Silencing of Aγ-Globin Gene Expression during Adult Definitive Erythropoiesis Mediated by GATA ...

13 Pages·2008·1.04 MB·English

MOLECULARANDCELLULARBIOLOGY,May2008,p.3101–3113 Vol.28,No.10
0270-7306/08/$08.00(cid:1)0 doi:10.1128/MCB.01858-07
Copyright©2008,AmericanSocietyforMicrobiology.AllRightsReserved.
Silencing of A(cid:2)-Globin Gene Expression during Adult Definitive
Erythropoiesis Mediated by GATA-1–FOG-1–Mi2 Complex
(cid:3) (cid:1)
Binding at the 566 GATA Site †
Susanna Harju-Baker,1§‡ Fl´avia C. Costa,1‡ Halyna Fedosyuk,1
Renee Neades,1 and Kenneth R. Peterson1,2*
DepartmentsofBiochemistryandMolecularBiology1andAnatomyandCellBiology,2Universityof
KansasMedicalCenter,KansasCity,Kansas66160
Received11October2007/Returnedformodification20November2007/Accepted8March2008
D
Autonomous silencing of (cid:1)-globin transcription is an important developmental regulatory mechanism ow
controllingglobingeneswitching.Anadultstage-specificsilenceroftheA(cid:1)-globingenewasidentifiedbetween n
(cid:2)730and(cid:2)378relativetothemRNAstartsite.AmarkedcopyoftheA(cid:1)-globingeneinsertedbetweenlocus lo
control region 5(cid:1) DNase I-hypersensitive site 1 and the (cid:3)-globin gene was transcriptionally silenced in adult a
d
(cid:4)-globinlocusyeastartificialchromosome((cid:4)-YAC)transgenicmice,butdeletionofthe352-bpregionrestored e
expression. This fragment reduced reporter gene expression in K562 cells, and GATA-1 was shown to bind d

withinthissequenceatthe(cid:2)566GATAsite.Further,theMi2protein,acomponentoftheNuRDcomplex,was fr
o
observedinerythroidcellswithlow(cid:1)-globinlevels,whereasonlyaweaksignalwasdetectedwhen(cid:1)-globinwas m
expressed. Chromatin immunoprecipitation of fetal liver tissue from (cid:4)-YAC transgenic mice demonstrated
h
that GATA-1, FOG-1, and Mi2 were recruited to the A(cid:1)-globin (cid:2)566 or G(cid:1)-globin (cid:2)567 GATA site when t
t
(cid:1)-globinexpressionwaslow(day18)butnotwhen(cid:1)-globinwasexpressed(day12).Thesedatasuggestthat p
:
during definitive erythropoiesis, (cid:1)-globin gene expression is silenced, in part, by binding a protein complex //
m
containingGATA-1,FOG-1,andMi2atthe(cid:2)566/(cid:2)567GATAsitesoftheproximal(cid:1)-globinpromoters.
c
b
.
a
Thehuman(cid:4)-globinlocusconsistsoffivefunctionalgenes, embryonic ε- and fetal (cid:2)-globin genes during definitive eryth- s
m
arranged spatially as 5(cid:5)-ε-G(cid:2)-A(cid:2)-(cid:6)-(cid:4)-3(cid:5) in the order in which ropoiesis (17, 59), and competition plays a major role in si-
.
they are expressed during development. Globin gene expres- lencingtheadult(cid:4)-globingeneduringprimitiveandfetalde- or
g
sion is regulated, in part, by the locus control region (LCR), finitiveerythropoiesis(70).Anunderstandingofthemolecular /

whichphysicallyconsistsofatleastfiveDNaseI-hypersensitive basis underlying interaction of these regulatory motifs is not o
n
site (HSs) located 6 to 22 kb upstream to the ε-globin gene. complete. Although autonomous silencing of (cid:2)-globin tran-
M
The embryonic ε-globin gene is transcribed during the first 6 scription during development has been documented (17, 19, a
weeks of primitive erythropoiesis in the embryonic yolk sac. 29,39,66),thesequencesencodingthesilencerelement(s)and rc
Duringtheswitchtofetaldefinitiveerythropoiesisintheliver, the mechanism(s) of repression have only recently been ex- h

theε-globingeneissilencedandtheG(cid:2)-andA(cid:2)-globingenes plored(64,68,69). 3
0
areactivated.Aroundthetimeofbirth,asecondswitchoccurs, Silencers bind repressor protein complexes that interfere ,
2
to adult definitive erythropoiesis. The site of hematopoiesis with promoter activity, thereby down-regulating gene expres- 0
moves to the bone marrow, the (cid:2)-globin genes are silenced, sion (61). Recent data demonstrate that two direct repeat 1
9
and the (cid:6)- and (cid:4)-globin genes are activated (67). Previous (DR1) elements located in the proximal ε-globin promoter b
studieshavedemonstratedthattwononexclusivemechanisms bind a novel repressor activity in definitive erythroid cells y

are involved in the (cid:2)- to(cid:4)-globin gene switch, competition calleddirectrepeaterythroid-definitiveprotein(DRED)(71). g
u
between the two genes for interaction with the LCR and au- A similar DR1 sequence exists 5(cid:5) to the A(cid:2)-globin gene (49). e
s
tonomous silencing of the (cid:2)-globin genes (6, 13, 20). In the Thenondeletional(cid:3)117Greekhereditarypersistenceoffetal t
competition model, the gene closer to the LCR has a higher hemoglobin(HPFH)pointmutation(25)altersthissequence,
probabilityofinteractionwiththeLCRandismoreabundantly resultingincontinuedexpressionofA(cid:2)-globininadulthumans
transcribed,unlessitisautonomouslysilenced(28,29,53,70). and human (cid:4)-globin locus yeast artificial chromosome
Autonomous silencing plays a major role in repressing the ((cid:4)-YAC)transgenicmicebearingthismutation(51).Purifica-
tionofDREDbindingactivityrevealedthatitisamultiprotein
complexcomposedofatleastfiveproteins.DR1-specificbind-
*Correspondingauthor.Mailingaddress:DepartmentofBiochem-
ingactivityconsistsoftwonuclearorphanreceptors,testicular
istryandMolecularBiology,MS3030,UniversityofKansasMedical
receptor 2 (TR2) and TR4, which are expressed in both em-
Center, 3901 Rainbow Boulevard, Kansas City, KS 66160. Phone:
[phone].Fax:[phone].E-mail:[email protected]. bryonicandadulterythroidcells(68).Thesetwoproteins(and
§Presentaddress:CenterforLungBiology,UniversityofWashing- others) collaboratively repress transcription of the ε- and
tonSchoolofMedicine,Seattle,WA98109. (cid:2)-globin genes. Recent evidence demonstrates that the DR
‡Co-firstauthors:theseauthorscontributedequallytothiswork.
sequenceelementautonomouslymediatesdefinitivestage-spe-
†Supplementalmaterialforthisarticlemaybefoundathttp://mcb
.asm.org/. cific (cid:2)-globin gene silencing (49, 69). COUP-TFII (NF-E3) is
(cid:1)Publishedaheadofprinton17March2008. anorphanreceptorthathasbothrepressorandactivatorprop-
3101
3102 HARJU-BAKER ET AL. MOL.CELL.BIOL.
ertiesandmaybeinvolvedinglobingeneswitchingbyrepress- Xenopus cells, and some subunits of this complex were also
ing ε-globin expression in fetal erythroid cells (21). In mice, found in Drosophila, Caenorhabditis elegans, and Arabidopsis,
COUP-TFII binds to the same direct repeats of the ε- and suggestingthatitiswidelyconservedduringevolution(2,78).
(cid:2)-globinpromotersasDRED,possiblyassistinginrepression TheNuRDcomplexisa2-MDacomplexandiscomprisedof
of expression from these genes. SSP, an activator of (cid:2)-globin at least seven polypeptides, including HDAC1, HDAC2,
gene expression, is a heteromeric complex consisting of CP2 MBD3, Mi2 (CHD), MTA, RbAp46, and RbAp48 (15, 78).
andanerythroid-specificprotein,NF-E4(24,32,77,80).This Functionsofthecomplexincludenucleosomeremodelingand
complexbindsthestageselectorelementintheproximalpro- histonedeacetylaseactivities(2,9,36,73).Ithasbeenassoci-
moterofthe(cid:2)-globingenes(32).AtruncatedformofNF-E4, atedwithepigeneticmechanismsoftranscriptionalrepression
p14 NF-E4, may interact with CP2 later in development, in- during development. Based on these data, we hypothesized
hibiting binding of the SSP complex to the stage selector ele- thatNuRDandFOG-1interactwithGATA-1toformacom-
ment,resultinginrepressionof(cid:2)-globingeneexpression(79). plex that may be involved in remodeling (cid:2)-globin gene-proxi-
Finally, DNA methylation by an MBD2-containing complex mal chromatin into a repressed state, leading to silencing of
maintains(cid:2)-globinsuppressioninadulterythroidcells(64). transcriptionduringtheadultstageoferythropoiesis.Chroma-
D
Genetic analyses showed that (cid:2)-globin gene-flanking se- tinimmunoprecipitationanalysissupportsthishypothesis.We o
quences encompass numerous regions with either positive or showthatGATA-1,FOG-1,andMi2arerecruitedtothe(cid:3)566 w
n
negative regulatory function (27, 66). When a marked A(cid:2)- A(cid:2)-globin and analogous (cid:3)567 G(cid:2)-globin GATA sites when lo
globingene(A(cid:2)m)wasinsertedbetweenLCR5(cid:5)HS1andthe (cid:2)-globinisnotexpressedorisexpressedatlowlevelsbutnot a
ε-globingenein(cid:4)-YACtransgenicmice(A(cid:2)m5(cid:5)ε(cid:4)-YAC),it when(cid:2)-globinistranscribedathighlevels. de
wassilencedduringadultdefinitiveerythropoiesis(29).Incon- d

trast, when a marked (cid:4)-globin gene ((cid:4)m) was inserted in this fr
o
samelocation,itwasexpressedthroughoutontogeny.Compe- MATERIALSANDMETHODS m
titionwiththe(cid:4)-globingeneforinteractionwiththeLCRdoes (cid:5)1sA(cid:1)m5(cid:1)(cid:3)(cid:4)-YACconstruct.A213-kbYACconstructcarryingthehuman h
notaccountfortheA(cid:2)m-globinsilencing,becausethegenewas (cid:4)-globinlocuswithamarkedA(cid:2)-globingene(A(cid:2)m5(cid:5)ε(cid:4)-YAC)wasusedto tt
silenced even in the absence of the (cid:4)-globin gene (29). The develop the (cid:7)1s A(cid:2)m 5(cid:5) ε (cid:4)-YAC (Fig. 1A) (29). This YAC was originally p:
nonnative location of the A(cid:2)m-globin gene also does not ex- eosftitmheate(cid:4)d-gtloobbien2l4o8cuksbrinegsiiozne(w4a6s,4m7,a5d2e,7a4v)a,iluanbtleilaonnetahrelygcloombipnlegteenseeqsueernvceer //m
plainthedown-regulation,sincethe(cid:4)m-globingeneinsertedin (http://globin.cse.psu.edu)(10).The(cid:4)-YACcontainsapproximately187kbof cb
thesamelocationwasexpressedthroughoutontogeny.There- thehuman(cid:4)-globinlocusflankedbyYACarms.TheA(cid:2)m-globingeneisa5.4-kb .a
fore, the A(cid:2)m-globin gene-proximal sequences must be SspI fragment (Fig. 1B) (GenBank file U01317 coordinates 38683 to 44077) s
insertedintoanEcoRVsiteatGenBankcoordinate15185(29).Themarkinthe m
responsible for the developmental silencing of this gene.
ToidentifytheA(cid:2)m-globingenesilencer,aseriesoftrunca- tAr(cid:2)amns-cgrloipbtiinongesntaertissiate6u-btiplizdeedlettoiodnifafetre(cid:1)n2ti1atteob(cid:1)et2w6eerenlattriavnesctoriptthseoAri(cid:2)g-inglaotbining .or
tionswasdesignedbasedonprevious(cid:2)-globinpromoteranal- fromthenormallylocated(cid:2)-globingenesandtheA(cid:2)m-globin(62). g/

ysis (66). A 352-bp deletion ((cid:7)1s) encompassing sequences Tocreatea(cid:7)1sA(cid:2)m5(cid:5)ε(cid:4)-YAC,theA(cid:2)m5(cid:5)ε(cid:4)-YACwasmodifiedusingthe o
n
from(cid:3)730to(cid:3)378relativetotheA(cid:2)-globinmRNACAPsite “pop-in,pop-out”homologousrecombinationmethodinSaccharomycescerevi-
was introduced into the A(cid:2)m-globin gene of the A(cid:2)m 5(cid:5) ε sAi(cid:2)aem-(g5lo).biAng3e5n2e-b((cid:3)p7d3e0lettoio(cid:3)n37w8a;sGienntrBoadnukcecdooirnditnhaete5s(cid:5)3-8d6is8t3altop3ro9m05o0t)e.rPloafsmthide Ma
(cid:4)-YAC, and the resultant (cid:7)1s A(cid:2)m 5(cid:5) ε (cid:4)-YAC was used to pHS1(H3/RV)/A(cid:2)m(StuI/RI),agiftfromQiliangLi,wasutilizedfortheyeast rc
establish transgenic mice. Our data demonstrate that A(cid:2)m- mutagenesis. This plasmid contains a fragment of the (cid:4)-LCR 5(cid:5) HS1 from h

globin was expressed during adult definitive erythropoiesis. HindIIItoEcoRV(GenBankcoordinates13769to15185)subclonedintothe 3
0
FurtheranalysisofthethisregionrevealedthataGATAsite HindIIIsiteofpRS406(Stratagene,Carlsbad,CA)andafragmentoftheA(cid:2)m- ,
located at (cid:3)566 was bound by GATA-1 in non-(cid:2)-globin-ex- gthloisbicnongsetnreucftrolamckSetduIthtoeE35c2o-RbpI(SGspenI-BSatunIkdciosotarldipnraotmeso3te9r05re0gtioon40o8f3t6h)e.TAh(cid:2)ums-, 20
pressing cells but lost in cells expressing (cid:2)-globin at a high globingenebutcontainedtheregiondownstreamof5(cid:5)HS1thattheA(cid:2)m-globin 19
level. Thus, the A(cid:2)m-globin gene promoter region from (cid:3)730 gene was originally integrated into. The plasmid was linearized with SphI b
to(cid:3)378functionsasa(cid:2)-globingenesilencerinadultdefinitive (GenBankcoordinate14100).YeastcarryingtheA(cid:2)m5(cid:5)ε(cid:4)-YACwassphero- y
plastedandtransformedwith4(cid:8)gofthelinearizedplasmid.YACtransforma- g
erythropoiesis,andthissilencingismediatedbyGATA-1.
tion, screening of positive clones, purification, and mouse transgenesis were u
GATA-1isamemberofafamilyofzincfingertranscription e
performedasdescribedpreviously(26,29,46,51,54). s
factors that plays a seminal role in the development and dif- Structuralanalysis.Transgeneintegrityandcopynumberstructuralanalyses t
ferentiation of several cell types, including erythrocytes, ofF-generationanimalswereperformedasdescribedelsewhere(seeMaterials
2
megakaryocytes,eosinophils,andmastcells(55,63).(cid:4)-Globin andMethodsinthesupplementalmaterial)(23,26,29,46,51,54).APCR-based
approachwasalsoutilizedtoconfirmthe(cid:7)1sA(cid:2)m5(cid:5)εregionstructure.The
locus transcriptional activation can be resolved into discrete
primer sequences and expected size fragments are shown in Table S1 in the
molecular steps involving the formation of distinct GATA-1–
supplementalmaterial.
cofactor assemblies at the promoters and the LCR (33). Re- RNase protection assays. Total mRNA was isolated from developmentally
centdatasuggestthatGATA-1canactbothasanactivatorand stagedmousefetuses,K562humanerythroleukemiacells,andMEL585mouse
as a repressor of gene transcription. Interaction between erythroleukemiacellsusingthePromegaRNagentsRNAisolationkit.RNase
protectionassays(RPAs)wereperformedasdescribedpreviously(29,30).
GATA-1 and the YY1 protein is involved in the silencing of Antibodydetectionof(cid:1)-and(cid:4)-globinproteinchains.Bloodfromadultmice
the ε-globin gene in primate and nonprimate species (58). was used to prepare slides to assess (cid:2)- and (cid:4)-globin chain synthesis. Details
Morerecently,bindingofaGATA-1–FOG-1–NuRDcomplex areprovidedinmaterialsandmethodsinthesupplementalmaterial.
was shown to silence hematopoietic genes in erythroid cells Cellculture.Murineerythroleukemia(MEL)cells(76)andK562cellswere
culturedinRPMImedium(Mediatech,Herndon,VA)supplementedwith10%
(31,63).FOG-1servesasthebridgingfactorbetweenGATA-1
heat-inactivatedfetalbovineserum,100(cid:8)Mnonessentialaminoacidmix,2mM
and the NuRD chromatin-remodeling complex (31). The
L-glutamine,and1mMsodiumpyruvateat37°Cin5%CO2.InductionofK562
NuRD complex was originally identified in mammalian and cellswasperformedbyculturingwith3mMhexamethylenebisacetamideand10
VOL.28,2008 GATA-1-MEDIATED REPRESSION OF (cid:2)-GLOBIN GENE EXPRESSION 3103
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FIG. 1. Constructs.(A)Schematicdiagramofthe213-kb(cid:4)-YAC.A187-kbEcoRIgenomicfragmentcontainingthehuman(cid:4)-globinlocusis h
shownasaline.YACvectorsequencesand(cid:4)-likeglobingenesequences(ε,G(cid:2),A(cid:2),(cid:10)(cid:4),(cid:6),and(cid:4))areshownasopenboxes.Thelocationofthe ttp
LCRisnoted;arrowsabovethelinedenoteHSs.ThelocationoftheA(cid:2)m/(cid:7)1sA(cid:2)m-globingeneinsertionisindicatedbyanarrowbelowtheline. :
/
SfiI restriction sites are displayed below the locus; the line above the locus delineates the 115-kb SfiI fragment used in assessment of (cid:4)-YAC /m
transgene integrity. YAC components are represented by black boxes: TRP1 and LYS2, YAC selectable markers for tryptophan and lysine c
prototrophy,respectively;ARS1,yeastautonomousreplicatingsequence(originofreplication);CEN1,centromere;MMTneo,mammaliancell b
selectable marker for G418 resistance. The sequence of the YAC insert (containing a 6-kb gap) is available on the Globin Gene Server .a
(http://globin.cse.psu.edu/). (B) 5.4-kb SspI A(cid:2)m-globin gene. The 5.4-kb SspI A(cid:2)m-globin gene utilized for modifying the (cid:4)-YAC contains s
m
approximately0.7kbof5(cid:5)flankingsequencesand3.2kbof3(cid:5)flankingsequences.Thetranscriptionstartsiteisindicatedbyanarrow.Restriction
.
enzyme sites used in previous experiments for promoter truncations are shown below the line (66). The numbers indicate distance from the o
A(cid:2)m-globingene5(cid:5)CAPsite.Canonicalbindingsitesforerythroid-specifictranscriptionfactorswiththeirapproximatelocationsareshownabove rg
theline.TwopredictedGATAbindingsitesinthe(cid:7)1sregionareshownbyarrows,withthefootprintedsitemarkedwithanasterisk.Previously /
suggestedpositiveandnegativeregulatoryregionsareindicatedby((cid:1))and((cid:3)),respectively(66).The352-bp(cid:7)1sdeletionfrom(cid:3)730to(cid:3)378 o
n
isindicatedatthebottom.
M
a
r
c
h

(cid:8)Mheminfor3days(14)priortonuclearextractpreparationorRNAextrac- AntibodiesagainstmurineGATA-1andGATA-2wereusedinshiftinhibition 30
tion. experiments(SantaCruzBiotechnology). ,

Phenylhydrazinetreatmentofmice.AdultC57BL/6miceatleast6weeksold Transient-transfectionassays.AseriesofpGL2plasmids(Promega,Madison, 2
0
weregiven60mg/kgofbodyweightofphenylhydrazine(10mg/mlinphosphate- WI)wascreatedfortransient-transfectionassays.PCRprimersweredesignedto 1
bufferedsaline;P-6926;Sigma-Aldrich,St.Louis,MO)viaintraperitonealinjec- amplify the 352-bp (cid:7)1s region from an A(cid:2)-globin gene plasmid and to add 9
tionforthreeconsecutivedays.Miceweresacrificed4daysposttreatment,and restrictionenzymesitestobothendsofthefragment.Thesameprimersutilized b
spleens were harvested and processed for nuclear extract preparation as de- fortheDNaseIfootprintingassaywereemployedforsubcloningthe(cid:7)1sinsert y
scribedelsewhere(seematerialsandmethodsinthesupplementalmaterial). intothepGL2vectors.The5(cid:5)endofthesense-strandprimercontainedaSacI g
Nuclear extract preparation. Nuclear extracts were prepared as described site,whereasthe5(cid:5)endoftheantisenseprimercarriedaNheIsite.Theamplified u
e
elsewhere(seematerialsandmethodsinthesupplementalmaterial)(5).Protein 5(cid:5)-SacI-(cid:7)1s-NheI-3(cid:5)fragmentwasligatedtoSacI/NheI-digestedpGL2plasmids s
concentrationsweredeterminedusingtheBio-Rad(Hercules,CA)quickBrad- (pGL2-promoterandpGL2-control).Correctcloneswereidentifiedbyrestric- t
fordassay. tionenzymedigestionanalysis,followedbyDNAsequencing.Thesequencing
DNaseIfootprinting.DNaseIfootprintingassayswereperformedessentially primersutilizedwereasfollows:pGL2primer1,5(cid:5)-TGTATCTTATGGTACTG
asdescribedelsewhere(seematerialsandmethodsinthesupplementalmaterial) TAACTG-3(cid:5); pGL2primer2, 5(cid:5)-CTTTATGTTTTTGGCGTCTTCCA-3(cid:5) (Pro-
(12).TheprimersemployedfortheDNaseIfootprintingassaywereasfollows: mega).
forwardDelta1S,5(cid:5)-GCGAGCTCTATTTTTCTAAGATGA-3(cid:5);reverseDelta K562cells(1(cid:9)107)werecotransfectedwith1(cid:8)gpSV(cid:4)-Galplasmid(Pro-
1S,5(cid:5)-TAGCTAGCTGTCTGTAGCTCCAG-3(cid:5).Thefootprintingexperiments mega)and17.5(cid:8)gofthepGL2plasmidsin0.4mlDulbecco’smodifiedEagle
wereperformedatleastthreetimes. mediumusingaBio-RadGenePulserelectroporatorsetat950-(cid:8)Fcapacitance
Electromobilitygelshiftassays.Electromobilityshiftassays(EMSAs)were and300V.Afterelectroporation,thecellsweredirectlyculturedfor24has
performedasdescribedelsewhere(seeMaterialsandMethodsinthesupple- describedabove.Cellswerecollected,washedwithphosphate-bufferedsaline,
mental material) (30, 37). The oligonucleotides utilized for EMSAs were as andlysedwith(cid:4)-galactosidase((cid:4)-Gal)assaylysisreagent(Promega).Lysates
follows:(cid:3)6795(cid:5)doubleGATA,5(cid:5)-TTCAAATAGGTACGGATAAGTAGATA wereanalyzedforproteinconcentrationusingtheBio-RadquickBradfordassay,
TTGAGGTAAGCATT-3(cid:5) (GenBank coordinates 38715 to 38754); (cid:3)566 3(cid:5) (cid:4)-GalactivitywasassayedusingthePromega(cid:4)-Galassay,andluciferaseactivity
single GATA, 5(cid:5)-ACTATTGAGAAATTAAGAGATAATGGCAAAAGTCAC wasmeasuredusingthePromegaLARIIreagent,allasdescribedbytherespec-
AAAG-3(cid:5)(GenBankcoordinates38828to38867).PotentialGATA-bindingsites tivemanufacturers.(cid:4)-Galandluciferasesignalswerenormalizedtotheprotein
areinbold.Themutatedoligonucleotideswerethesame,excepttheunderlined concentration.Luciferasesignalswerefurthernormalizedtothe(cid:4)-Galsignals.
TwaschangedtoG.Asacontrol,theSp1andEgroligonucleotides(sc-2502and ChIPassay.Chromatinimmunoprecipitation(ChIP)assayswereperformed
sc-2529,respectively)wereused(SantaCruzBiotechnology,SantaCruz,CA). as described previously (38) with the some modifications (see materials and
3104 HARJU-BAKER ET AL. MOL.CELL.BIOL.
methodsinthesupplementalmaterial).Fetalliverfromwild-type(cid:4)-YACtrans- locus extending from 5(cid:5) HS3 through the (cid:4)-globin gene; at
genic mice at 12 and 18 days postconception (E12 and E18) were utilized. least one of these transgenes extended through the H500
Cross-linking of protein-protein and protein-DNA complexes within the cells
HPFHbreakpoint.
wasperformedwith1%formaldehyde(freshparaformaldehyde).Chromatinwas
To confirm that the individual wild-type A(cid:2) (A(cid:2)wt)-, A(cid:2)m-,
sonicatedtoasizerangebetween200and1,000bp.Thesampleswereprecleared
with species-matched normal serum. Immunoprecipitations were carried out and (cid:7)1s A(cid:2)m-globin gene structures were intact within these
withGATA-1-,FOG-1-,andMi2-specificantibodiesorisotype-matchedimmu- mouse lines, Southern blot hybridization and PCR analyses
noglobulinG(IgG)(rabbit,mouse,orgoat)andproteinGconjugatedtoagarose wereperformedtoassesstransgeneintegrity.Southernblotsof
beadscontainingsalmonspermDNA(Upstate,Temecula,CA).Theimmuno-
SphI-orHpaI-digestedgenomicDNAswerehybridizedwitha
precipitatewaswashed,thecross-linkswerereversed,andthegenomicDNAwas
purified.RecruitmentofGATA-1,FOG-1,andMi2proteinswasmeasuredby 0.5-kb SmaI-XmnI 5(cid:5) HS1 probe to detect the (cid:4)-globin locus
real-time quantitative PCR, using gene-specific primers (see Table S2 in the fragments(datanotshown).Wild-type(cid:4)-YACDNAsamples
supplementalmaterial). showedtheexpected23.7-kbHpaIand10.4-kbStuIfragments
Real-timePCR.ChIPsampleswereanalyzedinduplicatebyreal-timePCR for the 5(cid:5) HS1 region. The A(cid:2)m-globin gene insertion intro-
withSYBRGreendyeusingaRocheLightCycler(RocheAppliedScience,Palo
duced additional HpaI and StuI recognition sites into the 5(cid:5)
Alto,CA).Toallowcomparisonamongprimersets,inputsamplesfromeach
conditionweredilutedseriallyfrom1:10to1:10,000andusedasstandardsforall HS1-ε-globin region. The predicted 5.2-kb HpaI and 6.4-kb D
PCRsamples.EnrichmentofaproteinbindingtoaspecificDNAsequencewas StuI A(cid:2)m-globin gene fragments were observed. The (cid:7)1s de- o
calculatedusingthecrossing-pointmethod(35).Thedatashownaretheaver- letionintheA(cid:2)m-globingeneremoved0.4kbofthe5(cid:5)region w
asagmesploef.dEurprolircabtaersrerseuplrtsesfernotmstaatndleaarsdtdtwevoiaitniodnespefrnodmenttheexpmeeraimn.ePntCsRfoprriemacehr of the A(cid:2)m-globin gene insert. Therefore, the expected HpaI nlo
sequencesareprovidedinTableS2inthesupplementalmaterial. fragmentfor(cid:7)1swas4.8kbinsteadof5.2kb.All(cid:7)1slineshad a
PreparationofnuclearlysatesandWesternblottinganalysis.Nuclearextracts this fragment. Similarly, the (cid:7)1s deletion removed a StuI site de
were prepared from K562 and MEL cell lines as described elsewhere (see from the A(cid:2)m-globin gene, resulting in a 15-kb fragment that d

materialsandmethodsinthesupplementalmaterial)(34).Westernblottingwas wasobservedintheselines.ThesedataindicatedthattheA(cid:2)m- fr
performedaccordingtostandardprocedures(seematerialsandmethodsinthe o
and(cid:7)1sA(cid:2)m-globingeneinsertswereinthecorrectlocation. m
supplementalmaterial)(5).
Antibodies.Seematerialsandmethodsinthesupplementalmaterialforin- APCR-basedapproachwasalsoutilizedtoconfirmthe(cid:7)1s h
formationonantibodiesemployedandsources. A(cid:2)m 5(cid:5) ε region structure (see Fig. S2A and B in the supple- tt
p
mental material). Primer set B (see Table S1 in the supple- :
/
/
mentalmaterial)wasdesignedtoannealtothepromoterand m
RESULTS
exon2oftheA(cid:2)m-,(cid:7)1sA(cid:2)m-,andA(cid:2)wt-globingenes(seeFig. c
b
(cid:5)1sA(cid:1)m5(cid:1)(cid:3)(cid:4)-YACtransgenestructuredeterminationand S2A in the supplemental material). The mark in the A(cid:2)m- .
a
copynumberanalysis.ToidentifypotentialA(cid:2)-globinsilencer globingene(and(cid:7)1sA(cid:2)m)isa6-bpdeletioninthe5(cid:5)untrans- s
m
elements,aseriesofdeletionsintheA(cid:2)-globingenepromoter latedregion(UTR)oftheA(cid:2)-globingene(62),whichremoves
.
and 5(cid:5) and 3(cid:5) flanking regions was planned for introduction aDdeIrestrictionenzymesite.Digestionofawild-type(cid:2)-glo- or
into the A(cid:2)m 5(cid:5) ε (cid:4)-YAC (Fig. 1A). The first deletion, from bin gene PCR product with DdeI results in three fragments, g/

(cid:3)730 to (cid:3)378 relative to the mRNA start site of the A(cid:2)m- 175bp,107bp,and20bpinsize,whereastheA(cid:2)m-globingene o
n
globin gene, was introduced into the A(cid:2)m 5(cid:5) ε (cid:4)-YAC by PCR product gives only two fragments, 195 bp and 107 bp in
M
homologous recombination in yeast (Fig. 1B). The resultant size.Additionally,twosetsofprimersthatflankthe5(cid:5)and3(cid:5) a
(cid:7)1sA(cid:2)m5(cid:5)ε(cid:4)-YACwasusedtoproducetransgenicmice.Five junctions of the A(cid:2)m- and (cid:7)1s A(cid:2)m-globin gene inserts were rc
transgenic lines were established after identification of designed. The 5(cid:5) junction primer set A (see Table S1 in the h

3
foundersbyPCRoftailbiopsyDNAs. supplemental material) produced a 793-bp fragment for the
0
Long-range restriction enzyme mapping structural analysis A(cid:2)m-globingeneanda426-bpfragmentforthe(cid:7)1sA(cid:2)m-globin ,
2
was performed using 11 radioactively labeled DNA probes geneduetothe352-bp(cid:7)1sdeletion(seeFig.S2B,toppanel, 0
spanning the locus from 5(cid:5) HS3 through the HPFH6 break- in the supplemental material). The 3(cid:5) junction is common to 1
9
point on Southern blots of pulsed-field gels (see Figure S1 in both A(cid:2)m- and (cid:7)1s A(cid:2)m-globin genes, and thus, PCR with b
thesupplementalmaterial)(47,51).Transgenecopynumbers primer set C (see Table S1 in the supplemental material) y

g
weredeterminedaspreviouslypublished(23,46).Itisimpor- generated a 629-bp fragment for both genes (see Fig. S2B,
u
tanttodetermineboththecopynumberandcontinuityofthe bottom panel, in the supplemental material). Wild-type e
s
YACsequencestoaccuratelycalculatequantitativecopynum- (cid:4)-YACdidnotgiveaproductwitheitherprimersetAorC,as t
ber-corrected gene expression levels. Only genes that are expected. The PCR analysis resulted in the predicted frag-
linkedtotheLCRwillbeexpressedatphysiologicallevels(52). ments for all the (cid:7)1s A(cid:2)m 5(cid:5) ε (cid:4)-YAC lines. Together, these
Line1carriedfourdifferent-sizecopiesofthelocusextending data demonstrate that the (cid:7)1s A(cid:2)m-globin gene inserts are in
fromtheLCRthroughtheHPFH3breakpoint.Line2hadtwo thecorrectlocationwithinthe(cid:7)1sA(cid:2)m5(cid:5)ε(cid:4)-YACs.
copies of the locus extending from the LCR through the A(cid:1)m-Globin is expressed in adult blood of (cid:5)1s A(cid:1)m 5(cid:1) (cid:3)
HPFH3region.Line3carriedfourcopiesoftheYACextend- (cid:4)-YAC transgenic lines. Analysis of the expression of human
ingfromtheLCRthroughtheHPHF6breakpoint.However, (cid:4)-likeglobinsinthe(cid:7)1sA(cid:2)m5(cid:5)ε(cid:4)-YACtransgenicmicewas
inaddition,thislineboretwocopiesoftheYACwithadele- performed by RPAs as described in Materials and Methods.
tion of the (cid:6)-globin gene and downstream region. Therefore, Samples of hematopoietic tissues and blood were collected
the copy numbers utilized for calculating per-copy expression fromdevelopmentallystagedembryos,fetuses,andadultsand
levelsforthislineweresixfortheglobingenesupstreamofthe analyzed using antisense RNA probes for human ε-, (cid:2)-, and
breakpoint and four for the (cid:4)-globin gene. Line 4 had two (cid:4)-globins and for the murine (cid:11)- and (cid:12)-globins. The mouse
copiesoftwodifferentsizesextendingfromtheLCRthrough (cid:11)-likeglobinsservedasinternalcontrolstoquantitatehuman
theHPFH6breakpoint.Line5hadfourcopiesofthecomplete (cid:4)-like globin transgene expression levels. The (cid:2)-globin ribo-
VOL.28,2008 GATA-1-MEDIATED REPRESSION OF (cid:2)-GLOBIN GENE EXPRESSION 3105
probe was synthesized from an A(cid:2)m-globin gene DNA tem-
plate,pT7A(cid:2)m(170)(4,62).Whenthisprobeannealswiththe
wild-type A(cid:2)-globin (A(cid:2)wt-globin) gene transcript, double-
stranded regions are formed at exon 2 and exon 1/5(cid:5) UTR,
except where the mark is located in the 5(cid:5) UTR. Therefore,
RNasedigestionwillgeneratea170-bpbandforexon2,which
iscommonforboththeA(cid:2)m-andA(cid:2)wt-globingenes.However,
theA(cid:2)wt-globinexon1/5(cid:5)UTRprotectedfragmentisdigested
into two bands, 118 bp plus 26 bp. The smaller band is not
usuallyvisualizedonanRPAimage.TheA(cid:2)m-globinexon1/5(cid:5)
UTR protected fragment is 138 bp in size, since it is not
cleaved by RNase. All of the (cid:7)1s lines expressed (cid:2)-globin,
whereasthepositivecontrolwild-type(cid:4)-YACadultblooddid
not show any (cid:2)-globin expression (Fig. 2). Wild-type (cid:4)-YAC D
E12 fetal liver expressed a high level of A(cid:2)wt-globin and a o
w
smaller amount of (cid:4)-globin. Although the (cid:4)-globin band in- n
tensities look variable and lower in one instance in the wild- lo
type (cid:4)-YAC adult blood control than in the (cid:7)1s lines, the a
d
average (cid:4)-globin expression in the (cid:7)1s lines was 25.3%, cor- e
d
rectedfortransgenecopynumberandnormalizedtoper-copy
f
mouse (cid:11)-globin expression, compared to 56.4% for the wild- ro
type(cid:4)-YACcontrol(Fig.2). m

Antibody detection experiments with adult transgenic h
t
mouse blood smears demonstrated expression of both (cid:2)- and tp
(cid:4)-globinproteinsinerythroidcellsofthe(cid:7)1sA(cid:2)m5(cid:5)ε(cid:4)-YAC ://
m
lines and in the positive-control (cid:3)117 A(cid:2)m Greek HPFH
c
(cid:4)-YAC line but not in wild-type (cid:4)-YAC transgenics (Fig. 3). b
FIG. 2. Human (cid:2)- and (cid:4)-globin transcription in adult blood of Thus,proteinexpressionreflectedthemRNAsynthesisprofile .a
(cid:7)1s A(cid:2)m 5(cid:5) ε (cid:4)-YAC transgenic mouse lines. Total RNA isolated s
foreachoftheselinesduringtheadultstageofdevelopment. m
from the blood of adults and an E12 fetal liver (FL; wild-type
(cid:4)-YAC control) was subjected to RNase protection assays. Anti- A representative developmental profile RPA for (cid:7)1s line 1 .o
senseRNAprobesforhuman(cid:2)-and(cid:4)-globinandmouse(cid:11)-globin hematopoietic tissues is shown in Fig. 4A. ε- and A(cid:2)wt-globin rg
(Mo (cid:11)) were utilized. Protected fragments are displayed on the expression were not detected at any developmental stage, /
right:(cid:4),(cid:4)exon2,205bp;(cid:2)ex2,A(cid:2)exon2,170bp;A(cid:2)mex1,A(cid:2)m o
exon1,138bp;A(cid:2)wtex1,A(cid:2)wtexon1,118bp;Mo(cid:11),129bp.The which was similar to ε- and A(cid:2)wt-globin expression patterns n
constructsareshownabovethepanel:numberedlanes,(cid:7)1sA(cid:2)m5(cid:5) measuredfortheA(cid:2)m5(cid:5)ε(cid:4)-YACmice(29).Levelsofhuman M
ε (cid:4)-YAC lines; ((cid:1)), wild-type (cid:4)-YAC controls; M, pBR322 MspI transgene expression corrected for copy number and normal- ar
molecularweightmarker. ized to copy number-corrected murine (cid:11)-like globin gene ex- ch
pressionareshowngraphicallyinFig.4BandC.(cid:4)-Globingene 3
0
expressionwasnormalduringearlyfetaldefinitiveerythropoi- ,

2
0
1
9

b
y

g
u
e
s
t
FIG. 3. (cid:2)-and(cid:4)-globinchainexpressionintransgenicmouseadultblood.Adultbloodsmearsfromtransgenicmicecarryingthewild-type
(cid:4)-YAC, (cid:3)117 A(cid:2)m Greek HPFH (cid:4)-YAC, or (cid:7)1s A(cid:2)m 5(cid:5) ε (cid:4)-YAC were stained for (cid:2)- and (cid:4)-globin protein chains (top and bottom panels,
respectively)usingindirectimmunolabeling.
3106 HARJU-BAKER ET AL. MOL.CELL.BIOL.
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.
FIG. 4. (A)Human(cid:4)-likeglobinexpressionin(cid:7)1sA(cid:2)m5(cid:5)ε(cid:4)-YACmousetissues.TotalRNAisolatedfromthehematopoietictissuesofstaged a
s
embryos,fetuses,andadultswassubjectedtoRNaseprotectionassays.AntisenseRNAprobesforhumanε-,(cid:2)-,and(cid:4)-globinandmouse(Mo) m
(cid:11)-and(cid:13)-globinwereutilized.ProtectedfragmentsareasdescribedinthelegendtoFig.2withthefollowingadditions:ε,εexon2,188bp;Mo .
(cid:13),151bp.Developmentaldayspostconceptionareindicatedabovethepanels.Differenttissuesareindicatedabovethelanes:YS,yolksac;FL, or
fetalliver;Bl,blood.Thenumbersdenotemultiplesamplesfromthesameline.(B)Quantitationofhuman(cid:4)-globinexpressionin(cid:7)1sA(cid:2)m5(cid:5)ε g
(cid:4)-YAClines.Per-copy(cid:4)-globintransgeneexpressionwascalculatedasapercentageofmurine(cid:11)-likeglobingeneexpression,alsocorrectedfor o/
endogenouscopynumber.Averageandstandarddeviationareshownforthreedifferentlinesofwild-type(cid:4)-YACmice(whitebars)andA(cid:2)m5(cid:5) n
ε(cid:4)-YACmice(graybars)andforall(cid:7)1sA(cid:2)m5(cid:5)ε(cid:4)-YACmice(blackbars).(C)Quantitationofhuman(cid:2)-globinexpressionin(cid:7)1sA(cid:2)m5(cid:5)ε(cid:4)-YAC M
lines.(cid:2)-GlobinexpressionwascalculatedandisrepresentedasdescribedinthelegendforpanelB. a
r
c
h

esis, but the level in adult blood was decreased compared to some(cid:2)-globinwhentheycarrythe(cid:4)-YAC(8,54).K562cells 3
0
that for the control transgenics (Fig. 4B). In fact, the level of express predominantly ε-globin and some (cid:2)-globin but no ,
2
(cid:4)-globinexpressionintheselineswasevenlowerthanthatin (cid:4)-globin(1,14,48,65).Followinginduction,K562cellsdisplay 0
the A(cid:2)m 5(cid:5) ε (cid:4)-YAC lines (29); expression was 38% in the increased(cid:2)-globinproduction(1,14,48,65).Wealsoutilized 1
9
former versus 59% in the latter (Fig. 4B). Expression of the nuclearextractpreparedfromthespleensofphenylhydrazine- b
(cid:2)-globin gene in the adult blood may repress transcription treated transgenic mice, which express high levels of (cid:2)-globin y

fromtheadult(cid:4)-globingene,presumablybecausetheLCRis due to the reticulocytosis resulting from hemolytic anemia g
u
engagingthe(cid:2)-globingenepromoterandinteractinglesswith (18).Onlyasinglefootprintwasobservedincellsthatdonot e
s
the(cid:4)-globingenepromoter.TheA(cid:2)m-globingenewasstrongly express any (cid:2)-globin or have a low expression level (CCRF- t
expressed during fetal definitive erythropoiesis and in adult CEM, MEL 585, and uninduced K562 cells) (Fig. 5; see also
blood(Fig.4C).Theseresultssuggestthatthe(cid:3)730to(cid:3)378 Fig. S3 in the supplemental material). The footprint was not
regioncontainsanA(cid:2)-globin-specificsilencer,sincedeletionof seenincellsthatexpress(cid:2)-globin(inducedK562cellsorphe-
thisregionallowsA(cid:2)-globinexpressionduringadultdefinitive nylhydrazine-treatedmousespleen)(Fig.5).Themoststriking
erythropoiesis. difference in the footprint intensity was observed between in-
A GATA site in the 352-bp (cid:5)1s fragment is footprinted in ducedanduninducedK562cells.UninducedK562cellnuclear
vitroincellsexpressinglowlevelsof(cid:1)-globinmRNA.Invitro extracts produced a clear footprint at the (cid:3)566 GATA site,
DNase I-footprinting studies were done utilizing probes cov- whichdisappeareduponinduction.Thefootprintedsequence
eringthe352-bp(cid:7)1sfragmentfrom(cid:3)730to(cid:3)378andnuclear (5(cid:5)-aagaGATAatggc-3(cid:5))waspredictedtobeastrong,canoni-
extracts from CCRF-CEM cells, MEL 585 mouse erythroleu- cal GATA-1 binding site (WGATAR) by MatInspector anal-
kemia cells, and uninduced or induced K562 cells. CCRF- ysis (57). This site was also present in the G(cid:2)-globin gene
CEMisahumanT-cell-lymphoblast-likecelllinethatdoesnot promoter; however, we did not utilize primers to test this
expressanyglobingenes(22).MEL585cellsaremurineeryth- regionoftheG(cid:2)-globingenepromoter.Asecondpairofnon-
roleukemiacellsthatexpresspredominantly(cid:4)-globinbutalso canonicalGATAsitesfurtherupstreamofthepromotercen-
VOL.28,2008 GATA-1-MEDIATED REPRESSION OF (cid:2)-GLOBIN GENE EXPRESSION 3107
ernblotanalysisdemonstratedadecreaseinGATA-1protein
ininducedK562cells,although(cid:4)-actinandSp1proteinlevels
werenotdecreased(seeFig.S4inthesupplementalmaterial).
The similar levels of these two proteins in uninduced and
inducedK562cellsinequaltotalproteinaliquotsasmeasured
by the Bradford assay (see Materials and Methods) indicate
that these extracts are qualitatively similar. Within a popula-
tion of K562 cells, (cid:2)-globin transcription is generally low but
measurable in uninduced cells and increases approximately
1.4-fold in induced cells (data not shown) (65). Thus, the
higher GATA-1 concentration in uninduced cells may aid in
occupation of silencer GATA sites, and following induction,
onlyactivatorGATAsitesareboundbythemorelimitedlevel
of GATA-1. The MEL 585 data corroborate the uninduced D
K562 data, since (cid:2)-globin is poorly expressed in transgenic o
w
derivativesofthisadulterythroidphenotypecellline(54),and n
theextractsfromphenylhydrazine-treatedmousespleenssup- lo
porttheinducedK562data,since(cid:2)-globinishighlyinducedin a
d
these cells. The (cid:3)679 GATA sites also appeared to weakly e
d
shift, but use of a mutant GAGA oligonucleotide did not
f
r
ablate this shift, indicating that the observed shift was due to o
m
nonspecific binding of protein. In addition, a GATA compet-

itor or GATA antibodies did not affect the shift at this site h
t
(data not shown). A single base pair mutation in the (cid:3)566 tp
:
GATAsite(T3G)ablatedtheshift.Competitionassayswith //
m
coldcompetitorsorantibodiesconfirmedthatGATA-1binds
c
tothe(cid:3)566GATAsiteinuninducedcells(Fig.6B).Addition b
.
ofoligonucleotideswithSp1/Egrproteinbindingsitesoracold a
s
GAGA competitor did not affect the shift, whereas a cold m
FIG. 5. InvitroDNaseIfootprintingofthe352-bp(cid:3)730to(cid:3)378 (cid:3)566 GATA oligonucleotide efficiently inhibited the shift. .o
(cid:7)1sfragment.The352-bp(cid:3)730to(cid:3)378fragmentwasPCRamplified Preincubation with a GATA-1 antibody abolished the shift, rg
usinganend-labeledsense-strandprimer.Proteinbindingexperiments whereas a GATA-2 antibody had no effect, indicating that /
o
were performed using labeled fragment and various nuclear extracts GATA-1wastheproteininthenuclearlysateresponsiblefor n
(shlaobwenledtoatbhoeverigthhet ofifguthree)a.uTtohreadseioqgureanpchewoifththtehefo(cid:3)ot5p6r6inGtA(FTPA-1s)ities the shift of the (cid:3)566 GATA site-containing oligonucleotides M
basesunderlined. (Fig. 6B). These data suggest that the (cid:3)566 GATA site is ar
c
boundbyGATA-1andthatGATA-1isinvolvedinrepression h
of(cid:2)-globingeneexpression. 3
tered at (cid:3)679 was also predicted by MatInspector, but it did The (cid:5)1s fragment is a silencer of gene expression. K562 0,
cellswereelectroporatedwithpSV(cid:4)-GalandaseriesofpGL-2 2
notappeartobeboundbyproteinsintheseexperiments(see 0
plasmids. The pGL-2-series reporter constructs (Promega) 1
Fig.S3inthesupplementalmaterial).Therefore,weconclude
9
thattheWGATARsequencelocatedat(cid:3)566bindsarepres- carry a luciferase gene linked to simian virus 40 (SV40) pro- b
sor in cells that do not express A(cid:2)-globin or that express it at moterandenhancerfragments.ThepGL-2promoterconstruct y

lowlevels. has an SV-40 promoter driving the luciferase gene, whereas g
u
GATA-1 binds to the distal A(cid:1)-globin gene promoter when the pGL-2 control vector has in addition an SV40 enhancer e
(cid:1)-globingeneexpressionisrepressed.SinceaGATAbinding downstream to the luciferase reporter gene. The 352-bp (cid:7)1s st
sitewasfoundtobefootprintedincellsinwhich(cid:2)-globingene fragment was inserted 5(cid:5) to the promoter of these two con-
transcripts were at a low level or not present, we wanted to structs(Fig.7A).Luciferaseactivitywascorrectedtothepro-
confirm whether this site was bound by GATA-1 or GATA-2 tein concentration and normalized to (cid:4)-galactosidase activity
proteinsunderconditionsof(cid:2)-globinrepressionorexpression. to control for electroporation efficiency. Expression was in-
We performed EMSAs with 40-mer double-stranded DNA creased approximately fivefold whether or not the SV40 en-
fragments which contained the (cid:3)566 (footprinted) or (cid:3)679 hancer was present (Fig. 7B). Previous data have shown that
(notfootprinted)GATAsites.WealsoutilizedDNAoligonu- thisenhancerdoesnotincreasethelevelofgeneexpressionin
cleotide probes in which the putative GATA binding site was K562cellsbutonlytheprobabilityofexpression(75).Ourdata
ablatedbychangingtheTtoaG(GATA3GAGA).Thedata revealed that the (cid:3)730 to (cid:3)378 (cid:7)1s fragment functions as a
showedthatonlythewild-typeGATAsiteat(cid:3)566wasshifted silencer following transient transfection and cell culture of
usinguninducedMEL585andK562extracts(conditionsofno uninduced K562 cells (Fig. 7B). In support of these data, we
or low (cid:2)-globin expression) (Fig. 6A). Although this is a per- recently produced (cid:4)-YAC transgenic mice containing a point
fect GATA site, the induced K562 nuclear extracts did not mutation in the (cid:3)566 A(cid:2)-globin GATA site. The founders
showsignificantGATA-1bindingactivity.Interestingly,West- showed a weak HPFH phenotype (data not shown), demon-
3108 HARJU-BAKER ET AL. MOL.CELL.BIOL.
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.
a
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.
o
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/

o
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M
a
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h

3
0
,

2
0
1
9

b
y

g
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FIG. 6. (A)EMSAofGATAfactorbindingsiteswithinthesilencersequence.Four40-merdouble-strandedDNAprobespreparedbyend e
labelingwereincubatedwithvariousnuclearextracts.Theprobesandnuclearextractsourcesareindicatedabovethefigure.TheGAGAprobes s
haveasinglenucleotidechange,fromGATAtoGAGA.Thenuclearextractswereasfollows:((cid:3)),nonuclearextract;M(cid:3),uninducedMEL585; t
K(cid:3),uninducedK562;K(cid:1),HMBA-hemin-inducedK562;P,phenylhydrazine-treatedmousespleen.(B)Competitionandantibodyshiftablation
EMSAsofthe(cid:3)566GATAsite.UninducedMEL585andK562nuclearextractswereincubatedwiththe(cid:3)566GATAprobe.Variouscompetitors
orantibodies(Ab)wereaddedtothemixture(labeledabovethefigure).
stratingthatthe(cid:3)566GATAsitewithinthisfragmentiswhere the immunoprecipitations. Figure 8A shows that the (cid:3)566
therepressorcomplexbinds. region of the A(cid:2)-globin gene was enriched for binding of the
The GATA-1 protein binds to the (cid:2)566 GATA site of the GATA-1 protein in E18 fetal liver samples ((cid:2)-globin re-
A(cid:1)-globin gene in vivo when (cid:1)-globin is not expressed. To pressed)comparedwithresultsintheratIgGcontrolsamples.
confirm that the GATA-1 protein binds to the (cid:3)566 GATA BindingoftheGATA-1proteinwasnotobservedinE12fetal
siteofA(cid:2)-globininvivo,weemployedChIPassaysusingE12 liver samples, where (cid:2)-globin was expressed. A negative con-
andE18fetalliversamplesfromwild-type(cid:4)-YACtransgenic trolincludedinallexperiments,mouseglyceraldehyde-3-phos-
mice((cid:2)-globinexpressedandnotexpressed,respectively).Anti- phatedehydrogenasereal-timePCRwiththesameChIPsam-
GATA-1andnormalratIgG(control)antibodieswereusedin ples, showed an absence of binding by GATA-1 in E12 and
VOL.28,2008 GATA-1-MEDIATED REPRESSION OF (cid:2)-GLOBIN GENE EXPRESSION 3109
that FOG-1 is required for repression mediated by GATA-1
(31).InthisworkweshowedthattheGATA-1proteinbindsto
the A(cid:2)-globin gene at the (cid:3)566 GATA site. Using the ChIP
assay,wenextexaminedwhetherFOG-1wasrecruitedtothe
(cid:3)566 GATA site in the absence of (cid:2)-globin expression. Our
analysis with anti-FOG-1 and normal goat IgG antibodies in
E12 fetal liver samples revealed no significant occupancy of
FOG-1atthe(cid:3)566sitewhen(cid:2)-globinwasexpressed(Fig.8B).
In contrast, in E18 fetal liver where (cid:2)-globin was silent, re-
cruitmentofFOG-1wasobserved(Fig.8B).
TheMi2proteinassociateswithGATA-1andFOG-1atthe
A(cid:1)-globin gene (cid:2)566 GATA binding site. The Mi2 protein,
which is one of the protein subunits of NuRD or MeCP1
complexes,hasbeenshowntoassociatewithGATA-1(31,63).
D
Thus, we hypothesized that Mi2 might interact with GATA-1 o
at the A(cid:2)-globin (cid:3)566 GATA silencer. To first assess the w
n
presence of the Mi2 protein in erythroid cell lines, Western lo
blotanalysiswasperformedwithnuclearextractsofMELand a
d
K562cellsusinganti-Mi2antibody(seeFig.S4inthesupple- e
mental material). K562 and MEL cells were treated with d

hemin and HMBA to induce (cid:2)-globin expression or left un- fr
o
treated. Mi2 protein showed a stronger signal in nuclear ex- m
tractsofcellswherethelevelof(cid:2)-globinexpressionwaslowor h
absent (uninduced MEL and K562 cells) than in cells where tt
p
(cid:2)-globin expression was induced (hemin- and HMBA-treated :
/
/
K562cells;seeFig.S4inthesupplementalmaterial).Although m
FIG. 7. Transient transfection of K562 cells with pGL2-(cid:7)1s con- we detected the presence of the Mi2 protein in the induced c
b
structs.Theconstructsaredepictedbydrawingsatthetop(A),andthe erythroidcellline,thelevelwasincreasedincellswithlowor .
relativeluciferaseunitscorrespondingtoeachconstructareshownat a
no(cid:2)-globinexpression,suggestingthatthepresenceoftheMi2 s
the bottom (B). Luciferase activity was corrected for total protein m
concentration and transfection efficiency as measured by cotransfec- protein might be associated with the repression of (cid:2)-globin .
tionwitha(cid:4)-Galreporterconstruct(pSV(cid:4)-Gal).ThepGL2promoter geneexpression. or
(pGL2-p) contains only the luciferase reporter gene driven by the WenextutilizedChIPtotestwhethertheMi2proteinbinds g/
SV40 promoter, denoted by white and medium-gray boxes, respec- invivotothe(cid:3)566GATAsiteoftheA(cid:2)-globingeneandifthis o
tively. The pGL2 control (pGL2-c) carries in addition a SV40 en- n
hancer,shownasalight-graybox.pGL2-p-(cid:7)1sisthesameaspGL2-p binding correlated with the transcriptional repression or acti- M
withthe(cid:3)730to(cid:3)378fragment(dark-graybox)ligatedintotheSacI vationoftheA(cid:2)-globingene.Immunoprecipitationwascarried a
andNheIsitesofpGL2-p.pGL2-c-(cid:7)1sisthesameaspGL2-cwiththe out using antibodies against Mi2 and normal rabbit IgG as a rc
(cid:7)1s fragment ligated into the SacI and NheI sites of pGL2-c. The control. Specific recruitment of the Mi2 protein to the (cid:3)566 h
resultsaretheaverageandstandarddeviationforthreeseparateex- GATA site of the A(cid:2)-globin gene was observed only when 3
perimentswithtwotothreereplicatespersample. 0
(cid:2)-globin expression was repressed (E18 fetal liver) (Fig. 8C). ,
2
No recruitment of Mi2 was observed in those samples where 0
(cid:2)-globinexpressionwasactive(E12fetalliver,Fig.8C).These 1
9
E18 samples (data not shown). Similar results were observed resultsprovidestrongevidencethatMi2isassociatedwiththe b
for FOG-1 and Mi2 (data not shown; discussed below). As a transcriptionalrepressionofA(cid:2)-globingeneexpressionbegin- y

g
positive control, we included ChIP data that demonstrated ning during late fetal liver definitive erythropoiesis in vivo,
u
recruitment of GATA-1 to the (cid:3)2.8 GATA site upstream of presumably through recruitment to the (cid:3)566 GATA site by e
s
the GATA-2 gene using E12 samples (see Fig. S5 in the sup- colocalizationwithGATA-1.SinceMi2isawell-characterized t
plemental material), a well-documented region where subunitoftheNuRDorMeCP1repressorcomplexes,itspres-
GATA-1 binds as an activator (45). The signal range was enceindicatesthatothercomponentsoftheserepressorcom-
similar to that measured at the (cid:3)566 A(cid:2)-globin GATA site plexes may function at this newly identified silencer region.
withE18fetalliversamples(Fig.8A).Finally,aregion2.3kb Together our findings strongly suggest that FOG-1 mediates
downstreamofthe(cid:3)566A(cid:2)-globinGATAsitewasincludedas thebindingoftheMi2proteintotheGATA-1proteinandthat
anadditionalnegativecontrol(seeFig.S5inthesupplemental this complex binds to the (cid:3)566 GATA site of the A(cid:2)-globin
material). The data showed an absence of GATA-1 (and gene. The binding of a GATA-1–FOG-1–Mi2 (NuRD or
FOG-1 and Mi2) binding in the same E18 samples where we MeCP1?) repressor complex to this silencer region plays an
demonstratedGATA-1(andFOG-1andMi2)recruitmentto important role in the transcriptional repression of (cid:2)-globin
the (cid:3)566 A(cid:2)-globin GATA site. These findings demonstrate expressionduringadultdefinitiveerythropoiesis.
that GATA-1 is recruited to this region only when (cid:2)-globin RecruitmentoftheGATA-1–FOG-1–Mi2repressorcomplex
expressionissilenced. to the analogous G(cid:1)-globin (cid:2)567 GATA site silencer. To an-
FOG-1 interacts with the GATA-1 protein in repressing alyzewhetherthesamerepressorcomplexwasrecruitedtothe
transcription of the A(cid:1)-globin gene. Previous studies showed analogous(cid:3)567GATAsiteoftheG(cid:2)-globingene,ChIPassays
3110 HARJU-BAKER ET AL. MOL.CELL.BIOL.
D
o
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9

b
FIG. 8. ChIPanalysisofthe(cid:3)566A(cid:2)-globinGATAsite(leftcolumn)andthe(cid:3)567G(cid:2)-globinGATAsite(rightcolumn)infetalliversamples y
from day E12 and E18 conceptuses of wild-type (cid:4)-YAC transgenic mice. The relative occupancy of the (cid:3)566 region of the A(cid:2)-globin gene g
(GenBankcoordinates38772to38937fromaccessionfileGI455025)orthe(cid:3)567regionoftheG(cid:2)-globingene(GenBankcoordinates33992to u
e
33845fromaccessionfileGI455025)bytheGATA-1(A),FOG-1(B),orMi2(C)protein(graybars)isshownincomparisontotheIgG(control)
s
samples(whitebars).ChIPwascarriedoutusingisotype-matchedIgG,GATA-1,FOG-1,andMi2antibodies(SantaCruzBiotechnology,Santa t
Cruz,CA).Theresultsaretheaveragesforatleasttwoexperiments,andeachexperimentwasperformedinduplicate.Mouseglyceraldehyde-
3-phosphatedehydrogenaseprimerswereusedasanegativecontrolforallreal-timePCR(datanotshown).Thespecificityoftheprimersfor
A(cid:2)-globinwasconfirmedbyrestrictionenzymedigestionofthePCRproducts.TheasteriskindicatesaStudentttestvalue(P)of(cid:1)0.025.
usingGATA-1,FOG-1,andMi2antibodiesandspecificprim- GATA-1, FOG-1, and Mi2 proteins to the G(cid:2)-globin gene
ers was carried out as described above (Fig. 8). Our results (cid:3)567 GATA site was weaker than that observed for the A(cid:2)-
showed that the GATA-1, FOG-1, and Mi2 proteins were globingene,suggestingthatdifferentmechanismsareinvolved
recruited to this GATA site only when (cid:2)-globin gene expres- intheregulationoftheexpressionofthetwo(cid:2)-globingenes.
sionwassilent(E18fetalliversamplesfromwild-type(cid:4)-YAC
transgeniclines)(Fig.8)andnotwhenthe(cid:2)-globingenewas
DISCUSSION
expressed (E12 fetal liver samples from wild-type (cid:4)-YAC
transgeniclines),inaparallelmannertothe(cid:3)566GATAsite A number of regulatory mechanisms operate to coordinate
of the A(cid:2)-globin gene. Interestingly, the binding of the temporalandtissue-specificexpressionofthe(cid:4)-likeglobingenes,