| Target gene experiment description: |
We also searched for putative MIR335 short- and long-range Enhancers:to this end, we combined chromatin state modeling data from ENC ODE ChIP-seq experiments showing enrichment in H3K4me1 and/or H3K27ac, histone modifications found on inactive and active Enhancers, respectively (Fig. S5 A), with ENCODE genome-wide chromosome conformation capture of Hi-C data (Rao et al., 2014). The Hi-C data reveal multiple pairwise interactions of these predicted enhancers with the MIR335 promoter (Fig. S5, BD), providing six MIR335-predicted distal enhancer sites (regions r6r11; Fig. S5 D). Acetylation of H3K27 on MIR335 enhancers detected in ASCLMNA(R482W) after adipogenic induction suggests interac-tions with the MIR335 promoter by chromatin looping (Zhang et al., 2013). We tested this hypothesis by two-color FISH using distinctly labeled promoter and enhancer probes. Probes were generated to cover the MIR335 gene and promoter as well as the distal enhancer element r11 located ?200 kb upstream of MIR335. In both undifferentiated and differentiated ASCs that were either native or expressing WT LMNA, these promoter and enhancer elements displayed a relatively low incidence of colocalization on the majority of alleles (?80%; Fig.?6, C and D). After induction of differentiation, however, this proportion markedly increased to 56% of alleles in the LMNA(R482W) mutants, reflecting interaction of a high proportion of MIR335 promoter and enhancer sites (Fig. 6, C and D).;We also searched for putative MIR335 short- and long-range Enhancers:to this end, we combined chromatin state modeling data from ENC ODE ChIP-seq experiments showing enrichment in H3K4me1 and/or H3K27ac, histone modifications found on inactive and active Enhancers, respectively (Fig. S5 A), with ENCODE genome-wide chromosome conformation capture of Hi-C data (Rao et al., 2014). The Hi-C data reveal multiple pairwise interactions of these predicted enhancers with the MIR335 promoter (Fig. S5, BD), providing six MIR335-predicted distal enhancer sites (regions r6r11; Fig. S5 D). Acetylation of H3K27 on MIR335 enhancers detected in ASCLMNA(R482W) after adipogenic induction suggests interac-tions with the MIR335 promoter by chromatin looping (Zhang et al., 2013). We tested this hypothesis by two-color FISH using distinctly labeled promoter and enhancer probes. Probes were generated to cover the MIR335 gene and promoter as well as the distal enhancer element r11 located ?200 kb upstream of MIR335. In both undifferentiated and differentiated ASCs that were either native or expressing WT LMNA, these promoter and enhancer elements displayed a relatively low incidence of colocalization on the majority of alleles (?80%; Fig.?6, C and D). After induction of differentiation, however, this proportion markedly increased to 56% of alleles in the LMNA(R482W) mutants, reflecting interaction of a high proportion of MIR335 promoter and enhancer sites (Fig. 6, C and D). |
