-arrestin1/mir-326 transcription unit is epigenetically

Research Article120573-Arrestin1miR-326 Transcription Unit Is EpigeneticallyRegulated in Neural Stem Cells Where It Controls Stemnessand Growth Arrest

Agnese Po1 Federica Begalli1 Luana Abballe2 Vincenzo Alfano1 Zein Mersini Besharat1

Giuseppina Catanzaro2 Alessandra Vacca2 Maddalena Napolitano1 Marco Tafani2

Felice Giangaspero34 Franco Locatelli5 Elisabetta Ferretti24 and Evelina Miele56

1Department of Molecular Medicine Sapienza University of Rome Rome Italy2Department of Experimental Medicine Sapienza University of Rome Rome Italy3Department of Radiological Oncological and Anatomopathological Sciences Sapienza University of Rome Rome Italy4IRCCS Neuromed Pozzilli Italy5Department of HematologyOncology and Stem Cell Transplantation Bambino Gesu Childrenrsquos Hospital IRCCS Rome Italy6Center for Life NanoScienceSapienza Istituto Italiano di Tecnologia Rome Italy

Correspondence should be addressed to Elisabetta Ferretti elisabettaferrettiuniroma1it

Received 4 November 2016 Revised 2 January 2017 Accepted 15 January 2017 Published 12 February 2017

Academic Editor Jinsong Zhang

Copyright copy 2017 Agnese Po et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Cell development is regulated by a complex network of mRNA-encoded proteins and microRNAs all funnelling onto themodulation of self-renewal or differentiation genes How intragenic microRNAs and their host genes are transcriptionallycoregulated and their functional relationships for the control of neural stem cells (NSCs) are poorly understood We propose herethe intragenic miR-326 and its host gene 120573-arrestin1 as novel players whose epigenetic silencing maintains stemness in normalcerebellar stem cells Such a regulation is mediated by CpG islands methylation of the common promoter Epigenetic derepressionof 120573-arrestin1miR-326 by differentiation signals or demethylating agents leads to suppression of stemness features and cell growthand promotes cell differentiation120573-Arrestin1 inhibits cell proliferation by enhancing the nuclear expression of the cyclin-dependentkinase inhibitor p27 Therefore we propose a new mechanism for the control of cerebellar NSCs where a coordinated epigeneticmechanism finely regulates 120573-arrestin1miR-326 expression and consequently NSCs stemness and cell growth

1 Introduction

Neural stem cells (NSCs) are believed to foster a hierar-chical developmental program in which self-renewal andplurimultipotency are responsible for the expansion andorthe maintenance of an uncommitted cell population poolNSCs are ready to undergo the cascade of lineage restrictionand subsequent terminal differentiation under the action ofregulatory morphogenic cues [1] The early postnatal murinecerebellum containsmultipotent NSCs which can be isolatedand expanded in vitro NSCs maintain their undifferentiatedphenotype under mitogenic signals and are able to differenti-ate into the different kind of neurons [2]

Recent evidences have highlighted the crucial role ofmicroRNAs (miRNAs) in conferring neural cell identitiesduring neural induction neuronal differentiation and sub-type specification [3] MiRNAs are widespread throughoutthe genome where they can be found in either intergenic orintragenic (especially intronic) regions [4] Transcription ofboth intergenic and intragenic miRNAs may be regulated bytheir ownpromoters whether some intragenicmiRNAs sharepromoters with their host genes that generate pre-miRNAand mRNA both arising from the same transcript [5 6]The consequent spatial and temporal coexpression implies afunctional relationship between the intragenic miRNA andits host gene

HindawiStem Cells InternationalVolume 2017 Article ID 5274171 11 pageshttpsdoiorg10115520175274171

2 Stem Cells International

We previously identified miR-326 as a miRNA that isrequired to differentiate cerebellar granule cell progenitors(GCPs) to mature granule cells [7]This miRNA is embeddedwithin the first intron of 120573-arrestin1 (120573arr-1) gene and shareswith its host gene the same regulatory sequences [8 9]Since stem cell commitment to GCPs is a crucial event incerebellar development [2 10] we raised the question ofmiR-326 expression and regulation in cerebellar NSC Wereport here thatmiR-326 expression is further downregulatedin NSC prior to progenitor commitment along with thedownregulation of its host gene Therefore we investigatedthe expression functions and regulation of miR-326120573arr-1transcription unit in NSCs

2 Materials and Methods

Unless otherwise indicated media and supplements werepurchased from Gibco-Invitrogen (Carlsbad CA) chemicalswere purchased from Sigma-Aldrich (St Louis MO) andcommercial products were used according to the manufac-turersrsquo instructionsprotocols

21 Cell Culture

211 Neural Stem Cells Culture Mouse cerebella were ob-tained from postnatal 4-day-old wild-type BL6mice (CharlesRiver) with the approval of Institutional Review Board [89] Briefly tissues were collected in HBSS supplementedwith 05 glucose and penicillin-streptomycin grossly trit-urated with serological pipette and treated with DNAse Ito a final concentration of 004 for 20 minutes Cellswere mechanically dissociated using pipettes of decreasingbore size to obtain a single-cell suspension Neural stemcells (NSCs) were derived and enriched through selectivemedium (SM) DMEMF12 supplemented with 06 glucose25mgmL insulin 60mgmL N-acetyl-L-cysteine 2mgmLheparin 20 ngmL EGF 20 ngmL bFGF (Peprotech RockyHill NJ) penicillin-streptomycin and B27 supplement with-out vitamin A For clonogenicity assay cells were plated atclonal density (1-2 cellsmm2) into 96-well plate To inducedifferentiation NSCs were plated onto D-poly-lysine-coateddishes in differentiation medium (DFM DMEMF12 withN2 supplement and 2mgmL heparin 06 glucose and60mgmL N-acetyl-L-cysteine containing 1 Calf Serumand PDGF 10 ngmL (Sigma P3076) or RA 2mM (SigmaR2625) for 48 hours)

212 Overexpression Studies Amaxa nucleofector (Lonza)was used to transfect plasmids according to manufacturerrsquosprocedure pcDNA3 120573-arrestin1 HA was a gift from RobertLefkowitz (Addgene plasmid 14693) [11] miR-326 vectorand its negative control were purchased from GeneCopoeia(MmiR3333-MR01)

213 Knockdown Experiments Silencing of 120573-arrestin1 wasperformed using ON-TARGETplus SMARTpool (L40976-00-005) from Thermo Scientific after testing each single

siRNA of the pool alone or in combination for its specificityto avoid OFF-target effects

214 Cell Proliferation Cell proliferation was evaluated byBrdU incorporation as previously described [2] More indetail after 24-hour pulse with BrdU NSCs were plated onpoly-lysine-coated Lab-Tek chamber slides (coverslips) andallowed to adhere for 3 hours Cells were fixed with 4paraformaldehyde permeabilized with 01 Triton X-100and BrdU detection (Roche) was performed Nuclei werecounterstained with the Hoechst reagent Cells were countedin triplicate and the number of BrdU-positive nuclei wasannotated

NSC growth was measured by MTT assay (Promega)according to manufacturerrsquos instructions Each sample wasmeasured in triplicate and repeated at least three times

For methylation analysis NSCs were treated with 10120583M5-azacytidine (5-AZA) for four days

22 RNA Isolation and Gene Expression Analysis Total RNAwas purified and reverse transcribed as previously described[12] Quantitative RT-PCR (qRT-PCR) analysis was per-formed using the ABI Prism 7900HT Sequence DetectionSystem (Thermo Scientific) using best coverage TaqMangene expression assays specific for each analyzed mRNAEach amplification was performed in triplicate and theaverage of the three threshold cycles was used to calcu-late the amount of transcripts (Thermo Scientific) mRNAquantification was expressed in arbitrary units as previ-ously described [13] mRNA quantification was expressedin arbitrary units as the ratio of the sample quantity tothe calibrator or to the mean values of control samples Allvalues were normalized to three endogenous controls 120573-actin 1205732-microglobulin and Hprt miRNA expression levelswere evaluated on RNA samples using specific stem-loopprimers to achieve retrotranscription (Thermo Scientific)and was followed by a quantitative RT-PCR using miRNA-specific TaqMan probes (Thermo Scientific) in a ABI Prism7900HT Sequence Detection System (Thermo Scientific)miRNA expression levels were normalized to RNAU6B [14]

23 Western Blot Assay Cells were lysed using RIPA buffer(Tris-HCl pH 76 50mM deoxycholic acid sodium salt 05NaCl 140mM NP40 1 EDTA 5mM NaF 100mM sodiumpyrophosphate 2mM and protease inhibitors) Lysates wereseparated on 8 acrylamide gel and immunoblotted usingstandard procedures The following antibodies were usedanti-120573-arrestin1 K-16 (sc-8182 Santa Cruz BiotechnologyCA) anti-mouse Nanog (Cosmo Bio Co Japan) anti-mouse120573III-tubulin (MAB 1637Millipore) anti-actin I-19 (sc-1616 Santa Cruz Biotechnology CA) anti-GAPDH (ab8245Abcam) anti-Sp1 1C6 (sc-420X Santa Cruz BiotechnologyCA) anti-p27 (F-8) (sc-1641 Santa Cruz Biotechnology)anti-Sox2 (MAB4343 Millipore Billerica MA) anti-Oct4(ab19857 Abcam) anti-mouse 120573III Tubulin (MAB 1637Millipore Billerica MA) anti-GFAPMAB360 (Millipore Bil-lerica MA) and anti-PARP p85 fragment (G7342 Promega)(anti-parp-C) HRP-conjugated secondary antibodies (Santa

Stem Cells International 3

Cruz Biotechnology CA) were used in combination withenhanced chemiluminescence (ECL Amersham AmershamUK) For nucleuscytoplasmic fractionation cells were lysedon ice with buffer containing 20mM Hepes pH 74 20glycerol 50mM KCl 1mM EDTA 5 NP 40 and proteaseinhibitors After centrifugation the cytoplasmic fractionswere collected (supernatant) The pelleted nuclear fractionwas lysed with buffer containing 20mM Hepes pH 7425 glycerol 400mM NaCl 1mM EDTA 1mM EGTA andprotease inhibitors

24 In Situ Hybridization and Immunofluorescence Assay Allreagents used prior to probe hybridization were preparedwith DEPC-treated water (diethyl pyrocarbonate SigmaD5758) to prevent RNAse contamination

Cells were fixed with 4 PFA for 10 minutes at roomtemperature After 3 washes with PBS cells were incubatedfor 2 minutes with 10 120583gmL proteinase K (Sigma P2308) at37∘C

Acetylation was then performed to enhance signal tobackground ratio using 12 triethanolamine (Sigma 90279)00018N HCl and 025 acetic anhydride (Sigma A6404)with constant stirring for 10 minutes at room temperatureCells were washed 3 times with PBS and prehybridizationwas performed using 50 formamide (Sigma F9037) 5XSSCbuffer (FLUKA cat S6639-1L) 01 Tween-20 92mM cit-ric acid (Sigma C1909) 50120583gmL heparin (Sigma H4784)and 500 120583gmL yeast RNA (Sigma R6750) for 3 hours at62∘CHybridization probe (double DIG-labeled Exiqon) wasdiluted in hybridization buffer at a final concentration of25 nM denaturated at 85∘C for 5 minutes cooled on ice andthen incubated on cells at 62∘C for 16 hours

Samples were incubated with 01x SSC buffer for 3 hoursat 67∘C and washed 3 times with 01M Tris-HCl pH 75 and015M NaCl Nonspecific antibody binding was performedwith 05 blocking reagent (Roche 11096176001) 5 SheepSerum (Sigma S3772) in 50mM Tris-HCl pH 75 and 5mMEDTA for 2 hours at room temperature in a humidifiedchamber Fluorescein conjugated anti-DIG was incubatedat the dilution of 1 200 in blocking buffer for 16 hoursat 4∘C After antibody incubation samples were washed3 times with 01M Tris-HCl pH 75 and 015M NaCl andcoverslips weremounted using fluorescentmounting (DAKOS3023)

Immunofluorescence was performed as previously des-cribed [13] More in detail NSCs were cultured in Lab-Tekchamber slides fixed in 4 paraformaldehyde for 20minat room temperature permeabilized with 01 Triton X-100 cells and incubated in blocking buffer (PBS with 1BSA) for 30min and then with anti-HA (sc-7392 SantaCruz) overnight in blocking solution at 4∘C 488-conjugatedanti-rabbit secondary antibody was purchased from Molec-ular Probes (Invitrogen) Nuclei were counterstained withHoechst (H6024 Sigma) At least 300 nuclei were countedin triplicate Fluorescence was visualized and images wereacquired with Carl Zeiss microscope (Axio Observer Z1)using ApoTome technology and AxioVision Digital ImageProcessing Software

25 Analysis of CpG Islands Methylation miR-326120573arr-1regulatory regionwas retrieved byRulai database (httprulaicshleduTRED)

CpG islands were identified in the regulatory regionsof miR-326120573arr-1 by using database of CpG islands andAnalytical Tool (DBCAT) software

The primers for methylation specific PCR were designedby using Methyl Primer Express Software v10 ThermoFisher Scientific Primers were first tested and validated usingmouse Universal Methylated Mouse DNA Standard (ZymoResearch)

251 Bisulphite Treatment and PCR Amplification DNAextraction was performed using the Qiamp DNA mini kit(Qiagen)The obtained DNAwas quantified using Nanodropspectrophotometer (Thermo Scientific) and treated with Epi-Tect Bisulfite kit (Qiagen) The converted DNA was used toPCR-amplify the 120573arr-1 regulatory region with the followingprimers

MeFw TTTTTATTTTTTGGGCGCGTATACMeRw GTCCAAACTAAAAAATCCCCGACUnFw TTTTTATTTTTTGGGTGTGTATATGTUnRw CATCCAAACTAAAAAATCCCCAAC

As positive control we used mouse Universal MethylatedMouse DNA Standard (Zymo Research)

26 Statistical Analysis Statistical analysis of cellular experi-mental triplicates was performed using StatView 41 software(Abacus Concepts Berkeley CA) Statistical differences wereanalyzed by MannndashWhitney119880 test for nonparametric valuesand a 119901 value of le005 was considered significant The resultsare expressed as mean plusmn SD from at least three experiments

3 Results

31 miR-326 and 120573arr-1 Expression Inversely Correlates withStemness First we evaluated expression levels of miR-326and of its host gene 120573arr-1 in NSCs (Figures 1(a)ndash1(d))NSCs displayed as compared to the bulk preneurosphere cellpopulation (T0) very low levels of bothmature and precursormiR-326 forms and of 120573arr-1 (Figures 1(a)ndash1(d)) togetherwith an enhanced expression of the Nanog stemness marker(Figure 1(d))

These results suggest that loss of miR-326120573arr-1 locusexpression is associated with a ldquostem-like phenotyperdquo Thisnotion was further supported by the observation that theexpression of miR-326 and 120573arr-1 increased in NSCs exposedto differentiation stimuli for example retinoic acid (RA) orplatelet-derived growth factor (PDGF) (Figures 2(a)ndash2(d))Under these conditions NSCs were able to differentiate intodistinct lineages as indicated by the increased expressionof both neuronal differentiation markers 120573III-tubulin (120573III-tub) and NeuN and astrocytic differentiation markers Gfapand S100 (Figures 2(c) and 2(d)) and downmodulated theexpression of stemness related markers Nanog Oct4 andSox2 (Figures 2(c) and 2(d))


Pri-miR-326 Pre-miR-326

lowast

lowast

0

05

1

15

mRN

A le

vels

T0NSC

(a)

miR-326

T0NSC

lowast

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vels

(b)

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arr-1

lowast

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05

1

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A le

vels

(c)

T0 NSC

arr-1

Nanog

Actin

(d)

Figure 1 miR-326 and 120573arr-1 are downregulated in cerebellar NSCs compared to the bulk cerebellar population (a) mRNA expressionlevels of the primary (pri-miR-326) and precursor (pre-miR-326) of miR-326 in NSCs compared to bulk cells (T0) (b) Mature miR-326levels in NSCs compared to bulk cells (T0) (c) 120573arr-1 mRNA levels in NSCs compared to bulk cells (T0) (d) Western Blot (WB) analysisof endogenous 120573arr-1 and Nanog in NSCs compared to bulk cells (T0) Actin as loading control (LC) (a)ndash(d) data are means plusmn SD from 3independent experiments lowast119901 lt 005

32 Epigenetic Inactivation of miR-326120573arr-1 Locus Next weinvestigated the mechanism responsible for miR-326120573arr-1locus inactivation in NSCs There is increasing evidence thatepigenetic regulation of stem cells including ldquoCpG islandrdquomethylation is crucial in the preservation of their stemnessby controlling the transcription switch onoff of specificdevelopmental genes [15]

Indeed the presence of several CpG islands in the regionspanning from the first exon to part of the first intron(Figure 3(a) and Supplementary Figure 1 see SupplementaryFigure 1 in the Supplementary Material available onlineat httpsdoiorg10115520175274171) suggested a DNAmethylation-dependent control Treatment of NSCs withthe 51015840-azacytidine (5-AZA) demethylating agent induced asignificant increase of miR-326 its precursor transcripts and120573arr-1 levels (Figure 3(b)) and impaired stemness features

while it increased cell differentiation (Figures 3(c) and 3(d))Usingmethylation specific PCRwe evaluated themethylationstatus of selected CpG islands We found that these CpGislands were methylated in NSCs but not in the startingbulk population (T0) (Figure 3(e)) Moreover when NCSswere treated with 5-AZA the analyzed CpG islands losttheir methylation status (Figure 3(f)) strongly suggestingthat the upregulation of 120573arr-1 and miR-326 was due to thedemethylation of their promoter region (Figure 3(b))

These findings highlight that the methylation status of120573arr-1miR-326 CpG islands is a mechanism to silence theirexpression in cerebellar NSCs

33120573arr-1Negatively RegulatesNSCs Self-Renewal via Increas-ing p27 Nuclear Expression Levels The above presented datasuggested a role of miR-326 and 120573arr-1 in the establishment


RA PDGF

SMDFM

lowast

lowast

0

25

5

miR

-326

leve

ls

(a)D

FMSM

Merge NucleimiR-326

(b)

Nanog Oct4 Sox2 NeuN Gfap S100

lowast

lowastlowast

lowast

SMDFM

Stemness Neuronal Astrocytic

Differentiation

lowastlowast lowast

lowast

arr-1 III-tub0

25

5

mRN

A le

vels

(c)

SM DFM

Nanog

Oct4

Sox2

Gfap

arr-1

III-tub

Gapdh

(d)

Figure 2 miR-326 and 120573arr-1 are upregulated in differentiated NSCs (a) miR-326 levels evaluated in NSC exposed to differentiation stimuli(DFM)with retinoic acid (RA) or platelet-derived growth factor (PDGF) for 48 hours Data aremeans plusmn SD from 3 independent experimentslowast119901 lt 005 (b) Fluorescent in situ hybridization (ISH) staining of miR-326 of NSCs grown in stemness conditions (SM) and after RA-induceddifferentiation (DFM) for 48 hours Nuclei are counterstained with Hoechst Scale bar 10120583m Representative ISH images from 4 independentexperiments (c) mRNA expression levels of 120573arr-1 along with stemness and differentiation markers of NSC in SM and after differentiation(DFM) (d) Western blot (WB) analysis of endogenous 120573arr-1 and markers of stemness (Nanog Sox2 and Oct4) and differentiation (120573III-tub and Gfap) of NSCs in SM and after differentiation (DFM) Gapdh as loading control (LC) ((c) and (d)) Data are means plusmn SD from 3independent experiments lowast119901 lt 005

of a ldquodifferentiated phenotyperdquo miR-326 is known to controlseveral morphogenic signals that sustain stemness such asthe Hedgehog pathway and the Notch pathway [7 16] andwe found it is able to regulate Gli2 and Smo expression alsoin NSCs (data not shown) Indeed miR-326 overexpressionin NSCs (Figure Supplementary 2A) significantly reducedclonogenicity (Figure Supplementary 2B) and cell viability(Figure Supplementary 2C)

On the other hand previous studies have shown that120573arr-1 functions as a cytoplasm-nucleus shuttling proteinthat interacts with p300CBP to activate the transcription ofthe cyclin-dependent kinase (CDK) inhibitor Cdkn1bp27kip(p27) a major determinant of cell cycle exit [16] To gaininsight into 120573arr-1 functions in neuronal cells stemness anddifferentiation we overexpressed 120573arr-1 (Figures 4(a) and4(b)) and found a strong impairment of the expression levels


TSS

First exon First intronChr 11

CpG island regionCpG sites

minus1321 +176

arr-1

(a)


Ctr5-AZA

arr-1

lowast

lowast

lowast

0

25

5

mRN

A le

vels

miR-326

lowast

0

25

5

miR

NA

leve

ls

(b)


Ctr5-AZA

Stemness Neuronal AstrocyticDifferentiation

III-tub0

25

5

mRN

A le

vels

lowast lowastlowast

lowastlowast

lowast

lowast

(c)

Nanog

Oct4

Sox2

Gfap

arr-1

III-tub

Actin

minus + 5-AZA

(d)

Methyl

Unmethyl

T0 NSC

(e)

Methyl

Unmethyl

minus + 5-AZA

(f)

Figure 3 CpG island methylation of miR-326120573arr-1 regulatory sequence controls 120573arr-1 and miR-326 expression (a) Schematicrepresentation of mouse miR-326120573arr-1 regulatory regions highlighting the CpG islands regions (light blue) and the CpG islands sites (blue)derived fromDBCAT software (b) mRNA expression levels of 120573arr-1 and precursor forms ofmiR-326 andmiR-326 expression levels in NSCsafter 4 days of 5-azacytidine (5-AZA) treatment Data aremeansplusmn SD from3 independent experiments lowast119901 lt 005 ((c)-(d))mRNAexpressionlevels (c) andWestern blot analysis (d) of endogenous 120573arr-1 stemness markers (Nanog Oct4 and Sox2) and differentiation markers (120573III-tub and Gfap) after 4 days of 5-azacytidine (5-AZA) treatment in NSCs Actin as LC Data are means plusmn SD from 3 independent experimentslowast119901 lt 005 (e)Methylation specific PCRof the screened region inNSCs compared to the starting bulk population (T0) (f)Methylation specificPCR of the screened region in NSCs after 5-AZA treatment ((e) and (f)) Data are representative images from 3 independent experiments

of the stemnessmarkers Nanog Oct4 and Sox2 (Figures 4(b)and 4(c)) impaired clonogenicity together with decreasedproliferation rate and cell viability (Figures 4(d) and 4(e))and increased cell apoptosis (Figure 4(f)) suggesting that120573arr-1 may have a role both in the regulation of stemnessfeatures and in the regulation of cell cycle Indeed we

found that when shifted to differentiation medium NSCsincreased 120573arr-1 level was paralleled by an activation of p27transcription and the increase of p27 protein in the nucleusas observed in nucleouscytoplasmic fractionation experi-ment (Figure 5) Accordingly p27 mRNA and the nuclearprotein cell fraction levels were increased by exogenously


Moc

k

NucleiMerge Anti-HA

oe H

A-

arr-1

(a)

Actin

Nanog

minus +

Sox2

Oct4

oe HA-arr-1

arr-1

(b)

Nanog Oct4 Sox2

lowastlowast

lowast

0

05

1

15

mRN

A le

vels

MockHA-arr-1

(c)

MockHA-arr-1

MockHA-arr-1

lowastlowast

0

10

20

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40

Num

ber o

f col

onie

s

0

50

100

150

o

f Brd

U+

cells

(d)

lowast

MockHA-arr-1

0

05

1

15

MTT

(AU

)

(e)

+

Parp-c

Gapdh

minus oe HA-arr-1

(f)

Figure 4 120573arr-1 overexpression impairs NSCs clonogenicity and proliferation (a) Representative image of immunofluorescence stainingof NSCs after overexpression of HA-120573arr-1 Nuclei are counterstained with Hoechst Scale bar 5120583m (b) WB analysis of endogenousstemnessmarkers in NSCs after overexpression of HA-120573arr-1 Loading control actin RepresentativeWestern blot images from 3 independentexperiments (c) mRNA expression levels of stemness markers in NSCs after overexpression of HA-120573arr-1 (d) Oncosphere forming assay(number of colonies left panel) and bromodeoxyuridine (BrdU) uptake (right panel) in NSCs after ectopic expression of HA-120573arr-1 (e) Cellviability (MTT assay) in NSC after ectopic expression of HA-120573arr-1 (f) WB analysis of cleaved Parp (Parp-c) in NSCs after overexpression ofHA-120573arr-1 Loading control Gapdh Representative Western blot images from 3 independent experiments ((c) (d) and (e)) Data are meansplusmn SD from 3 independent experiments lowast119901 lt 005


p270

25

5

mRN

A ex

pres

sion

lowast

SMDFM

(a)

DFMNucCyto

+ +

Gapdh

p27

Sp1

arr-1

minus minus

(b)

Figure 5120573arr-1 controls NSCs proliferation via p27 expression (a) p27mRNA expression levels evaluated by qRT-PCR inNSCs grown understemness conditions (SM) and after differentiation (DFM) for 48 hours Data are means plusmn SD from 3 independent experiments lowast119901 lt 005(DFM versus SM) (b) Western blot showing subcellular localization of endogenous 120573arr-1 and p27 in NSC cultured in SM or DFM for 48hours 120573arr-1 and p27 proteins levels were assessed in cytosolic (Cyto) and nuclear (Nuc) fractions Gapdh and Sp1 protein levels were usedas loading controls and markers for purity of Cyto and Nuc fractions respectively Representative Western blot images from 3 independentexperiments

expressed 120573arr-1 (Figures 6(a) and 6(b)) and decreased bysiRNA-mediated abrogation of 120573arr-1 expression (Figures6(c) and 6(d))

Altogether these results show that 120573arr-1 expressioninhibits the cell cycle via activation of p27 thus blocking cellstemness features in NSCs

4 Discussion

In this study we propose a model in which epigeneticsilencing of the intragenic miR-326 and its host gene 120573arr-1maintains physiological neuronal stemness While miR-326and 120573arr-1 are highly expressed in differentiated cells wherethey induce growth arrest in NSCs they are kept at low levelsthrough CpG hypermethylation Coherently modulation ofthe expression of 120573arr-1 through overexpression regulatedthe differentiation and growth rate of cerebellar NSCs Thereactivation of the locus miR-326120573arr-1 enhances the cellcycle inhibitor p27 while inhibiting proliferative signalling(eg Hh or Notch by miR-326) thus resulting in stem celldifferentiation and growth arrest (Figure 7)

These findings suggest a new circuitry composed of miR-326120573Arr-1 morphogenic molecules and cell cycle modifierswhich control neural stemness status

In particular here we identify 120573arr-1 as a new player inthe coordinated control of cerebellar NSCs 120573arr-1 was firstidentified as a gene encoding a scaffold protein that regulates

G-protein-coupled receptor (GPCR) through interactionwith cytoplasmic proteins linking GPCRs to intracellularsignaling pathways [17 18] Our observations of the roleof 120573arr-1 in NSCs are consistent with the growth arrestobserved in GCPs where 120573arr-1 directly affects gene expres-sion translocating into the nucleus where it interacts withtranscription cofactors at the promoters of the target gene p27[19]

Consistently previous studies investigated the role of thisCDK-inhibitor in suppressing self-renewal and proliferationwhile driving differentiation of NSCs [17 18 20 21] andcerebellar progenitors [22 23] A further link between p27and stemprogenitor cell differentiation is provided by thep27-destabilizing effect induced by REST a repressor ofneuronal differentiation resulting in maintenance of cellproliferation and blockade of neuronal differentiation [24]In this way loss of p27 maintains a high turnover of self-renewing cells by coupling the ability to control both cellcycle and undifferentiated status Of note 120573arr-1 overex-pression in NSCs affected also cell viability and increasedcell apoptosis suggesting that such scaffold adaptor proteincould have other possible functions in controlling differentmechanisms besides the cell growth arrest

Our findings underline a coordinated epigenetic mecha-nism that finely regulates miR-326120573arr-1 expression and cellgrowth in neural stem cells Indeed the same promoter drivesthe expression of 120573arr-1 and miR-326 through an epigeneticcontrol in cerebellar NSC


p27

lowast

lowast

MockHA-arr-1

arr-10

2

4

6m

RNA

expr

essio

n

(a)

+ +

NucCyto

Gapdh

Sp1

p27

HA-arr-1

arr-1

minus minus

(b)

siCtrsiArrb1

p27

lowast

lowast

arr-10

05

1

15

mRN

A ex

pres

sion

(c)

p27

Actin

+

arr-1

siarr-1DFM+ +minus

minus minus

(d)

Figure 6 120573arr-1 controls NSCs proliferation via p27 expression (a) 120573arr-1 and p27 mRNA expression levels in NSCs transfected with theHA-120573arr-1 plasmid for 48 hrs (b) Subcellular localization of 120573arr-1 and p27 proteins in NSCs transfected with the HA-120573arr-1 plasmid andanalyzed 48 hrs after transfection Proteins are shown in the cytosolic (Cyto) and nuclear (Nuc) fractions Sp1 nuclear loading control Gapdhcytoplasmic loading control Representative Western blot images from 3 independent experiments (c) 120573arr-1 and p27 mRNA expressionlevels in NSCs transfected with control siRNA (siCtr) or 120573arr-1 siRNA (si120573arr-1) cultured for 24 hrs in DFM (d) Western blot showingendogenous 120573arr-1 and p27 protein levels in NSCs transfected with control siRNA (siCtr) or 120573arr-1 siRNA (si120573arr-1) cultured for 24 hrsin DFM Representative Western blot images from 3 independent experiments ((a) and (c)) Data are means plusmn SD from 3 independentexperiments lowast119901 lt 005


NSC

NSC maintenance

Nanog

ProliferationStemness

NSC differentiation

miR-326

StemnessProliferation

Nanog

miR-326miR-326p27 p27

Hedgehog

NotchHedgehog

Notch

-arrestin1-arrestin1

-arrestin1 -arrestin1

miR-326

uarr

uarr

uarr

uarr

darrdarr

darr

darr

Figure 7 miR-326 and 120573-arrestin1 are epigenetically silenced and control stemness features in cerebellar NSCs We propose a model inwhich epigenetic silencing of the intragenic miR-326 and its host gene 120573-arrestin1 maintains physiological neuronal stemness In cerebellarNSCs miR-326 and 120573-arrestin1 are epigenetically silenced through hypermethylation of CpG islands on their common promoter leadingto a permissive molecular environment for the expression of prostemness and proproliferative cues Upon differentiation or demethylatingtreatment the promoter of the locus miR-326120573-arrestin1 is demethylated and thus the expression levels of miR-326 and 120573-arrestin1 areupregulated While miR-326 targets molecules belonging to the Hedgehog pathway and the Notch pathway thus hampering stemnessproperties 120573-arrestin1 acts as a cofactor for the transcriptional activation of p27 leading to cell cycle arrest

The ability of miR-326120573arr-1 to be epigenetically regu-lated also links this transcript to the processes preservingthe stemness status Indeed increasing evidence supports thecrucial role of epigenetic regulation of stem cells includingNSCs [25] Such a regulation includes the mechanismsresponsible for the maintenance of repressive chromatinstates at specific loci consisting of CpG islands methylation[26]

Accordingly in our study miR-326 and 120573arr-1 are amongthe silenced genes in NSCs whose regulation contributes tobalance the opposing forces of cell growth and differentiationIndeed the miR-326120573arr-1 promoter is characterized bymethylated or demethylated CpG islands in NSC or differ-entiated cells respectively

Interestingly in line with this miR-326 and 120573arr-1 havebeen described to be key molecules in multiple sclerosispathogenesis being crucial for CD4+ T cell survival anddifferentiation Indeed 120573arr-1 enhanced the expression of theprotooncogene Bcl2 via the acetylation of histone H4 at theBcl2 promoter [27] while miR-326 promoted interleukin 17-(IL-17-) producing T helper cells (TH-17) differentiation bytargeting Ets-1 a negative regulator of TH-17 differentiation[28] Of note CD4+ T cells from patients with multiplesclerosis had much higher Arrb1 expression [27] and miR-326 expression was highly correlated with disease severity inpatients with multiple sclerosis [28]

Even if no specific studies of 120573arr-1miR-326 coregulationhave been conducted inmultiple sclerosis our results together

with the abovementioned observations allow speculatingthat the 120573arr-1miR-326 transcription unit is regulated inother stem or progenitor cells during differentiation par-ticipating in the control of cell fate development anddisease

5 Conclusions

Wedescribe in the present work the role and regulation of thelocusmiR-326120573arr-1miR-326 and its host gene120573arr-1 repre-sent a novel miRNAprotein network that controls cerebellarNSCs through the modulation of morphogenic signals andcell cycle modifiers both regulating stemness and suggestedto be involved in the maintenance of self-renewal feature ofNSCs

In conclusion our findings describe a bivalent signal(miRNA and hosting protein encoding gene) convergingupon the coordinated inhibition of normal stem cell func-tions

Disclosure

Federica Begallirsquos current address is Center for Cell Signallingand Inflammation (CCSI)Department ofMedicine ImperialCollege London Elisabetta Ferretti and Evelina Miele are co-last authors


Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by grants fromMinistry of Univer-sity and Research Sapienza University of Rome and ItalianSpace Agency (ASI)

References

[1] L Harris O Zalucki M Piper and J I-T Heng ldquoInsights intothe biology and therapeutic applications of neural stem cellsrdquoStem Cells International vol 2016 Article ID 9745315 18 pages2016

[2] A Po E Ferretti E Miele et al ldquoHedgehog controls neuralstem cells through p53-independent regulation of Nanogrdquo TheEMBO Journal vol 29 no 15 pp 2646ndash2658 2010

[3] L Stappert B Roese-Koerner and O Brustle ldquoThe role ofmicroRNAs in human neural stem cells neuronal differentia-tion and subtype specificationrdquo Cell and Tissue Research vol359 no 1 pp 47ndash64 2015

[4] A Rodriguez S Griffiths-Jones J L Ashurst and A BradleyldquoIdentification of mammalian microRNA host genes and tran-scription unitsrdquo Genome Research vol 14 pp 1902ndash1910 2004

[5] S Baskerville and D P Bartel ldquoMicroarray profiling of microR-NAs reveals frequent coexpression with neighboring miRNAsand host genesrdquo RNA vol 11 no 3 pp 241ndash247 2005

[6] Y-K Kim and V N Kim ldquoProcessing of intronic microRNAsrdquoThe EMBO Journal vol 26 no 3 pp 775ndash783 2007

[7] E Ferretti E De Smaele E Miele et al ldquoConcerted microRNAcontrol of Hedgehog signalling in cerebellar neuronal progen-itor and tumour cellsrdquo The EMBO Journal vol 27 no 19 pp2616ndash2627 2008

[8] A M Monteys R M Spengler J Wan et al ldquoStructure andactivity of putative intronic miRNA promotersrdquo RNA vol 16no 3 pp 495ndash505 2010

[9] J W Mandell G Glass E P Gianchandani et al ldquoDephos-phorylation of 120573-arrestin 1 in glioblastomasrdquo Journal of Neu-ropathology and Experimental Neurology vol 68 no 5 pp 535ndash541 2009

[10] A Lee J D Kessler T-A Read et al ldquoIsolation of neural stemcells from the postnatal cerebellumrdquo Nature Neuroscience vol8 no 6 pp 723ndash729 2005

[11] L M Luttrell S S G Ferguson Y Daaka et al ldquo120573-arrestin-dependent formation of 1205732 adrenergic receptor-src proteinkinase complexesrdquo Science vol 283 no 5402 pp 655ndash661 1999

[12] N Garg A Po E Miele et al ldquoMicroRNA-17-92 cluster isa direct Nanog target and controls neural stem cell throughTrp53inp1rdquo The EMBO Journal vol 32 no 21 pp 2819ndash28322013

[13] E Ferretti E De Smaele A Po et al ldquoMicroRNA profiling inhuman medulloblastomardquo International Journal of Cancer vol124 no 3 pp 568ndash577 2009

[14] E Miele F R Buttarelli A Arcella et al ldquoHigh-throughputmicroRNA profiling of pediatric high-grade gliomasrdquo Neuro-Oncology vol 16 no 2 pp 228ndash240 2014

[15] K Williams J Christensen and K Helin ldquoDNA methylationTET proteinsmdashguardians of CpG islandsrdquo EMBO Reports vol13 no 1 pp 28ndash35 2012

[16] B Kefas L Comeau D H Floyd et al ldquoThe neuronalmicroRNA miR-326 acts in a feedback loop with Notch andhas therapeutic potential against brain tumorsrdquo The Journal ofNeuroscience vol 29 no 48 pp 15161ndash15168 2009

[17] J Kang Y Shi B Xiang et al ldquoA nuclear function of 120573-arrestin1in GPCR signaling regulation of histone acetylation and genetranscriptionrdquo Cell vol 123 pp 833ndash847 2005

[18] S R Parathath L A Mainwaring A Fernandez-L C G Gul-dal Z Nahle and A M Kenney ldquo120573-arrestin-1 links mitogenicsonic hedgehog signaling to the cell cycle exit machinery inneural precursorsrdquoCell Cycle vol 9 no 19 pp 4013ndash4024 2010

[19] L Ma and G Pei ldquo120573-arrestin signaling and regulation oftranscriptionrdquo Journal of Cell Science vol 120 pp 213ndash218 2007

[20] E M Neuhaus A Mashukova J Barbour D Wolters and HHatt ldquoNovel function of 120573-arrestin2 in the nucleus of maturespermatozoardquo Journal of Cell Science vol 119 no 15 pp 3047ndash3056 2006

[21] P S Knoepfler ldquoArrestinrsquo the hedgehog Shh limits its ownsignaling via 120573-Arrestin1rdquo Cell Cycle vol 9 no 21 pp 4260ndash4261 2010

[22] C Menchon M J Edel and J C I Belmonte ldquoThe cellcycle inhibitor p27 Kip1 controls self-renewal and pluripotencyof human embryonic stem cells by regulating the cell cycleBrachyury and Twistrdquo Cell Cycle vol 10 no 9 pp 1435ndash14472011

[23] Y-L Zheng B-S Li P Rudrabhatla et al ldquoPhosphorylationof p271198701198941199011 at Thr187 by Cyclin-dependent kinase 5 modulatesneural stem cell differentiationrdquo Molecular Biology of the Cellvol 21 no 20 pp 3601ndash3614 2010

[24] K Miyazawa T Himi V Garcia H Yamagishi S Sato and YIshizaki ldquoA role for p27Kip1 in the control of cerebellar granulecell precursor proliferationrdquoThe Journal of Neuroscience vol 20no 15 pp 5756ndash5763 2000

[25] M A Lopez-Ramirez and S Nicoli ldquoRole of mirnas and epi-genetics in neural stem cell fate determinationrdquo Epigenetics vol9 no 1 pp 90ndash100 2014

[26] M J Boland K LNazor and J F Loring ldquoEpigenetic regulationof pluripotency and differentiationrdquo Circulation Research vol115 no 2 pp 311ndash324 2014

[27] Y Shi Y Feng J Kang et al ldquoCritical regulation of CD4+T cell survival and autoimmunity by 120573-arrestin 1rdquo NatureImmunology vol 8 no 8 pp 817ndash824 2007

[28] C Du C Liu J Kang et al ldquoMicroRNA miR-326 regulatesTH-17 differentiation and is associated with the pathogenesis ofmultiple sclerosisrdquoNature Immunology vol 10 no 12 pp 1252ndash1259 2009

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


We previously identified miR-326 as a miRNA that isrequired to differentiate cerebellar granule cell progenitors(GCPs) to mature granule cells [7]This miRNA is embeddedwithin the first intron of 120573-arrestin1 (120573arr-1) gene and shareswith its host gene the same regulatory sequences [8 9]Since stem cell commitment to GCPs is a crucial event incerebellar development [2 10] we raised the question ofmiR-326 expression and regulation in cerebellar NSC Wereport here thatmiR-326 expression is further downregulatedin NSC prior to progenitor commitment along with thedownregulation of its host gene Therefore we investigatedthe expression functions and regulation of miR-326120573arr-1transcription unit in NSCs

2 Materials and Methods

Unless otherwise indicated media and supplements werepurchased from Gibco-Invitrogen (Carlsbad CA) chemicalswere purchased from Sigma-Aldrich (St Louis MO) andcommercial products were used according to the manufac-turersrsquo instructionsprotocols

21 Cell Culture

211 Neural Stem Cells Culture Mouse cerebella were ob-tained from postnatal 4-day-old wild-type BL6mice (CharlesRiver) with the approval of Institutional Review Board [89] Briefly tissues were collected in HBSS supplementedwith 05 glucose and penicillin-streptomycin grossly trit-urated with serological pipette and treated with DNAse Ito a final concentration of 004 for 20 minutes Cellswere mechanically dissociated using pipettes of decreasingbore size to obtain a single-cell suspension Neural stemcells (NSCs) were derived and enriched through selectivemedium (SM) DMEMF12 supplemented with 06 glucose25mgmL insulin 60mgmL N-acetyl-L-cysteine 2mgmLheparin 20 ngmL EGF 20 ngmL bFGF (Peprotech RockyHill NJ) penicillin-streptomycin and B27 supplement with-out vitamin A For clonogenicity assay cells were plated atclonal density (1-2 cellsmm2) into 96-well plate To inducedifferentiation NSCs were plated onto D-poly-lysine-coateddishes in differentiation medium (DFM DMEMF12 withN2 supplement and 2mgmL heparin 06 glucose and60mgmL N-acetyl-L-cysteine containing 1 Calf Serumand PDGF 10 ngmL (Sigma P3076) or RA 2mM (SigmaR2625) for 48 hours)

212 Overexpression Studies Amaxa nucleofector (Lonza)was used to transfect plasmids according to manufacturerrsquosprocedure pcDNA3 120573-arrestin1 HA was a gift from RobertLefkowitz (Addgene plasmid 14693) [11] miR-326 vectorand its negative control were purchased from GeneCopoeia(MmiR3333-MR01)

213 Knockdown Experiments Silencing of 120573-arrestin1 wasperformed using ON-TARGETplus SMARTpool (L40976-00-005) from Thermo Scientific after testing each single

siRNA of the pool alone or in combination for its specificityto avoid OFF-target effects

214 Cell Proliferation Cell proliferation was evaluated byBrdU incorporation as previously described [2] More indetail after 24-hour pulse with BrdU NSCs were plated onpoly-lysine-coated Lab-Tek chamber slides (coverslips) andallowed to adhere for 3 hours Cells were fixed with 4paraformaldehyde permeabilized with 01 Triton X-100and BrdU detection (Roche) was performed Nuclei werecounterstained with the Hoechst reagent Cells were countedin triplicate and the number of BrdU-positive nuclei wasannotated

NSC growth was measured by MTT assay (Promega)according to manufacturerrsquos instructions Each sample wasmeasured in triplicate and repeated at least three times

For methylation analysis NSCs were treated with 10120583M5-azacytidine (5-AZA) for four days

22 RNA Isolation and Gene Expression Analysis Total RNAwas purified and reverse transcribed as previously described[12] Quantitative RT-PCR (qRT-PCR) analysis was per-formed using the ABI Prism 7900HT Sequence DetectionSystem (Thermo Scientific) using best coverage TaqMangene expression assays specific for each analyzed mRNAEach amplification was performed in triplicate and theaverage of the three threshold cycles was used to calcu-late the amount of transcripts (Thermo Scientific) mRNAquantification was expressed in arbitrary units as previ-ously described [13] mRNA quantification was expressedin arbitrary units as the ratio of the sample quantity tothe calibrator or to the mean values of control samples Allvalues were normalized to three endogenous controls 120573-actin 1205732-microglobulin and Hprt miRNA expression levelswere evaluated on RNA samples using specific stem-loopprimers to achieve retrotranscription (Thermo Scientific)and was followed by a quantitative RT-PCR using miRNA-specific TaqMan probes (Thermo Scientific) in a ABI Prism7900HT Sequence Detection System (Thermo Scientific)miRNA expression levels were normalized to RNAU6B [14]

23 Western Blot Assay Cells were lysed using RIPA buffer(Tris-HCl pH 76 50mM deoxycholic acid sodium salt 05NaCl 140mM NP40 1 EDTA 5mM NaF 100mM sodiumpyrophosphate 2mM and protease inhibitors) Lysates wereseparated on 8 acrylamide gel and immunoblotted usingstandard procedures The following antibodies were usedanti-120573-arrestin1 K-16 (sc-8182 Santa Cruz BiotechnologyCA) anti-mouse Nanog (Cosmo Bio Co Japan) anti-mouse120573III-tubulin (MAB 1637Millipore) anti-actin I-19 (sc-1616 Santa Cruz Biotechnology CA) anti-GAPDH (ab8245Abcam) anti-Sp1 1C6 (sc-420X Santa Cruz BiotechnologyCA) anti-p27 (F-8) (sc-1641 Santa Cruz Biotechnology)anti-Sox2 (MAB4343 Millipore Billerica MA) anti-Oct4(ab19857 Abcam) anti-mouse 120573III Tubulin (MAB 1637Millipore Billerica MA) anti-GFAPMAB360 (Millipore Bil-lerica MA) and anti-PARP p85 fragment (G7342 Promega)(anti-parp-C) HRP-conjugated secondary antibodies (Santa















3 Results





lowast

lowast

0

05

1

15

mRN

A le

vels

T0NSC

(a)

miR-326

T0NSC

lowast

0

05

1

15

mRN

A le

vels

(b)

T0NSC

arr-1

lowast

0

05

1

15

mRN

A le

vels

(c)

T0 NSC

arr-1

Nanog

Actin

(d)








RA PDGF

SMDFM

lowast

lowast

0

25

5

miR

-326

leve

ls

(a)D

FMSM

Merge NucleimiR-326

(b)


lowast

lowastlowast

lowast

SMDFM


Differentiation

lowastlowast lowast

lowast

arr-1 III-tub0

25

5

mRN

A le

vels

(c)

SM DFM

Nanog

Oct4

Sox2

Gfap

arr-1

III-tub

Gapdh

(d)





TSS



minus1321 +176

arr-1

(a)


Ctr5-AZA

arr-1

lowast

lowast

lowast

0

25

5

mRN

A le

vels

miR-326

lowast

0

25

5

miR

NA

leve

ls

(b)


Ctr5-AZA


III-tub0

25

5

mRN

A le

vels

lowast lowastlowast

lowastlowast

lowast

lowast

(c)

Nanog

Oct4

Sox2

Gfap

arr-1

III-tub

Actin

minus + 5-AZA

(d)

Methyl

Unmethyl

T0 NSC

(e)

Methyl

Unmethyl

minus + 5-AZA

(f)





Moc

k

NucleiMerge Anti-HA

oe H

A-

arr-1

(a)

Actin

Nanog

minus +

Sox2

Oct4

oe HA-arr-1

arr-1

(b)

Nanog Oct4 Sox2

lowastlowast

lowast

0

05

1

15

mRN

A le

vels

MockHA-arr-1

(c)

MockHA-arr-1

MockHA-arr-1

lowastlowast

0

10

20

30

40

Num

ber o

f col

onie

s

0

50

100

150

o

f Brd

U+

cells

(d)

lowast

MockHA-arr-1

0

05

1

15

MTT

(AU

)

(e)

+

Parp-c

Gapdh

minus oe HA-arr-1

(f)



p270

25

5

mRN

A ex

pres

sion

lowast

SMDFM

(a)

DFMNucCyto

+ +

Gapdh

p27

Sp1

arr-1

minus minus

(b)




4 Discussion








p27

lowast

lowast

MockHA-arr-1

arr-10

2

4

6m

RNA

expr

essio

n

(a)

+ +

NucCyto

Gapdh

Sp1

p27

HA-arr-1

arr-1

minus minus

(b)

siCtrsiArrb1

p27

lowast

lowast

arr-10

05

1

15

mRN

A ex

pres

sion

(c)

p27

Actin

+

arr-1

siarr-1DFM+ +minus

minus minus

(d)



NSC

NSC maintenance

Nanog


NSC differentiation

miR-326


Nanog

miR-326miR-326p27 p27

Hedgehog

NotchHedgehog

Notch



miR-326

uarr

uarr

uarr

uarr

darrdarr

darr

darr







5 Conclusions



Disclosure



Competing Interests


Acknowledgments


References




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















3 Results





lowast

lowast

0

05

1

15

mRN

A le

vels

T0NSC

(a)

miR-326

T0NSC

lowast

0

05

1

15

mRN

A le

vels

(b)

T0NSC

arr-1

lowast

0

05

1

15

mRN

A le

vels

(c)

T0 NSC

arr-1

Nanog

Actin

(d)








RA PDGF

SMDFM

lowast

lowast

0

25

5

miR

-326

leve

ls

(a)D

FMSM

Merge NucleimiR-326

(b)


lowast

lowastlowast

lowast

SMDFM


Differentiation

lowastlowast lowast

lowast

arr-1 III-tub0

25

5

mRN

A le

vels

(c)

SM DFM

Nanog

Oct4

Sox2

Gfap

arr-1

III-tub

Gapdh

(d)





TSS



minus1321 +176

arr-1

(a)


Ctr5-AZA

arr-1

lowast

lowast

lowast

0

25

5

mRN

A le

vels

miR-326

lowast

0

25

5

miR

NA

leve

ls

(b)


Ctr5-AZA


III-tub0

25

5

mRN

A le

vels

lowast lowastlowast

lowastlowast

lowast

lowast

(c)

Nanog

Oct4

Sox2

Gfap

arr-1

III-tub

Actin

minus + 5-AZA

(d)

Methyl

Unmethyl

T0 NSC

(e)

Methyl

Unmethyl

minus + 5-AZA

(f)





Moc

k

NucleiMerge Anti-HA

oe H

A-

arr-1

(a)

Actin

Nanog

minus +

Sox2

Oct4

oe HA-arr-1

arr-1

(b)

Nanog Oct4 Sox2

lowastlowast

lowast

0

05

1

15

mRN

A le

vels

MockHA-arr-1

(c)

MockHA-arr-1

MockHA-arr-1

lowastlowast

0

10

20

30

40

Num

ber o

f col

onie

s

0

50

100

150

o

f Brd

U+

cells

(d)

lowast

MockHA-arr-1

0

05

1

15

MTT

(AU

)

(e)

+

Parp-c

Gapdh

minus oe HA-arr-1

(f)



p270

25

5

mRN

A ex

pres

sion

lowast

SMDFM

(a)

DFMNucCyto

+ +

Gapdh

p27

Sp1

arr-1

minus minus

(b)




4 Discussion








p27

lowast

lowast

MockHA-arr-1

arr-10

2

4

6m

RNA

expr

essio

n

(a)

+ +

NucCyto

Gapdh

Sp1

p27

HA-arr-1

arr-1

minus minus

(b)

siCtrsiArrb1

p27

lowast

lowast

arr-10

05

1

15

mRN

A ex

pres

sion

(c)

p27

Actin

+

arr-1

siarr-1DFM+ +minus

minus minus

(d)



NSC

NSC maintenance

Nanog


NSC differentiation

miR-326


Nanog

miR-326miR-326p27 p27

Hedgehog

NotchHedgehog

Notch



miR-326

uarr

uarr

uarr

uarr

darrdarr

darr

darr







5 Conclusions



Disclosure



Competing Interests


Acknowledgments


References




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



lowast

lowast

0

05

1

15

mRN

A le

vels

T0NSC

(a)

miR-326

T0NSC

lowast

0

05

1

15

mRN

A le

vels

(b)

T0NSC

arr-1

lowast

0

05

1

15

mRN

A le

vels

(c)

T0 NSC

arr-1

Nanog

Actin

(d)








RA PDGF

SMDFM

lowast

lowast

0

25

5

miR

-326

leve

ls

(a)D

FMSM

Merge NucleimiR-326

(b)


lowast

lowastlowast

lowast

SMDFM


Differentiation

lowastlowast lowast

lowast

arr-1 III-tub0

25

5

mRN

A le

vels

(c)

SM DFM

Nanog

Oct4

Sox2

Gfap

arr-1

III-tub

Gapdh

(d)





TSS



minus1321 +176

arr-1

(a)


Ctr5-AZA

arr-1

lowast

lowast

lowast

0

25

5

mRN

A le

vels

miR-326

lowast

0

25

5

miR

NA

leve

ls

(b)


Ctr5-AZA


III-tub0

25

5

mRN

A le

vels

lowast lowastlowast

lowastlowast

lowast

lowast

(c)

Nanog

Oct4

Sox2

Gfap

arr-1

III-tub

Actin

minus + 5-AZA

(d)

Methyl

Unmethyl

T0 NSC

(e)

Methyl

Unmethyl

minus + 5-AZA

(f)





Moc

k

NucleiMerge Anti-HA

oe H

A-

arr-1

(a)

Actin

Nanog

minus +

Sox2

Oct4

oe HA-arr-1

arr-1

(b)

Nanog Oct4 Sox2

lowastlowast

lowast

0

05

1

15

mRN

A le

vels

MockHA-arr-1

(c)

MockHA-arr-1

MockHA-arr-1

lowastlowast

0

10

20

30

40

Num

ber o

f col

onie

s

0

50

100

150

o

f Brd

U+

cells

(d)

lowast

MockHA-arr-1

0

05

1

15

MTT

(AU

)

(e)

+

Parp-c

Gapdh

minus oe HA-arr-1

(f)



p270

25

5

mRN

A ex

pres

sion

lowast

SMDFM

(a)

DFMNucCyto

+ +

Gapdh

p27

Sp1

arr-1

minus minus

(b)




4 Discussion








p27

lowast

lowast

MockHA-arr-1

arr-10

2

4

6m

RNA

expr

essio

n

(a)

+ +

NucCyto

Gapdh

Sp1

p27

HA-arr-1

arr-1

minus minus

(b)

siCtrsiArrb1

p27

lowast

lowast

arr-10

05

1

15

mRN

A ex

pres

sion

(c)

p27

Actin

+

arr-1

siarr-1DFM+ +minus

minus minus

(d)



NSC

NSC maintenance

Nanog


NSC differentiation

miR-326


Nanog

miR-326miR-326p27 p27

Hedgehog

NotchHedgehog

Notch



miR-326

uarr

uarr

uarr

uarr

darrdarr

darr

darr







5 Conclusions



Disclosure



Competing Interests


Acknowledgments


References




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


RA PDGF

SMDFM

lowast

lowast

0

25

5

miR

-326

leve

ls

(a)D

FMSM

Merge NucleimiR-326

(b)


lowast

lowastlowast

lowast

SMDFM


Differentiation

lowastlowast lowast

lowast

arr-1 III-tub0

25

5

mRN

A le

vels

(c)

SM DFM

Nanog

Oct4

Sox2

Gfap

arr-1

III-tub

Gapdh

(d)





TSS



minus1321 +176

arr-1

(a)


Ctr5-AZA

arr-1

lowast

lowast

lowast

0

25

5

mRN

A le

vels

miR-326

lowast

0

25

5

miR

NA

leve

ls

(b)


Ctr5-AZA


III-tub0

25

5

mRN

A le

vels

lowast lowastlowast

lowastlowast

lowast

lowast

(c)

Nanog

Oct4

Sox2

Gfap

arr-1

III-tub

Actin

minus + 5-AZA

(d)

Methyl

Unmethyl

T0 NSC

(e)

Methyl

Unmethyl

minus + 5-AZA

(f)





Moc

k

NucleiMerge Anti-HA

oe H

A-

arr-1

(a)

Actin

Nanog

minus +

Sox2

Oct4

oe HA-arr-1

arr-1

(b)

Nanog Oct4 Sox2

lowastlowast

lowast

0

05

1

15

mRN

A le

vels

MockHA-arr-1

(c)

MockHA-arr-1

MockHA-arr-1

lowastlowast

0

10

20

30

40

Num

ber o

f col

onie

s

0

50

100

150

o

f Brd

U+

cells

(d)

lowast

MockHA-arr-1

0

05

1

15

MTT

(AU

)

(e)

+

Parp-c

Gapdh

minus oe HA-arr-1

(f)



p270

25

5

mRN

A ex

pres

sion

lowast

SMDFM

(a)

DFMNucCyto

+ +

Gapdh

p27

Sp1

arr-1

minus minus

(b)




4 Discussion








p27

lowast

lowast

MockHA-arr-1

arr-10

2

4

6m

RNA

expr

essio

n

(a)

+ +

NucCyto

Gapdh

Sp1

p27

HA-arr-1

arr-1

minus minus

(b)

siCtrsiArrb1

p27

lowast

lowast

arr-10

05

1

15

mRN

A ex

pres

sion

(c)

p27

Actin

+

arr-1

siarr-1DFM+ +minus

minus minus

(d)



NSC

NSC maintenance

Nanog


NSC differentiation

miR-326


Nanog

miR-326miR-326p27 p27

Hedgehog

NotchHedgehog

Notch



miR-326

uarr

uarr

uarr

uarr

darrdarr

darr

darr







5 Conclusions



Disclosure



Competing Interests


Acknowledgments


References




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


TSS



minus1321 +176

arr-1

(a)


Ctr5-AZA

arr-1

lowast

lowast

lowast

0

25

5

mRN

A le

vels

miR-326

lowast

0

25

5

miR

NA

leve

ls

(b)


Ctr5-AZA


III-tub0

25

5

mRN

A le

vels

lowast lowastlowast

lowastlowast

lowast

lowast

(c)

Nanog

Oct4

Sox2

Gfap

arr-1

III-tub

Actin

minus + 5-AZA

(d)

Methyl

Unmethyl

T0 NSC

(e)

Methyl

Unmethyl

minus + 5-AZA

(f)





Moc

k

NucleiMerge Anti-HA

oe H

A-

arr-1

(a)

Actin

Nanog

minus +

Sox2

Oct4

oe HA-arr-1

arr-1

(b)

Nanog Oct4 Sox2

lowastlowast

lowast

0

05

1

15

mRN

A le

vels

MockHA-arr-1

(c)

MockHA-arr-1

MockHA-arr-1

lowastlowast

0

10

20

30

40

Num

ber o

f col

onie

s

0

50

100

150

o

f Brd

U+

cells

(d)

lowast

MockHA-arr-1

0

05

1

15

MTT

(AU

)

(e)

+

Parp-c

Gapdh

minus oe HA-arr-1

(f)



p270

25

5

mRN

A ex

pres

sion

lowast

SMDFM

(a)

DFMNucCyto

+ +

Gapdh

p27

Sp1

arr-1

minus minus

(b)




4 Discussion








p27

lowast

lowast

MockHA-arr-1

arr-10

2

4

6m

RNA

expr

essio

n

(a)

+ +

NucCyto

Gapdh

Sp1

p27

HA-arr-1

arr-1

minus minus

(b)

siCtrsiArrb1

p27

lowast

lowast

arr-10

05

1

15

mRN

A ex

pres

sion

(c)

p27

Actin

+

arr-1

siarr-1DFM+ +minus

minus minus

(d)



NSC

NSC maintenance

Nanog


NSC differentiation

miR-326


Nanog

miR-326miR-326p27 p27

Hedgehog

NotchHedgehog

Notch



miR-326

uarr

uarr

uarr

uarr

darrdarr

darr

darr







5 Conclusions



Disclosure



Competing Interests


Acknowledgments


References




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Moc

k

NucleiMerge Anti-HA

oe H

A-

arr-1

(a)

Actin

Nanog

minus +

Sox2

Oct4

oe HA-arr-1

arr-1

(b)

Nanog Oct4 Sox2

lowastlowast

lowast

0

05

1

15

mRN

A le

vels

MockHA-arr-1

(c)

MockHA-arr-1

MockHA-arr-1

lowastlowast

0

10

20

30

40

Num

ber o

f col

onie

s

0

50

100

150

o

f Brd

U+

cells

(d)

lowast

MockHA-arr-1

0

05

1

15

MTT

(AU

)

(e)

+

Parp-c

Gapdh

minus oe HA-arr-1

(f)



p270

25

5

mRN

A ex

pres

sion

lowast

SMDFM

(a)

DFMNucCyto

+ +

Gapdh

p27

Sp1

arr-1

minus minus

(b)




4 Discussion








p27

lowast

lowast

MockHA-arr-1

arr-10

2

4

6m

RNA

expr

essio

n

(a)

+ +

NucCyto

Gapdh

Sp1

p27

HA-arr-1

arr-1

minus minus

(b)

siCtrsiArrb1

p27

lowast

lowast

arr-10

05

1

15

mRN

A ex

pres

sion

(c)

p27

Actin

+

arr-1

siarr-1DFM+ +minus

minus minus

(d)



NSC

NSC maintenance

Nanog


NSC differentiation

miR-326


Nanog

miR-326miR-326p27 p27

Hedgehog

NotchHedgehog

Notch



miR-326

uarr

uarr

uarr

uarr

darrdarr

darr

darr







5 Conclusions



Disclosure



Competing Interests


Acknowledgments


References




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


p270

25

5

mRN

A ex

pres

sion

lowast

SMDFM

(a)

DFMNucCyto

+ +

Gapdh

p27

Sp1

arr-1

minus minus

(b)




4 Discussion








p27

lowast

lowast

MockHA-arr-1

arr-10

2

4

6m

RNA

expr

essio

n

(a)

+ +

NucCyto

Gapdh

Sp1

p27

HA-arr-1

arr-1

minus minus

(b)

siCtrsiArrb1

p27

lowast

lowast

arr-10

05

1

15

mRN

A ex

pres

sion

(c)

p27

Actin

+

arr-1

siarr-1DFM+ +minus

minus minus

(d)



NSC

NSC maintenance

Nanog


NSC differentiation

miR-326


Nanog

miR-326miR-326p27 p27

Hedgehog

NotchHedgehog

Notch



miR-326

uarr

uarr

uarr

uarr

darrdarr

darr

darr







5 Conclusions



Disclosure



Competing Interests


Acknowledgments


References




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


p27

lowast

lowast

MockHA-arr-1

arr-10

2

4

6m

RNA

expr

essio

n

(a)

+ +

NucCyto

Gapdh

Sp1

p27

HA-arr-1

arr-1

minus minus

(b)

siCtrsiArrb1

p27

lowast

lowast

arr-10

05

1

15

mRN

A ex

pres

sion

(c)

p27

Actin

+

arr-1

siarr-1DFM+ +minus

minus minus

(d)



NSC

NSC maintenance

Nanog


NSC differentiation

miR-326


Nanog

miR-326miR-326p27 p27

Hedgehog

NotchHedgehog

Notch



miR-326

uarr

uarr

uarr

uarr

darrdarr

darr

darr







5 Conclusions



Disclosure



Competing Interests


Acknowledgments


References




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


NSC

NSC maintenance

Nanog


NSC differentiation

miR-326


Nanog

miR-326miR-326p27 p27

Hedgehog

NotchHedgehog

Notch



miR-326

uarr

uarr

uarr

uarr

darrdarr

darr

darr







5 Conclusions



Disclosure



Competing Interests


Acknowledgments


References




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Competing Interests


Acknowledgments


References




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

-arrestin1/mir-326 transcription unit is epigenetically

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