Subnuclear domain proteins in cancer cells support the functions of RUNX2 in the DNA damage response.

© 2015. Published by The Company of Biologists Ltd.

Subnuclear domain proteins in cancer cells support transcription factor RUNX2 functions in DNA damage response Seungchan Yang1, Alexandre J. C. Quaresma1,2, Jeffrey A. Nickerson1, Karin M. Green3, Scott A. Shaffer3, Anthony N. Imbalzano1, Lori A. Martin-Buley4, Jane B. Lian1,4, Janet L. Stein1,4, Andre J. van Wijnen1,5 and Gary S. Stein1,4* 1

Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA

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Institute of Biomedicine, Department of Biochemistry and Developmental Biology, University of Helsinki, Finland

Journal of Cell Science

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Department of Biochemistry and Molecular Pharmacology & Proteomics and Mass Spectrometry Facility, University of Massachusetts Medical School, Worcester, MA 4

Department of Biochemistry & Vermont Cancer Center, University of Vermont Medical School, Burlington, VT, USA 5

Departments of Orthopedic Surgery & Biochemistry and Molecular Biology, Mayo Clinic, 200 First Street S.W., MSB 3-69, Rochester, MN 55905 *To Whom Correspondence Should Be Addressed: Gary S. Stein, Department of Biochemistry, University of Vermont College of Medicine, 89 Beaumont Avenue, Burlington, VT, USA, P: 802-656-4874; F: 802-656-2140; E: [email protected] Running Title: RUNX2 Scaffolding in DNA Damage Keywords: RUNX2, DNA damage response, nuclear matrix, proteomics, cancer, breast, prostate, osteosarcoma, INTS3, BAZ1B Contract Grant Sponsor: National Institutes of Health grant P01 CA082834, P01 AR048818 and R01 AR039588. Author Contributions: SY, JAN, ANI, JBL, JLS, AVW and GSS planned and designed the study. SY performed the experiments, with help from AQ and JAN for confocal microscopy, and KMG and SAS for mass spectrometry. Data were analyzed and interpreted by SY, KMG, SAS, AQ, JAN, ANI, JBL, JLS, AVW and GSS. SY drafted the manuscript, with help from JAN, ANI, JBL, JLS, AVW, LM-B, SAS and GSS. All authors read and approved the final manuscript.

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JCS Advance Online Article. Posted on 20 January 2015

ABSTRACT Cancer cells exhibit modifications in nuclear architecture and transcriptional control. Tumor growth and metastasis are supported by RUNX-family transcriptional scaffolding proteins, which mediate assembly of nuclear matrix–associated gene regulatory hubs. We used proteomic analysis to identify RUNX2-dependent protein-protein interactions associated with the nuclear matrix in bone, breast and prostate tumor cell types and found that RUNX2 interacts with three distinct proteins that respond to DNA damage: RUVBL2, INTS3 and BAZ1B. Subnuclear foci

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RUNX2, INTS3 and BAZ1B form UV-responsive complexes with the serine 139-


containing these proteins change in intensity or number following UV irradiation. Furthermore,

its expression in chemotherapy-resistant and/or metastatic tumors.

phosphorylated isoform of H2AX (γH2AX). UV irradiation increases the interaction of BAZ1B with γH2AX and decreases histone H3, lysine 9 acetylation levels (H3K9-Ac), which mark accessible chromatin. RUNX2 depletion prevents the BAZ1B/γH2AX interaction and attenuates loss of H3K9 and H3K56 acetylation. Our data are consistent with a model in which RUNX2 forms functional complexes with BAZ1B, RUVBL2 and INTS3 to mount an integrated response to DNA damage. This proposed cytoprotective function for RUNX2 in cancer cells may clarify

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INTRODUCTION Nuclei are highly structured and the spatial organization that supports nuclear metabolism is increasingly well characterized. This nuclear architecture encompasses two interconnected structures: chromatin and a nuclear matrix (Berezney et al., 1995; van Driel et al., 1995; Stenoien et al., 1998; Zink et al., 2004; Kubben et al., 2010; Markaki et al., 2010; Meldi and Brickner, 2011; Simon and Wilson, 2011). Molecular complexes that perform and regulate transcription,


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RNA processing, DNA replication, DNA repair, and apoptosis; and organize chromatin structure are localized within distinct nuclear domains. The ultrastructure of the nuclear matrix is well characterized and, in addition to protein and DNA, requires RNA to maintain its integrity (Nickerson et al., 1997). The protein composition of the isolated nuclear matrix was initially analyzed using two-dimensional gel electrophoresis (Fey and Penman, 1988; Dworetzky et al., 1990; Getzenberg et al., 1991). This approach indicated that the nuclear matrix is comprised of at least 200 proteins and that its composition is partially cell-type specific. Several dozen nuclear matrix proteins have been identified, including Numa, Matrin, HnRNPs, NMP1, and NMP2 (Fey and Penman, 1988; Berezney et al., 1995; van Driel et al., 1995; Pratap et al., 2011), revealing that the nuclear matrix contains both structural and regulatory components. Molecular characterization of the nuclear matrix protein NMP2 (Bidwell et al., 1993; Merriman et al., 1995) established that this protein is a member of the RUNX (AML/PEBP2alpha/CBFA) family of lineage-specific transcription factors, which control tissue development and have pathological roles in cancer (Zaidi et al., 2007a). RUNX proteins are localized at specific subnuclear domains through peptide-targeting sequences (Zeng et al., 1997; Zeng et al., 1998). They participate in the scaffolding of macromolecular protein/protein complexes that control gene transcription by supporting chromatin-related epigenetic mechanisms (Zaidi et al., 2001). These mechanisms involve formation of multiple complexes with proteins that mediate histone modifications or chromatin remodeling (Delcuve et al., 2009), including protein histone acetyl transferases (HATs, such as p300), histone deacetylases (e.g., HDACs), co-regulators (e.g., TLE-1/groucho), YAP and SMADs (Zaidi et al., 2002; Zaidi et al., 2004) Disruption of RUNX-protein subnuclear targeting alters transcriptional programs and compromises cell growth and differentiation (Zaidi et al., 2006), reflecting pathological linkage to acute myelogenous leukemia, and breast and prostate cancers (Zaidi et al., 2007b). 3

RUNX2/NMP2 is a master regulator of skeletal development (Lian et al., 2004; Lian et al., 2006; Kuo et al., 2009), and has been linked to bone cancer and metastases from other cancers (Pratap et al., 2011). Because RUNX2 is a rate-limiting, scaffolding protein, that is critical for the molecular organization of both transcriptional and epigenetic complexes at multiple target genes within matrix-associated subnuclear domains, establishing which nuclear matrix proteins are linked to the diverse biological functions of RUNX2 is a key objective. In this study, we used advanced protein mass spectrometry and proteomic analysis to identify proteins that are recruited to the nuclear matrix in a RUNX2-dependent manner and that associate with RUNX2 in bone,

separate and distinct functions may associate with RUNX2 to form a novel, multifunctional integrated protein complex involved in the UV-induced DNA damage response. Our data indicate that RUNX2 supports the scaffolding of these proteins into an integrated complex that is coupled to the DNA damage response. This finding suggests that RUNX2 is a direct participant and regulator of the DNA damage response, thus providing a new molecular dimension in our understanding of DNA repair and its dysregulation in cancer.


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breast and prostate cancer cells. Strikingly, we found that three proteins with previously reported

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RESULTS Definition of a RUNX2-dependent proteome associated with the nuclear matrix RUNX2 localizes in subnuclear domains (Fig. 1A) that are associated with the nuclear matrix (Zaidi et al., 2001). To investigate RUNX2-related protein complexes in this nucleaseresistant chromatin compartment by proteomic analysis and analyze the extent to which the protein profile of this compartment is RUNX2 dependent, we used siRNA-mediated knockdown


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of RUNX2 in Saos2 osteosarcoma cells. We transiently transfected cells with siRNA targeting RUNX2 and examined subcellular localization of RUNX protein by confocal immunofluorescence microscopy. The number of RUNX2-containing nuclear foci was diminished in cells transfected with RUNX2-targeting siRNA (siRUNX2), but gross nuclear morphology was maintained (Fig. 1A). Cells were then fractionated and proteins from different subcellular compartments were analyzed by SDS-PAGE and Western blot, using markers to validate the fractionation of cytoplasmic, chromatin, and nuclear matrix compartments (e.g., GAPDH was used as a cytoplasmic marker, acetylated histone H3 as a chromatin marker, and lamin B as a nuclear matrix marker) (Fig. 1B). siRNA-mediated depletion of RUNX2 greatly reduced RUNX2 protein levels in whole cell lysates and chromatin fractions, and rendered it undetectable in the nuclear matrix compartment. RUNX2 knockdown did not, however, alter the overall composition of the most abundant nuclear proteins residing in the nuclear matrix fraction as visualized by staining the gel with Coomassie blue (data not shown). Mass spectrometry analysis of the nuclear matrix proteome in bone cancer cells Nano-liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) was used to examine the molecular consequences of RUNX2 depletion on the proteomic profile of nuclear matrix proteins. Proteins in the nuclear matrix fraction from control- and siRNA-transfected cells were separated by SDS-PAGE and processed for nanoLC-MS/MS (Dzieciatkowska et al., 2014). In total, >1000 proteins were identified from the product ion spectra using the Swissprot database (Table S1 in supplementary material), and >700 nuclear proteins were selected based on Gene Ontology (GO) analysis (Fig. 1C). To assess differences in the relative protein abundance in the nuclear matrix fraction between cells transfected with nontargeting control- or RUNX2-targeting siRNA, we used semi-quantitative spectral counting (Zybailov et al., 2005; Lundgren et al.,

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2010). With this approach, we found that >100 proteins in the nuclear matrix fraction are downregulated >2-fold in cells transfected with siRNA targeting RUNX2 (Fig. 1C). Of this subset, we selected proteins that exhibited the greatest relative change when RUNX2 was depleted via siRNA knockdown or that are known to have a role in transcriptional regulation, chromatin remodeling and/or histone modification (Figs. 1D and 1E, and Table S2 in supplementary material). For example, in cells with a 40-fold reduction in RUNX2 levels, INTS3 levels exhibit a >80-fold decrease. Because the transcription factor RUNX2 may modulate the levels of distinct proteins in the

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nuclear matrix by regulating transcription or protein/protein interactions, we examined mRNA expression of selected proteins by reverse transcription–quantitative polymerase chain reaction (RT-qPCR) analysis. As expected, RUNX2 mRNA levels in cells transfected with RUNX2siRNA were decreased 3–4-fold compared to levels in cells transfected with nontargeting siRNA. However, mRNA expression levels corresponding to the proteins identified in our proteomic screen did not display significant changes due to transfection with siRNA targeting RUNX2 (Fig. 2A). This suggests that the differences in protein abundance observed by proteomic analysis (see


Fig. 1D) are not due to RUNX2-dependent transcriptional regulation. RUNX2-dependent proteins that were detected by at least five unique peptides in nanoLC-MS/MS-based protein profiling and showed promise as regulatory contributors by preliminary bioinformatic analysis, were confirmed to be localized in the nuclear matrix compartment by Western blot analysis (Fig. 2B). Proteins of interest, PRMT1 and CTCF, were also included in the analysis. Collectively, these data indicate that changes in nuclear matrix association of proteins upon RUNX2 depletion are mediated by post-transcriptional mechanisms (e.g., protein/protein interactions, subcellular compartmentalization). We selected three RUNX2-interacting proteins, RUVBL2, INTS3, and BAZ1B, for further studies, because their functions are broadly related to chromatin organization, DNA repair, and/or formation of protein/protein complexes that may be relevant to the molecular pathology of cancer cells. To support our proteomic identification, we examined the endogenous cellular localization of these proteins in Saos2 cells using immunofluorescence confocal microscopy (Fig. 2C). Nuclear foci were seen using primary antibodies that target RUVBL2, INTS3, and BAZ1B (Fig. 2C), consistent with the possibility that these proteins localize in the nuclear matrix in association with RUNX2. 6

Selective interactions of RUNX2 with RUVBL2, INTS3, and BAZ1B We examined whether RUNX2 changes the nuclear matrix association of RUVBL2, INTS3 and BAZ1B proteins by their recruitment via protein-protein interactions (Fig. 3A). Coimmunoprecipitation experiments revealed that endogenous RUVBL2 and BAZ1B each exhibit interactions with RUNX2 in osteosarcoma cell lines, Saos2 and U2OS (Fig. 3B). Endogenous RUVBL2 protein present in lysates of Saos2 and U2OS cells was effectively coimmunoprecipitated with M70 antibody, which recognizes a C-terminal domain in RUNX2. BAZ1B was also co-immunoprecipitated using S19 antibody, which interacts with the N-

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terminal region of RUNX2 (Fig. 3B). We did not detect a clear interaction between endogenous RUNX2 and INTS3 using either the C- or N-terminal–directed RUNX2 antibodies, but were able to readily detect RUNX2-INTS3–complexed proteins in RUNX2, RUVBL2 co-expression analysis (see below). Because RUVBL2 is a molecular chaperone (Izumi et al., 2010), we tested if RUVBL2 can recruit RUNX2 and the other two identified proteins, INTS3 and BAZ1B in U2OS cells. We transiently co-transfected cells with plasmids expressing Flag-RUVBL2 and either full-length (aa


1–528) or C-terminal deleted (aa 1–376) RUNX2 protein. The exogenously expressed FLAGRUVBL2 co-immunoprecipitated protein from both RUNX2 constructs, as well as endogenous INTS3 and BAZ1B (Fig. 3C). This result suggests that RUVBL2 can form a complex in vivo, and that the C-terminal region of RUNX2 may not be necessary for the interaction of RUNX2 with RUVBL2, INTS3 or BAZ1B. To further support the finding that the N-terminal region of RUNX2, containing the RHD, is responsible for these interactions, we examined protein-protein interactions using glutathione-S-transferase (GST) pull-down assays. GST, GST plus the RUNX2 RHD, or GST plus the RUNX2 C-terminal domain downstream of the RHD were immobilized on beads and incubated with Saos2 cell lysate. After washing, samples were recovered and separated using PAGE, then visualized by Western blot analysis. The results indicate that RUVBL2, INTS3 and BAZ1B proteins each interact with the DNA-binding Runthomology domain (aa 107–241) of RUNX2, whereas no interaction was seen in assays that did not include this N-terminal RUNX2 domain (Fig. 3D). The signal from BAZ1B was stronger than those from INTS3 and RUVBL2; this could be due to technical variation in antibody interactions or biological differences in the “strength” of protein interactions. These results

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indicate that the conserved DNA binding domain, RHD, of RUNX2 is important for interactions with RUVBL2, INTS3, and BAZ1B. The identification of RUNX2 complexes containing RUVBL2, INTS3, and BAZ1B in osteosarcoma cells, that express RUNX2 endogenously, raises the question of whether the same complexes form in other cell types that express RUNX2 as part of a pathological process. To address this, we examined RUNX2-related protein/protein interactions in PC3 cells, a metastatic prostate cancer cell line, and MDA-MB-231 cells, a metastatic breast cancer cell line, by coimmunoprecipitation analysis. First, we fractionated cells and conducted Western blot analysis;

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we found that RUVBL2, INTS3, and BAZ1B are detected in the nuclear matrix of both PC3 and MDA-MB-231 cells (Fig. 4A). Endogenous interactions of RUNX2 with two of the three RUNX2 binding proteins were analyzed by co-immunoprecipitation using cell lysates from PC3 or MDA-MB-231 cells and N- or C-terminal directed RUNX2 antibodies (Fig. 4B). The results suggest that RUNX2 complexes from both PC3 and MDA-MB-231 cells obtained using M70, the C-terminal directed RUNX2-antibody, contain RUVBL2. In immunoprecipitate obtained using S19, the N-terminal directed RUNX2-antibody, a strong RUNX2 band was seen and a


band corresponding to BAZ1B was also present. Taken together, the co-immunoprecipitation data suggest that RUNX2 interacts with RUVBL2 and BAZ1B in PC3 and MDA-MB-231 cells. The interaction of RUNX2 with INTS3 appears to be more tenuous and requires elevated levels of RUVBL2 and/or RUNX2. RUNX2 association with RUVBL2, INTS3, and BAZ1B in subnuclear domains points to a novel role for RUNX2 in DNA damage response Next we examined the subcellular localization of RUNX2 in relation to each of the three proteins in Saos2 cells by confocal immunofluorescence microscopy. We observed colocalization of RUNX2 and any of the three proteins in only a few subnuclear domains (Fig. 5A). Because the interactions of RUVBL2, INTS3, or BAZ1B with RUNX2 appear to be, at least in part, interdependent, we performed gene ontology analysis to assess whether these proteins have common functions or participate in shared pathways (Figs. 5B and 5C). This analysis revealed that the three proteins are involved in cellular responses to DNA damage and are linked to histone protein H2AX (gene symbol: H2AFX) through either a RUVBL2-INTS3 network or a BAZ1B network (Fig. 5B). The finding from our studies that these two networks intersect is novel, and the implication is that RUNX2 is involved in H2AX-dependent genomic 8

surveillance mechanisms. H2AX is a key sensor of genomic stress: it is phosphorylated on serine 139 (referred to as γ-H2AX) by DNA-dependent protein-kinases, such as ataxia telangiectasia mutated [ATM] and ATM-related [ATR] (Figs. 5B and 5C). To further characterize this relationship, we directly investigated molecular and cellular responses involving this set of four proteins upon induction of DNA damage using UV-irradiation, an accessible laboratory method for damaging DNA in a well-defined manner. RUNX2 colocalizes with γ-H2AX in a limited number of distinct foci

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transfected with RUNX2 or nontargeting siRNA were UV irradiated and analyzed by confocal


To determine if RUNX2 is involved in the nuclear response to DNA damage, cells

6A, right). The total protein level of γ-H2AX in irradiated cells increases after UV exposure,

immunofluorescence microscopy. In cells transfected with nontargeting siRNA, the number of RUNX2 foci with RUVBL2 or BAZ1B increased modestly 60 min after UV irradiation, whereas the RUNX2 foci that exhibit co-localization with INTS3 adjacent to one of the nucleoli are constitutively present regardless of UV (Fig. 6A, left). RUNX2 depletion by RNA interference does not change the subnuclear localization of any of the three proteins after UV irradiation, albeit that the localization of INTS3 in the nucleus is transiently altered 30 min after UV (Fig.

however, RUNX2, RUVBL2, INTS3, and BAZ1B levels do not change (Fig. 6C). This suggests that foci for RUVBL2, INTS3 and BAZ1B change in number or appearance upon UV exposure, but can form independently of RUNX2 (Fig. 6A). Because BAZ1B binds both γ-H2AX (Barnes et al., 2010) and RUNX2 (Fig. 3), we examined whether RUNX2 associates with γ-H2AX in situ. We found that both large and small RUNX2 foci containing γ-H2AX are present at 30 min after UV irradiation (Fig. 6B). The partial co-localization of RUNX2 with γ-H2AX is consistent with our observation that the γ-H2AX binding protein, BAZ1B, interacts with RUNX2. To investigate whether RUNX2 plays a role in formation of γ-H2AX foci, we examined RUNX2-knockdown in Saos-2 cells (Figs. 6 and 7). Importantly, the total levels of γ-H2AX are increased in siRUNX2-transfected cells compared to cells transfected with negative-control siRNA (siNS) (Fig. 6C). This finding is consistent with similar observations in RUNX2-null cells (Zaidi et al., 2007b). As expected, UV irradiation increases the percentage of cells that exhibit nuclear γ‑H2AX foci (Fig. 7B), as well as the number of foci per cell in nontargeting siRNA-treated control cells (Fig. 7C). However, in RUNX2-depleted cells, there is a basal level

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of γ-H2AX foci in the majority of the cells (Figs. 7A and 7B), which increases after UV treatment (Figs. 7C and 7D). Taken together, our results suggest that RUNX2 depletion enhances formation of γ-H2AX foci in the nucleus. Because RUNX2 interacts with the γ-H2AX binding protein BAZ1B, we examined whether RUNX2 and γ-H2AX participate in protein-protein interactions within a larger complex. Saos2 cells were transfected with FLAG-H2AX expression construct, then FLAG-H2AX was immunoprecipitated from cell lysates. As expected, BAZ1B did co-immunoprecipitate with FLAG-H2AX (Fig. 7E). The immunoprecipitates also contained INTS3 and RUNX2, but signal


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above background was only seen in lysates from UV-irradiated cells. BAZ1B is a protein kinase that phosphorylates H2AX on tyrosine 142 (Y142) (Xiao et al., 2009). Indeed, phosphorylation of Y142 is observed in H2AX immunoprecipitates (Fig. 7E). These results suggest that RUNX2 binds to BAZ1B and γ-H2AX in response to UV. RUNX2 decreases histone H3 acetylation on Lysine 9 in response to UV irradiation Because of the interrelationships between RUNX2, BAZ1B, γ-H2AX and the cellular response to UV, we further explored the biological function of RUNX2 in UV-induced DNA damage response. For example, other post-translational modifications of histones occur in addition to phosphorylation of H2AX in response to DNA damage, including decreased acetylation of H3K9 and H3K56 by activation of HDACs or inhibition of acetyltransferases (e.g., GCN5) (Yu et al., 2011). In osteosarcoma cells transfected with nontargeting siRNA and exposed to UV irradiation, acetylation of H3K9 and H3K56 decreases, while phosphorylation of H2AX S139 increases (Figs. 8A and 8B). H2AX Y142 phosphorylation appears to be slightly decreased when the signal intensity is normalized relative to total H2AX recovery (Fig. 8B). RUNX2 depletion via siRNA knockdown modestly attenuates the UV-dependent reductions in acetylation of both H3K9 and H3K56. The results of RUNX2 knockdown on UV-dependent phosphorylation of H2AX are more dramatic. We saw an increase in H2AX S139 phosphorylation after UV irradiation and the abolishment of the UV-related reduction in Y142 phosphorylation in cells transfected with RUNX2-siRNA (Figs. 8A and 8B). The latter finding supports the concept that RUNX2 modulates BAZ1B functions linked to H2AX phosphorylation and the response to UV irradiation. Because BAZ1B-mediated phosphorylation of Y142 in H2AX triggers apoptosis (Xiao et al., 2009), we examined cell survival in response to UV. Cells with decreased levels of RUNX2 appear to be protected from UV-induced cell loss compared to 10

nontargeting siRNA–transfected control cells (siNS) (Fig. 8C). Finally, immunoprecipitation analysis of Saos2 cells cotransfected with the FLAG-H2AX expression construct and RUNX2targeting or nontargeting siRNA, and treated with UV irradiation indicates that the BAZ1B interaction with H2AX, which is normally increased by UV exposure, is actually diminished in the absence of RUNX2 (Fig. 8D). In conclusion, the combined results of this study are consistent with a model in which RUNX2 participates with BAZ1B and other associated proteins in cell


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survival in response to UV-induced DNA damage.

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DISCUSSION RUNX2 is a nuclear-matrix–associated transcription factor with biological activities in normal development or cancer that are determined by the dynamic association of interacting proteins to form different functional complexes depending on the physiological context. To understand this proteome of RUNX2-interacting proteins at the nuclear matrix, we employed a novel approach that combines the depletion of RUNX2 as a molecular scaffolding protein and


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high-resolution proteomic analysis of changes in the protein profile of the nuclear matrix-related subnuclear fractions in cancer cell lines. Analysis of this subnuclear proteome in control cells containing RUNX2 revealed that, as expected, the nuclear matrix contains a large number of well-established structural molecules (e.g., Lamin A/C and B, Numa, Matrin-3) and many proteins involved in RNA metabolism. These observations are entirely consistent with results from a number of recent proteomic studies that characterize nuclear matrix proteins from embryonic stem cells, tumor cell types, and plant cells (Oehr; Calikowski et al., 2003; Barboro et al., 2009; Albrethsen et al., 2010; Nasrabadi et al., 2010; Warters et al., 2010). The main finding of the current study is the identification of three proteins that were shown to have shared, RUNX2-dependent functions in UV-related DNA-damage response. Interestingly, the nuclear matrix has been reported to provide a platform for DNA repair (Mullenders et al., 1990; Zaalishvili et al., 2000; Atanassov et al., 2005). One protein identified by our proteomic analysis is RUVBL2, a member of the AAA+ family (ATPase associated with diverse cellular activities) of DNA helicases (Izumi et al., 2010). Human RUVBL2 is the apparent homologue of the bacterial RUVB protein, which encodes a DNA helicase essential for homologous recombination and DNA double-strand break repair. Recently, RUVBL2 has also been identified in chromatin remodeling complexes including INO80, SRCAP, Uril, and Tip60 (Gorynia et al., 2011). The second protein, INTS3, was initially characterized as an RNA polymerase II Cterminal domain binding factor involved in the 3′ processing of small nuclear RNAs (Inagaki et al., 2008). Recently, INTS3 has been identified as a key component of DNA damage response (Huang et al., 2009; Skaar et al., 2009). BAZ1B, the third protein, is a novel tyrosine-protein kinase related to the bromodomain family. It contains a structural motif characteristic of proteins that bind acetylated lysines and are involved in chromatin-dependent regulation of transcription (Xiao et al., 2009). BAZ1B phosphorylates H2AX at Tyr142, which plays a pivotal role in DNA 12

repair and acts as a molecular marker that distinguishes between apoptotic and repair responses to genotoxic stress. The simultaneous identification of these three proteins as prominent RUNX2-dependent nuclear-matrix proteins suggested that they may share a RUNX2-related function. RUVBL2, INTS3, BAZ1B, and RUNX2 form a complex that regulates DNA repair following UV induced DNA damage In these experiments we found that the association of endogenous RUNX2 with BAZ1B


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attenuated γ-H2AX foci formation, and resulted in higher rates of cell death after UV treatment than in RUNX2-depleted cells. The DNA-damage marker γ-H2AX relays signals from DNA lesions and recruits DNA damage repair machinery as well as chromatin remodelers to maintain genome stability. When DNA damage signal transduction is disrupted, however, cells fail to repair damaged DNA. We propose a model in which RUNX2 binds to γ-H2AX complex with BAZ1B in response to DNA damage and inhibits BAZ1B. Although cellular DNA-damage responses mediated by BAZ1B have not been well characterized, the molecular function of BAZ1B in response to DNA damage has been described (Xiao et al., 2009). According to the authors, phosphorylation of H2AX at Tyr142 by BAZ1B inhibits phosphorylation of H2AX at Ser139. So, the balance between survival and apoptosis can be maintained by two different, but neighboring phosphorylation sites, Ser139 and Tyr142, of H2AX. However, our data indicated that in spite of increased phosphorylation at Tyr142, RUNX2-knockdown cells did not show higher levels of apoptosis as reported by others. Instead, these cells had higher survival rates after UV treatment than cells transfected with nontargeting siRNA. We attribute this result to the fact that the Saos2 cells used in our study do not express p53. The tumor suppressor p53 plays a key role in DNA damage response including arresting cells in G1 and G2 phases (Decraene et al., 2001) and activating wildtype p53–induced phosphatase 1 (WIP1), to reduce Ser139 phosphorylation of H2AX (Cha et al., 2010). For future studies, it would be interesting to evaluate the effects of treatments that induce dsDNA breaks, rather than the relatively benign single-strand breaks expected from the UV-irradiation levels used in the experiments described here.

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Histone modification in DNA damage response: Histone acetylation Eukaryotic cells protect their genome from UV-induced DNA damage through a sequence of molecular processes that include damage recognition, chromatin opening, DNA repair, and chromatin sealing. Since chromatin is a multi-component complex of DNA and proteins, and the arrangement of nucleosomes throughout the genome is highly variable, this repair process has not been exhaustively characterized. Recently, however, histone acetylation was identified as a key player involved in opening chromatin to provide access for the DNA repair machinery. Histone H3 and H4 acetylation changes were observed after UV irradiation in yeast as well as

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human cells (Yu et al., 2011). Cells can modulate histone acetylation levels by recruiting either histone acetyltransferases (HATs) or histone deacetylases (HDACs) depending on the cellular context. H3K79 methylation is another important posttranslational modification involved in opening and sealing chromatin following UV irradiation. DNA-damage-dependent histone modification happens not only in the main nucleosome components, but also in a variant histone H2AX with phosphorylation on S139, γ-H2AX. In conclusion, we have defined RUNX2-dependent protein/protein interactions that are


associated with the nuclear matrix and discovered three proteins involved in the DNA damage response—BAZ1B, RUVBL2, INTS3—that require RUNX2 for scaffolding into a multifunctional complex. This complex supports histone displacement, DNA unwinding, and stabilization of single-stranded DNA to mount an integrated response to DNA damage in breast, prostate, and bone cancer cells. ACKNOWLEDGMENTS We thank the members of our laboratory, especially Jason Dobson and Shirwin Pockwinse for stimulating discussions and/or general support. This work was supported, in whole or in part, by National Institutes of Health Grants P01 CA082834 and P01 AR048818. The contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health.

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FIGURE LEGENDS Figure 1. Proteomic analysis of RUNX2-related nuclear matrix proteins in RUNX2knockdown cells. (A) Immunofluorescence staining of Saos2 cells transfected with either nontargeting siRNA (siNS) or RUNX2-targeting siRNA (siRUNX2). (B) Proteins in whole cell lysates, W; cytoplasmic extracts, C; DNase I/salt extracts, D; and nuclear matrix fraction, N, were resolved by 15% SDS-PAGE and analyzed by Western blot using primary antibodies specific for the indicated proteins. GAPDH, Histone H3, and Lamin B were used as markers for

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(Fibrillarin) and B23 (Nucleophosmin) are nuclear-matrix components that are expected to be


cytoplasmic extracts, DNase I/salt extracts, and nuclear matrix fraction, respectively. FBR

136 proteins out of 207 RUNX2-dependent nuclear proteins were identified as ‘downregulated’

recovered in both DNase I/salt extracts and the nuclear matrix fraction. (C) Workflow for the proteomic screening of RUNX2-dependent nuclear matrix proteins. 1093 proteins were identified by mass spectrometry-assisted fingerprinting of nuclear matrix fraction prepared from Saos2 cells transfected with nontargeting siRNA (siNS) or RUNX2 targeting siRNA (siRUNX2). 721 were identified as nuclear proteins by Gene Ontology (GO) analysis. Spectral counting obtained from mass spectrometry was used to compare the relative fold-change of protein levels;

nuclear proteins by RUNX2 knockdown (see Table S1 in supplementary materiald). A functional subset of proteins including chromatin remodelers, epigenetic regulators, transcriptional controllers, and most RUNX2-dependent nuclear proteins were selected by further screening for proteins identified via ≥5 peptides (Table S2 in supplementary material). (D) The graph shows the log (base 2) fold-change in protein levels between nuclear matrix fractions of cells transfected with RUNX2-targeting siRNA or non targeting siRNA determined via spectral counting obtained from mass spectrometry analysis. Results for a functional subset of proteins including transcription regulators, chromatin remodelers, and histone modifiers is shown. The number of unique peptides identified by mass spectrometry is indicated, as are the biological functions of proteins: T for transcription regulator, C for chromatin remodeler, and H for histone modifier. (E) Functions and fold-decreases due to RUNX2 knockdown of representative proteins from the mass spectrometry analysis of nuclear matrix proteins from siRNA-transfected Saos2 cells. Fold decrease in protein levels were calculated by dividing the spectral counts for an identified protein by the sum of the spectral counts per sample.

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Figure 2. Quantitative analysis of RUNX2-dependent proteins. (A) The levels of messenger RNA for the indicated genes from Saos2 cells treated with nontargeting (siNS) or RUNX2-targeting siRNA (siRUNX2) were analyzed by quantitative real time polymerase chain reaction (qRT-PCR). Two independent biological duplicates were performed and error bars indicate the standard deviations. (B) Western blot analysis of subcellular localization of proteins identified by mass spectrometry confirms that all are present in the nuclear matrix fraction (N) of Saos2 cells, (C) Immunofluorescence staining of RUVBL2, INTS3, and BAZ1B proteins in Saos2 cells. Size bar and the thickness of z-section are 5 µm and 0.2 µm, respectively.

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Differential interference contrast (DIC) and DAPI images were placed in top- and bottom-right corner of each image, respectively. Figure 3. Interaction of RUNX2 with RUVBL2, INTS3, and BAZ1B. (A) The functional domains of RUNX2 RUVBL2, INTS3, and BAZ1B and the location of peptide sequences identified by mass spectrometry. The peptide fragment identified by mass spectrometry is indicated as closed bar. Functional domains in each peptide are indicated: RHD, Runt homolog domain; NMTS, nuclear matrix targeting sequences; AAA, ATPase associated with a variety of


cellular activities; KD, kinase domain; DDT, DNA binding homeobox and different transcription factors; PHD, plant homeo domain; BRD, bromodomain. (B) Co-immunoprecipitation of RUNX2 with interacting proteins was analyzed by Western blot. To detect RUNX2-RUVBL2, INTS3, or -BAZ1B endogenous interactions, 5 mg of whole cell lysates from Saos2 or U2OS cells were immunoprecipitated with 5 µg of anti-RUNX2 antibodies or 5 µg of normal rabbit IgG as a negative control. Immunoprecipitation products were then analyzed by Western blot, using anti-RUVBL2, -INTS3, or -BAZ1B antibodies. Note that no clear immunoprecipitation products were seen using anti-INTS3 antibodies and the results are not shown. (C) Coimmunoprecipitation of Flag-RUVBL2 protein with full-length RUNX2 (WT: 1–528 a.a.) or C-terminal deleted mutant (ΔC: amino acid 1–376 a.a.). U2OS cells were transiently cotransfected with a Flag-RUVBL2 expression construct and either full-length or C-terminal deleted RUNX2 construct. Whole cell lysates were incubated with anti-Flag M2 agarose beads (Sigma). Washed beads were subjected to SDS-PAGE and analyzed by Western blot using specific antibodies against the indicated proteins. Asterisks (*) mark bands caused by nonspecific interactions. (D) Bacterially expressed GST (G), GST fused to the Runt homolog domain of RUNX2, 107~241 a.a. (GST-R), or GST fused to the C-terminal of RUNX2, 240~528 16

a.a. (GST-C) proteins were immobilized on glutathione–beads and incubated with whole cell lysates from Saos2 cells. After extensive washing, proteins bound to the beads were eluted in protein sample buffer and analyzed by Western blotting with antibodies against the indicated proteins. Figure 4. Interaction of RUNX2 with RUVBL2, INTS3, and BAZ1B in metastatic prostate PC3 or breast MDA-MB-231 cells. (A) Biochemical fractionation of PC3 and MDAMB-231 cells. Proteins in whole cell lysates, W; cytoplasmic extracts, C; DNase I/salt extracts, D; and nuclear matrix fraction, N, from PC3 or MDA-MB-231 cells were resolved on 8 % or 15%

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SDS-PAGE and analyzed by Western blot with the indicated primary antibodies. (B) Coimmunoprecipitation of RUNX2 with interacting proteins was analyzed by Western blot. To detect RUNX2-RUVBL2, -INTS3, or -BAZ1B endogenous interactions, 5 mg of whole cell lysates from Saos2 or U2OS cells were immunoprecipitated with 5 µg of anti-RUNX2 antibodies or 5 µg of normal rabbit IgG as a negative control. Proteins were separated via SDS-PAGE, blotted and immunoblots were probed with anti-RUVBL2, -INTS3, or -BAZ1B antibodies. Antibodies against GAPDH, Histone H3, and Lamin B were used as markers for cytoplasmic


extracts, C; DNase I/salt extracts, D; and nuclear matrix fraction, N; respectively. Figure 5. RUNX2 association with RUVBL2, INTS3, and BAZ1B in vivo. (A) Immunofluorescence staining of RUNX2 (Alexa 488, green) with RUVBL2, INTS3, or BAZ1B (Alexa 555, red) in Saos2 cells was analyzed by confocal microscopy. Size bar and the thickness of z-section are 5 µm and 0.2 µm, respectively. DIC and DAPI images were placed in the top- and bottom-right corner of each image, respectively. (B) The functional protein networks of RUVBL2, INTS3, and BAZ1B were analyzed using STRING (version 9.0). Boxes below the network diagrams show a simplified version of the model. (C) Reported biological functions of RUNX2, RUVBL2, INTS3, and BAZ1B, with references. Figure 6. UV effect on RUNX2 and interacting proteins. Saos2 cells were transfected with nontargeting (siNS) or RUNX2-targeting siRNA (siRUNX2), and were UV-irradiated (300 J/m2) or not treated with radiation (NT). (A) and (B) Confocal microcopy analysis of coimmunofluorescence staining. Unirradiated (NT) cells and 30 or 60 minutes post-UVirradiation cells were permeabilized, fixed, and stained with RUNX2 antibody and RUVBL2, INTS3, BAZ1B (A), or γ-H2AX (B) antibodies. Size bar and the thickness of z-section are 5 µm and 0.2 µm, respectively. DIC and DAPI images were placed in the top- and bottom-right corner 17

of each image, respectively. (C) Western blot analysis of H2AX, γ-H2AX, RUNX2, RUVBL2, INTS3, BAZ1B, and GAPDH in whole cell lysates from unirradiated and UV-irradiated Saos2 cells. UV-irradiated cells were harvested 5 or 30 min after UV irradiation (300 J/m2). Figure 7. RUNX2 association with γ-H2AX by UV irradiation. Saos2 cells transfected with non-targeting siRNA (siNS) or RUNX2-targeting siRNA (siRUNX2) were not treated with radiation (NT) or UV-irradiated (300 J/m2). (A) After UV-irradiation, cells were incubated for 30 or 60 min, then permeabilized, fixed, and co-stained with RUNX2 (green) and γ-H2AX (red)

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antibodies and analyzed by confocal microscopy. Size bar and the thickness of z-section is 5 µm and 0.2–0.3 µm, respectively. (B) Cells showing γ-H2AX foci in the nucleus were counted. The graph shows the ratio of the number of cells with nuclear γ-H2AX foci to the total cells in the field; 3–14 cells were counted per field, and at least 9 fields were counted per treatment. (C) The average number of γ-H2AX foci per nucleus was counted and plotted; at least 36 cells were analyzed for each data point. The error bar indicates standard deviation. (D) Representative confocal images from 30 min after UV irradiation are shown. (E) Saos2 cells were transfected with FLAG-H2AX expression construct, UV irradiated or left untreated, then FLAG-H2AX


protein was immunoprecipitated and analyzed by Western blot. Since the intensity of ECL signal from Ser139 phosphorylation on H2AX from input lanes was low, we included longer exposures of the blot for Ser139 phosphorylation (P-S139-H2AX) and total H2AX input lanes. The upper band from the P-S139-H2AX and H2AX blots corresponds to overexpressed FLAG-H2AX and the lower band is from endogenous H2AX. *Long indicates data from a longer exposure of blots for ECL detection. Figure 8. RUNX2-dependent histone modification in response to UV. (A) Saos2 cells transfected with non-targeting siRNA (siNS) or RUNX2-targeting siRNA (siRUNX2) were unirradiated (control) or UV (300J/m2)-irradiated. Then, cells were further incubated for 60 min, and K56 and K9 acetylation of histone H3, and S139 and Y142 phosphorylation of histone H2AX were analyzed by Western blot. (B) The graph shows a quantification of the Western blot data in (A) created using Image J software. Each band from acetylation or phosphorylation of H3 or H2AX was measured and normalized to total H3 or H2AX levels. The image represents analysis of at least 3 independent Western blots; error bars represent SD. (C) Saos2 cells transfected with siNS or siRUNX2 were unirradiated (Control) or UV-irradiated (UV, 300 J/m2), and images of cells were analyzed by Nikon phase contrast microscopy (10x). (D) 18

Immunoprecipitation analysis of UV-irradiated (UV, 60 min) or unirradiated (–) Saos2 cells cotransfected with FLAG-H2AX expression plasmid and the indicated siRNA using anti-FLAG


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antibody. Immunoprecipitation with normal IgG was used as a control.

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MATERIALS AND METHODS Cell culture and UV irradiation Cells were grown at 37°C with 10% CO2. Saos2 and U2OS cells were cultured with McCoy’s 5A medium with 15% FBS (Saos2) or 10% FBS (U2OS). MDA-MB-231 cells were cultured with alpha-MEM with 10% FBS. PC3 cells were cultured with T-medium with 10% FBS. L-Glutamine and PS mixture (100 units/ml penicillin plus 100 µg/ml streptomycin) were

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MA). For UV irradiation, cells were washed with PBS and irradiated in the UV-crosslinker


added to culture media for all cells (all cell culture reagents were from Thermo Fisher, Waltham,

Thermo Fisher) and incubated with Oligofectamine (Thermo Fisher) for 30 min at room

model XL-1000 (Spectronics, Westbury, NY) with 300 J/m2. Culture media were added immediately after irradiation. siRNAs, expression plasmids, and transfection conditions Nontargeting control siRNA (siNS) (D-001210–01-20) and RUNX2-targeting siRNA (siRUNX2) #4 were purchased from Thermo Fisher. The target sequence for RUNX2 (#4) is AAGGUUCAACGAUCUGAGAUUUU. siRNAs (20 nmol) were diluted in Opti-MEM (0.5 mL,

temperature. Cells were washed twice with PBS, and then washed in Opti-MEM just prior to transfection. Cells were incubated for 4 h incubation at 37°C with the siRNA-Oligofectamine transfection mixture. 3X-FBS–containing growth media were then added to cultures and siRNAtransfected cells were harvested 48 h later. For transient transfection appropriate plasmids were transfected into subconfluent Saos2 or U2OS cells using FuGENE6 or X-tremeGENE transfection reagent (Roche, Basel Switzerland). Expression plasmid for FLAG-H2AX was gifted from Dr. Michael Rosenfeld (University of California, San Diego). Expression plasmid for FLAG-RUVBL2 was purchased from Addgene (addgene.org). All transfections were equalized for total DNA by adding empty plasmid. Biochemical fractionation Cells were washed twice with cold PBS, harvested by scraping, then centrifuged for 10 min at 800 x g, 4°C. After gentle aspiration of PBS from the pellet, the cells were extracted in cytoskeletal buffer (10 mM Pipes, pH 6.8/100 mM NaCl/300 mM sucrose/3 mM MgCl2/1 mM EGTA/Protease inhibitor cocktail (Roche)/1 mM AEBSF/2 mM VRC/25 nM MG132) 20

containing 0.1% Triton X-100 for 5 min at 4°C to remove the soluble proteins. The mixture was centrifuged for 5 min at 800 x g, 4°C, and the supernatant was collected as ‘cytoplasmic extract’. The pellet was resuspended in cytoskeletal buffer with 10,000 U/mL RNase-free DNase I (Sigma-Aldrich, St. Louis, MO) for 1 h at 30°C. Chromatin was then removed by salt extraction with 2 M ammonium sulfate to a final concentration of 250 mM for 10 min at room temperature and centrifuged for 5 min at 800xg, 4°C. The supernatant was collected as ‘DNase I/salt extract’. The remaining pellet was resuspended in protein sample buffer and collected as a ‘nuclear matrix fraction’. For proteomic analysis, equal volume fraction of each step of extraction and nuclear


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matrix fraction was loaded on SDS-polyacrylamide gels. In-gel trypsin digestion SDS-PAGE was performed with 4–15% linear gradient polyacrylamide gels containing TrisHCl (Bio-Rad, Hercules, CA). After electrophoresis, Coomassie-stained gel lanes were cut into 15 bands, each representing a unique molecular weight region, processed for in-gel digestion. Briefly control and siRUNX2 gel lanes were processed and transferred to 0.5 mL tubes. In some cases a “short gel” was run: nuclear matrix fractions were electrophoresed ~2 cm by SDS-PAGE, Coomassie stained, then the entire protein band was processed for in-gel digestion. All gel pieces were de-stained twice with 200 µL 25 mM ammonium bicarbonate in 50% acetonitrile for 30 min at 37°C, and reduced with 80 µL 7.6 mg/mL dithiothreitol (DTT) for 10 min at 60°C. Excess solvent was removed and gel pieces were alkylated with 80 µL 18.5 mg/mL iodoacetamide for 1 h at room temperature. The excess solvent was removed and gel pieces were washed twice with 200 µL of 25 mM ammonium bicarbonate in 50% acetonitrile for 15 min at 37°C and shrunk with 50 µL acetonitrile for 10 min at room temperature. Excess acetonitrile was removed and the gel pieces were dried in a speedvac. Finally, 10 µL 10 ng/µL trypsin (Promega, Madison, WI) and 10 µL 25 mM ammonium bicarbonate were added to each tube. Additional 25 mM ammonium bicarbonate was added until the gel pieces were fully swollen (~10–50 µL). Samples were then incubated for 4 h at 37°C and peptides recovered by collecting excess solvent. Gel pieces were extracted twice more with 50 µL 50% acetonitrile/5% formic acid, vortexing, centrifuging, and incubating for 15 min. Solvent was removed and combined with the previous recovered solvent, for a total of 3 extractions. Combined extracts were dried on a speedvac.

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Nanoflow LC-MS/MS and database search Samples were reconstituted in 20 µL 2% acetonitrile, 0.1% (v/v) formic acid, 0.01% (v/v) trifluoroacetic acid in water and analyzed using two different instrument platforms. Peptide samples from one full set of SDS gel pieces were injected (5 µL) using a NanoAcquity (Waters Corporation, Milford, MA) UPLC and loaded onto a Waters Symmetry C18 (180 μm i.d. × 2 cm) trapping column at a flow rate of 5 μL/min for 3 min. Peptides were then separated by in-line gradient elution using a 75 μm i.d. × 10 cm Waters BEH 130 (1.7 μm) analytical column, at a

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2% acetonitrile, 0.1% (v/v) formic acid, 0.01% (v/v) trifluoro acetic acid; mobile phase B: 98%


flow rate of 300 nL/min using a linear gradient from 3 to 90% B over 95 min (mobile phase A:

for both acquisition types. Precursor ions analyzed were subjected to dynamic exclusion for 30

acetonitrile, 0.1% formic acid, 0.01% trifluoro acetic acid). Peptides were eluted into an LTQ (Thermo Scientific) linear ion trap mass spectrometer operating in positive-ion electrospray and data-dependent acquisition modes. One mass spectrum was acquired over the m/z 400–2,000 range followed by serial tandem mass spectrometry (i.e. MS/MS) on the seven most abundant signals. Precursor ion isolation width was 2.0 Da, collision energy was 35%, ion population targets were 10,000 for MS and 5,000 for MS/MS, and maximum ion fill times were 200 msec

sec using a window of –0.5 to +1.5 m/z. The repeat count was 1 with a 30 sec delay; ions at m/z 371 and 445 were also excluded from MS/MS. Another set of similarly prepared samples were analyzed using a Proxeon Easy nanoLC (Thermo Scientific) system directly configured to an LTQ-Orbitrap Velos (Thermo Scientific) hybrid mass spectrometer. Peptide samples (2 µL) were loaded at 4 µL/min for 7 minutes onto a custom-made trap column (100 µm i.d. fused silica with Kasil frit) containing 2 cm of 200Å, 5 µm Magic C18AQ particles (Michrom Bioresources, Auburn, CA). Peptides were then eluted using a custom-made analytical column (75 µm I.D. fused silica) with gravity-pulled tip and packed with 25 cm 100Å, 5 µm Magic C18AQ particles (Michrom). Peptides were eluted with a linear gradient as described above. Mass spectrometry data were acquired using a data-dependent acquisition routine of acquiring one mass spectrum from m/z 350 -2,000 in the Orbitrap (resolution, 60,000; ion population, 1.0 x 106; maximum ion injection time, 500 msec) followed by tandem mass spectrometry in the linear ion trap (LTQ) of the 10 most abundant precursor ions observed in the mass spectrum. MS/MS data were acquired using a precursor isolation width of 22

2.0 Da, a collision energy of 35%, an ion population of 5,000, and a maximum ion fill time 50 msec. Charge state rejection of singly-charged ions and dynamic exclusion was utilized (–0.1 to +1.1 Da window, repeat count 1 (30 sec delay)) to minimize data redundancy and maximize peptide identification. The raw data files were processed using Extract MSN software (Thermo Scientific) and searched against the human index of the SwissProt database (version 09/24/11) with Mascot (version 2.3.02; Matrix Science, London, UK) and X! Tandem (The GPM (www.thegpm.org); version Cyclone (2010.12.01.1)) software packages. LTQ Orbitrap Velos data were searched

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using a parent mass tolerance of 15 ppm and a fragment mass tolerance of 0.5 Da. LTQ data utilized a parent tolerance of 1.2 Da and a fragment tolerance of 1.0 Da. Full tryptic specificity with 2 missed cleavages was considered and variable modifications of acetylation (protein Nterm), cyclization of N-terminal S-carbamoylmethylcysteine (peptide N-term), and oxidation (methionine) and fixed modification of carbamidomethylation (cysteine) were considered. All search results were loaded into Scaffold software (Version 3.3.1; Proteome Software, Portland, OR) for comparative analyses using spectral counting of tandem mass spectra and full annotation


of the data (Searle, 2010). Peptide identifications were accepted if they could be established at greater than 95.0% probability by the Peptide Prophet algorithm (Keller et al., 2002) following Scaffold delta-mass correction. Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least 2 identified peptides; protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii et al., 2003). Normalized spectral counts were calculated by dividing the spectral counts for an identified protein by the sum of the spectral counts per sample. Quantitative gene expression analysis RNA was isolated using Trizol reagent (Thermo Fisher Scientific) and trace DNA removed using the DNA-free RNA kit (Zymo Research, Irvine, CA). cDNA was synthesized using Superscript III (Thermo Fisher Scientific) and amplified using gene-specific primers (Supplementary Table 2) and iTAQ SYBR green supermix (Bio-Rad Laboratories). Reactions were run and data collected on an ABI PRISM 700 system (Thermo Fisher Scientific). Primers for PCR are displayed in supplementary Table 1.

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Immunoprecipitation Confluent cells on 100 mm diameter plates were harvested and solubilized in lysis buffer [10 mM Tris/HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 0.1 % Nonidet P40, 1 mM DTT and 1 mM PMSF]. Insoluble material was removed by centrifugation. The supernatants were incubated for 12 h at 4°C with 2 μg of anti-RUNX2 (M70 or S19; Santa Cruz Biotechnology, Santa Cruz, CA), anti-Flag (M2; Sigma-Aldrich), and a mixture of anti-rabbit and anti-goat IgG (Santa Cruz Biotechnology). Following the addition of 30 μL Protein A/G–agarose beads (Santa Cruz Biotechnology), mixtures were incubated for 2 h at 4°C with rotation. Immune complexes


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were washed three times with lysis buffer; the agarose beads were then boiled for 10 min in sample buffer. Immunoprecipitates were run in 8% acrylamide SDS-PAGE or 4–20% gradient precast gels (Bio-Rad), followed by Western blot analysis with RUVBL2, INTS3, BAZ1B, H2AX, phosphor-Ser139-H2AX antibodies. GST pull-down assays The proteins containing N-terminal glutathione S-transferase (GST) fused in-frame to the RUNX homologues were obtained by expression in Escherichia coli BL21 strain as previously reported (Pande et al., 2013). 1 μg of GST alone; GST fused N-terminal RHD domain, or Cterminal RUNX2 proteins were incubated with Glutathione Sepharose 4B (GE Healthcare) beads in 500 μL binding buffer (20 mM Tris pH 8.0, 100 mM KCl, 0.5% NP-40, 10 mM EDTA, 0.05 mM PMSF, 1 mM DTT) for 30 min at 4°C with rotation. The beads were washed four times with 500 μL binding buffer for 5 min at 4°C, and incubated with 1 mg of whole cell lysates from confluent Saos2 cells for 12 h at 4°C with rotation. After four washes with binding buffer, each for 5 min at 4°C, the beads were resuspended in 50 μL of sample buffer and boiled for 10 min at 95°C. The proteins that were retained with the beads were run in 8% acrylamide SDS-PAGE and analyzed by Western blot with specific antibodies against RUVBL2 (Santa Cruz Biotechnology), INTS3 (Abcam, Cambridge, UK), and BAZ1B (Abcam). Confocal microscopy To prepare cells for immunofluorescence confocal microscopy analysis, Saos2 cells were washed in Hanks balanced salt solution (Thermo Fisher Scientific), and were incubated in 0.5% Triton X-100 in cytoskeletal buffer for 10 minutes. This step removes soluble proteins from both the cytoplasm and nucleus. Cells were later fixed in 4% formaldehyde in cytoskeletal buffer for

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50 minutes on ice. Antibody staining: cells were stained with antibodies as described (Wagner et al., 2003). Primary antibody dilutions were as follows: rabbit polyclonal antibody against RUNX2 (M70), (1:100; Santa Cruz Biotechnology); mouse monoclonal antibody against RUVBL2 (B9), (1:40; Santa Cruz Biotechnology); mouse monoclonal antibody against BAZ1B (1:40; Santa Cruz Biotechnology); goat polyclonal antibody against INTS3 (1:500; Abcam); mouse monoclonal antibody against phosphorylated S139 of H2AX (1:100; Millipore). All antibodies were incubated overnight at 4°C. The following Alexa-fused secondary antibodies (Thermo Fisher Scientific) were used: Alexa Fluor 488 goat anti-rabbit IgG (1:2000); Alexa

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Fluor 568 goat anti-mouse IgG (1:2000); Alexa Fluor 594 donkey anti-goat IgG (1:2000); Alexa Fluor 488 donkey anti-rabbit IgG (1:2000). Coverslips were mounted with ProlongGold (Thermo Fisher Scientific). Images were collected using a Leica TCS SP5 confocal microscope using x 63 oil lens (numerical aperture = 1.4) at optimum zoom, fixed pinhole size (100 µm) and optimum Z-plane interval (from 0.2–0.3 µm z-stack), depending on the sample. The 405, 488 and 561excitation lasers were used to excite DAPI, Alexa 488, 568, and 594 respectively, and were activated sequentially during image collection. Images were analyzed using Image J and


Photoshop 5.0.

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van Driel, R., Wansink, D. G., van Steensel, B., Grande, M. A., Schul, W. and de Jong, L. (1995). Nuclear domains and the nuclear matrix. Int. Rev. Cytol. 162A, 151-189. Wagner, S., Chiosea, S. and Nickerson, J. A. (2003). The spatial targeting and nuclear matrix binding domains of SRm160. Proc Natl Acad Sci U S A 100, 3269-3274. Warters, R. L., Cassidy, P. B., Sunseri, J. A., Parsawar, K., Zhuplatov, S. B., Kramer, G. F. and Leachman, S. A. (2010). The nuclear matrix shell proteome of human epidermis. J. Dermatol. Sci. 58, 113-122. Xiao, A., Li, H., Shechter, D., Ahn, S. H., Fabrizio, L. A., Erdjument-Bromage, H., IshibeMurakami, S., Wang, B., Tempst, P., Hofmann, K. et al. (2009). WSTF regulates the H2A.X DNA damage response via a novel tyrosine kinase activity. Nature 457, 57-62. Yu, S., Teng, Y., Waters, R. and Reed, S. H. (2011). How chromatin is remodelled during DNA repair of UV-induced DNA damage in Saccharomyces cerevisiae. PLoS Genet 7, e1002124. Zaalishvili, T. M., Gabriadze, I. Y., Margiani, D. O., Philauri, V. R. and Surguladze, N. M. (2000). Participation of Poly(ADP-ribose)-polymerase of nuclear matrix in DNA repair.

Biochemistry. Biokhimiia 65, 659-661. 29

Zaidi, S. K., Sullivan, A. J., van Wijnen, A. J., Stein, J. L., Stein, G. S. and Lian, J. B. (2002). Integration of Runx and Smad regulatory signals at transcriptionally active subnuclear sites. Proc Natl Acad Sci U S A 99, 8048-8053. Zaidi, S. K., Javed, A., Choi, J. Y., van Wijnen, A. J., Stein, J. L., Lian, J. B. and Stein, G. S. (2001). A specific targeting signal directs Runx2/Cbfa1 to subnuclear domains and contributes to transactivation of the osteocalcin gene. J. Cell Sci. 114, 3093-3102. Zaidi, S. K., Sullivan, A. J., Medina, R., Ito, Y., van Wijnen, A. J., Stein, J. L., Lian, J. B. and Stein, G. S. (2004). Tyrosine phosphorylation controls Runx2-mediated subnuclear

Accepted manuscript

targeting of YAP to repress transcription. EMBO J. 23, 790-799. Zaidi, S. K., Javed, A., Pratap, J., Schroeder, T. M., J, J. W., Lian, J. B., van Wijnen, A. J., Stein, G. S. and Stein, J. L. (2006). Alterations in intranuclear localization of Runx2 affect biological activity. J. Cell. Physiol. 209, 935-942. Zaidi, S. K., Young, D. W., Javed, A., Pratap, J., Montecino, M., van Wijnen, A., Lian, J. B., Stein, J. L. and Stein, G. S. (2007a). Nuclear microenvironments in biological control and cancer. Nat. Rev. Cancer 7, 454-463.


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30

Zybailov, B., Coleman, M. K., Florens, L. and Washburn, M. P. (2005). Correlation of relative abundance ratios derived from peptide ion chromatograms and spectrum counting for


Accepted manuscript

quantitative proteomic analysis using stable isotope labeling. Anal. Chem. 77, 6218-6224.

31

A

siRunx2

B

siNS

RUNX2

siRunx2 siNS W C D N W C D

N

RUNX2

GAPDH H3(Acetyl) Lamin B FBR B23

C Total nuclear matrix proteins (1093)

W: whole cell lysate C: cytoplasmic extract D: DNase I extract N: nuclear matrix

D

7 6

Gene Ontology

Spectral counting RUNX2-dependent proteins (207) Spectral counting

Log2 decrease

Nuclear proteins (721)

5 4 3 2 1 0

Downregulated by siRunx2 (136)

RUNX2 INTS3 CTCF GATA6 CNOT1 L3MBTL3 RBBP4 WHSC1 ROD1 PRMT1 RUVBL2 KLF5 PHF5A POLR1E CEBPZ RUNX3 EBNA1BP2 BAZ1B DDX3X PFH2 PINX1


Accepted manuscript

Size bar = 5 m *Thickness of z section = 0.2 m

Number of peptides 2 7 2 2 2 2 5 4 8 3 18 2 3 6 31 6 6 13 6 4 5 Transcription control T T T T T T T T T T T C C C C C Chromatin remodeler Histone modifier

H

H

E

*Calculated by dividing the spectral counts for an identified protein by the sum of the spectral counts per sample.

Figure 1


C

B

4

Saos2 W

C

D

N

RUNX2

3

RUNX3 2

ROD1 RUVBL2

1

BAZ1B INTS3

U

N X IN 2 TS C 3 T G CF AT C A6 N L3 OT M 1 B W TL3 H SC R 1 O PR D1 R MT U 1 VB L KL 2 PH F5 F R 5A U N BA X3 Z1 PH B F2

0

R

Fold decrease by siRunx2

Accepted manuscript

A

PRMT1 CTCF

RUVBL2

INTS3

BAZ1B

GAPDH W: whole cell lysate C: cytoplasmic extract D: DNase I extract N: nuclear matrix

Size bar = 5 µm, thickness of z section = 0.2 µm

Figure 2

A

B U2OS

Saos2

RUNX2 RHD

IP

NMTS

Input

115-RTDSPNFLCVLPSHWR-131

IgG

Input

M70

IgG

IP M70

RUVBL2

RUVBL2 AAA

RUNX2

Accepted manuscript

417-KGTEVQVDDIKRVYSLFLDESR-438

IP Input

INTS3

IgG

IP S19

Input

IgG

S19

BAZ1B

5-KGKGAAAAAAASGAAGGGGGGAGA-30

RUNX2

BAZ1B KD

PHD

DDT

BRD


1069-GGLGYVEETSEFEAR-1083

D

C

RUNX2

C

+ +

RUNX2 WT RUNX2 C Flag-RUVBL2

RUVBL2 * nonspecific signal

BAZ1B

C

INTS3 RUVBL2

INTS3

WT *

RUNX2

C

ut

+ +

*

BAZ1B

WB

+ -

WB

+ -

Inp

IP: Flag

Coomassie blue

Beads only

376

-

R

G

RHD

1

INTS3

G

T-C

528

GS

1

NMTS

T-R

NMTS

T

RHD

GS

WT

RHD

GS

RUNX2

250 150 100 75 GST-C

50

GST-R

37 Size (kDa)

Figure 3

A RUNX2 RUVBL2 INTS3 BAZ1B

W

C

D

N

W

C

D

B

PC-3

N

Input

MDA-MB-231 IP

IP IgG M70

RUVBL2

RUVBL2

RUNX2

RUNX2

BAZ1B RUNX2

Input

IP IgG S19

Input

IgG

Input

IgG

M70

IP

BAZ1B

S19

RUNX2


Accepted manuscript

GAPDH LaminB H3(Acetyl)

MDA-MB-231

PC-3

Figure 4

A RUNX2 RUVBL2

RUNX2 BAZ1B

RUNX2 INTS3

Accepted manuscript

Size bar = 5 µm *Thickness of z section = 0.2 µm


B

C

INTS3-RUVBL2 INTS3

BAZ1B BAZ1B

RUVBL2 H2AX

Protein RUNX2

RUVBL2

INTS3 BAZ1B

Biological process Transcription regulation Osteoblast differentiation Bone metastasis DNA damage DNA recombination DNA repair Growth regulation Transcription regulation DNA damage repair DNA damage Transcription regulation

H2AX

References Long, 2012 Pratap et al., 2011 Kashiwaba et al., 2010 Jha and Dutta, 2009 Ruthernburg et al., 2005 Skaar et al., 2009 Barnett and Krebs, 2011 Xiao et al., 2009

Figure 5

A

siNS 30 min

60 min

Not Treated

30 min

60 min

BAZ1B

Accepted manuscript RUNX2

RUNX2 INTS3

RUNX2 RUVBL2

Not Treated

siRUNX2

RUNX2 Journal of Cell RUNX2 Science -H2AX -H2AX

B

C

siNS Not Treated

30 min

60 min

Western blot siRUNX2 siNS NT 5 30 NT 5 30 Post UV (min) -H2AX H2AX RUNX2

siRUNX2

GAPDH RUVBL2 INTS3 BAZ1B

Figure 6

A

RUNX2 siNS siRUNX2

Merged (RUNX2/ -H2AX) siNS siRUNX2

30 min 60 min

Size bar = 5 m, Thickness of z section = 0.2 m

60 40 20 0

siNS siRUNX2

80 60

20 0

NT 30 min 60 min Time Post UV

Input –

30 min post UV siNS

siRUNX2

40

NT 30 min 60 min Time Post UV

E

D

RUNX2

80

C

-H2AX

siNS siRUNX2

UV

IgG

IP: FLAG – UV

DAPI

100 cells with -H2AX foci (%)


B

Number of -H2AX foci/nuclei

Accepted manuscript

Post UV

Not Treated

-H2AX/DAPI siNS siRUNX2

Size bar = 5 m *Thickness of z section = 0.2 m

BAZ1B INTS3 RUNX2

WB

P-S139-H2AX H2AX P-Y142-H2AX *Long

Figure 7

A

Post UV 60 min

siRunx2 siNS cont UV cont UV

Acet-H3-K56

Accepted manuscript

RUNX2

Control

siRunx2

0.5

0

0

2

10 1

5 0

0

control UV control UV

H2AX

0.5

15


P-H2AX-Y142

1


P-H2AX-S139

1


Relative level of modification

H3

GAPDH

siNS

siRunx2

D

IP: FLAG-H2AX siNS IgG

–

UV

siRunx2 –

UV

BAZ1B FLAG-H2AX

72 hrs

Postof UVCell Science Journal

siNS

Acet-H3-K56 / Acet-H3-K9 / P-H2AX-S139 / P-H2AX-Y142 / total H3 total H3 total H2AX total H2AX 1.5 1.5 20 3

Acet-H3-K9

C

B

Figure 8

Spatially distinct functions of Clb2 in the DNA damage response.

Targeting the DNA Damage Response in Cancer.

RUNX2: A Master Bone Growth Regulator That May Be Involved in the DNA Damage Response.

WRNIP1 functions upstream of DNA polymerase η in the UV-induced DNA damage response.

Targeting DNA damage response in cancer therapy.

DNA damage response in adult stem cells.

Roles of RNA-Binding Proteins in DNA Damage Response.

Elevated DNA damage response in pancreatic cancer.

Phosphorylation-Dependent Regulation of the DNA Damage Response of Adaptor Protein KIBRA in Cancer Cells.

Doxycycline and therapeutic targeting of the DNA damage response in cancer cells: old drug, new purpose.

Potential negative regulation of damage-recognition proteins in cisplatin-resistant HeLa cells in response to DNA damage.

The catalytic topoisomerase II inhibitor dexrazoxane induces DNA breaks, ATF3 and the DNA damage response in cancer cells.

DNA damage response genes and the development of cancer metastasis.

The DNA damage response induces antigen presenting cell-like functions in fibroblasts.

Role of polycomb group proteins in the DNA damage response--a reassessment.

The role of the cancer stem cell marker CD271 in DNA damage response and drug resistance of melanoma cells.

J domain independent functions of J proteins.

Bufalin induces cell death in human lung cancer cells through disruption of DNA damage response pathways.

DNA damage response (DDR) via NKX3.1 expression in prostate cells.

Autophagy in DNA damage response.

MDM4 regulation by the let-7 miRNA family in the DNA damage response of glioma cells.

Protein degradation pathways regulate the functions of helicases in the DNA damage response and maintenance of genomic stability.

Targeting the epigenetics of the DNA damage response in breast cancer.

The Role of PALB2 in the DNA Damage Response and Cancer Predisposition.