We found that SNF2H repressed expression of cytokine mRNA (IL-2, IL-17A, IL-5, IL-13) in activated cells. By contrast, SNF2H activated expression of IL-3 mRNA. SNF2H bound to the tested loci in both the EL4 cell line and primary T cells, suggesting the regulation was direct. SNF2H decreased accessibility at some binding sites within the IL2 locus, and increased accessibility within some IL3 binding sites. We also found that loss of the ISWI ATPase SNF2H reduced protein expression of ACF1, a binding partner for SNF2H.
The major role for SNF2H on cytokine expression in our study was repression. This is in contrast to our previous work identifying BRG1 as an activator of expression of Th2 genes (Wurster and Pazin, 2008
). SNF2H is an ATPase in the ISWI subfamily, while BRG1 is in the SWI/SNF subfamily. SWI/SNF family members have been more frequently linked to gene activation, though repression has also been found. ISWI family members are more frequently linked to repression, though there is also evidence for a role in gene activation. We found little if any change in SNF2H binding upon stimulation of cells, by contrast, we previously found BRG1 binding to target loci could change rapidly upon stimulation (Wurster and Pazin, 2008
). We found that SNF2H binding was regulated during development and T helper differentiation. Thus, the SNF2H-mediated chromatin accessibility correlated with gene expression, while the SNF2H binding pattern did not. Consistent with these binding kinetics, SNF2H exerts effects on both the basal and activated chromatin structure of target genes. We found SNF2H binding at distal regions as well as promoter regions, similar to our previous findings with BRG1.
Unexpectedly, we found that SNF2H has opposing effects on cytokine gene expression; SNF2H repressed most of the tested genes, but activated one. These changes in activity correlated with accessibility, suggesting a simple model that accessibility enables transcription. We do not know why SNF2H has opposite effects on the chromatin structure of these loci. Examination of the extent of histone acetylation or histone methylation did not reveal a simple classification of the activation and repression targets.
One hypothesis for the opposing effects of SNF2H is that the SNF2H partners could be different at these loci. SNF2H is known to bind several proteins, including ACF1, BPTF, WSTF, BAZ2A, BAZ2B. Perhaps the identity of the binding partner is different at loci where SNF2H is a coactivator, leading to a different remodeling result. We do detect more ACF1 at CNSa than IL-2 -1.8k and promoter. We were unable to reliably measure BPTF at any of the tested loci. Another hypothesis is that the relative amounts of other remodeling enzymes determines the outcome. CNSa has more BRG1 binding than do the IL2 loci. We have not measured NuRD complexes containing Mi2/CHD3/CHD4 ATPases in this study.
These findings reveal that SNF2H, a chromatin remodeling ATPase not previously examined in lymphocytes, can regulate cytokine gene expression in a direct manner. SNF2H is essential for early development in mouse (Stopka and Skoultchi, 2003
), while the fly homolog is required for early development and cell viability (Deuring et al., 2000
). In budding yeast, flies, frogs and mice, ISWI proteins can repress gene expression (Deuring et al., 2000
; Dirscherl et al., 2005
; Fazzio et al., 2001
; Fyodorov et al., 2004
; Landry et al., 2008
; Sherriff et al., 2007
). However, ISWI proteins have also been linked to gene activation in several species and cell-free biochemical systems (Badenhorst et al., 2002
; Barak et al., 2003
; Ito et al., 1997
; Landry et al., 2008
; LeRoy et al., 1998
; Mizuguchi et al., 1997
). Moreover, an ISWI binding protein has been reported to be recruited by histone H3K4 trimethylation, a mark frequently found at active promoters (Wysocka et al., 2006
). Analysis of other untested remodeling ATPases such as CHD7, which is linked to immunodeficiency (Gennery et al., 2008
), will help us to understand the chromatin remodeling landscape and how it determines gene expression programs in lymphocytes.