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Multivalent Chromatin Engagement and Inter-Domain Crosstalk Regulate MORC3 Atpase

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Multivalent Chromatin Engagement and Inter-Domain Crosstalk Regulate MORC3 Atpase Article Multivalent Chromatin Engagement and Inter- domain Crosstalk Regulate MORC3 ATPase Graphical Abstract Authors Forest H. Andrews, Qiong Tong, Kelly D. Sullivan, ..., Joaquin M. Espinosa, Scott B. Rothbart, Tatiana G. Kutateladze Correspondence [email protected] In Brief MORC3 is upregulated in Down syndrome and is linked to cancer. Using genetic, biochemical, and structural analyses, Andrews et al. show that the MORC3 CW domain associates with methylated histone H3 and negatively regulates MORC3 DNA-dependent ATPase activity by binding to the MORC3 ATPase domain. Highlights Accession Numbers d MORC3 is upregulated in Down syndrome and is linked to 5SVX cancer 5SVY 5SVI d The MORC3 CW domain binds to methylated histone H3K4 GSE78942 GSE84531 d The CW:H3K4me interaction is essential for MORC3 recruitment to chromatin d The CW domain negatively regulates MORC3 DNA- dependent ATPase activity Andrews et al., 2016, Cell Reports 16, 3195–3207 September 20, 2016 ª 2016 The Author(s). http://dx.doi.org/10.1016/j.celrep.2016.08.050 Cell Reports Article Multivalent Chromatin Engagement and Inter-domain Crosstalk Regulate MORC3 ATPase Forest H. Andrews,1,5 Qiong Tong,1,5,6 Kelly D. Sullivan,1,4 Evan M. Cornett,2 Yi Zhang,1 Muzaffar Ali,1 JaeWoo Ahn,1 Ahway Pandey,1,4 Angela H. Guo,3 Brian D. Strahl,3 James C. Costello,1 Joaquin M. Espinosa,1,4 Scott B. Rothbart,2 and Tatiana G. Kutateladze1,7,* 1Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO 80045, USA 2Center for Epigenetics, Van Andel Research Institute, Grand Rapids, MI 49503, USA 3Department of Biochemistry and Biophysics, The University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA 4Linda Crnic Institute for Down Syndrome, University of Colorado School of Medicine, Aurora, CO 80045, USA 5Co-first author 6Present address: Wuhan Institute of Physics and Mathematics Chinese Academy of Sciences, Wuhan 430071, China 7Lead Contact *Correspondence: [email protected] http://dx.doi.org/10.1016/j.celrep.2016.08.050 SUMMARY kemia nuclear bodies (PML-NBs), where it regulates p53 activity essential for cellular senescence in human and mouse fibro- MORC3 is linked to inflammatory myopathies and blasts (Mimura et al., 2010; Takahashi et al., 2007). Accumulating cancer; however, the precise role of MORC3 in evidence suggests a role of MORC3 in transcription, because it normal cell physiology and disease remains poorly promotes gene silencing when bound to SUMO-2 (Rosendorff understood. Here, we present detailed genetic, et al., 2006). biochemical, and structural analyses of MORC3. MORC3 is a large, 939-residue multi-modular protein. It con- We demonstrate that MORC3 is significantly upregu- tains an N-terminal gyrase, heat shock protein 90 (Hsp90), histi- dine kinase, and MutL (GHKL)-type ATPase domain, which is lated in Down syndrome and that genetic abnormal- MORC3 conserved in other human MORC family members (MORC1, ities in are associated with cancer. The CW MORC2, and MORC4) and is present in a diverse array of chro- domain of MORC3 binds to the methylated histone matin-modified enzymes and chaperones (reviewed in Dutta and H3K4 tail, and this interaction is essential for recruit- Inouye, 2000; Li et al., 2013). Although the catalytic activity of ment of MORC3 to chromatin and accumulation in MORC3 has not been established, the ATP-hydrolyzing function nuclear bodies. We show that MORC3 possesses of orthologous and homologous Morc proteins was found to be intrinsic ATPase activity that requires DNA, but it is essential for heterochromatin condensation and gene silencing negatively regulated by the CW domain, which inter- in Arabidopsis thaliana and for chromatin remodeling in response acts with the ATPase domain. Natively linked CW to DNA damage in mammalian cells (Li et al., 2013; Moissiard impedes binding of the ATPase domain to DNA, re- et al., 2012; Pastor et al., 2014). The ATPase domain in sulting in a decrease in the DNA-stimulated enzy- MORC3 is followed by a CW-type zinc finger and a C-terminal coiled-coil region. The MORC3 CW domain has been shown to matic activity. Collectively, our studies provide a interact with histone H3 peptides (Li et al., 2012; Liu et al., molecular framework detailing MORC3 functions 2016); however, the functional significance of this interaction re- and suggest that its modulation may contribute to mains unclear. The biological role of the coiled-coil region is un- human disease. known, though it may mediate protein dimerization. Reports have demonstrated that MORC3 is misregulated in INTRODUCTION the brain of a Down syndrome (DS) mouse model (Ling et al., 2014) and anti-MORC3 antibodies are found in most patients Microrchidia 3 (MORC3) is a member of the Morc family of four with cancer-associated DM (Fiorentino et al., 2013), yet the pre- proteins evolutionarily conserved throughout the eukaryotic cise role of MORC3 in normal cellular processes and in disease kingdom. MORC3, also called nuclear matrix protein 2 (NXP2), has not been determined. Here, we present detailed genetic, was originally identified as a nuclear matrix-associated protein biochemical, and structural analyses of MORC3. We report on with RNA-binding activity (Kimura et al., 2002). It is one of the histone H3K4me- and DNA-binding functions of MORC3, inter- most common autoantigens present in inflammatory myopa- domain crosstalk, and intrinsic catalytic activity of this ATPase. thies, with one-fourth of juvenile dermatomyositis (DM) patients Our mechanistic and mutagenesis studies reveal an intricate being found positive for anti-MORC3 antibodies (Gunawardena multivalent engagement of MORC3 with chromatin and regula- et al., 2009). Clinical studies reveal elevated MORC3 expression tion of the ATPase activity by DNA and CW and suggest a in leukocytes following anti-cancer chemotherapy (Gonza´ lez- possible link between impaired MORC3 histone-binding activity Ferna´ ndez et al., 2012). MORC3 localizes to promyelocytic leu- and disease. Cell Reports 16, 3195–3207, September 20, 2016 ª 2016 The Author(s). 3195 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Figure 1. MORC3 Is Consistently Upregulated in Trisomy 21 Cells (A) Box-and-whisker plots of MORC2, MORC3, and MORC4 expression levels across tissue types from the GTEx Portal. (B–D) Box-and-whisker plots of MORC3, MORC2, and MORC4 expression in D21 and T21 fibroblasts (B), lymphoblastoid cell lines (C), and patient monocytes and T cells (D). The mRNA expression values are in RPKM. Benjamini-Hochberg adjusted p values were calculated using DESeq2. (E) Venn diagram of differentially expressed genes in DS from human fibroblasts, human lymphoblastoids, and mouse brains. See also Figure S1. RESULTS AND DISCUSSION MORC2 and MORC4, which are located on chr22 and chrX, respectively, using the Genotype-Tissue Expression (GTEx) Por- MORC3 Is Markedly Upregulated in DS tal. We found that all three genes are expressed ubiquitously Because the MORC3 gene is encoded on chromosome 21 across tissues and to similar levels (Figure 1A). To determine (chr21), trisomy of which causes DS, the most common chromo- whether MORC3 is subject to inter-individual and tissue-specific somal abnormality in humans, we examined whether MORC3 variation, we next performed RNA sequencing (RNA-seq) on a contributes to the disease pathology. We first analyzed and panel of 12 age- and gender-matched euploid (D21) and T21 compared expression of MORC3 and two other family members, fibroblasts and measured MORC3, MORC2, and MORC4 3196 Cell Reports 16, 3195–3207, September 20, 2016 expression levels. We found that MORC3 is substantially upre- log rank test), and uterine corpus endometrial carcinoma gulated in T21 cells compared to D21 cells, and this discernible (p = 0.036, log rank test) (Figure S1B). A possible association upregulation is conserved across different individuals (Fig- of MORC3 antibody-positive idiopathic inflammatory myopa- ure 1B). In contrast, MORC2 and MORC4 expression levels re- thies and cancer has also been suggested (Ichimura et al., mained unchanged in T21 and D21 cells. 2012). Collectively, the genetic abnormalities found in DS, DM, To test whether MORC3 upregulation is conserved in a and cancer patients point to a physiological importance of different tissue, we carried out RNA-seq on a panel of six age- MORC3, yet our knowledge of its function at the molecular level matched female D21 and T21 lymphoblastoid cells. Like in fibro- is limited. blasts, MORC3, but not MORC2, was upregulated in T21 lymphoblastoid cells relative to D21 cells (Figure 1C). MORC4 Binding of MORC3-CW to the Histone H3 Tail Is was also strongly upregulated in T21 lymphoblastoid cells; how- Modulated by PTMs ever, this upregulation appeared to be unique to these cell lines In an effort to better understand the mechanism of action of and potentially confounded by the near lack of MORC4 expres- MORC3, we carried out biochemical and structural analyses of sion in D21 cells. Finally, to examine MORC3 expression levels its functional modules: the CW domain and the ATPase domain in naive patient samples, we isolated monocytes and T cells (Figure 2A). While the CW domain of MORC3 was shown to asso- from 17 individuals (10 T21 and 7 D21), extracted RNA, and per- ciate with the histone H3 tail (Li et al., 2012), its preference for formed RNA-seq. Analysis of the RNA-seq data revealed and regulation by posttranslational modifications (PTMs) have consistent upregulation of MORC3, but not MORC2 or not been characterized. To determine the specific binding part- MORC4, in individuals with T21 (Figure 1D). Furthermore, the ner and assess the effect of PTMs on binding activity of observed upregulation level of MORC3 was equal to or greater MORC3-CW, we tested the CW domain in a high-throughput than that of genes known to contribute to DS pathology, histone peptide microarray (Figures 2B–2D).
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