X Inactivation in Mammals

Done by Rana Abdulnaser AL-Hakimi

Definitions. Imprinted X Chromosome Inactivation.XIST Regulator.Why only the Xp is inactivated?Reactivation X Chromosome. Random X Chromosome Inactivation. Non-random X Chromosome Inactivation.

CONTENTS

X- chromosome inactivation in mammals

XCI is a dosage-compensation mechanism that evolved to equalize expression levels of x-linked genes in female (2x) and male (1x) by transcriptional silencing of one x-chromosome in female mammalian cells.

It is responsible for initiating X inactivation in cis: an X-chromosome fragment that carries a Xic can become

inactivated, whereas one in which the Xic is missing cannot.

The Xic is also involved in ‘counting’, whereby only a single X is kept active per two sets of autosomes in a cell, and all other Xic-carrying chromosomes are inactivated.

DEFINITIONS

X- inactivation center (XIC)

Reciprocal translocation Deletion Normal

Conserved structure, low sequence conservation (except for repea).

17 kb – 19 kb spliced, untranslated, nuclear transcriptXist is essential for the initiation of inactivation in cis RNA expressed from and “coats” the inactive X chromosome in

cis.Xist can only induce silencing during an early developmental

time. Conserved “A” repeats ensure silencing function... How?Multiple Xist domains required for coating… How?

Xist (X- inactivation specific transcript) RNA

This process occur in the early stage of embryo development

approximately in 2-cell stage until the blastocyst stage and continues in the trophectoderm and the primitive endoderm.. The cell undergo to inactivate x parental only and the x maternal stay active..

IMPRINTING X INACTIVATION

In mouse

Why only the imprinted X inactivation found in XP not in XM ?? because of Rnf12 that inherited only from mother germ line and this factor act as XIST activator and work in trans ..

XIST-Independent In this step of imprinting x- inactivation the non-genic repeat

regions, such as long interspersed elements (LINEs) and short interspersed repetitive elements (SINEs), is silenced.

The repeat silencing precedes genic silencing in imprinted X-inactivation.

The X-linked repeat elements preinactivated and inherited from the paternal germ line.

Imprinted X-inactivation originates from meiotic sex chromosome inactivation (MSCI) in male spermatogenesis and that the pre-inactivated X-chromosome is inherited from father to daughter.

IMPRINTED XCI OCCURS IN TWO STEPS

MSCI

Meiotic silencing is a general silencing mechanism, which represses unsynapsed chromatin in male germ cells and has been termed meiotic silencing of unsynapsed chromatin (MSUC).

MSCI consists of two genetically separable steps:

1

MDC1-INDEPENDENT recognition of the unsynapsed axis, in which the ATR–TOPBP1 complex phosphorylates H2AX

2

MDC1-DEPENDENT chromosome-wide spreading of the ATR–TOPBP1 complex and the cH2AX signal to the entire chromatin.

MSCI PROCESS

gH2AX signal occurs at the unsynapsed axes of sexChromosomes.

MDC1 (mediator of DNA damage checkpoint protein 1) mediates the spreading of the gH2AX signal over the chromosome-wide domain through directly recruits the ATR–TOPBP1 complex to amplify the gh2ax signals in meiotic silencing.

XY body formation The chromosome-wide accumulation of gH2AX on sex chromosomes is concomitant with the formation of a distinct heterochromatin domain called the XY body.

This step Occurs at the four cell stage.The XIST expression lead to chromosome XP-inactivation.

XIST expressed from XP . XIST starts coating the XP .

The number of XIST recruit the Polycomb repressive complex 2 (pcr2) to the xi.

XIST- DEPENDENT

In mouse

What happens in this step?

1

2

3

PCR2 recruite histon h2 lysine 27 trimethylation (H3k27 me3).

TSIX RNA expressed from XM.

4

5

They thought that imprinted x-inactivation doesn’t occur in human, human x chromosome was investigated(Ilse M. van den Berg.,et al 2009) in three levels:

XIST expression in early human embryos.

XIST- DEPENDENT

In human

1

At the morula stage only a fraction of unstable XIST signals was expressed and this indicating absence of XCI male preimplantation.

Inmale

Majority of cells had pinpoint signals for XIST RNA at the 8-cell stage. The XIST signal gradually accumulated to a full cloud on one of the X chromosomes at the late morula and blastocyst stages.

In female

Transcriptional activity on the XIST-coated chromosomal

region.

activity was investigated by Cot1 RNA FISH staining, which highlights areas of ongoing hnRNA transcription; trancriptionally silent nuclear compartments, such as an Xi chromosome, are devoid of Cot1 RNA.

!

2

Inmale

In female (A–C) Cells of a female lastocyst embryo stained for Cot1 RNA (red in A) and XIST RNA (green in B) showing depleted regions of Cot-1 RNA around the XIST signals indicating the position of the Xi chromosome (merged in C). (D–F) Representative cell of a female blastocyst with staining for the X centromeres and XIST RNA (D; Xcen in magenta, XIST in green) together with Cot1 (red in E). Transcription of Cot1 RNAwas absent in a region that overlaps with XIST RNA staining (F), whereas the active X without XIST staining overlaps witha Cot1-positive region. (G–J) Female blastocyst cell with two X centromeres (cyan in G) has a single XIST cloud on one X chromosome (green in H) and monoallelic expression of CHIC1 on the other X chromosome (red in I, merged in J).

Chromatin conformational changes on the inactive

X -chromosome to further explore.

1. The chromatin state of X chromosomes was carried out with antibodies that detect

Hypoacetylation of lysine 9 on histone H3 (H3K9ac) accumulation of trimethylation of lysine 27 on H3 (H3K27Me3). The enrichment of the histone variant acroH2A.

2. The gender identification of the embryo used DNA FISH.

4

Inmale

Embryos did not show accumulation Of H3K27Me3 or macroH2aAnd no specific exclusion of H3K9ac was observed.

double staining showed a single region where accumulated H3K27Me3 formed an exact overlay with the region of H3K9 hypoacetylation.(A–C) indicating the presence of an Xi. Furthermore,macroH2A enrichment and accumulation of H3K27Me3(D–F) colocalized exactly in blastocyst cells. Up to 30% of the analyzable cells in blastocysts showed a double immunostaining of chromatin marks that are specific for XCI (either H3K27Me3 together with macroH2A or H3K27Me3 in a depleted region of H3K9ac). The other 70% of the cells had no visible accumulation(or depletion, in the case of H3K9ac) of either antibody, and single accumulations were rarely found (<5%).

In female

In marsupials

Imprinted XCI is found in all tissues of marsupialsXCI is not random but is paternally imprinted.Marsupials exhibit dosage compensation by wide silencing of the X. No hypermethylation is found Only hypoacetylation.

X-CHROMOSOME REACTIVATION

This reactivation process allows both Xp and Xm get an equal chance to be subjected to random XCI in the future embryo-proper.The over expression of TSIX is sufficient for reactivation of XP .. The experiment done by transgenic TSIX in extra-embryonic tissue placenta (which always have imprinting inactivation..This cell was reactivated by TSIX.

The first instance of X-reactivation occurs at the blastocyst stage between embryonic day (E)3.5 and E4.5. This coincides with the time when blastocysts implant into the uterus and shortly after the first distinct cell lineages become

apparent. X-chromosome reactivation doesn’t occur in the trophectoderm and primitive

endoderm, which will later give rise to extra-embryonic tissues like the placenta. The characteristic signs of the X-reactivation process are the downregulation of Xist expression. disappearance of the accumulation of Polycomb proteins Ezh2 and Eed and their

associated histon H3 lysine 27 di/tri-methylation mark (H3K27me2/3) from the paternal X-chromosome.

A new study suggests that reactivation of some X-linked genes and of repeat sequences might even occur before the chromosome-wide removal of Xist RNA and H3K27me3 from the inactive X-chromosome.

X-CHROMOSOME REACTIVATION

Binding of Oct4, Sox2 and Nanog has been demonstrated to Xist intron 1 in ES cells lead to downregulation of Xist expression from the inactive X-chromosome.

Furthermore, Oct4, Klf4, c-Myc and Rex-1 bind DXPas34 and Oct4, Sox2 and Klf4 bind Xite, both of which are enhancers of Tsix, the non-coding antisense regulator gene of Xist during X-inactivation.

Depletion of Oct4 from ES cells by RNAi knockdown or inducible downregulation results in upregulation of Xist from both X-chromosomes in female cells and depending on experimental conditions even from male ES cells.

Conversely Tsix is downregulated after Oct4, Rex1 or c-Myc knockdown.

THE FUNCTION OF PLURIPOTENCY FACTORS IN X-REACTIVATION

Nanog in particular seems to be important for X-reactivation

in blastocysts, as Nanog-mutant female embryos

fail to erase the characteristic H3K27 trimethylation from

the inactive X in the inner cell mass .Nanog-mutant ES cells show some Xist upregulation

albeit at lower levels than after Oct4 depletion.

Xist activators

  JPX. RNF12. H19. RepA (recruits PRC2)..

(RepA-PCR2)..Target the H3k27me3 modification on the Xist promoter to facilitate Xist transcription.

Xist inhibators

(Plulripotency factors )

Tsix activators (Xite, DXPas34).

Xist regulatory factors

TsixTSIX expression blocks loading of the RepA-PRC2 complex onto the 5 end of Xist.Implicated in the direct regulation of DNA methylation.Recruiting the RNAI machinery.

RANDOM-X INACTIVATION

Random X inactivation occurs in the early female embryo, where both the maternal and the paternal X chromosome have an equal chance of becoming inactivated.

Sensing: Xpr which is a heterochromatic region brings the XICs together.

Counting/choice: The number of XICs are counted, and one X chromosome is randomly chosen to remain active while the other is targeted for inactivation. This was done by XICs pairing.

RANDOM X-INACTIVATION STEPS.

Initiation

X-chromosome pairing via the interaction between oct4 and ctcf that lead to distribution of tanscription and pluripotent factors on the two X– chromosome this lead to:

Xist expression from one X-chromosome and Tsix repression from the same allele.

In the other chromosome Tsix recruits the DNA methylatransferase dnmt3a which methylate Xist promoter leading to Xist silencing and the cell now have X activation . In the active X the Tsix regulate the Xist silencing by directly suppress Xist or indirectly by suppress Xist activators

RepA RNA is expressed from the future Xi and recruits the PRC2 complex. Jpx and Rnf12 are upregulated from both the Xi and Xa.

Pairing .. Counting.. Choice

Spreading is the process of compacting and inactivating the X

chromosome. A gene designated XIST with the X inactivation center encoded

an RNA that coat the X chromosome and promotes compaction- beginning at the XIC and progressing toward both ends until the entire x chromosome is inactivated, when spreading is finished the compacted factor could bar body.

Spreading

factors that found during spreading process

YY1(YingYang1) (hnRNPu)RNA –U (heterogeneous nuclear riboncleo protein U )SATB1 (special A+T rich binding protein 1) LINE(long interspersed elements )PCL2 (Polycomblike 2)

Factors Figures

YY1(YingYang1)

( hnRNPu )RNA –U (heterogeneous nuclear riboncleo protein U )

LINE(long interspersed elements )

SATB1 (special A+T rich binding protein 1)

PCL2 (Polycomblike 2)

1 1,5

2

2

3

3

4

5

The bar body is replicated just like other chromosome, but both copies of the replicated bar body remain highly compacted and essentially inactivated.Thus, once an embryonic cell undergoes X-inactivation, all cells that drive from it will have the same chromosome inactivation .

Maintenance

NON RANDOM X-INACTIVATION

Primary non random x-inactivation cause by the mutations in Xist or Tsix. When the mutation found in Tsix the wild type become always the activated chromosome. When the mutation found in Xist gene the wild type become always x-inactivated chromosome. According to experiment the mice that inherited mutation in Tsix from their father, born healthy, but those inherited mutation from their mother only 18% of them survived.

PrimaryNon random

X-inactivation

NON RANDOM X-INACTIVATION

Secondary nonrandom inactivation wherebycells expressing one of the two x-chromosome are selected against that the selective death of cells that inactivate the incorrect number of X- chromosome because the fate of X-chromosome doesn't determined before silencing .

SecondaryNon-random

X-inactivation

References   Panning, B. 2008 X-chromosome inactivation: the molecular basis of silencing: Journal of Biology.7:30

Ilse, M. den Berg, V. Joop ,S. E, Laven. Stevens, M. Jonkers, I. Jan Galjaard, R Gribnau, J. and J. Hikke van Doorninck 2009 X Chromosome Inactivation Is Initiated in Human Preimplantation Embryos:The American Journal of Human Genetics. 84: 771–779.  Kalantry, S. 2011 Recent Advances in X-Chromosome Inactivation: NIH Public Access Author Manuscript. 226(7): 1714–1718.

Payer, B. Jeannie T, Lee . Namekawa H, S. 2011 X-inactivation and X-reactivation: epigenetic hallmarks of mammalian reproduction and pluripotent stem cells: Hum Genet. 130: 265–280.

     

References Jeon, Y. Sarma, K. and T Lee, J. 2012 New and Xisting regulatory mechanisms of X chromosome inactivation: Current Opinion in Genetics & Development. 22:62–71.

Valerie Gendre, A. and Heard, E. 2011Fifty years of X- activationResearch:Development .138: 5049-5055.

Augui, S. Elphège P. Nora. and Heard, E. 2011 Regulation of X-chromosome inactivation by the X-inactivation centre: Nature Reviews Genetics. 12: 429-442 . E Senner, C.Brockdorff, N. Xist gene regulation at the onset of X inactivation 2009:Current Opinion in Genetics & Development.19(2): 122-126. T. Lee, J. 2011 Gracefully ageing at 50, X-chromosome inactivation becomes a paradigm for RNA and chromatin control: Nature Reviews Molecular Cell Biology. 12: 815-826.