LSH Controls DNA Methylation Primarily By Promoting UHRF1 DNA Accessibility

DNA methylation is one of the most important epigenetic modifications,which plays a critical role in gene transcription,imprinting and tumorigenesis.LSH is the only member in the SNF2 chromatin remodeling enzyme family which is known to play a role in regulation of DNA methylation.Previous studies have revealed a significantly reduced DNA methylation in the LSH knockout mice and implicated a critical role for LSH in de novo DNA methylation,but the detailed mechanisms by which LSH regulates DNA methylation remain to be fully elucidated.In this study we first generated multiple LSH knockout cell lines including He La,HCT116,MCF7 and NIH3T3 by CRISPR-Cas9.Subsequent quantitative measurements of global levels of cytosine methylation(5m C)by HPLC demonstrated that LSH knockout resulted in significantly reduced DNA methylation in all cell lines.Genome-wide RRBS methylation analysis revealed that the level of DNA methylation declined at all genetic elements.To determine if LSH regulates DNA maintenance methylation,we generated DNA methyltransferase DNMT3A and DNMT3B double knockout He La cells(DNMT3A^-/-/3B^-/-)and DNMT3A,DNMT3B and LSH triple knockout cells(DNMT3A^-/-/3B^-/-/LSH^-/-).Subsequent DNA methylation alaysis indicates that while DNMT3A/DNMT3B double knockout only moderately reduced the level of DNA methylation in He La cells,DNMT3A/DNMT3B/LSH triple knockout led to a severe reduction of DNA methylation,indicating that LSH positively regulate DNA methylation primarily through DNMT1.We found that loss of LSH led to a reduced association of UHRF1 with replicating DNA and a diminished H3 ubiquitination.Biochemical studies revealed that LSH interacts with UHRF1 but not DNMT1.Interestingly,we found that knockdown of UHRF1 also led to reduced LSH chromatin association,suggesting that LSH and UHRF1 gain access to chromatin in a cooperative manner,presumably through a direct protein-protein interaction.RNA-seq analysis revealed a positive correlation between the reduction of DNA methylation level and up-regulation of gene expression.Taken together,we provide evidence that LSH promotes DNMT1-mediated DNA maintenance methylation primarily by enhancing UHRF1 DNA accessibility.