DNA Methylation Regulates Alternative Polyadenylation via CTCF and the Cohesin Complex

Dysregulation of DNA methylation and mRNA alternative cleavage and polyadenylation (APA) are both prevalent in cancer. Through various mechanisms, these processes promote carcinogenesis by altering the production of oncogenes and tumour suppressors. Although promoter DNA methylation is most studied, the majority of differentially methylated regions occur in non-promoter contexts, including the gene 3' ends, where APA occurs. Using genome-wide methylation sequencing and poly(A)-sequencing we identified a correlation between DNA methylation and APA. As such, we sought to define the mechanism regulating this relationship. Here, we show that DNA methylation regulates APA using CCCTC binding factor (CTCF) binding and recruitment of the cohesin complex. Using data of cancer subtypes available from The Cancer Genome Atlas we authenticated this correlation in vivo. This mechanism of DNA methylation-regulated APA demonstrates the impact of aberrant DNA methylation on transcriptome diversity in cancer. Overall design: Cultured cells were fixed with 1% formaldehyde for 15 min and quenched with 0.125 M glycine. Chromatin was isolated by the addition of lysis buffer, followed by disruption with a Dounce homogenizer. Lysates were sonicated and the DNA sheared to an average length of 300-500 bp. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by ethanol precipitation. Pellets were resuspended and the resulting DNA was quantified on a NanoDrop spectrophotometer. Extrapolation to the original chromatin volume allowed quantitation of the total chromatin yield. An aliquot of chromatin (30 ug) was precleared with G (for Pol2Ser2 and Pol2Ser5) or A (for CTCF, RAD21, SMC1, and H3K27Ac) agarose beads (Invitrogen). Genomic DNA regions of interest were isolated using 4ug of antibody against each protein. Complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. Illumina sequencing libraries were prepared from the ChIP and Input DNAs by the standard consecutive enzymatic steps of end-polishing, dA-addition, and adaptor ligation. After a final PCR amplification step, the resulting DNA libraries were quantified and sequenced on Illumina's NextSeq 500 (75 nt reads, single end). Cultured cells were fixed with 1% formaldehyde for 15 min and quenched with 0.125 M glycine. Chromatin was isolated by the addition of lysis buffer, followed by disruption with a Dounce homogenizer. Lysates were sonicated and the DNA sheared to an average length of 300-500 bp. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by ethanol precipitation. Pellets were resuspended and the resulting DNA was quantified on a NanoDrop spectrophotometer. Extrapolation to the original chromatin volume allowed quantitation of the total chromatin yield. An aliquot of chromatin (30 ug) was precleared with G (for Pol2Ser2 and Pol2Ser5) or A (for CTCF, RAD21, SMC1, TotalRNAPol2, and H3K27Ac) agarose beads (Invitrogen). Genomic DNA regions of interest were isolated using 4ug of antibody against each protein. Complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. Illumina sequencing libraries were prepared from the ChIP and Input DNAs by the standard consecutive enzymatic steps of end-polishing, dA-addition, and adaptor ligation. After a final PCR amplification step, the resulting DNA libraries were quantified and sequenced on Illumina's NextSeq 500 (75 nt reads, single end).