CMTR1 is recruited to transcription start sites and has enhanced influence over ribosomal protein and histone genes [ChIP-seq]

We investigated the gene-specificity of mRNA cap methyltransferase CMTR1 and its impact in RNA expression. In this ChIP-seq experiment, we investigated the genome-wide chromatin binding profiles of CMTR1 in mouse embryonic stem cells. We demontrated that CMTR1 binds to the transcription start site (TSS) correlating with RNA polymerase II (RNAP II) levels, with predominant binding at histone genes and ribosomal protein (RP) genes. Moreover, the repression of CMTR1 expression resulted in a loss of RNAP II binding at the TSS. Overall design: CMTR1 and RNMT ChIP (chromatin immunoprecipitation) libraries were prepared from the CRISPR-Cas9 HA-GFP knock-in (KI) mouse embryonic stem cell lines were generated in which endogenous RNMT and CMTR1 were expressed as N-terminal HA-GFP fusion proteins. The control ChIP and RNAP II ChIP libraries were prepared from the parental stem cell line E14 of the CMTR1/RNMT KI cell lines. For RNAP II ChIP, cells treated with the control scramble siRNA and CMTR1 specific siRNAs for 36 hours. Cells were cross-linked by 1% formaldehyde for 10 min. Then the glycine was added to quench the reaction. The cell lysates were prepared as described (Varshney et al., 2018).The chromatin was sonicated in Bioruptor (Diagenode) at high power for 20 min (30 s on/off, 20 cycles). 25-50 micrograms of the sonicated chromatin were used for each IP. 50µl (10%) from each diluted extract for IP were taken as the inputs. For ChIP using anti-HA beads, 20µL of anti-HA magnetic beads (Pierce) were added to lysates from CMTR1/RNMT KI cells (as IPs) and E14 cells (as control) for 4°C overnight incubation. For RNAP II ChIP, 2µg of anti-Rpb1 (CST 14958) were added to lysates from E14 cells for 4 °C overnight reaction before the incubation with protein G Dynabeads (Life Technologies) for additional 2 hours. The beads were washed and eluted as described (Varshney et al., 2018) before the proteinase K treatment and reverse crosslinking at 42 °C overnight. The eluted DNA was purified by the QIAquick PCR purification kit (QIAGEN).