DNMT3B maintains mCA landscape and regulates mCG status of bivalent promoters in human embryonic stem cells

In mammalian cells, DNMT3B is known as a de novo DNA methyltransferase. However, its preferential target sites for DNA methylation are largely unknown. By studying CA methylation (mCA) and various histone mark distributions in human embryonic stem cells (hESC), we set up a connection between mCA, H3K36me3, and DNMT3B. We found that mCA, H3K36me3 and DNMT3B signals in hESC are distributed in a 3-level pattern: low level at promoter region, intermediate level before first splicing junction and high level afterward. Knocking out DNMT3B (KO) in hESC demolished mCA, and this further confirmed that DNMT3B is the main enzyme which maintains mCA. Furthermore, the mCA could not be maintained after we deleted the H3K36me3 binding domain, PWWP domain, in DNMT3B. This observation suggests that DNMT3B maintains mCA through its histone interaction. This is the first study that describes the detailed landscape of mCA and its connection with DNMT3B. In contrast to mCA, we only observed a minor reduction of global mCG level from 83.7% to 79.2% after DNMT3B KO. 5256 de novo hypomethylated regions (dnHMRs) were found in the KO cell, in which 57.8% overlapped annotated promoter sites. Intriguingly, most of these promoter dnHMRs were bivalent, possessing both H3K4me3 and H3K27me3. We call them spurious bivalent promoters. Gene ontology (GO) analysis associated spurious bivalent promoters with development and cell differentiation. Overall, we found the importance of DNMT3B for shaping mCA landscape and maintaining the fidelity of bivalent promoter landscape in hESC.