Figure 2: ATP-dependent SWI/SNF complexes bind ECRs in the Wt1 locus.

(a) Sequence alignment of the Wt1 locus from mouse and human. Peak heights indicate degree of sequence homology; pink bars above the peaks denote evolutionary conserved regions (ECRs); yellow represents the 5′ untranslated region (UTR); blue indicates exon 1. Boxed regions were selected for further analysis by ChIP. Numbers above boxed regions denote ECR approximate distances upstream (−) or downstream (+) of the transcription start site (TSS). (b–d) ChIP-qPCR data using chromatin derived from embryonic hearts at E11.5 and anti-BRG1, anti-BRM or anti-BAF180 antibodies. Note that no enrichment was detected in non-ECR elements ((−)ECR) confirming ChIP signal specificity. (e) Sequential ChIP (re-ChIP) with an anti-BAF180 antibody using embryonic heart-derived chromatin pulled-down with anti-BRG1. (f,g) ChIP-qPCR data using chromatin derived from adult intact (no MI) hearts and anti-BRG1 or anti-BRM antibodies. (h,i) ChIP-qPCR data using chromatin derived from adult primed- (+Tβ4), injured-hearts at day 4 post-MI and anti-BRG1 or anti-BRM antibodies. (j,k) ChIP-qPCR data using chromatin derived from adult non-primed, injured-hearts at day 4 post-MI and anti-BRG1 or anti-BRM antibodies. (l) Wt1 immunostaining in post-MI adult hearts showing expanded subepicardial space (indicated by double-arrow), but weak re-expression of Wt1 in the epicardium (arrowhead). Three independent experiments per antibody were performed using 20 hearts at E11.5 and three adult hearts per experiment. ChIP results are presented as fold enrichment over input, whereas re-ChIP results are present in fold enrichment over the level of ChIP with negative control IgG antibody. All error bars are data±s.d. Significant differences (P values) were calculated using two-tailed Student’s t-test (*P≤0.05; **P≤0.01). bp, base pairs; kb, kilobases; my, myocardium; ses, subepicardial space. Scale bar 100 μm.