Abstract 2367 Characterizing the DNA binding behavior of the retroelement silencing factor MORC2

To protect genome integrity, cells have acquired defense mechanisms against invasive genetic material, such as retroelements that integrate into host genomes. The ATPase MORC2 (Microrchidia family CW-type zing finger 2) is critical for the transcriptional silencing of retroelements in the human genome. Loss of MORC2 is known to increase expression of the LINE-1 retrotransposon and integrated proviral DNA from the retrovirus HIV-1. MORC2 is hypothesized to contribute to transcriptional silencing by limiting DNA accessibility through DNA compaction; however, the molecular mechanism by which this is achieved remains unclear. To understand how MORC2 engages DNA, we purified full-length human MORC2 and characterized its DNA binding behavior in vitro. Protein-DNA crosslinking mass spectrometry experiments uncovered a previously unknown DNA binding region between the N- and C-terminal domains. It was previously shown that the N-terminal ATPase domain of MORC2 homodimerizes upon ATP binding. Here, we define a second homodimerization interface at the MORC2 C-terminus by size exclusion chromatography coupled to multi-angle light scattering. Dimerization of the N- and C-terminal domains could allow MORC2 to encircle DNA substrates within the lumen of the dimers, which would represent a unique mode of DNA binding. Indeed, pull-down assays with plasmid DNA show that MORC2 can capture circular DNA substrates when bound to the ATP analog AMP-PNP. We also observe that MORC2 can capture two DNA substrates simultaneously using pseudo-circular DNA-bound beads in pull-down assays. DNA binding by MORC2 appears to diminish MORC2's rate of ATP hydrolysis, which in turn may prolong MORC2 enclosure around DNA substrates. Finally, mutations to the MORC2 DNA binding region prevent capture of circular DNA substrates in pull-down assays and lose gene silencing activity in cells as measured by RNAseq. Taken together, this study suggests that MORC2 contributes to gene silencing by encircling DNA like a clamp, which may limit DNA accessibility and thereby impede transcription. NF is supported by the NSF-GRFP. Research in the Vos Lab is supported by the Smith Family Foundation, Alex's LemonadeFoundation Crazy 8 Initiative, and the NIH Director's New Innovator Award (1DP2GM146254-01).