Previously, we found that chromatin remodeling was required for Th2 cytokine gene expression. We found that transcription factors recruited the SWI/SNF remodeling enzyme BRG1 to specific sites in Th2 cells. BRG1 was required before and after Th2 differentiation. We found the ISWI remodeling enzyme SNF2H could activate and repress gene expression in T cells. Progress: T helper cell differentiation and activation require specific transcriptional programs accompanied by changes in chromatin structure. However, little is known about the chromatin remodeling enzymes responsible. We performed genome-wide analysis to determine the general principles of BRG1 binding, followed by analysis of specific genes to determine whether these general rules were typical of key T cell genes. BRG1 binding was to localized, enhancer-sized regions, a finding subsequently confirmed by two other labs. We found that binding of the remodeling protein BRG1 was programmed by both lineage and activation signals. BRG1 binding positively correlated with gene activity at protein-coding and microRNA (miRNA) genes. BRG1 binding was found at promoters and distal regions, including both novel and previously validated distal regulatory elements. Distal BRG1 binding correlated with expression, and novel distal sites in the Gata3 locus possessed enhancer-like activity, suggesting a general role for BRG1 in long-distance gene regulation. One of these regions was tested by an independent lab, and confirmed. BRG1 recruitment to distal sites in Gata3 was impaired in cells lacking STAT6, a transcription factor that regulates lineage-specific genes. Together, these findings suggest that BRG1 interprets both differentiation and activation signals and plays a causal role in gene regulation, chromatin structure, and cell fate. Our findings suggest that BRG1 binding is a useful marker for identifying active cis-regulatory regions in protein-coding and miRNA genes. We investigated gene regulation at the IL-3/GM-CSF gene cluster. We found BRG1, a SWI/SNF remodeling ATPase, bound a novel distal element, CNSa. BRG1 binding was strongest in differentiated, stimulated T helper cells, paralleling IL-3 and GM-CSF expression. Depletion of BRG1 reduced IL-3 and GM-CSF transcription. BAF-specific SWI/SNF subunits bound to this locus and regulated IL-3 expression. CNSa was in closed chromatin in fibroblasts, open chromatin in differentiated T helper cells, and moderately open chromatin in nave (undifferentiated) T helper cells;BRG1 was required for the most open state. CNSa increased transcription of a reporter in an episomal expression system, in a BRG1-dependent manner. The NF-kB subunit RelA/p65 bound CNSa in activated T helper cells. Inhibition of NF-kB blocked BRG1 binding to CNSa, chromatin opening at CNSa, and activation of IL-3 and GM-CSF. Together, these findings suggest CNSa is a distal enhancer that binds BRG1 and NF-kB. This publication is in the NCBI pipeline for PMC, and so may not be listed in the bibliography: NF-kappaB and BRG1 bind a distal regulatory element in the IL-3/GM-CSF locus. Wurster AL, Precht P, Pazin MJ. Mol Immunol. 2011 Aug 8. Epub ahead of print PMID: 21831442 This publication (invited review) is in press (to appear online by October 2011), so it cannot listed in the bibliography: ATP-dependent chromatin remodeling in T cells Andrea L. Wurster and Michael J. Pazin Biochemistry and Cell Biology, Manuscript ID 2011-0042 Genome-wide BRG1 binding data in 6 murine T cell subsets (Raw sequence reads, aligned tags BRG1 binding regions and ChIP-seq stats for all T helper subsests) were deposited in GEO, and are publicly available at accession number GSE23719. Comprehensive gene expression data in 4 murine T helper subsets, WT and BAF180 deficient cells, are at GEO (GSE31676) and will be publicly released upon publication of the BAF180 described work below. Our results provide new information on the regulation of T cell fate and function by chromatin remodeling enzymes. This work could be important in understanding the biological basis of, and identifying therapeutic targets for, autoimmune disorders, allergy and atopy, and vaccine response. Future: We will publish our study of BAF180, a SWI/SNF subunit specific to the PBAF form of the SWI/SNF complex in T helper cells using conditional knockout technology. No further experiments are planned.
