The G9a/CHCHD2/Sirt1 regulatory module acts on RNase H1 to control R-loop formation at rDNA sites

Abstract

R-loops are both regulators in many cellular processes and threats to genome integrity. Understanding the mechanism underlying regulation of R-loops is very important. Inspired by the findings of RNase H1-mediated R-loop formation, we focus our interest on the regulation of RNase H1 recruitment and expression. Here we report that G9a not only boosts the recruitment of RNase H1 to reduce R-loop accumulation at the rDNA site, but also positively regulates RNase H1 expression, whereas CHCHD2 prevents RNase H1 from being recruited to rDNA site and acts as a repressive transcription factor to inhibit the expression of RNase H1 to increase R-loop formation. We also found that G9a methylated the promoter of the RNase H1 gene, which inhibited the binding of CHCHD2. By contrast, when G9a was knocked down, the recruitment of CHCHD2 and Sirt1 to the RNase H1 promoter increased, which co-inhibited the RNase H1 transcription. Furthermore, G9a could directly bind CHCHD2, possibly decreasing free CHCHD2. Sirt1 could interact with CHCHD2 and functioned as a repressor suppressing transcription of the RNase H1 gene. Knockdown of Sirt1 led to binding of more G9a to the RNase H1 promoter. Taken together, we demonstrate that G9a regulates the expression of RNase H1 to maintain the steady-state balance of R-loops by suppressing CHCHD2 and Sirt1 corepressors being recruited to the target gene promoter.