NF-κB and BRG1 bind a distal regulatory element in the IL-3/GM-CSF locus

Abstract

We investigated gene regulation at the IL-3/GM-CSF gene cluster. We found BRG1, a SWI/SNF remodeling ATPase, bound a 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 naïve (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-κB subunit RelA/p65 bound CNSa in activated T helper cells. Inhibition of NF-κB 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-κB.

Introduction

GM-CSF and IL-3 are two tightly linked and closely related proinflammatory cytokines that have arisen by a gene duplication event. Both cytokines are involved in the activation and survival of multiple myeloid lineages. Clinically, IL-3 and GM-CSF are used to replenish white blood counts in patients after chemotherapy. However, inappropriately elevated expression of IL-3 and GM-CSF is also associated with inflammatory disorders such as arthritis and psoriasis. The major biological source of GM-CSF and IL-3 appears to be activated, differentiated T cells, with little or no specificity with respect to T cell subsets. IL-3 is also produced by mast cells and eosinophils, while GM-CSF is also produced by monocytes and macrophages.

The transcriptional regulation and corresponding changes in the chromatin structure of the IL-3/GM-CSF gene cluster have been extensively studied (Cockerill, 2004). These are separated by only 13 kb in the mouse genome and located within one megabase of the Th2 cytokine cluster (IL-4, IL-13, and IL-5). The expression of IL-3 and GM-CSF in T cells is highly regulated; expression is limited to differentiated T cells, and requires T cell activation. Several enhancers, both upstream and intergenic, have been identified in the IL-3/GM-CSF locus. Transcription factors associated with T cell activation (NFAT and NF-κB) and otherwise (Sp1 and GATA) bind to and regulate the activity of these elements (Cakouros et al., 2001, Duncliffe et al., 1997, Holloway et al., 2003, Johnson et al., 2004). Changes in nucleosome mobility as detected by the generation of DNase I hypersensitive sites (DHS) following T cell activation, is associated with the promoters and enhancers of the IL-3/GM-CSF locus (Holloway et al., 2003, Johnson et al., 2004). Chromatin reorganization extends across a 3 kb region around the intergenic GM-CSF enhancer region and is associated with gene activity (Bert et al., 2007). Although the tissue specific expression pattern of IL-3 and GM-CSF largely overlap they are not identical, as evidenced by the exclusive expression of GM-CSF in myeloid cells, indicating these genes can be regulated independently (Bert et al., 2007). The recent identification of an insulator element located between IL-3 and GM-CSF may provide a means to segregate the regulatory elements associated with this gene cluster (Bowers et al., 2009).

Although chromatin structure changes within the IL-3/GM-CSF locus have been well documented during both T cells development and after T cell activation, less is known about the enzymes that catalyze these changes. A recent study demonstrated that in early thymocyte development the IL-3/GM-CSF locus exists in an epigenetically silent state as defined both by nuclease accessibility and histone modifications (Mirabella et al., 2010). As the T cells develop into a mature T cells and T cell blasts the cytokine locus acquires a more active chromatin structure marked by DHS, active histone modifications and the expression of inducible noncoding RNA's (Mirabella et al., 2010). BRG1, a remodeling enzyme, has been identified as a regulator functioning at the GM-CSF promoter. However in one study BRG1 recruitment to the promoter was reduced following T cell activation, while in another BRG1 was enriched (Brettingham-Moore et al., 2008, Holloway et al., 2003). A role for BRG1 in remodeling events outside of the proximal promoter regions has not been reported, thought distal BRG1 binding has been reported in a T cell line and primary T cells (Precht et al., 2010). ISWI, another type of remodeling enzyme, has also been found to regulate gene expression in T cells (Landry et al., 2011, Precht et al., 2010). In the T cell line EL4, ISWI activated expression of IL-3, while repressing expression of IL-2, IL-5, IL-13, and IL-17A (Precht et al., 2010). At these loci, remodeling enzyme binding is found at promoters and at distal regions (De et al., 2011, Precht et al., 2010, Wurster and Pazin, 2008).

Changes in chromatin structure are catalyzed by chromatin remodeling enzymes. ATP-dependent remodeling enzymes reposition, unfold, displace and assemble nucleosomes, while other classes of remodeling enzymes covalently modify histone proteins or DNA. ATP-dependent remodeling can directly alter gene expression in cell-free systems and in cells (Saha et al., 2006). Remodeling may alter binding of transcription factors and RNA polymerase, transcription factor and RNA polymerase function, and alter higher-order or long-range chromatin structure. Remodeling enzymes are thought to bind DNA non-specifically, and are recruited by interactions with transcription factors, modified histones and non-coding RNA (Biddie and Hager, 2009, Clapier and Cairns, 2009, Hassan et al., 2001, Ho and Crabtree, 2010, Neely et al., 1999, Tarakhovsky, 2010, Wysocka et al., 2006, Yudkovsky et al., 1999). ATP-dependent remodeling enzymes are often multi-protein complexes, classified by their ATPase subunit into subfamilies such as SWI/SNF, ISWI and Mi2 (Saha et al., 2006).

BRG1 is an ATPase in the SWI/SNF subfamily, and is essential for embryonic development (Bultman et al., 2000). In cell-free systems SWI/SNF enzymes can displace, unfold and slide nucleosomes (Lorch et al., 2006, Schnitzler et al., 1998, Whitehouse et al., 1999). Mammalian SWI/SNF has been found in BAF and PBAF complexes containing a small number of distinct subunits and many common subunits (Lemon et al., 2001, Nie et al., 2000, Yan et al., 2005), as well as complexes specific to ES cells and neurons (Ho et al., 2009, Lessard et al., 2007).

BRG1 has been found to play an important role in T cell development (Chi, 2004). BRG1 also plays an important role in macrophages (Ramirez-Carrozzi et al., 2006) and differentiated T helper cells, including T helper 1 (Th1), T helper 2 (Th2) and T helper 17 cells (De et al., 2011, Letimier et al., 2007, Wurster and Pazin, 2008, Zhang and Boothby, 2006). Genome-wide analysis of BRG1 binding during Th differentiation suggested BRG1 activated many genes in each fate, in response to activation-specific and lineage-specific signals (De et al., 2011). Distal regulatory elements are frequently involved in Th gene regulation, and may be sites for remodeling enzyme function (Agarwal and Rao, 1998a, De et al., 2011, Jones and Flavell, 2005, Lee et al., 2003, Placek et al., 2009, Wurster and Pazin, 2008). Distal regulatory elements may play a widespread, if underappreciated, role in gene regulation, and distal chromatin structure may be better correlated with gene activity than promoter chromatin structure (Heintzman et al., 2009, Visel et al., 2009).

Here, we asked whether the SWI/SNF subunit BRG1 is required for IL-3/GM-CSF gene expression and remodeling of the cytokine locus. We found that knockdown of BRG1 expression in primary effector T cells impaired the expression of both cytokines. BRG1-containing BAF complexes bound to multiple known regulatory elements in the IL-3/GM-CSF cytokine cluster, in an inducible manner; little if any binding was found in naïve cells. Comparative sequence analysis revealed the existence of additional conserved noncoding sequence (CNS) regions 25–40 kb downstream of the cytokine cluster; one in particular, CNSa, binds BRG1 to an especially high degree. We detected changes in chromatin accessibility at CNSa when BRG1 expression was reduced, suggesting a dependence on BRG1 for establishing an active chromatin conformation at this site. Activation-induced recruitment of BRG1 to CNSa appears to depend at least in part on the NFκb pathway. Finally, CNSa appears to possess BRG1-dependent enhancer activity in a chromatin-based reporter assay. BRG1 appears to be an important regulator of chromatin structure and gene expression in IL-3/GM-CSF locus and was a useful marker in the identification of a novel, distal regulatory element.