the development of multicellular systems requires that multipotent progenitors differentiate into specialized lineage - restricted daughter cells .
the adoption of a particular cell fate by multipotent cells is orchestrated by networks of transcription factors , which act to coordinate changes in gene expression commensurate with the ultimate function of the cell fate in question .
commitment of multipotent cells to a particular lineage often requires the silencing of gene products that are incompatible with the function of end - product cells .
for instance , during hematopoiesis , erythroid and myeloid lineage genes are silenced during the generation of lymphocyte - biased progenitors ( miyamoto et  al . , 2002 ) and b cell and myeloid - affiliated genes
understanding the regulation of cell fate decisions in hematopoiesis should provide insights into the development of a wide array of multicellular systems and lead to strategies to enhance or limit the generation of particular cell types .
these include ikaros and pu.1 , which promote the generation of lymphoid - biased precursors , and early b cell factor-1 ( ebf1 ) , pax5 , and the e2a isoforms e12 and e47 ( encoded by  the  tcf3 locus ) , which coordinate the differentiation of lymphoid progenitors into lineage - restricted pro - b cells ( reviewed in mandel and grosschedl , 2010 ) . given that each of these factors is essential for generating b cell lineage precursors , much work has been devoted to identifying regulated and coregulated target genes .
ebf1 , pax5 , and tcf3 gene products synergize to activate the expression of the pre - bcr components 5 and vpreb and the b cell signaling protein ig- ( encoded by igll1 , vpreb1 , and cd79a , respectively ) ( reviewed in busslinger , 2004 ; hagman and lukin , 2006 ) .
notably , pax5 , ebf1 , and tcf3 gene products are each proposed to suppress differentiation of alternative fates ( ikawa et  al . , 2004 ; nutt et  al . , 1999 ; pongubala et  al . ,
regard , pax5 is regarded as the dominant determinant of b cell commitment , because deletion of pax5 in pro - b cells or mature peripheral b cells allows these cells to adopt alternative fates ( cobaleda et  al . , 2007 ; mikkola et  al . , 2002 ) .
a key but unresolved question is whether e12 and e47 and/or ebf1 promote b cell lineage restriction by collaborating with pax5 or whether these factors are components of distinct transcriptional circuits important for acquiring and perhaps maintaining b cell identity . in the thymus ,
the t  cell program is initiated when the earliest defined t  cell precursors ( etps ) encounter ligands for the notch receptor family ( sambandam et  al . , 2005 ) .
stimulation of notch1 on etps by the notch ligand delta - like-4 ( dl4 ) promotes the expression of t - cell - affiliated transcription factors including tcf1 ( encoded by tcf7 ) , which in turn promotes the expression of many genes required for t  cell function ( weber et  al . , 2011 ) .
however , early t  cell development is also controlled by the zinc finger transcription factor gata3 .
indeed , gata3 is essential for very early t  cell development in the thymus beginning at the etp stage ( hendriks et  al . , 1999 ;
taghon et  al . , 2007 ) , and optimal notch1 expression may require gata3 ( wei et  al . , 2011 ) .
suppression of the t  cell fate in b cells is thought to occur through the pax5-driven repression of notch1 ( souabni et  al . , 2002 ) .
however , we showed previously that ebf1 prevents myeloid and t  cell differentiation when introduced into pax5 progenitors ( pongubala et  al . , 2008 ) .
the latter observation suggests that pax5-independent transcriptional pathways may also regulate b cell lineage restriction , while also raising questions about the mechanism(s ) employed by ebf1 to constrain t  cell differentiation . here
, we utilize a series of gain- and loss - of - function approaches to uncover the transcriptional mechanism underpinning ebf1-mediated suppression of t  cell development .
our findings indicate that ebf1 limits early t  cell differentiation by directly repressing gata3 transcription and suggest that ebf1 silences gata3 expression by promoting repressive histone modifications across gata3 regulatory regions .
these data identify a transcriptional circuit critical for preventing t  cell differentiation and adopting the b cell fate .
lymphoid - biased progenitors in the bone marrow ( bm ) , also referred to as common lymphoid progenitors ( clps ) ( kondo et  al . , 1997 ) , can be subdivided into several subpopulations .
more mature b - cell - lineage - biased progenitors within this heterogeneous population are also termed pre - pro - b cells and are characterized by progressive loss of t  cell potential coincident with expression of the surface proteins b220 and/or ly6d ( inlay et  al . , 2009 ; rumfelt et  al . , 2006 ) .
other researchers have employed a 5 transgene to mark b - cell - lineage - biased precursors in these pools ( mansson et  al . , 2008 ) .
given the rarity of these cells ( less than 0.2% of all bm cells ) and the diverse approaches used to resolve these populations , we developed a flow cytometric strategy based on differential surface expression of b220 and ly6d on lymphoid - biased progenitors defined previously as lineage(lin)cd19il-7rflt3sca1c - kit ( allman et  al . , 2003 ) . with this approach we resolved three populations of il-7rflt3sca1c - kit cells defined as b220ly6d , b220ly6d , and
b220ly6d in both wild - type and ebf1 mice ( figure  1a and figure  s1a available online ) .
coexpression of b220 and ly6d correlated with increased ebf1 , pax5 , and rag1 expression ( figure  s1b ) , and when sorted from wild - type mice , the b220ly6d subset possessed fewer cells with t  cell lineage potential ( figure  1b ; inlay et  al .
notably , however , although ebf1 b220ly6d precursors were not substantially altered in number or phenotype compared to their wild - type counterparts ( figure  1a ) , they exhibited increased t  cell lineage potential in both bulk and single - cell cultures ( figures 1b and 1c ) .
these data extend past analyses ( zandi et  al . , 2008 ) by showing that ebf1 constrains t  cell potential as b - cell - lineage - biased b220ly6d progenitors give rise to b - cell - lineage - restricted precursors . to test directly
whether ebf1 prevents very early t  cell differentiation independently of pax5 , we sorted bm progenitors from pax5 fetal liver , transduced these cells with control or ebf1-expressing retrovirus , and then resorted transduced ( gfp ) cells onto op9-dl4 stromal cells .
as shown ( figure  2a ) , despite stimulation with dl4 , upon enforced ebf1 expression , pax5 progenitors generated substantially fewer t lineage cells compared to controls . enforcing ebf1 expression also prevented t  cell development from ebf1 and pax5 lsks transduced with an active allele for notch1 ( icn1 ) ( figures 2b and s2 ) .
likewise , ebf1 prevented t  cell differentiation from progenitors transduced with the notch1 target tcf7 ( encoding tcf1 ) ( figure  2c ; weber et  al . , 2011 ) .
these data indicate that ebf1 can override t - cell - lineage - promoting signals mediated by the notch1-tcf1 pathway and further support the notion that ebf1 limits cell - intrinsic t  cell potential in lymphoid precursors independently of pax5 . to test whether ebf1 represses
the expression of essential t  cell lineage genes , we transduced an ebf1 progenitor cell line corresponding to pre - pro - b cells with ebf1 ( pongubala et  al . , 2008 ) and analyzed changes in gene expression on affymetrix microarrays . in these experiments ,
transcripts for gata3 declined reproducibly , whereas notch1 and tcf7 transcript levels as well as the t  cell lineage regulator bcl11b were not altered or increased slightly ( figure  s3a ) .
we repeated these analyses but with quantitative rt - pcr and locus - specific probes in ebf1 progenitors transduced with virus encoding ebf1 and/or pax5 .
transduction with ebf1 or pax5 led to the expected upregulation of transcripts for igll1 and vpreb1 encoding components of the pre - bcr , and cotransduction with ebf1 and pax5 resulted in a synergistic increase in abundance of these transcripts ( figure  3a ) .
ebf1 transduction did not perturb tcf7 transcription substantially and appeared to drive a modest increase in notch1 expression that was overridden by coexpression of pax5 . in sharp contrast , ebf1 transduction consistently induced an 80% decline in gata3 transcript abundance ( figure  3a , top left ) .
pax5 transduction , by comparison , led to only a modest reduction in gata3 transcripts , and cells cotransduced with ebf1 and pax5 mirrored transduction with ebf1 alone .
introduction of ebf1 into a pax5 pro - b cell line ( schaniel et  al . , 2002 ) also repeatedly resulted in a 50% decline in gata3 transcripts ( figure  3b ) , and ebf1 expression continued to promote the downregulation of gata3 transcripts in cells cotransduced with icn1 or tcf1 ( figure  3c ) .
next , we analyzed gata3 , notch1 , and tcf7 transcript abundance in wild - type and ebf1 lincd19il-7rflt3sca1c - kitb220 bm lymphoid progenitors subdivided based on differential ly6d surface expression ( see figure  1 ) .
remarkably , wild - type b220ly6d progenitors exhibited a 7- to 10-fold decrease in gata3 transcripts compared to cells within the less mature b220ly6d fraction ( figure  3d ) , indicating that decreased gata3 expression correlates with loss of t  cell lineage potential .
furthermore , ebf1 b220ly6d precursors exhibited a robust increase in gata3 transcripts compared to their wild - type counterparts to levels found in wild - type b220ly6d progenitors .
in contrast , we observed only a modest increase in notch1 , tcf7 transcripts in b220ly6d precursors lacking ebf1 .
as expected , expression of the canonical ebf1 target igll1 was severely decreased in ebf1 b220ly6d progenitors ( figure  3d , right - most panel ) .
these data , together with the data in figure  1 , show that deletion of ebf1 results in increased gata3 transcripts and a corresponding increase in t  cell lineage potential in b - cell - lineage - biased lymphoid progenitors , implicating ebf1-mediated repression of gata3 as an important event in early b cell development .
given that early b cell development involves collaborative interactions between ebf1 and the e12 and e47 transcription factors encoded by the tcf3 gene ( lin et  al .
, 2010 ; sigvardsson et  al . , 1997 ) , we tested whether ebf1-driven downregulation of gata3 mrna expression required tcf3-encoded proteins .
for these experiments we employed 4-hydroxytamoxifen ( 4-oht)-responsive fusion proteins consisting of either the e47 or ebf1 coding regions fused to a mutated ligand - binding domain of estrogen receptor- ( referred to as e47:er or ebf1:er , respectively ) ( kikuchi et  al . , 2008 ; xu and kee , 2007 ) .
these constructs  were introduced into tcf3 or ebf1 progenitors and routinely resulted in nearly 100% gfp cells ( not shown ) .
notably , whereas induction of e47:er in tcf3 progenitors led to only a 2-fold decrease in gata3 transcripts 12  hr later ( figure  s3b ) , induction of ebf1:er expression resulted in a 5-fold decrease in gata3 mrna within 12  hr , similar to experiments with ebf1 progenitors ( figure  s3c ) .
moreover , after only 4  hr , induction of ebf1:er in tcf3 progenitors led to a significant loss in gata3 transcript abundance ( figure  s3c , left ) .
as expected , in this system induced ebf1:er caused robust increases in igll1 transcripts ( figure  s3c , right ) .
these data indicate that silencing of gata3 expression by ebf1 does not strictly require e2a proteins .
first , we tested whether inhibition of protein synthesis with cycloheximide ( chx ) prevents ebf1-driven decreases in gata3 transcripts .
gata3 downregulation still occurred when transduced tcf3 or ebf1 progenitors were preincubated with chx for 8  hr before adding 4-oht ( figure  s3d ) .
likewise , increases in igll1 transcripts were also intact in chx - pretreated cells ( figure  s3e ) . to test further the possibility that ebf1 mediates silencing of gata3 indirectly by activating an unknown transcriptional repressor , we determined whether conversion of ebf1 into an obligate repressor prevented ebf1-mediated downregulation of gata3 . to this end
we fused the dna binding domain of ebf1 to the  drosophila engrailed repression domain ( fang et  al .
, 2007 ; vickers and sharrocks , 2002 ) . the resulting ebf1-engrailed protein readily repressed transcription of igll1 when introduced into ebf1 pre - pro - b cells ( figure  4a , left )
however , ebf1-engrailed continued to decrease transcript abundance for gata3 similar to wild - type ebf1 ( figure  4a , right ) . together
we utilized the ecr - browser database ( http://ecrbrowser.dcode.org ) to determine whether the gata3 locus contains conserved ebf1 binding sequences .
we noted two such sites within the gata3 locus , one upstream of the gata3 promoter 1b (  site a , 
2,350 bases from the transcriptional start site 1b ) and a second site within the second intron (  site b ,  3,290 bases downstream of transcriptional start site 1b ) ( figure  s4a ) .
notably , past data indicate that the intronic regions of gata3 play fundamental roles in regulating its expression ( hwang et  al . , 2002 ) .
we performed chip experiments with ebf1 antibodies and pcr primers flanking either putative ebf1 binding site ( table s1 ) .
target populations included cd19 bm b lineage cells , the ebf1 progenitor cell line , and wild - type cd3 splenic t  cells .
we observed a 4- to 6-fold enrichment in ebf1 binding at each site in cd19 bm cells but not ebf1 progenitors or peripheral t  cells ( figure  4b ) .
we also failed to detect ebf1 binding from a gene desert region on mouse chromosome 6 in ebf1 progenitors and cd19 bm cells ( not shown ) .
in addition , a 24 base pair biotin - labeled dna probe encompassing  site a  led to formation of a protein - dna complex in electrophoretic mobility shift assays using nuclear extracts from 293 t cells transfected with a his6-tagged version of ebf1 , which was super - shifted by an anti - histidine antibody .
this complex failed to form when we used a probe in which the putative ebf1 site was mutated and was reduced substantially upon inclusion of an unlabeled wild - type cd79a probe containing a canonical ebf1 binding site ( figure  s4b ) .
these data further support a model whereby ebf1 mediates the direct repression of gata3 transcription . to probe the mechanism underlying repression of gata3
, we tested whether ebf1 promotes epigenetic changes proximal to ebf1 binding sites associated with transcriptional repression .
first , we explored the possibility that ebf1 promotes dna methylation of cpg residues . though the gata3 promoter and second intron both contain prominent cpg islands adjacent to the ebf1 binding sites ( attema et  al . , 2007 )
, by sodium bisulfite sequencing we did not detect changes in the methylation status of these regions in multipotent lymphoid progenitors versus pro - b cells ( not shown ) . because gene silencing can be mediated by histone modifications independently of dna methylation ( kondo et  al . , 2008 ) , we also evaluated repression - associated histone - methylation signatures upon ectopic ebf1 expression .
we performed chip experiments with a h3k27me3-specific antibody , because stable transcriptional silencing is tightly associated with trimethylation of h3k27 ( table s1 ; barski et  al . , 2007 ) .
cd19 bm cells were highly enriched for h3k27me3 marks in the gata3 locus , although this was not the case for ebf1 progenitors ( figure  4c ) .
again , the gene desert region on chromosome 6 was included as an additional negative control ( data not shown ) .
furthermore , transduction of ebf1 progenitors with ebf1 readily induced the acquisition of h3k27me3 marks on chromatin surrounding both ebf1 binding sites ( figure  4d ) .
these data suggest that  ebf1 orchestrates epigenetic changes at the gata3 locus by directly or indirectly recruiting polycomb ( pcg ) group complexes that impart repressive h3k27me3 modifications to this region . we sought to test whether ectopic expression of progenitors with gata3 would bypass ebf1-mediated suppression of t  cell differentiation . to this end
we cotransduced ebf1 progenitors with ebf1 and gata3 before sorting these cells on op9-dl4 cultures .
however , consistent with past work indicating that transduction of lymphoid progenitors with gata3 readily induces apoptosis ( taghon et  al . , 2007 ) , there was a progressive decline in viable gata3-positive cells after transduction ( figure  s5a ) .
we also considered generating transgenic mice as an alternative approach to achieving increased gata3 expression .
however , given that gata3 expression is regulated by distal enhancer elements ( hosoya - ohmura et  al .
consequently , we tested whether blocking ebf1 binding to gata3 regulatory sites prevented ebf1-driven suppression of early t  cell differentiation .
indeed , we reasoned that a failure of ebf1 to bind to and repress the gata3 locus should result in increased gata3 expression , thereby providing an alternative approach for increasing gata3 expression in lymphoid progenitors . to this end , we designed a retroviral construct ( pmx-6zfp ) to express a synthetic hexa - modular zinc - finger protein ( 6zfp ) specific for an 18 base region including the last seven nucleotides of the 8 base core ebf1 binding site between exons 1a and 1b (  site a  ) of the gata3 locus ( figure  s5b ) .
we were unable to engineer a zn - finger protein to block binding of ebf1 to  site b  because of inadequate target site overlap requirements ( see experimental procedures ) .
the individual modular domains of 6zfp were linked via a polydactyl zinc finger assembly strategy for recognizing extended sequences ( gonzalez et  al . , 2010 ) .
a vp64 transcriptional activation domain was included to counter any potential stalling effect of 6zfp on endogenous transcription ( beerli et  al . , 2000 ) , and a hemagglutinin ( ha ) tag was added for assessing 6zfp binding to particular dna segments .
control constructs included retroviruses lacking a polypeptide encoding insert ( pmx ) and a second construct encoding an unrelated nonfunctional peptide ( pmx - ss ) .
significantly , after transduction of wild - type lsks with pmx-6zfp , the 6zfp protein readily occupied 
site a  as predicted , but we were unable to detect occupancy of 6zfp at  site b  or at canonical ebf1 binding sites within the cd79a or igll1 loci ( figure  5a ) .
in contrast , pmx - transduced cells failed to exhibit 6zfp enrichment at any of these sites .
pax5 progenitors were transduced with yfp - marked ebf1 virus and/or one of the aforementioned pmx constructs and then  cultured on op9-dl4 stromal cells for 7  days . as shown ( figure  5b ) , whereas ebf1 expression alone led to the expected reduction in thy1.2cd25 t lineage cells , cotransduction with ebf1 and pmx-6zfp but not with ebf1 and either control pmx constructs restored numbers of t lineage cells to 50% of that observed in cells transduced with pmx-6zfp alone ( pmx - ss data not shown ) .
as expected , 6zfp expression led to decreased enrichment for ebf1 between exons 1a and 1b without affecting ebf1 binding between exons 2 and 3 ( figure  5c ) .
moreover , cotransduction with pmx-6zfp and ebf1 restored gata3 mrna levels to 50% of ebf1-transduced cells ( figure  5d ) , and pmx-6zfp expression alone increased gata3 mrna levels 2-fold compared to cells transduced with pmx ( not shown ) .
notably , the increased levels of gata3 induced with 6zfp expression correlated with decreased differentiation of b220cd19 b lineage cells from wild - type fetal liver cells cotransduced with ebf1 ( figure  6a ) , suggesting that elevated gata3 mrna levels , in part due to reduced ebf1 occupancy within  site a ,  perturbed early b cell development . to test this hypothesis directly ,
we introduced pmx or pmx-6zfp into bm lsks  and clps sorted from either vav transgenics or vavgata3 mice and then sorted gfp cells from these cultures onto op9 stromal cells . strikingly , although 6zfp prevented b  cell differentiation when introduced into gata3-competent lymphoid progenitors , early b cell differentiation was restored upon deletion of gata3 ( figure  6b ) .
altogether these findings indicate that direct ebf1-mediated repression of gata3 transcription is critical for extinguishing t  cell lineage  choice and allowing lymphoid progenitors to adopt the b cell fate . finally , to probe whether ebf1 can repress gata3 in t - lineage - committed cells , we assessed whether survival of gata3-dependent tcf3 1.f9 thymoma cells ( xu and kee , 2007 ) is perturbed upon introducing ebf1 .
transduction of 1.f9 cells with ebf1 led to a 5-fold reduction of gata3 transcript abundance in these cells ( figure  s6a ) . whereas control virus did not affect viability over 8  days , introducing ebf1 decreased cell viability to less than 20% within 4  days ( figure  s6b ) .
consistent with past data ( xu and kee , 2007 ) , loss of viability correlated with accumulation of the active form of proapoptotic caspase 3 ( figures s6c ) . moreover , both cell death and active caspase 3 generation were reversed upon enforced coexpression of ebf1 and gata3 in 1.f9 cells ( figures s6d and s6e ) .
these data further support a model whereby ebf1 constrains t  cell differentiation by silencing gata3 expression and further suggest that ebf1 may limit t  cell development by perturbing gata3-dependent survival mechanisms .
our results provide insights into the transcriptional circuitry responsible for generating early b - cell - lineage - restricted precursors . whereas deletion of ebf1 in b - cell - lineage - biased lymphoid progenitors catalyzed an increase in gata3 transcript abundance concurrent with a rejuvenation in their t  cell potential , enforcing ebf1 repressed t  cell differentiation and gata3 mrna levels , even in pax5-deficient progenitors and despite ectopic notch1 and tcf1 activity .
perhaps most notably , perturbation of ebf1 binding at a gata3 regulatory region interfered with ebf1-driven suppression of early t  cell differentiation , concomitantly preventing b cell differentiation due to increased gata3 expression .
ebf1 is an indispensible component of the transcriptional regulatory framework required for generating pro - b cells . within this network of transcription factors ,
e2a proteins activate the expression of ebf1 and foxo1 , and subsequently ebf1 works in concert with e12 and e47 and foxo1 to promote pax5 expression ( lin et  al . , 2010 ) .
although these previous studies demonstrate synergism between these transcription factors to activate certain key b - cell - specific molecules , they also raise questions about how specific components of this network might collaborate to constrain alternative fates . because pax5 is essential for establishing and maintaining b cell identity ( cobaleda et  al . , 2007 ;
2002 ) , one view would be that b cell lineage restriction is mediated chiefly and perhaps exclusively by pax5 .
however , our results highlight the ability of ebf1 to suppress t  cell differentiation of pax5-deficient progenitors without an immediate decline in notch1 transcripts . furthermore , unlike many genes that are upregulated by ebf1 , our data suggest that ebf1 represses gata3 transcription without input from e2a proteins . therefore , we propose that b cell lineage restriction is controlled by two or more separate transcriptional pathways , with pax5 and ebf1 repressing notch1 and gata3 expression , respectively . moreover , in light of past data indicating that e2a proteins are unique among b cell regulatory transcription factors in their ability to repress erythroid differentiation ( ikawa et  al . , 2004 ) , b cell lineage commitment may be facilitated  by a variety of transcriptional and temporally distinct mechanisms . in this model , e2a proteins would silence erythroid and perhaps myeloid potentials as lymphoid - biased progenitors develop , ebf1 would silence t  cell potential as well as residual myeloid potentials during adoption of the b cell fate , and pax5 would silence alternative fates after the generation of pro - b cells .
this viewpoint is consistent with recent data suggesting that suppression of alternative fates during t  cell commitment also requires the activity of multiple transcriptional pathways ( zhang et  al . , 2012 ) .
further work , including an evaluation of the potential role played by ebf1 in maintaining mature b cell identity and the role of other transcriptional regulators and cofactors in this process , may shed additional light on whether maintenance of the b cell fate also requires input from multiple transcription factors .
although the role of gata3 in peripheral t  cell differentiation has been studied intensively ( amsen et  al . , 2007 ; fang et  al . , 2007
) , recent studies together with our findings emphasize the essential role played by gata3 in very early stages of t  cell development . in the thymus ,
notch1 signaling in early t lineage cells induces the expression of additional regulators including hes1 and tcf7 . in turn
, tcf1 promotes the expression of numerous genes required for t  cell development and function ( weber et  al . , 2011 ) , but tcf7-deficient mice continue to generate t  cells , albeit with decreased numbers ( schilham et  al . , 1998 ) .
by contrast , gata3-deficient progenitors fail to generate t lineage cells beyond the etp stage of early thymocyte development ( hendriks et  al .
indeed , recent data suggest that gata3 activity may be essential for optimal notch1 expression in etps , as shown by the fact that gata3 binds to the notch1 promoter in early thymocytes and that deletion of gata3 in thymocytes reduces notch1 transcripts ( wei et  al . , 2011 ) .
moreover , gata3 occupies distinct sets of regulatory regions at different stages of thymocyte development ( zhang et  al . , 2012 ) , suggesting that gata3 regulates many diverse aspects of t  cell development downstream of notch1 activation .
the latter viewpoint is supported by our data showing that ebf1 prevents t  cell differentiation despite ectopic notch1 activity or enforced tcf1 expression .
altogether , these collective observations indicate that gata3 constitutes a prime target for regulatory pathways that silence the t  cell fate in early b lineage cells .
the mechanisms governing transcriptional activation of ebf1 target genes have been studied intensively ( hagman et  al . , 1995 ; maier et  al .
by contrast , how ebf1 represses transcription of certain loci is only beginning to be addressed ( gao et  al . , 2009 ) .
recent analyses of ebf1 occupancy of genes across the b cell genome suggest that the vast majority of ebf1 targets are located in transcriptionally active regions ( treiber et  al . , 2010 ) .
therefore , the default mode for ebf1 may be as a transcriptional activator . however , other ebf1 target loci were found in transcriptionally inactive regions , and ebf1 has also been shown to downregulate the expression of the e - protein regulators id2 and id3 ( thal et  al . , 2009 ) .
therefore , ebf1 may repress additional important loci during establishment and maintenance of the b cell fate .
the use of synthetic zn fingers such as our 6zfp construct to evaluate the role of defined cis elements may prove useful in studying other region - specific protein - dna interactions in a wide variety of biological and experimental contexts .
however , interpretation of data derived from this approach could be confounded by off - target dna binding events .
here , 6zfp did not appear to bind to alternative ebf1 binding sites within the gata3 , cd79a , or igll1 locus and led to the predicted outcomes in gata3 mrna levels and t and b cell differentiation .
we considered performing chip - seq experiments to determine the identity of these alternative elements , but we suspect strongly that the functional relevance of these sites would be obscure at best .
indeed , recent genome - wide analyses of dna occupancy by ebf1 suggest that endogenous transcription factors may also bind to a wide range of elements with unknown functional relevance ( treiber et  al . , 2010 ) .
therefore , although synthetic proteins containing six tandem zn fingers such as 6zfp may prove to possess minimal off - target effects ( gabriel et  al . , 2011 ) , validating the specificity of such proteins with a particular biological outcome may require the use of cells bearing appropriate mutations .
finally , although the precise molecular details remain unclear , we hypothesize that the switch between activator and repressor functions of ebf1 at different loci reflects differential spatio - temporal interactions of ebf1 with coactivator and corepressor complexes , which in turn would be dictated by diverse proximal and/or distal cis elements .
our results show that ebf1 binding within the gata3 locus is associated with repressive h3k27me3 marks on nearby chromatin .
these results implicate ebf1 in the active recruitment of polycomb group ( pcg ) repressor complexes .
currently the chief mediator of h3k27me3 modifications in eukaryotes is the protein enhancer of zeste 2 ( ezh2 ) , a member of the polycomb repressor complex 2 ( prc2 ) ( cao and zhang , 2004 ) .
prc2 activity in turn attracts prc1 , which prevents transcriptional initiation by rna polymerase ii ( dellino et  al . , 2004 ) .
therefore , it is tempting to suggest that ebf1 binding at the gata3 locus directly or indirectly initiates mobilization of prc2 complexes to this region , thereby blocking interactions with coactivators and stabilizing chromatin modifications associated with long - term transcriptional silencing . assuming that this sequence of events is indeed contingent on sequence - specific corepressors , competitive blockade of their cognate dna sites via our zn - finger protein strategy might neutralize repressive effects of ebf1 at the gata3 locus .
furthermore , given that histone modification is a dynamic and reversible process ( tagoh et  al .
, 2004 ; zhang et  al . , 2012 ) , it will be informative to test whether induced deletion of ebf1 in b - lineage - committed cells causes the reversal of trimethylation on h3k27 residues in gata3 regulatory regions , possibly leading to gata3 expression .
the consequences of such aberrant expression would give us further clues into the biological significance of gata3 silencing in b cells .
c57bl/6 ( b6 ) and b6.ly5 females ( 68  weeks of age ) were from the nci animal facility ( frederick , md ) .
ebf1 mice were kindly provided by b. kee ( university of chicago ) with permission from r. grosschedl ( max planck institute , freiburg ) .
pax5 mice were kindly provided by m. busslinger ( research institute of molecular pathology , vienna ) .
gata3 and vav mice were kindly provided by j. zhu ( national institutes of health ) and d.  kioussi ( national institute for medical research , london ) , respectively .
all animal experiments were performed according to protocols approved by the office of regulatory affairs of the university of pennsylvania in accordance with guidelines set forth by nih and by the local ethics committee at the university of oxford and the united kingdom home office .
primary progenitors and progenitor cell lines were cultured as described previously ( pongubala et  al . , 2008 ; xu and kee , 2007 ) .
for expression of synthetic zn - finger polypeptides , the predicted 6zfp mini - gene sequence was synthesized by blue heron biotech , cloned into the control pmx - vp64-ires - gfp retroviral construct ( c. barbas , scripps research institute ) , and renamed pmx-6zfp .
a plasmid eliciting translation of an unrelated nonfunctional peptide ( pmx - ss ) was also provided by the barbas lab . a nuclear localization signal sequence ( nls ) and hemagglutinin ( ha ) tag are also included in the pmx vector backbone .
stained single - cell suspensions were stained and analyzed as described previously ( pongubala et  al . , 2008 ) .
b6.ly5 hosts were irradiated ( 900r ) 6  hr before intravenous transfer of day 14.5 fetal liver cells isolated from b6 or pax5 or ebf1 embryos .
bulk and clonal assays measuring t  cell differentiation were performed with op9-dl4 stromal cells as described previously ( weber et  al . , 2011 ) and in the supplemental information .
high - titer virus was generated by a capo4 transfection protocol ( pear et  al . , 1993 ) .
cultures were supplemented with fresh medium at 12  hr after infection and harvested for downstream applications at indicated time points after infection . for quantitative rt - pcr
, rna was purified from indicated cell types with the qiagen rneasy mini kit and reverse transcribed to cdna with ge first - strand cdna synthesis kit .
real - time pcr was performed with inventoried taqman probes for indicated genes and analyzed on an abi prism 7300 system ( applied biosystems ) .
cd3 thymocytes and cd19 bm cells were column selected ( miltenyi biotec ) . in case of control vector and ebf1-transduced ebf1 cells or cells cotransduced with
ebf1 and pmx / pmx-6zfp / pmx - ss viruses , viable gfp or yfpgfp cells were sorted either 24  hr or 7  days after infection as indicated .
chip was performed on indicated cell types ( 2   10 cells / assay ) with the chip - it kit ( active motif ) . in brief , cells were fixed for 10  min at room temperature with 1% formaldehyde , treated with glycine stop - fix solution , then lysed with dounce - homogenizer .
pelleted nuclei were digested in presence of protease inhibitor cocktail and chromatin was enzymatically sheared .
sheared chromatin was immunoprecipitated with 4  g anti - ebf1 ( a kind gift from r. grosschedl ) or anti - h3k27me3 ( 07 - 449 , millipore ) or anti - ha ( ab9110 , abcam ) .
after proteinase k digestion , dna was immediately used in quantitative real - time pcr ( sybr green , abi ) .
a negative control primer set ( active motif , catalog number 71011 ) for an 82 base pair gene desert region on mouse chromosome 6 was included in each immunoprecipitation .
nonenrichment for ebf1 or anti - h3k27me3 at this site served as a chip specificity control . a 48 base pair ( bp )
sequence spanning the ebf1 binding  site a  ( as confirmed by chip assays ) in gata3 genomic locus was selected . using the publicly available online resource called zinc finger tools ( http://www.scripps.edu/mb/barbas/zfdesign/zfdesignhome.php ) , an 18  bp potential target site including the ebf1 binding site was identified and the amino acid sequence of a hexa - modular zinc finger protein ( 6zfp ) predicted to bind to this site was designed ( table s2 ) .
because of unmet target site overlap requirements , designing 6zfp for a target site covering the intronic ebf1 binding  site b  was avoided as recommended ( mandell and barbas , 2006 ) .
the means of each data set were analyzed by student s t test , with a two - tailed distribution assuming equal sample variance .
 summarythe mechanisms underlying the silencing of alternative fate potentials in very early b cell precursors remain unclear . using gain- and 
 loss - of - function approaches together with a synthetic zinc - finger polypeptide ( 6zfp ) engineered to prevent transcription factor binding to a defined cis element , we show that the transcription factor ebf1 promotes b  cell lineage commitment by directly repressing expression of the t - cell - lineage - requisite gata3 gene . 
 ebf1-deficient lymphoid progenitors exhibited increased t  cell lineage potential and elevated gata3 transcript expression , whereas enforced ebf1 expression inhibited t  cell differentiation and caused rapid loss of gata3 mrna . 
 notably , 6zfp - mediated perturbation of ebf1 binding to a gata3 regulatory region restored gata3 expression , abrogated ebf1-driven suppression of t  cell differentiation , and prevented b cell differentiation via a gata3-dependent mechanism . 
 furthermore , ebf1 binding to gata3 regulatory sites induced repressive histone modifications across this region . 
 these data identify  a transcriptional circuit critical for b cell lineage commitment . 
Introduction
Results
Discussion
Experimental Procedures