Figure 5
CCAT1-L is required to maintain the chromatin looping at the MYC locus. (A) The chromatin region between MYC-515 and MYC-335 exhibits strong characteristics of a super-enhancer in HCT116 cells but not in H1 cells (histone modifications data were retrieved from ENCODE collection39). CCAT1-L, MYC-335 and MYC loci are highlighted in red. (B) Distribution of H3K27ac signal across enhancers (outer figure) and super-enhancers (inner figure) in HCT116 cells. Rank and H3K27ac signal of enhancers and super-enhancers were downloaded from the literature20. 387 super-enhancers (black points) were identified from uneven distribution of H3K27ac signal among normal enhancers (grey points), and the CCAT1-L-associated super-enhancer (red point) is ranked as #12 super-enhancers with high H3K27ac signals. (C, D) Knockdown of CCAT1-L reduced the chromatin looping at the MYC locus. The long-range interaction frequencies between three chromatin regions (MYC-335/MYC, MYC-335/MYC-515, and MYC/MYC-515) were reduced after knockdown of CCAT1-L as revealed by 3C assays in HT29 cells. Over 90% of CCAT1-L was depleted after the ASO treatment in 3C assays (data not shown). (E) Knockdown of CCAT1-L has no effect on the chromatin looping at the β-globin locus. The same HindIII restriction fragments were designed for 3C primers and PCRs were performed at the same time as C and D. (F) Knockdown of CCAT1-L reduced the chromatin looping at the MYC locus in HCT116 cell line with CCAT1-L in cis overexpression. The long-range interaction frequencies between the chromatin regions examined in B and C were also reduced after knockdown of CCAT1-L in the HCT116 cell line as revealed by 3C assays. In C-F, the relative abundance of each 3C PCR product was determined using ImageJ and labeled underneath. 3C experiments were repeated three times. Supportive data are included in Supplementary information, Figures S5 and S6.
