Mouse MORC3 is a GHKL ATPase that localizes to H3K4me3 marked chromatin Sisi Lia,1,2, Linda Yenb,1, William A. Pastorb, Jonathan B. Johnstonc, Jiamu Dud, Colin J. Shewb, Wanlu Liue, Jamie Hob, Bryan Stenderb, Amander T. Clarkb,e,f,g, Alma L. Burlingamec, Lucia Daxingerh,3, Dinshaw J. Patela,3, and Steven E. Jacobsenb,f,i,3 aStructural Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065; bDepartment of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, CA 90095; cDepartment of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158; dShanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201602, China; eMolecular Biology Institute, University of California, Los Angeles, CA 90095; fEli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA 90095; gJonsson Comprehensive Cancer Center, University of California, Los Angeles, CA 90095; hDepartment of Human and Clinical Genetics, Leiden University Medical Center, 2300 RC Leiden, The Netherlands; and iHoward Hughes Medical Institute, University of California, Los Angeles, CA 90095 Contributed by Steven E. Jacobsen, June 16, 2016 (sent for review May 16, 2016; reviewed by Shiv I. S. Grewal and Fei Lan) Microrchidia (MORC) proteins are GHKL (gyrase, heat-shock protein dimethylation and trimethylation marks (H3K4me2 and H3K4me3) 90, histidine kinase, MutL) ATPases that function in gene regulation in (11–13). Consistent with this hypothesis, human and murine MORC3 multiple organisms. Animal MORCs also contain CW-type zinc finger have been identified in mass spectrometry screens as H3K4me3 domains, which are known to bind to modified histones. We solved readers (12–14). Mutation of a critical tryptophan residue in the the crystal structure of the murine MORC3 ATPase-CW domain bound human MORC3 CW domain disrupts localization in the nucleus, to the nucleotide analog AMPPNP (phosphoaminophosphonic acid- suggesting that CW domain-mediated recognition of H3K4me3 is adenylate ester) and in complex with a trimethylated histone H3 also critical for MORC3 targeting to chromatin (6). lysine 4 (H3K4) peptide (H3K4me3). We observed that the MORC3 Here we report a 2.6-Å crystal structure of the MORC3 N-terminal ATPase domain forms a dimer when bound to AMPPNP. – We used native mass spectrometry to show that dimerization is ATP- (ATPase-CW cassette) AMPPNP (phosphoaminophosphonic – dependent, and that dimer formation is enhanced in the presence of acid-adenylate ester) H3K4me3 complex, which is a symmet- nonhydrolyzable ATP analogs. The CW domain uses an aromatic cage rical dimer. We show that MORC3 is an active ATPase that forms to bind trimethylated Lys4 and forms extensive hydrogen bonds with ATP-dependent dimers and uses the CW domain to make contacts the H3 tail. We found that MORC3 localizes to promoters marked by with the H3K4me3 peptide. Arg8 from the histone peptide is an- H3K4me3 throughout the genome, consistent with its binding to chored by hydrogen bonding in a pocket on the CW domain, and H3K4me3 in vitro. Our work sheds light on aspects of the molecular the trimethylated Lys4 side chain is engaged via an aromatic cage. dynamics and function of MORC3. In vivo, MORC3 localizes to H3K4me3-marked chromatin. These observations reveal molecular details of MORC action that likely X-ray crystallography | histone mark reader | ATPase | native mass spectrometry Significance he Microrchidia (MORC) family of ATPase proteins has been The Microrchidia (MORC) family of ATPases are important regu- Tshown to be an important regulator of gene silencing in multiple lators of gene silencing in multiple organisms but little is known organisms. This family was first described in mice, when it was dis- about their molecular behavior. In this study, we used crystal- covered that morc1 null males showed arrested spermatogenesis (1). lography and native mass spectrometry to show that MORC3 This arrest was later shown to be associated with transposon dere- forms dimers when it binds to nonhydrolyzable ATP analogues. pression, implicating murine MORC1 as a crucial mediator of trans- We also determined that the CW zinc finger-like domain of poson silencing (2). Arabidopsis thaliana MORC1andMORC6were MORC3 can bind euchromatic histone H3 lysine 4 (H3K4) meth- shown to mediate silencing of transposons in a manner largely – ylation and that MORC3 localizes to H3K4me3-marked chromatin. independent of changes in DNA methylation (3 5). Studies in The MORC3 crystal structure provides details as to the intermo- Caenorhabditis elegans, which lack DNA methylation, also concluded lecular interactions that allow dimerization and the binding to thatthesingleMORCgeneinthisorganismplaysaroleintransgene ATP and histones. This work reveals key molecular activities of silencing (4). Although the biological importance of MORC ATPases MORC3 that might apply to other MORC family members in in enforcing gene silencing across multiple organisms is clear, how eukaryotic organisms. they are targeted and how they function are poorly understood. The MORC ATPases share a similar domain arrangement. The Author contributions: S.L., L.Y., W.A.P., J.B.J., J.D., A.T.C., A.L.B., L.D., D.J.P., and S.E.J. N terminus contains a GHKL (gyrase, heat-shock protein 90, his- designed research; S.L., L.Y., W.A.P., J.B.J., J.D., C.J.S., J.H., and B.S. performed research; tidine kinase, MutL) type ATPase domain, and at the C terminus is L.Y. and J.D. contributed new reagents/analytic tools; S.L., L.Y., W.A.P., J.B.J., J.D., and W.L. a coiled-coil segment. MORCs have been reported to form func- analyzed data; and S.L., L.Y., J.D., D.J.P., and S.E.J. wrote the paper. tional homomultimers or heteromultimers, where multimerization Reviewers: S.I.S.G., National Institutes of Health; and F.L., Fudan University. is likely mediated by the N- and/or the C-terminal domains (4, 6, 7). The authors declare no conflict of interest. The coiled-coil region has been proposed to promote constitutive Data deposition: The atomic coordinates and structure factors have been deposited in the dimerization, whereas N-terminal ATPase head dimerization oc- Protein Data Bank, www.pdb.org (PDB ID codes 5IX1 and 5IX2). The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi. curs only on ATP binding (6). This is consistent with other GHKL nlm.nih.gov/geo (accession no. GSE78258). ATPases described in the literature, many of which have been 1 S.L. and L.Y. contributed equally to this work. reported to undergo ATP-dependent dimerization (8–10). Both 2Present address: Department of Biology, South University of Science and Technology of plant and animal MORCs are capable of forming nuclear bodies, China, Shenzhen, Guangdong 518055, China. and mutations that impair ATP binding and/or hydrolysis disrupt 3To whom correspondence may be addressed. Email: [email protected], nuclear body formation of human MORC3 (6). [email protected], or [email protected]. Animal MORCs also carry a CW-type zinc finger domain, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. which has been proposed to read histone H3 lysine 4 (H3K4) 1073/pnas.1609709113/-/DCSupplemental. E5108–E5116 | PNAS | Published online August 15, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1609709113 Downloaded by guest on September 28, 2021 are conserved in other eukaryotic MORC proteins, and suggest (Fig. 1A). The CW-type zinc finger has been identified as an PNAS PLUS an epigenetic mark associated with MORC3 localization in vivo. H3K4me reader module as assayed by structural and biochemical methods (11–13, 15). To gain insight into the molecular mecha- Results nism of MORC3, we solved the crystal structure of the MORC3 Overall Structure of the MORC3–AMPPNP–H3(1–15)K4me3 Complex. ATPase-CW domain cassette in complex with the nonhydrolyzable MORC3 is composed of an N-terminal ATPase domain followed ATP analog AMPPNP and H3(1–15)K4me3 peptide by single- by a CW-type zinc finger, a flexible linker segment without pre- wavelength anomalous diffraction. The structure of the complex dictable secondary structure, and a C-terminal coiled-coil domain (Fig. 1A) was refined to 2.6-Å resolution, yielding an R factor of A flexible linker MORC3 ATPase CW CC AMPPNP CW B CW A Zn2+ Zn2+ Mg2+ Mg2+ H3K4me3 H3K4me3 ATPase A ATPase B Complex A Complex B C B BIOCHEMISTRY F18 L14 I9 F18 F83 CW F83 ATPase Mol A Mol B D E D454 D424 AMPPNP Y217 R444 G270 Q271 W51 Q442 Mg2+ R405 T55 E184 K216 D215 Fig. 1. Structure of MORC3 ATPase-CW cassette in complex with AMPPNP and H3K4me3 peptide. (A) Overall crystal structure of MORC3–AMPPNP–H3K4me3 complex. The crystallized region is delineated by the red line. The ATPase and CW domains are shown in ribbon representation and colored green and magenta in monomer Mol A and cyan and orange in monomer Mol B, respectively. The bound peptides are shown in space filling representation, whereas the AMPPNP molecules are in stick representation. (B) The MORC3 dimer shown with one monomer in ribbon representation and the other monomer in elec- trostatic surface representation, demonstrating the extensive interactions between the two monomers. The N terminus of one monomer interacts with the other monomer, with Ile9 and Leu14 residues positioned within two small hydrophobic pockets. (C) Two aromatic residues, Phe18 and Phe83, of one monomer pack against the same residues in the other monomer, forming a hydrophobic core of the dimer interface. (D) The interactions between the ATPase domain and the CW domain. The hydrogen bonds are highlighted with dashed red lines. (E) The AMPPNP fits into a narrow binding pocket inside the ATPase domain.