Role of DNA methyltransferases 3A and 3B in inheritance of DNA methylation patterns

Proper propagation of epigenetic information during somatic cell divisions is critical for preserving gene expression patterns and cellular identity. However, the molecular mechanisms responsible for faithful inheritance of epigenetic marks are still poorly understood. In this thesis work, I have studied the inheritance of DNA methylation patterns through somatic divisions, focusing on the role of DNA methyltransferases 3A and 3B in this process.;DNA methylation patterns are established during development and then maintained through multiple somatic cell divisions by co-operative activity of the de novo and maintenance DNA methyltransferases - DNMT3A/3B and DNMT1, respectively. A key question that remains unresolved is how the de novo DNMT3A/3B enzymes assist in faithful inheritance of methylation patterns in somatic cells while guarding against aberrant de novo DNA methylation. Using sucrose density gradient analyses of fractionated chromatin, I have shown that almost all of the cellular contents of DNMT3A/3B, but not DNMT1, are strongly anchored to nucleosomes containing methylated DNA, allowing little free DNMT3A/3B to exist in the nucleus. This binding of DNMT3A/3B to nucleosomes does not require the presence of other known chromatin-associated proteins such as PCNA, HP1, MeCP2, EZH2, HDAC1, UHRF1 and G9a, but does require synergistic interactions of the conserved domains of DNMT3A/3B with the intact nucleosomes. The PWWP domain of DNMT3A assists such nucleosome binding through interaction with the H3K36me3 mark. Further, tight binding of DNMT3A/3B to nucleosomes in the presence of DNA methylation stabilizes these proteins. Drastic reduction of cellular DNA methylation levels results in a dramatic transcription-independent decrease of DNMT3A/3B proteins due to reduced nucleosome binding and subsequent degradation of the unstable free protein. Stabilization of DNMT3A/3B on nucleosomes in methylated regions promotes propagation of DNA methylation with DNMT3A/3B working synergistically in this maintenance process. Taken together, this thesis work presents an unexpected self-regulatory inheritance mechanism which not only ensures somatic propagation of methylated states by DNMT1 and DNMT3A/3B enzymes but also prevents aberrant de novo methylation by causing degradation of free DNMT3A/3B enzymes.