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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*

1Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA

2Institute of Biomedicine, Department of Biochemistry and Developmental Biology, University of Helsinki, Finland

3Department of Biochemistry and Molecular Pharmacology & Proteomics and Mass Spectrometry Facility, University of Massachusetts Medical School, Worcester, MA

4Department of Biochemistry & Vermont Cancer Center, University of Vermont Medical School, Burlington, VT, USA

5Departments 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: gary.stein@uvm.edu

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.

© 2015. Published by The Company of Biologists Ltd.Jo

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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

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

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

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

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

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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,

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).

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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,

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

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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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 nuclease-

resistant 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

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 down-

regulated >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

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 RUNX2-

siRNA 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.

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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). Co-

immunoprecipitation 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 co-

immunoprecipitated with M70 antibody, which recognizes a C-terminal domain in RUNX2.

BAZ1B was also co-immunoprecipitated using S19 antibody, which interacts with the N-

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 FLAG-

RUVBL2 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 Runt-

homology 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 co-

immunoprecipitation analysis. First, we fractionated cells and conducted Western blot analysis;

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

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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

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

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

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.

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

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

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

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nontargeting siRNA–transfected control cells (siNS) (Fig. 8C). Finally, immunoprecipitation

analysis of Saos2 cells cotransfected with the FLAG-H2AX expression construct and RUNX2-

targeting 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

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

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 C-

terminal 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

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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

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

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 multi-

functional 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 RUNX2-

knockdown 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

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

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

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;

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

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.

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

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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 MDA-

MB-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%

SDS-PAGE and analyzed by Western blot with the indicated primary antibodies. (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. 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-UV-

irradiation 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

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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)

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)

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Immunoprecipitation analysis of UV-irradiated (UV, 60 min) or unirradiated (–) Saos2 cells co-

transfected with FLAG-H2AX expression plasmid and the indicated siRNA using anti-FLAG

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

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

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

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,

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

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 siRNA-

transfected 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)

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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

matrix fraction was loaded on SDS-polyacrylamide gels.

In-gel trypsin digestion

SDS-PAGE was performed with 4–15% linear gradient polyacrylamide gels containing Tris-

HCl (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

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

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

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

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

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

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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

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 N-

term), 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

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 C-

terminal 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

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 561-

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

Total nuclear matrix proteins (1093)

Nuclear proteins (721)

RUNX2-dependent proteins (207)

Downregulated by siRunx2 (136)

Gene Ontology

Spectral counting

Spectral counting 0

1

2

3

4

5

6

7

RU

NX

2 IN

TS3

CTC

F G

ATA

6 C

NO

T1

L3M

BTL

3 R

BB

P4

WH

SC

1 R

OD

1 P

RM

T1

RU

VB

L2

KLF

5 P

HF5

A P

OLR

1E

CE

BP

Z R

UN

X3

EB

NA

1BP

2 B

AZ1

B

DD

X3X

P

FH2

PIN

X1

2 7 2 2 2 2 5 4 8 3 18 2 3 6 31 6 6 13 6 4 5 Number of peptides Transcription control

Chromatin remodeler Histone modifier

T T T T T T T T T T T C C C C C

H H

siRunx2 siNS R

UN

X2

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

A

Lamin B

RUNX2

GAPDHH3(Acetyl)

FBR

B23

W C D N W C D N siRunx2 siNS

C

E

D

W: whole cell lysateC: cytoplasmic extractD: DNase I extractN: nuclear matrix

B

Log 2 d

ecre

ase

Figure 1

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Fold

dec

reas

e by

siR

unx2

0

1

2

3

4

RUNX2

INTS3

CTCF

GATA6

CNOT1

L3MBTL3

WHSC1

ROD1

PRMT1

RUVBL2

KLF5

PHF5A

RUNX3

BAZ1B

PHF2

A

INTS3 BAZ1B RUVBL2 C

W C D N

Saos2

RUNX2

RUNX3

CTCF

GAPDH

PRMT1

BAZ1B

RUVBL2

INTS3

ROD1

B

Figure 2

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

W: whole cell lysateC: cytoplasmic extractD: DNase I extractN: nuclear matrix

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D

Input IgG S19

Saos2

RUNX2

RUVBL2

U2OS

Input IgG M70 IP

Input IgG M70 IP

BAZ1B

IP Input IgG S19

IP

RUNX2

Inpu

t

GST

-R

GST

-C

GST

BAZ1B

INTS3

RUVBL2

WB

GST-R

GST-C

250 150

100 75

50

37 Size (kDa)

Coo

mas

sie

blue

NMTS RHD

RUNX2

R

C

G

G

BAZ1B

RUNX2 WB

RUNX2 WT

RUNX2 C

Flag-RUVBL2 - - + - +

- + + - - - - - + +

IP: Flag

Bea

ds o

nly

RUVBL2

INTS3

WT

C

INTS3

NMTS RHD

RUNX2

RHD

WT

C 376 1

1 528

NMTS RHD

115-RTDSPNFLCVLPSHWR-131

RUNX2

417-KGTEVQVDDIKRVYSLFLDESR-438

AAA RUVBL2

5-KGKGAAAAAAASGAAGGGGGGAGA-30

INTS3

BRD PHD DDT KD BAZ1B

1069-GGLGYVEETSEFEAR-1083

Figure 3

A

C

B

*

*

* nonspecific signal

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Figure 4

RUNX2

GAPDH

PC-3

W C D N

BAZ1B INTS3

RUVBL2

LaminB H3(Acetyl)

MDA-MB-231

W C D N

A

Input IgG S19 IP

BAZ1B

RUNX2

Input IgG M70

RUVBL2

RUNX2

IP

RUVBL2 Input IgG M70

IP

RUNX2

Input IgG S19 IP

BAZ1B

RUNX2

PC-3 MDA-MB-231 B

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RUNX2 RUVBL2

RUNX2 BAZ1B

RUNX2 INTS3

Figure 5

A

C

B

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

INTS3 BAZ1BRUVBL2

H2AX H2AX

Protein Biological process References

RUNX2 Transcription regulation

Long, 2012 Pratap et al., 2011

Osteoblast differentiation Bone metastasis

RUVBL2

DNA damage Kashiwaba et al., 2010 Jha and Dutta, 2009

Ruthernburg et al., 2005

DNA recombination DNA repair Growth regulation Transcription regulation

INTS3 DNA damage repair Skaar et al., 2009

BAZ1B DNA damage Barnett and Krebs, 2011

Xiao et al., 2009 Transcription regulation

INTS3-RUVBL2 BAZ1B

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siNS R

UN

X2

RU

VB

L2

RU

NX

2 IN

TS3

RU

NX

2 B

AZ1

B

30 min 60 min Not Treated 60 min

siRUNX2 30 min Not Treated

Figure 6

A

CB

60 min

siNS Not Treated

RU

NX

2

-H2A

X

30 min

siRUNX2

RU

NX

2

-H2A

X

-H2AX

GAPDH

RUVBL2

RUNX2

H2AX

Post UV (min) NT 5 30 NT 5 30 siNS

INTS3

BAZ1B

siRUNX2

Western blot

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IP: FLAG

H2AX

P-S139-H2AX

RUNX2

BAZ1B

– UV IgG – UV Input

WB

INTS3

P-Y142-H2AX

*Long

0

20

40

60

80

100

NT 30 min 60 min

siNS siRUNX2

cel

ls w

ith

-H2A

X fo

ci (%

)

Time Post UV

-H2AX/DAPI

Not

Tre

ated

30

min

60

min

Pos

t UV

siNS siRUNX2 siNS siRUNX2 siNS siRUNX2

Merged (RUNX2/ -H2AX) RUNX2

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

Num

ber o

f -H

2AX

foci

/nuc

lei

0

20

40

60

80

NT 30 min 60 min

siNS siRUNX2

Time Post UV

siNS siRUNX2

30 min post UV

RU

NX

2

-H2A

X

DA

PI

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

A

B

E

C D

Figure 7

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Acet-H3-K56 / total H3

Acet-H3-K9 / total H3

P-H2AX-S139 / total H2AX

P-H2AX-Y142 / total H2AX

B siNS siRunx2

RUNX2

Acet-H3-K56

Acet-H3-K9

cont UV cont UV siNS siRunx2

GAPDH

H3

H2AX

P-H2AX-S139

Post UV 60 min A

0

0.5

1

1.5

cont

rol

UV

co

ntro

l U

V 0

0.5

1

1.5

cont

rol

UV

co

ntro

l U

V 0

5

10

15

20

cont

rol

UV

co

ntro

l U

V 0

1

2

3

cont

rol

UV

co

ntro

l U

V

Rel

ativ

e le

vel o

f mod

ifica

tion

P-H2AX-Y142

siNS siRunx2

Con

trol

Post

UV

72 h

rs

C

BAZ1B

FLAG-H2AX

IgG – UV – UV

siNS siRunx2

IP: FLAG-H2AX D

Figure 8

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