Fig. 4: The super-enhancer CCAT1-5L regulates MYC transcription via chromatin looping. | Nature

Fig. 4: The super-enhancer CCAT1-5L regulates MYC transcription via chromatin looping.

From: RIC-seq for global in situ profiling of RNA–RNA spatial interactions

Fig. 4

a, A snapshot of the RIC-seq, RNA-seq and H3K27ac signals around the CCAT1 locus. Northern blot probes, black lines. The chimeric interactions between CCAT1-5L and MYC are shown in red. b, CCAT1-5L, MYC promoter RNA and PVT1 are colocalized by smFISH in HeLa cells. 5L, CCAT1-5L. Scale bar, 5 μm. c, 3C-qPCR analysis of long-distance interactions at the CCAT1-5L, MYC and PVT1 loci upon knockdown of CCAT1-5L RNA with LNA mix. Western blot showing reduced MYC protein levels. Arrows indicate the potential ligation products of DpnII restriction fragments. d, HnRNPK interacts with CCAT1-5L by ChIRP-MS (top) and western blot (bottom). LacZ, control probe; ODD and EVEN, two different sets of antisense oligonucleotides to CCAT1-5L. e, HnRNPK monomer and dimer binding sites at CCAT1-5L, MYC and PVT1 RNA revealed by CLIP-seq. SCX acts as a negative control. f, HnRNPK occupies the CCAT1-5L, MYC and PVT1 loci, as shown by ChIP–qPCR. g, Co-IP of Flag-tagged hnRNPK (Flag–K) and HA-tagged hnRNPK (HA–K) protein from the transfected cell lysates. h, Co-IP of hnRNPK and Pol II in an RNA- and DNA-independent manner. i, ChIP–qPCR showing the occupancy of Pol II at the CCAT1-5L, MYC and PVT1 loci before or after hnRNPK knockdown. sihnRNPK, siRNA against hnRNPK; WT, siRNA-resistant hnRNPK; MT, hnRNPK (T389A/Q391A) mutant. j, A model of CCAT1-5L-mediated chromatin looping on the transcriptional activation of MYC. K, hnRNPK; dashed lines, dimerization. Data in c are mean ± s.e.m. Data in f and i are mean ± s.d.; n = 3 biological replicates, two-tailed, unpaired t-test. The experiments in b, d, g, h were independently repeated three times with similar results.

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