Here, we found that there is substantial cell specificity in both GATA3 binding and gene regulation by GATA3 even among the closely related T cell lineages. GATA3 regulates distinct key factors in the thymus and during T helper cell differentiation. Our data indicated that GATA3 facilitates both H3K4me1 and H3K4me2, and H3K27me3 at enhancers to mediate gene activation and repression, respectively.
Among the most striking aspects of our results is the very large number of genes to which GATA3 binds and the fact that it activates some, represses others and appears to have little or no effect on the level of expression of the rest. This suggests that GATA3 acts in a more subtle manner than simply as a “switch” to turn on the Th2 cell phenotype. Instead, it may control the activation of many genes that are critical for Th2 cell function while simultaneously repressing genes that might inhibit Th2 cell function or Th2 cell differentiation.
One challenge is to understand the importance of GATA3 binding to sites at which it does not appear to alter the expression of the bound genes. One possibility is that the local binding of GATA3 may regulate other genes through long-distance chromatin interaction, such as Th2 cytokine locus control region in the Rad50 gene. A second possibility is that these genes are regulated under the various conditions that the Th cells may encounter in vivo but which are not operative in vitro. A third possibility is that GATA3 binding does not directly activate or repress transcription of its target genes; instead, it prepares chromatin so that other factors that collaborate with GATA3 can bind and regulate transcription when they become available. Thus, the effects of deleting Gata3 could not be detected in the absence of these collaborating factors.
Our data indicate that the function of GATA3 exhibits two levels of specificity in different cells: one level at its binding site selection and the second level at its functional regulation of bound genes. At the binding level, GATA3 target genes demonstrate some lineage specificities even among the closely related T helper cell lineages. The lineage specificity of GATA3 binding in different cells cannot be attributed solely to differences in GATA3 expression and number of binding sites among these different cell types.
Since GATA3 is a sequence-specific DNA-binding protein, its cell type-specific binding suggests that GATA3 target recognition is regulated by other mechanisms in addition to the GATA3 sequence motif. Indeed, our motif analysis revealed that in addition to a primary WGATAA motif, GATA3 binding sites contained various secondary motifs including Ets, Runx, AP1, TCF11 and AREB6 motifs or only secondary motifs but lacked the primary WGATAA motif. There are several potential mechanisms that secondary motif-recognizing factors may be used to influence GATA3 function: (1) GATA3 physically interacts with another factor, which stabilizes the binding of both factors or destabilizes the binding of one factor; (2) GATA3 binding facilitates H3K4 methylation and creates a chromatin environment such that another factor can bind; (3) GATA3 binds to a site in open chromatin that is maintained by another factor; (4) GATA3 facilitates H3K27me3 modification at target region to inhibit binding or to suppress the activity of another factor. These non-exclusive ways of action may explain how GATA3 binding can be associated with both gene activation and repression. The functional collaboration between different transcription factors is not unique for GATA3 but has been found with other key regulators, for example, PU.1 in macrophage and EBF1 in B cells (Ghisletti et al., 2010
; Treiber et al., 2010
Lineage-specific cytokine and transcription factor genes are associated with distinct histone modification patterns in different T cell lineages (Ansel et al., 2006
; Hatton et al., 2006
; Roh et al., 2005
; Schoenborn et al., 2007
; Wei et al., 2009
). Active histone modifications at the Th2 cytokine locus depend on key transcription factors such as STAT6 and GATA3 (Yamashita et al., 2002
), suggesting that these factors may regulate transcription by modulating the chromatin structure of their target genes. Our genome-wide analyses indicated that deletion of Gata3
decreased H3K4me1 and H3K4me2 modifications at GATA3 binding sites associated with the genes that were positively regulated by GATA3. By contrast, deletion of Gata3
resulted in a decrease in H3K27me3 at the negatively regulated genes. We found that in Th2 cells, a substantial proportion of GATA3-bound genes had changed their epigenetic modifications but not their gene expression when Gata3
was deleted. These results indicate that the epigenetic changes are directly regulated by GATA3 binding and are not a consequence of transcriptional activation or repression. The modest changes in the histone modifications at GATA3 binding sites upon deletion of Gata3
suggest that other factors co-bound with GATA3 may play redundant roles in recruiting histone methyltransferases.
Histone modification may influence transcription by attracting different transcription co-factors. H3K4 methylation can recruit co-activators, e.g., the ATP-dependent chromatin remodeling complex, NURF (Wysocka et al., 2006
) and HATs (Wang et al., 2009
), whereas H3K27me3 signals can be recognized by the PRC1 polycomb repressor complex (Fischle et al., 2003
), which mediates transcriptional repression in Drosophila and humans. Therefore, our data support the model that GATA3 binding creates a chromatin environment that can make target sites of other transcription factors accessible or can be recognized by co-activators or co-repressors of transcription although it is also possible that in some cases, GATA3 may activate or repress transcription directly.
In summary, we have characterized GATA3 binding in many distinct T lymphocytes and analyzed GATA3-mediated gene regulation at a genome-wide level. Our dataset provides valuable information for further characterization of GATA3 target genes and cis-regulatory elements that GATA3 binds to as well as proteins that collaborate with GATA3 to regulate the differentiation and function of various T cell lineages.