(R3005) Mechanism of non-coding RNA regulation of DNMT3A and its relation to histones, regulatory proteins, and clinically relevant mutations

Jonathan Sandoval (UC santa Barbara)

De novo DNA methylation by DNMT3A is a fundamental epigenetic modification that contributes to transcriptional regulation during cellular development and differentiation.  Recent findings show that Fos Proto-Oncogene (Fos) extra-coding RNA (ecRNA) inhibits DNMT3A activity, Fos ecRNA knockdown results in neuronal hypomethylation and that Fos ecRNA expression is essential for fear memory formation in rats.  Thus, further highlighting the role of RNA in the modulation of genomic DNA methylation.  We show Fos ecRNA strongly correlates with Fos mRNA synthesis in primary cortical neurons on a single cell level and provide evidence that Fos ecRNA modulation of DNMT3A at these actively transcribed sites occurs in a sequence-independent manner.  DNMT3A is also regulated by interactions with histone H3 tails and regulatory proteins, and the crosstalk between these epigenetic systems ensures appropriate epigenetic regulation.  We provide insights into this crosstalk by assessing the relationship of regulatory RNAs, histone H3 tails and regulatory proteins in the simultaneous regulation of DNMT3A activity.  Using DNMT3L, Fos ecRNA or CHD RNA in the presence of synthetic histone H3 tails or reconstituted polynucleosomes, we show regulatory RNAs play dominant roles in the modulation of DNMT3A activity in DNMT3A-H3 tails-regulatory protein-RNA complexes.  Through mutational mapping and computational modeling we show that the interactions between DNMT3A and Fos-1 ecRNA occur at the tetramer interface of DNMT3A.  Furthermore, while substitutions to clinically relevant residues on the DNMT3A tetramer interface, such as R771A, disrupt this ecRNA-mediated regulation, formation of DNMT3A_CDR771A-DNMT3L heterotetramers restores the inhibition of enzymatic activity by Fos-1 ecRNA.  The regulation of epigenetic modifiers by non-coding RNAs is linked to diverse cancers including AML and UCEC.  Our results further highlight the significance of RNA molecules in directing DNMT3A function and additionally provide insights into the crosstalk and mechanism for the modulation of DNMT3A by H3 tails, regulatory proteins and RNAs.

Support or Funding Information

NSF award number 1808775