Herein, we provide a bi-molecular model in which the relative binding of two transcription factors, STAT5 and E2A, to Eκi control the initiation of Igk
germline transcription in pre-B cells. Central to this model is our demonstration that STAT5 binds as a tetramer to Eκi and recruits the PRC2 which contains Ezh2. Ezh2 then marks Eκi, as well as the adjacent Jκ cluster and Cκ, with H3K27me3. While other areas of the Igk
locus are not marked with H3K27me3, loss of Ezh2 is sufficient for escape from IL-7-mediated Igk
repression. This STAT5-mediated repression mechanism ensures that Igk
germline transcription is silenced as long as B cell progenitors are productively receiving proliferative signals through the IL-7R. Upon escape from IL-7R signaling, and the loss of activated STAT5, H3K27me3 is lost. The transition to open chromatin is regulated by pre-BCR-mediated induction of free nuclear E2A, which binds Eκi and promotes acquisition of H3K4me1 and H4Ac. In the absence of both STAT5 and E2A binding, the Eκi was devoid of the assayed histone modifications. These data suggest that STAT5 and E2A binding, and the complexes they recruit, are sufficient to account for transition of the Eκi region from a silenced to an active state46
. This conclusion is consistent with previous studies demonstrating promiscuous Igk
germline transcription in Stat5-/-
early B cell progenitors31
and observations that both E2A24
and the Eκi23
are required for opening chromatin at Igk
The epigenetic status of the Eκi region was controlled by two transcription factors, providing an explanation for the tight correlation between Igk
germline transcription and accessibility to recombination11
. In our genome-wide studies, the H3K27me3 mark conferred by STAT5 and Ezh2 binding correlates with transcriptional repression. It is interesting that STAT5 binding was associated with discrete H3K27me3 peaks over Eκi as well as Jκ and Cκ. This suggests that the chromatin in this region is not linear and that the Jκ and Cκ regions might be positioned over Eκi.
In bulk populations of B cell progenitors, the transition between a repressed and active Igk
locus appeared complete with little evidence of substantial heterogeneity. These data are consistent with recent observations that both Igk
alleles are transcriptionally active before recombination10
. Presumably, the mechanisms described here, which control Igk
transcription and accessibility, do not restrict recombination to one allele (allelic exclusion)36
. Furthermore, the strong co-segregation of the H3K4me1 and H4Ac marks suggest that there is not an intermediate “poised” state for Eκi in the context of Igk
recombination in pre-B cells22,46
Assessment of the relationships between STAT5 and H3K27me3 across the genome of proliferating pro-B cells revealed that STAT5 likely functions as a repressor of other genes. Analysis of H3K27me3 and STAT5 co-incident peaks within putative regulatory regions of genes revealed that all had tandem GAS motifs, and in all, the 3′ GAS was a partial motif. However, overall the most common nucleotides at each position constituted a canonical GAS motif. These data provide in vivo evidence that STAT5 recruitment to tetrameric binding sites in multiple genes is associated with epigenetic repression. Furthermore, the high correlation between STAT5 binding to tetrameric sites and H3K27me3 marks suggests that tetrameric binding to specific DNA sequences may be necessary for epigenetic repression.
The consequences of STAT5 mediated repression may vary depending upon the context in which STAT5 binding occurs. While Igk
transcription is induced upon entry into the pre-B cell pool, many genes binding both STAT5 and H3K27me3 in pro-B and large pre-B cells remained relatively repressed throughout development and peripheral maturation. This may reflect the fact that, in many cases, H3K27me3 is a stable mark as compared to other histone PTMs47
or that STAT5-mediated silencing co-occurs at some sites with other more stable mechanisms of gene repression.
Our data suggest that tetrameric STAT5 recruits Ezh2 which represses a significant subset of genes regulated by STAT5 during B lymphopoiesis. Furthermore, we predict that this mechanism is relevant to T lymphopoiesis as the TCRα enhancer contains potential STAT5 tetrameric sites abutting several conserved DNA binding motifs48
. However, it is unlikely that, in general, the presence of STAT5 tetramers, or of tetrameric binding sites, is sufficient to predict gene repression. This is suggested by observations that STAT5 tetramers can be associated with gene activation and multilineage leukemias43
. Furthermore, recent evidence indicates that STAT5 can also repress transcription by recruiting a histone deacetylase and by competing for binding with STAT349
. These examples indicate that understanding how and when STAT5 represses transcription will require a detailed understanding of how STAT5 recruits PRC2 and other repressive complexes.