includes the male regulatory gene DMRT1: a comparative(re)view on avian sex determination. Cytogenet Cell Genet 89: 67–78.

18. Shetty S, Griffin DK, Graves JAM (1999) Comparative painting reveals strongchromosome homology over 80 million years of bird evolution. ChromosomeRes 7: 289–295.

19. Lercher MJ, Urrutia AO, Hurst LD (2003) Evidence that the human Xchromosome is enriched for male-specific but not female-specific genes. Mol BiolEvol 20: 1113–1116.

20. Saifi GM, Chandra HS (1999) An apparent excess of sex- and reproduction-related genes on the human X chromosome. Proc Biol Sci 266: 203–209.

21. Zechner U, Wilda M, Kehrer-Sawatzki H, Vogel W, Fundele R, et al. (2001) Ahigh density of X-linked genes for general cognitive ability: a run-away processshaping human evolution? Trends Genet 17: 697–701.

22. Ross MT, Grafham DV, Coffey AJ, Scherer S, McLay K, et al. (2005) The DNAsequence of the human X chromosome. Nature 434: 325–337.

23. Storchova R, Divina P (2006) Nonrandom representation of sex-biased genes onchicken Z chromosome. J Mol Evol 63: 676–681.

24. Lyon MF (1961) Gene action in the X-chromosome of the mouse (Mus musculusL.). Nature 190: 372–373.

25. Graves JAM, Gartler SM (1986) Mammalian X chromosome inactivation:testing the hypothesis of transcriptional control. Somat Cell Mol Genet 12:275–280.

26. Cooper DW, Johnston PG, Graves JAM (1993) X-inactivation in marsupials andmonotremes. Sem Dev Biol 4: 117–128.

27. Johnston CM, Lovell FL, Leongamornlert DA, Stranger BE, Dermitzakis ET, etal. (2008) Large-scale population study of human cell lines indicates that dosagecompensation is virtually complete. PLoS Genet 4: e9.

28. Carrel L, Willard HF (2005) X-inactivation profile reveals extensive variability inX-linked gene expression in females. Nature 434: 400–404.

29. Brown CJ, Lafreniere RG, Powers VE, Sebastio G, Ballabio A, et al. (1991)Localization of the X inactivation centre on the human X chromosome in Xq13.Nature 349: 82–84.

30. Rastan S (1983) Non-random X-chromosome inactivation in mouse X-autosome translocation embryos–location of the inactivation centre. J EmbryolExp Morphol 78: 1–22.

31. Costanzi C, Pehrson JR (1998) Histone macroH2A1 is concentrated in theinactive X chromosome of female mammals. Nature 393: 599–601.

32. Heard E, Rougeulle C, Arnaud D, Avner P, Allis CD, et al. (2001) Methylationof histone H3 at Lys-9 is an early mark on the X chromosome during Xinactivation. Cell 107: 727–738.

33. Jeppesen P, Turner BM (1993) The inactive X chromosome in female mammalsis distinguished by a lack of histone H4 acetylation, a cytogenetic marker forgene expression. Cell 74: 281–289.

34. Graves JAM (1982) 5-azacytidine-induced re-expression of alleles on the inactiveX chromosome in a hybrid mouse cell line. Exp Cell Res 141: 99–105.

35. Mohandas T, Sparkes RS, Shapiro LJ (1981) Reactivation of an inactive humanX chromosome: evidence for X inactivation by DNA methylation. Science 211:393–396.

36. Lyon MF (2006) Do LINEs Have a Role in X-Chromosome Inactivation?J Biomed Biotechnol 2006: 59746.

37. Hore TA, Koina E, Graves JAM (2007) The region homologous to the X-chromosome inactivation centre has been disrupted in marsupial andmonotreme mammals. Chromosome Res 15: 147–161.

38. Davidow LS, Breen M, Duke SE, Samollow PB, McCarrey JR, et al. (2007) Thesearch for a marsupial XIC reveals a break with vertebrate synteny.Chromosome Res 15: 137–146.

39. Shevchenko AI, Zakharova IS, Elisaphenko EA, Kolesnikov NN, Whitehead S,et al. (2007) Genes flanking Xist in mouse and human are separated on the Xchromosome in American marsupials. Chromosome Res 15: 127–136.

40. Duret L, Chureau C, Samain S, Weissenbach J, Avner P (2006) The Xist RNAgene evolved in eutherians by pseudogenization of a protein-coding gene.Science 312: 1653–1655.

41. Graves JAM (1967) DNA synthesis in chromosomes of cultured leucocytes fromtwo marsupial species. Exp Cell Res 46: 37–57.

42. Wakefield MJ, Keohane AM, Turner BM, Graves JAM (1997) Histoneunderacetylation is an ancient component of mammalian X chromosomeinactivation. Proc Natl Acad Sci U S A 94: 9665–9668.

43. Loebel DA, Johnston PG (1996) Methylation analysis of a marsupial X-linkedCpG island by bisulfite genomic sequencing. Genome Res 6: 114–123.

44. Cooper DW (1971) Directed genetic change model for X chromosomeinactivation in eutherian mammals. Nature 230: 292–294.

45. Takagi N, Sasaki M (1975) Preferential inactivation of the paternally derived Xchromosome in the extraembryonic membranes of the mouse. Nature 256:640–642.

46. Huynh KD, Lee JT (2005) X-chromosome inactivation: a hypothesis linkingontogeny and phylogeny. Nat Rev Genet 6: 410–418.

47. Marahrens Y, Panning B, Dausman J, Strauss W, Jaenisch R (1997) Xist-deficient mice are defective in dosage compensation but not spermatogenesis.Genes Dev 11: 156–166.

48. Okamoto I, Arnaud D, Le Baccon P, Otte AP, Disteche CM, et al. (2005)Evidence for de novo imprinted X-chromosome inactivation independent ofmeiotic inactivation in mice. Nature 438: 369–373.

49. Mikkelsen TS, Wakefield MJ, Aken B, Amemiya CT, Chang JL, et al. (2007)Genome of the marsupial Monodelphis domestica reveals innovation in non-coding sequences. Nature 447: 167–177.

50. Kuroda Y, Arai N, Arita M, Teranishi M, Hori T, et al. (2001) Absence of Z-chromosome inactivation for five genes in male chickens. Chromosome Res 9:457–468.

51. Kuroiwa A, Yokomine T, Sasaki H, Tsudzuki M, Tanaka K, et al. (2002)Biallelic expression of Z-linked genes in male chickens. Cytogenet Genome Res99: 310–314.

52. McQueen HA, McBride D, Miele G, Bird AP, Clinton M (2001) Dosagecompensation in birds. Curr Biol 11: 253–257.

53. Itoh Y, Melamed E, Yang X, Kampf K, Wang S, et al. (2007) Dosagecompensation is less effective in birds than in mammals. J Biol 6: 2.

54. Ellegren H, Hultin-Rosenberg L, Brunstrom B, Dencker L, Kultima K, et al.(2007) Faced with inequality: chicken do not have a general dosagecompensation of sex-linked genes. BMC Biol 5: 40.

55. Teranishi M, Shimada Y, Hori T, Nakabayashi O, Kikuchi T, et al. (2001)Transcripts of the MHM region on the chicken Z chromosome accumulate asnon-coding RNA in the nucleus of female cells adjacent to the DMRT1 locus.Chromosome Res 9: 147–165.

56. Melamed E, Arnold AP (2007) Regional differences in dosage compensation onthe chicken Z chromosome. Genome Biol 8: R202.

57. Bisoni L, Batlle-Morera L, Bird AP, Suzuki M, McQueen HA (2005) Female-specific hyperacetylation of histone H4 in the chicken Z chromosome.Chromosome Res 13: 205–214.

58. Wrigley JM, Graves JAM (1988) Sex chromosome homology and incomplete,tissue-specific X-inactivation suggest that monotremes represent an intermediatestage of mammalian sex chromosome evolution. J Hered 79: 115–118.

59. Cooper DW, Edwards C, James E, Sharman GB, VandeBerg JL, et al. (1977)Studies on metatherian sex chromosomes. VI. A third state of an X-linked gene:partial activity for the paternally derived Pgk-A allele in cultured fibroblasts ofMacropus giganteus and M. parryi. Aust J Biol Sci 30: 431–443.

60. Johnston PG, G.B. S, James EA, Cooper DW (1978) Studies on metatherian sexchromosomes. VII. Glucose-6-phosphate dehydrogenase expression in tissuesand cultured fibroblasts of Kangaroos. Aust J Biol Sci 31: 415–424.

61. Koina E, Wakefield MJ, Walcher C, Disteche CM, Whitehead S, et al. (2005)Isolation, X location and activity of the marsupial homologue of SLC16A2, anXIST-flanking gene in eutherian mammals. Chromosome Res 13: 687–698.

62. Disteche CM (1999) Escapees on the X chromosome. Proc Natl Acad Sci U S A96: 14180–14182.

63. Yen ZC, Meyer IM, Karalic S, Brown CJ (2007) A cross-species comparison ofX-chromosome inactivation in Eutheria. Genomics.

64. Nguyen DK, Disteche CM (2006) High expression of the mammalian Xchromosome in brain. Brain Res 1126: 46–49.

65. Ohlsson R, Paldi A, Graves JAM (2001) Did genomic imprinting and Xchromosome inactivation arise from stochastic expression? Trends Genet 17:136–141.

66. Gimelbrant A, Hutchinson JN, Thompson BR, Chess A (2007) Widespreadmonoallelic expression on human autosomes. Science 318: 1136–1140.

67. Carrel L, Willard HF (1999) Heterogeneous gene expression from the inactive Xchromosome: an X-linked gene that escapes X inactivation in some human celllines but is inactivated in others. Proc Natl Acad Sci U S A 96: 7364–7369.

68. Disteche CM, Filippova GN, Tsuchiya KD (2002) Escape from X inactivation.Cytogenet Genome Res 99: 36–43.

69. Disteche CM (1995) Escape from X inactivation in human and mouse. Trendsin Genetics 11: 17–22.

70. Ciccodicola A, D’Esposito M, Esposito T, Gianfrancesco F, Migliaccio C, et al.(2000) Differentially regulated and evolved genes in the fully sequenced Xq/Yqpseudoautosomal region. Human Molecular Genetics 9: 395–401.

71. Graves JAM, Disteche CM (2007) Does gene dosage really matter? J Biol 6: 1.

Dosage Compensation in Platypus

PLoS Genetics | www.plosgenetics.org 12 July 2008 | Volume 4 | Issue 7 | e1000140

185

72. Smith CA, Sinclair AH (2004) Sex determination: insights from the chicken.Bioessays 26: 120–132.

73. Alsop AE, Miethke P, Rofe R, Koina E, Sankovic N, et al. (2005) Characterizingthe chromosomes of the Australian model marsupial Macropus eugenii (tammarwallaby). Chromosome Res 13: 627–636.

74. Liu WH, Saint DA (2002) Validation of a quantitative method for real time PCRkinetics. Biochemical & Biophysical Research Communications 294: 347–353.

75. Zhang Z, Schwartz S, Wagner L, Miller W (2000) A greedy algorithm foraligning DNA sequences. J Comput Biol 7: 203–214.

76. Kent WJ (2002) BLAT- the BLAST-like alignment tool. Genome Res 12:656–664.

77. Germer S, Holland MJ, Higuchi R (2000) High-throughput SNP allele-frequency determination in pooled DNA samples by kinetic PCR. GenomeResearch 10: 258–266.

78. van Raamsdonk CD, Tilghman SM (2001) Optimizing the detection of nascenttranscripts by RNA fluorescence in situ hybridization. Nucleic Acids Research29: e42.

Dosage Compensation in Platypus

PLoS Genetics | www.plosgenetics.org 13 July 2008 | Volume 4 | Issue 7 | e1000140

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APPENDIX 4: GENOME OF THE MARSUPIAL

MONODELPHIS DOMESTICA REVEALS

INNOVATION IN NON-CODING SEQUENCES

The following appendix is a publication from early 2007 that reports the genomic

sequence of the gray short-tailed opossum (Monodelphis domestica), the first marsupial

to have its genetic makeup fully characterised.

Mikkelsen TS, Wakefield MJ, Aken B, Amemiya CT, Chang JL, Duke S,

Garber M, Gentles AJ, Goodstadt L, Heger A, Jurka J, Kamal M, Mauceli E,

Searle SM, Sharpe T, Baker ML, Batzer MA, Benos PV, Belov K, Clamp M,

Cook A, Cuff J, Das R, Davidow L, Deakin JE, Fazzari MJ, Glass JL, Grabherr

M, Greally JM, Gu W, Hore TA, et al. (2007) Genome of the marsupial

Monodelphis domestica reveals innovation in non-coding sequences. Nature 447:

167-177.

My contribution to this 164-author publication was a number of localisation

experiments and bioinformatic analyses, most of which were reported in Chapter 2. My

demonstration that marsupials do not possess a homologue of XIST, and have

rearrangements in the region homologous to the X inactivation centre, was critical to the

section of this paper discussing X inactivation. As discussed in Chapter 6, this paper

predicts that the evolution of XIST within the eutherian mammals imposed restrictions

upon evolution of the X chromosome, suppressing genomic rearrangement and

enriching the insertion of LINE1 repeat elements.

187

Pages 188-199 cannot be shown as they derive from the copyrighted publication:

Mikkelsen TS, Wakefield MJ, Aken B, Amemiya CT, Chang JL, Duke S,

Garber M, Gentles AJ, Goodstadt L, Heger A, Jurka J, Kamal M, Mauceli E,

Searle SM, Sharpe T, Baker ML, Batzer MA, Benos PV, Belov K, Clamp M,

Cook A, Cuff J, Das R, Davidow L, Deakin JE, Fazzari MJ, Glass JL, Grabherr

M, Greally JM, Gu W, Hore TA, et al. (2007) Genome of the marsupial

Monodelphis domestica reveals innovation in non-coding sequences. Nature 447:

167-177. doi:10.1038/nature05805

This publication can be retrieved from: http://dx.doi.org/10.1038/nature05805