RNase H2 is a trimeric ribonuclease that specifically degrades the RNA moiety of DNARNA hybrids. Mutations in RNase H2 result in Aicardi–Goutières syndrome (AGS), a rare inflammatory disorder notable for being a ‘mendelian mimic’ of congenital viral brain infection. Some AGS-associated mutations in the RNase H2B subunit do not affect the catalytic activity of the RNase H2 in vitro and are clustered together on the surface of the complex, suggesting a possible role for these residues in mediating important interactions of RNase H2.

I set out to identify binding partner(s) of RNase H2B whose binding is impaired by this cluster of mutations, by screening for interactors of FLAG-tagged wild-type and mutant RNase H2B in HEK293T cells. In this work, I identify several members of a putative chromatin-silencing complex as novel RNase H2B binding partners whose binding is impaired by each mutation in this cluster, including: HDAC2, a histone deacetylase, LSD1, a histone demethylase, CoREST, a co-repressor of transcription, TFII-I, a transcription factor, and ZMYM3, an MYM-type zinc finger protein of unknown function.

By making several truncation mutants of ZMYM3, I show that a C-terminal region containing a ‘PXP’ repeat motif mediates its interaction with RNase H2, and that the PXP-containing proteins ZMYM2 and ZMYM4 interact with RNase H2, whereas the PXP-null proteins ZMYM1 and ZMYM6 do not. By systematic truncation of the zinc fingers of ZMYM3 I also demonstrate that the first zinc finger mediates its interaction with TFII-I, while the eighth and ninth zinc fingers are each sufficient for its interaction with LDS1, HDAC2 and CoREST. These interaction sites implicate ZMYM3 as a novel type of scaffold protein mediating interactions between deacetylase, demethylase and RNase H-type enzymes.

To better understand the function of ZMYM3, I generate a ZMYM3 knock-out mouse embryonic stem cell line, and show that ZMYM3 is not required for cell viability or DNA repair, and is not cell-cycle regulated. Given the association of ZMYM3 with various regulators of transcription, I hypothesised that ZMYM3 might be involved in regulating a set of transcripts through these associations. To test this, I compare the transcriptomes of ZMYM3 knock-out and wild-type mouse embryonic stem cell lines by RNA-Seq. While deletion of ZMYM3 has no effect on transcription globally in this context, ZMYM3-null cells express lower levels of the primary transcript of microRNA 142, a specific target of the AGS-associated dsRNA deaminase ADAR1. To better understand the in vivo function of ZMYM3, an ES cell line with a ZMYM3 exon flanked by loxP sites is generated, to allow the conditional deletion of ZMYM3 in a whole organism.

The association of deacetylase, demethylase and RNase H-type enzymes raises the possibility that histone modification and degradation of DNA-RNA hybrids may be coordinated, and is therefore of potential importance for the field of chromatin biology. As the roles of these associations remain unknown, several functional models of these interactions are proposed, with a discussion of the possible reasons why disruption of these interactions may result in Aicardi–Goutières syndrome.