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Potential target gene of INO80 chromatin remodeling complex -BRCA2- and CDKN1A- interacting protein (BCCIP)# 5 Jiaming Su, Yi Sui, Jian Ding, Yang Yang, Shuang Shen, Jingji Jin** (Jilin University, School of Life Sciences) Abstract: BRCA2- and CDKN1A-interacting protein (BCCIP) has been considered an important cofactor for BRCA2 in tumor suppression. We previously observed the down-regulation of BCCIP in multiple clinically diagnosed primary tumor tissues including ovarian cancer, renal cell carcinoma and 10 colorectal carcinoma. In this study, we first demonstrate that BCCIP expression was regulated by human INO80 (hINO80) chromatin remodeling complex in cells. In chromatin immunoprecipitation (ChIP) assays, we show that the catalytic subunit hINO80 protein and YY1, a subunit of hINO80 complex, co-localize the BCCIP promoter region (+0.25 kb downstream of the BCCIP), and the existence of hINO80 is required for recruiting the hINO80 complex to BCCIP promoter. RNAi 15 knockdown of hINO80, Arp8 or YY1 resulted in reduction of BCCIP expression both in mRNA and protein level. In contrast, over-expression of hINO80, Arp8 or Arp5 enhanced the luciferase activity of pGL4-BCCIP. Together, as a novel target gene, BCCIP is regulated by hINO80 chromatin remodeling complex. Our findings will provide: 1) a theoretical basis to further elucidate the collaborative functions between the hINO80 complex and BCCIP in human cells; 2) an experimental basis for 20 clarifying the role of BCCIP and BRCA2 in tumor suppression.

Key words: BCCIP; INO80; chromatin remodeling complex

0 Introduction BCCIP is a BRCA2- and CDKN1A (Cip1, p21)-interacting nuclear protein (1-2). Alternatively 25 spliced BCCIPα and BCCIPβ share N-terminal 258 amino acids which form the N-terminal acidic domain (NAD) and the internal conserved domain (ICD) (1). ICD of BCCIPs interacts with BRCA2. Knockdown BCCIP with siRNA markedly reduces BRCA2 and RAD51 nuclear foci and reduces homologous recombinational repair (HRR) of DNA double-strand breaks (DSBs), suggesting the important role of BCCIP in BRCA2- and RAD51-dependent response to DNA 30 damage and HRR (3). Over-expression of BCCIP delays G1 to S cell cycle progression and elevating p21 expression, suggesting the regulation of BCCIP in cell growth (4). In contrast, silencing BCCIP reduces p21 expression in a p53 dependent manner and impairs G1 to S checkpoint activation in response to ionizing radiation in HT1080 cells (5). Furthermore, knockdown BCCIP by siRNA in HT1080 cells leads to chromosomal polyploidization, 35 centrosome amplification and abnormal mitotic spindle formation, indicating an essential role of BCCIP in chromosome stability and cytokinesis (6). Together, BCCIP has been implicated in various biological processes in cells. Human INO80 (hINO80) complex is one of the most highly conserved chromatin remodelers. It shares eight core subunits with yeast INO80 (yINO80) complex, including a SNF2 40 ATPase-INO80 catalytic subunit, actin-related proteins Arp4, Arp5, Arp8, Tip49a and Tip49b AAA+ ATPases, and hIes2 and hIes6 (7). Like the other chromatin remodelers, hINO80 complex possesses ATPase and DNA nucleosomal sliding activities. Electron microscopy (EM) studies showed that the yINO80 complex formed an elongated embryo-like shape with

Foundations: Research Fund for the Doctoral Program of Higher Education of China (No. 20110061110020); National Natural Science Foundation of China (No. 31571316) Brief author introduction:Jiaming Su (1988-), male, PhD, Biochemistry and Molecular Biology Correspondance author: Jingji Jin (1962-), female, professor, Epigenetics. E-mail: [email protected]

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head-neck-body-foot topology in yeast (8,9). Although the complete crystal structure of hINO80 45 complex is still unknown, three modules that assemble on three distinct domains of the hINO80 protein have been reported. All revolutionarily conserved subunits assembled on the conserved helicase-SANT-associated/post-HSA (HSA/PTH) and ATPase domains of hINO80 protein. Both HSA/PTH and ATPase domains are essential for catalyzing the ATP-dependent nucleosome remodeling activity of the hINO80 complex (10). 50 Biochemical purifications revealed that YY1, a GLI-Krüppel family transcription factor, is tightly associated with the hINO80 chromatin remodeling complex (7). Although YY1 was initially thought to be metazoan specific subunit of the INO80 complex, it was subsequently characterized the orthologous of YY1 in Schizosaccharomyces pombe INO80 complex (11). In the hINO80 complex, YY1 with Arp4 and Arp8 together participate in assembling HSA/PTH module, 55 suggesting that like other conserved subunits, YY1 is also essential for maintaining the ATP-dependent nucleosome remodeling activity of the hINO80 complex (10). yINO80 is required for proper transcriptional regulation of many target genes (12), while in Drosophila, INO80 facilitates transcriptional repression of ecdysone-regulated genes during prepupal development (13). The master transcription factors play integral roles in the pluripotency circuitry of embryonic 60 stem cells (ESCs). Recent experimental evidences demonstrate that INO80 is required in ESC self-renewal, somatic cell reprogramming, and blastocyst development (14). In addition, genome-wide studies show that INO80 binds many, but not all, loci in the genome (15), however, it is unclear whether there is a direct correlation between INO80 binding and target gene expression levels. One possibility is that transcription factor like YY1, a subunit of the INO80 65 complex, plays a role in helping in recognition of INO80-target genes, and recruiting the INO80 complex to some target genes such as CDC6 and GRP78 (16). As an important cofactor for BRCA2 in tumor suppression, BCCIP has been implicated in many important cellular processes with obvious links to cancer. We and other colleagues have previously reported that BCCIP was downregulated in several cancer tissues such as renal cell 70 carcinoma, ovarian cancer and colorectal carcinoma (17,18). Therefore, it is important to elucidate the role of BCCIP in tumorigenesis, especially to know how the BCCIP is regulated in cells. In this study, using combined methods of chromatin immunoprecipitation (ChIP) assays, RNAi knockdown, luciferase reporter assay, immunofluorescence staining, qPCR and Western blots, we first demonstrate that the BCCIP regulated by hINO80 complex, suggesting that BCCIP might be 75 a novel target gene of the hINO80 complex. The regulation of BCCIP by hINO80 complex will shed light on new functions of BCCIP-hINO80 complex. 1 Materials and Methods 1.1 Antibodies Anti-YY1 (H414, sc-1703X) rabbit polyclonal antibody, rabbit total IgG (sc-2027) and mouse 80 total IgG (sc-2025) were obtained from Santa Cruz Biotechnology (U.S.A.). Anti-Flag (M2)-agarose and anti-Flag M2 (F3165) monoclonal antibody were from Sigma (U.S.A.). Anti-INO80 (NM_017553, residue 1-526 aa), anti-hArp8 (NM_022899, full length), and anti-GAPDH (NM_002046, full length) rabbit polyclonal antibodies were raised against bacterially expressed proteins (Jilin University). Anti-BCCIP (16043-1-AP) polyclonal antibody 85 was purchased from Proteintech Group (China, Wuhan). 1.2 Cell culture/Maintenance Human embryonic kidney (HEK) 293T cells and HeLa cells were maintained in Dulbecco’s modified Eagle’s Medium (GIBCO, Life TechnologiesTM, U.S.A.) supplemented with 10% fetal

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bovine serum (KangYuan Biology, China) and 1% Penicillin-Streptomycin mixture (Thermo

90 Fisher Scientific, U.S.A) at 37°C in the presence of 5% CO2. 1.3 Reverse Transcription PCR (RT-PCR) Total RNA was isolated using RNAiso Plus (Takara, Japan). Total RNA (1 μg) from each sample was used as a template to produce cDNA with PrimeScript 1st Strand cDNA Synthesis Kit (Takara). hINO80, Arp8, Arp5, hIes6, hIes2, YY1, BCCIP and GAPDH mRNA was measured by 95 quantitative real time PCR (qPCR) with EcoTM Real-Time PCR System (Illumina, Gene Company Limited (China, Hongkong). All PCR products were synthesized using the following program: initial denaturation step was conducted at 95°C for 30 seconds, followed by 35 cycles of denaturation at 95°C for 5 seconds, annealing at 60°C for 30 seconds and extension at 72°C for 30 seconds. Following primer sets were used for qPCR anaysis: INO80, 100 5’-CGGAATCGGCTTTTGCTA-3’ (forward) and 5’-TGTCGGCTGGTCAGTTGG-3’ (reverse), primers yielded a 292 bp product; Arp8, 5’-CCAGGCTGAGAAGGGTGATA-3’ (forward) and 5’-GCAGGAAGAGTGTCTGTGGC-3’ (reverse), yielded a 200 bp product; Arp5, 5’-GTACCGCTGGTGCTGGACAA-3’ (forward) and 5’-TGAAGCTCCAGGTTGACCGG-3’ (reverse), yielded a 236 bp product; Ies6, 5’-ATGGCGGCGCAAATTCCAAT-3’ (forward) and 105 5’-AATGGCAAAGGTTTGGCAGC-3’ (reverse), yielded a 247 bp product; Ies2, 5’-GGAGAAGCCCTGGAGTTGAG-3’ (forward) and 5’-GGAACACTCTTGGTCCCCAG-3’ (reverse), yielded a 199 bp product; YY1, 5’-CCCTCATAAAGGCTGCACAA-3’ (forward) and 5’-TGAACCAGTTGGTGTCGTTT-3’ (reverse), yielded a 147 bp product; BCCIP, 5’-TCAAGAGTTGGTTCTACGCTTC-3’ (forward) and 5’- CATGGGCAGAGCGATCTGT-3’ 110 (reverse), yielded a 111 bp product. The primer sets used for internal control GAPDH were as follows: 5’-ATCACTGCCACCCAGAAGAC-3’ (forward) and 5’- ATGAGGTCCACCACCCTGTT-3’ (reverse), yielded a 443 bp product. 1.4 Constructions of plasmids and Transient transfection Full-length cDNAs encoding the human INO80 (NM_017553), Arp5 (BC038402) and Arp8 115 (NM_022899) proteins were sub-cloned with Flag tags into pcDNA3.1(-). To generate the Flag-tagged hINO80, Arp5 and Arp8 proteins, cells were seeded in 6-well tissue culture plates (~3×105 cells/well). The next day, 2 µg of the plasmids were then transiently transfected using 6 µL PEI (Cat. 23966, Polysciences, China) according to the manufacturer’s recommendation. After 48 hours of transfection, the whole cell lysate was prepared and subjected to SDS-PAGE gel 120 (12%). The proteins were analyzed by western blot with Flag antibody. INO80-shRNA plasmids were also transfected using PEI. 1.5 Transient transfection and siRNA knockdown HEK293T cells were transfected with 0.2~0.8 μg pcDNA/Flag-INO80, pcDNA/Flag-Arp8, pcDNA/Flag-Arp5 and (pcDNA3.1 as control) for over-expression of hINO80, Arp5 and Arp8. 125 All siRNAs were ordered from Dharmacon (U.S.A.). HeLa cells were transfected with 15~20 pmol Arp8- (Customized), Arp5- (Customized) and non-targeting-siRNA (D-001206, as control), hINO80- (D-004176), hIes2- (D-009848) or hIes6-siRNA (D-019327) SMART pool for knocking down hINO80, Arp5, Arp8, hIes2 and hIes6. 48 hours after transfection, total RNA or whole-cell extract was prepared for RT-PCR and western blot analysis. In ChIP assay, 293T cells were 130 transfected with pBS-shINO80 plasmid (10 μg/10 cm culture plate) for knocking down hINO80. 1.6 Luciferase reporter assay A length of 391 bp (-353 bp to +38 bp) region of BCCIP promoter was introduced into pGL4-luc vector. Luciferase reporter assays were performed essentially as described (19). HEK293T cells were co-transfected with 0.8 μg of pGL4 (Promega) which encodes firefly luciferase, 2 ng of the

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135 control plasmid Renilla luciferase vector (Promega) which encodes renilla luciferase and effector plasmid expressing pGL4-BCCIP using PEI reagent (Polysciences, China). Total effector plasmid in each transfection was adjusted to 0.8 μg with empty vector. After 48 hours, pGL4-BCCIP transactivation activity was determined by measuring firefly and Renilla luciferase activities using the Dual-Luciferase Reporter assay kit (Promega, USA) and by normalizing firefly 140 to Renilla luciferase. 1.7 DNA microarray and Statistical analysis Specific genes knocked down with siRNA (include siINO80, siArp5, siArp8, siIes2 and siIes6) HeLa cells were stored in an RNA hold solution (ER501-01, Beijing Transgen Biotech Co., Ltd.) and sent to EMTD Science and Technology Development Co., Ltd. (Beijing, China) for DNA 145 microarray analysis. The Illumina microarray datasets were accessible from the National Center for Biotechnology Information Gene Expression Omnibus (GEO) data repository (http://www.ncbi.nlm.nih.gov/geo/) using the series accession number GSE68655. The online biological classification tool DAVID (Database for Annotation, Visualization and Integrated Discovery) was used to perform the enrichment analysis (20,21). DAVID was utilized to conduct 150 Gene Ontology (GO) (22) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) (23) pathway enrichment analysis of differentially expressed genes (DEGs). During the analysis, p<0.05 and FDR<0.02 genes used in the annotation were defined as statistically significant. 1.8 Immunofluorescence Staining HeLa cells were cultured and grown to ~30% confluence in 24-well plates containing a coverslips 155 (8D1007, Nest) on each well. Cells were then transfected with siINO80, siArp8 and siYY1 using RNAMAX (Invitrogen). 48 hours later, cells were permeabilized with 0.5% TritonX-100 in PBS buffer for 5 min, followed by blocking with 1% bovine serum albumin in PBS for 1 hour at 37°C. Then, cells were incubated with BCCIP antibody (1:200) at room temperature, then stained with FITC-conjugated secondary antibody (rabbit/green: 1:300, sc-2012). Cell nuclei were stained with 160 DAPI containing Vectashield (Vector Laboraries,Inc. Cat.No-H-1200). Fluorescence images were observed with Olympus BX40F Microscope (Olympus Corporation). 1.9 Chromatin Immunoprecipitation (ChIP) Assay One 10 cm dish containing 1 × 107 of HEK293T cells grown to 80% of confluence were used for each ChIP. ChIP assays were carried out essentially as described using INO80 and YY1 antibodies 165 (16). ChIP DNA was subjected to qPCR. Each experiment was performed 2-3 independent times. Both ChIP and no-Ab signal were normalized to total input. Seven primer sets were used in quantitative real time PCR (qPCR). The primer sets for qPCR on the promoter region of BCCIP were as follows: BCCIP -1.7 kb (-1769 bp ~ -1603 bp), 5’-GACAGCTTCCTCTCTTCTCCA-3’ (forward) and 5’-ATTTCGGTCTTTCCTGGGAGT-3’ (reverse); -1.2 kb (-1281 bp ~ -1093 bp), 170 5’- CCCTCCACTCTTCTCTCCAAA-3’ (forward) and 5’-CCCTCCACTCTTCTCTCCAAA-3’ (reverse); -0.7 kb (-860 bp ~ -694 bp), 5’-CTCGAACTCCTGACCTCTGGTG-3’ (forward) and 5’-CTGGAGCGCATTTCTTGACC-3’ (reverse); -0.2 kb (-205 bp ~ -53 bp), 5’- AGCTTTGGGAGGAGTGATGGAG-3’ (forward) and 5’- TAGCCAAGGGGGTGAGGAAC-3’ (reverse); +0.25 kb (+177 bp ~ +353 bp), 5’- TCATTGACGAGGTGAGAAGGAC-3’ (forward) 175 and 5’- GCAGTGTGTTAAAGACCGAGGAG-3’ (reverse); +2.9 kb (+2841 bp ~ +2984 bp), 5’- GCTGCCGTGTCTGGTTTATTTG-3’ (forward) and 5’- GCCAAGATTGCCATCTGTGAAG-3’ (reverse). 2 Results 2.1 BCCIP was screened as one of the potential target genes of hINO80 chromatin

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180 remodeling complex from gene expression profiles We previously identified a hINO80 ATP-dependent chromatin remodeling complex which is composed of N-terminal domain, the helicase-SANT-associated (HSA) module, and the SNF2 module (7). HSA and SNF2 modules form an enzyme core (Fig. 1A) and play a key role in regulating the whole enzyme activity of the INO80 complex (10). Although increasing evidence 185 suggests that hINO80 complex plays an important role in gene transcriptional regulation (15,24), the exact regulatory mechanisms are still unclear. In order to explore the genes regulated by hINO80 complex, HeLa cells with specific siRNA (siINO80, siArp8, Arp5, siIes2 and siIes6) knocked down (Fig. 1B) were sent to EMTD Science and Technology Development Co., Ltd. (Beijing, China) for gene expression profile analysis. From DNA microarray analyses using 190 Illumina Genomestudio, a total of 1932, 1445, 1235, 2707 and 2159 genes were shown to be differentially expressed among hINO80, Arp8, Arp5, hIes6 or hIes2 and NT siRNA knockdown HeLa cells, respectively (Fig. 1C). The shared and distinct gene expression in each population is depicted in a Venn diagram (Fig. 1D). Interestingly, a total of 133 genes are co-regulated by INO80, hIes2, hIes6 and Arp5 which are components of the SNF2 module. On the other hand a 195 total of 602 genes were co-regulated by INO80 and Arp8 which participate in assembling HSA module. Selected overlap of expressed genes including 8 down- and 6 up-regulated genes in INO80 complex knockdown HeLa cells are shown in Table 1. mRNA from different siRNA knockdown cells was measured with RT-qPCR. Compared to internal control GAPDH, selected gene mRNA including AMHR2, BCCIP, CDH1, CCL28, FGFBP1, GRTP1, TPD52 and WTIP 200 was down-regulated with siRNA knockdown of hINO80, Arp8, Arp5, hIes6, and hIes2 in HeLa cells. In contrast, CDKN1A (p21), EGFR, GAS1, MBD1, RAF1 and TGFBR1 mRNAs was up-regulated (data not shown).

Fig 1. BCCIP was found in overlapping expressed gene profiles in hINO80 complex knockdown HeLa cells.

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205 (A) Schematic diagram of functional domains of hINO80 complex. NTD, N-terminal domain; HSA, helicase-SANT-associated domain; SNF2_N, SNF2 family N-terminal domain; Ins, insertion domain; HELICc, Helicase C-terminal domain. (B) Relative mRNA levels in specific-siRNA knockdown HeLa cells.(C) Gene expression profiles in specific-siRNA knockdown Hela cells. Stacked column chart represents the total number of regulated genes in the indicated specific siRNA knockdown HeLa cells. Each bar in the stacked column chart is 210 composed of down- and up-regulated genes. (D) Overlapping of differentially expressed genes (DEGs) in hINO80, hIes2, hIes6, hArp5 and hArp8 siRNA knocdown HeLa cells. DEGs between the knockdown samples and siNT (Non-targeting siRNA) control were detected by the Illumina Custom differential expression algorithm that was conducted by the Company with software package Genomestudio V2011. Then, entrez gene IDs were exploited for Venn diagram plotting including 2159 genes for Ies2, 1932 genes for hINO80, 2707 genes for Ies6, 1235 genes 215 for Arp5 and 1445 genes for Arp8.

2.2 BCCIP was down-regulated in hINO80 complex knocked down HeLa cells. The University of California-Santa Cruz (UCSC) Genome Browser is a tool for viewing genomic data sets. Analysis of genes listed in Table 1 from the browser found that the GLI-Krüppel family 220 transcription factor YY1, a subunit of INO80 complex, restricted to the BCCIP transcriptional start site proximal region in different types of cell lines including human lung carcinoma type II epithelium-like A549, human liver hepatocellular carcinoma HepG2 and human colon cancer HCT116. To determine if BCCIP was a potential target gene of the hINO80 complex, we first examined the BCCIP expression level in hINO80 complex knockdown HeLa cells. The efficiency 225 of specific siRNA knockdown is as shown in Fig. 2B. Down-regulation of BCCIP by hINO80 complex was confirmed using RT-qPCR and immunofluorescence staining approaches in siINO80, siArp8 or siYY1 treated HeLa cells. As shown in Fig. 2A and C, BCCIP mRNA and protein expression levels were significantly decreased in INO80 complex knocked down HeLa cells.

230 Fig 2. Down regulation of BCCIP was demonstrated in hINO80 complex knockdown HeLa cells. (A) Depression of BCCIP protein in immunofluorescence stained cells. Indicated siRNA (15pmol) knockdown HeLa cells were stained by BCCIP antibody. DAPI staining shows total nuclei. (B) Verification of siRNA knockdown efficiency. hINO80, Arp8 and YY1 proteins were detected using indicated antibodies. (C) Decline of BCCIP expression in hINO80-, Arp8- or YY1 siRNA knockdown HeLa cells. The expression of BCCIP mRNA in 235 specific-siRNA knockdown HeLa cells was examined. *p<0.05, **p<0.01 (Student t-test).

2.3 Transactivation of BCCIP was enhanced by co-transfection of pGL4-BCCIP and

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INO80 complex in 293T cells To understand the regulatory interaction between hINO80 complex and BCCIP, the effects of 240 hINO80 complex on BCCIP-mediated transactivation were investigated. To do that, BCCIP promoter region (Fig. 3A, upper panel) was sub-cloned into pGL4 luciferase vector. In contrast to a basal-level pGL4-BCCIP-luciferase activity, co-transfection of Flag-tagged hINO80 or Arp5 enhanced BCCIP-luciferase activity in a dose-dependent manner (Fig. 3A, lower panel). To further confirm these results, pGL4-BCCIP protein expression levels were analyzed by Western 245 blot after co-transfection of pGL4-BCCIP and hINO80, Arp5 or Arp8. As shown in Fig. 3B, pGL4-BCCIP protein level was significantly increased in a dose-dependent manner with increasing the amount of INO80 complex, suggesting the roles of INO80 complex in regulating the BCCIP transactivation. Quantified protein levels are shown in Fig. 3C.

250 Fig 3. Luciferase activity of BCCIP was facilitated by over-expression of INO80 complex. (A) A dual luciferase assay. pGL4-BCCIP-luc (-353 bp to +38 bp) (upper panel) and INO80 or Arp5 pcDNA plasmids with 0, 0.2, 0.4 and 0.8 μg were co-transfected into HEK293T cells. 48hours after transfection, luciferase activity in the lysates was determined by a dual luciferase assay. Error bars represent data from three independent experiments (lower panel). (B) Higher expression of pGL4-BCCIP in hINO80 complex over-expressed 293T cells. BCCIP 255 proteins in co-transfected INO80-, Arp5- or Arp80 cDNAs and pGL4-BCCIP (48hours) were analyzed by Western blot with indicated antibodies. (C) Quantified proteins. Western blot images in B were quantified with densitometry using Quantity One Basic software (BioRad). Bar graph shows the standard error of the mean of three independent experiments. **p<0.01 in comparison with pcDNA3.1-transfected control (Student t-test).

260 2.4 INO80 complex binds to the BCCIP transcriptional start site-proximal region. Experimental results of knockdown and over-expression clearly show that BCCIP level in both mRNA and protein in cells is controlled by hINO80 complex. To clarify whether the BCCIP is transcriptionally regulated by hINO80 complex, seven primer sets were designed to yield ChIP DNA for BCCIP promoter proximal region as well as the region far away from the upstream/or

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265 downstream transcriptional start site (Fig. 4A, upper panel). The specificity of each primer sets was confirmed by qPCR and 2.5% DNA agarose (Fig. 4A, lower panel). Chromatin-immunoprecipitation (ChIP) assays were performed using hINO80 and YY1 antibodies in 293T cells. As shown in Fig. 4B and C, binding of hINO80 and YY1 was restricted at +0.25 kb downstream of the BCCIP transcriptional start site, representing the co-localization of hINO80 270 and YY1 on the BCCIP promoter region. Analysis of ChIP-qPCR products on 2.5% DNA agarose also revealed a significant enrichment both in hINO80 and YY1 ChIP at +0.25 kb downstream of the BCCIP transcriptional start site (Fig. 4D). To further confirm the INO80 complex binding to BCCIP promoter region, ChIP assays using INO80 and YY1 antibodies were carried out in pBS-shINO80 transfected 293T cells (Fig. 5A and B). As shown in Fig. 5C and D, knocking down 275 INO80 significantly reduced both INO80 (p<0.05 at -0.2 kb site and p<0.01 at +0.25 kb site, respectively) and YY1 (p<0.01 at +0.25 kb site) around the BCCIP transcriptional start site proximal region.

280 Fig 4. hINO80 complex localizes at BCCIP promoter. (A) Seven primer sets in the BCCIP locus used for amplifying ChIP DNA (upper panel). PCR products from each primer set were subjected to 2.5% agarose gel and visualized by ethidium bromide (lower panel). (B-C) Co-occupying of hINO80 and YY1 at BCCIP promoter region. ChIP assays were performed using hINO80 (B) or YY1 (C) antibodies. hINO80 or YY1 ChIP DNA was analyzed by qPCR. Bar graph shows the ratios of ChIP DNA signals (normalized to input) to IgG (also normalized 285 to input). Error bars represent the standard error of the mean of three independent experiments. (D) Enrichment of hINO80 complex at the +0.25 kb downstream of the BCCIP transcriptional start site. ChIP DNA amplified at +250 bp downstream of BCCIP transcriptional start site was subjected to 2.5% agarose gel and visualized by ethidium bromide (upper panel). Quantified PCR signals (Quantity One software) were analyzed by t-test (lower panel). Error bars indicate mean ± SE, and the significant difference is expressed as **p<0.01 (Student t-test). 290

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Fig 5. Binding of hINO80 complex to BCCIP promoter was reduced in siINO80-treated 293T cells. (A-B) Verification of hINO80 knockdown efficiency. 293T cells were transfected with pBS-shINO80 or pBS-Vector. 48 hours later, relative mRNA and protein levels were analyzed by RT-qPCR and western blot, respectively. 295 Quantified protein levels are shown in B (lower panel). (C-D) ChIP assays in hINO80 knockdown 293T cells. ChIP assays were performed using hINO80 or YY1 antibodies in pBS-shINO80-treated 293T cells. ChIP DNA was analyzed by qPCR. Bar graph shows the ratios of ChIP DNA signals (normalized to input) to IgG (also normalized to input). Error bars represent mean ± SE (n=3). *p<0.05, **p<0.01 in comparison with siNT control (Student t-test). 300 3 Discussion INO80 chromatin remodeling complex is highly conserved from yeast to human (25). Revolutionarily conserved subunits including Arp8, Arp5, Arp4, Ies2, Ies6, TIP4a/b and YY1 form an enzyme core which is composed of two modules assembled on the conserved HSA/PTH 305 and ATPase domains of the hINO80 protein. Although the exact functions of two modules remain unclear, several nuclear Arps such as Arp4 and Arp8 have been shown to bind to DNA and histones, suggesting that INO80 complex may contribute to the recognition of DNA and/ or nucleosome substrates (26,27), thereby regulating gene expression through chromatin structure alteration by its ATP-dependent nucleosome remodeling activity. Based on the analysis of 310 performed gene expression profiles from hINO80-, Arp5-, Arp8-, hIes6- and hIes2-knockdown HeLa cells (Fig. 1C and D), we verified that over thousands of genes were regulated in each gene expression profile. Interestingly, a total of 133 genes were co-regulated by silencing the subunits which composed of SNF2 module (ATPase domain) including hINO80, Arp5, hIes6 and hIes2; however, a total of 602 genes were co-regulated by silencing the subunits which composed of 315 HSA module such as hINO80 and Arp8. After KEGG pathway enrichment analysis, differentially expressed genes were enriched in multi-KEGG pathways including p53 signaling pathways and cell cycle (28), suggesting the importance of the INO80 complex in cellular biological processes. With Arp4 and Arp8 together, zinc-finger transcription factor YY1 participates in assembling of

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HSA/PTH module, suggesting the functions of YY1 in maintaining the nucleosome remodeling 320 activity of the INO80 complex. We previously reported that YY1 can recruit the hINO80 complex to some target genes such as CDC6 and GRP78, helping in recognition of INO80-target genes (16). Based on our screening experiments we predict that BCCIP may be a target gene for hINO80 complex. The following reasons are sufficient to support our hypothesis: 1) According to the gene expression profiles, BCCIP was co-regulated by HSA and SNF2 modules (Table 1); 2) BCCIP 325 interacts with tumor suppressor gene BRCA2 and closely related to cancer gene p21, and down regulation of BCCIP was found in several cancer tissues in our previous study (18); 3) YY1, a component of the HSA module of the INO80 complex, binds to the BCCIP transcriptional start site proximal region in different types of cancer cell lines including A549, HepG2 and HCT116 in UCSC Genome Browser; 4) Our recent data demonstrated that p21 was negatively regulated by 330 hINO80 complex in a p53-dependent manner (28). In this report, we first present evidence that BCCIP is a novel target gene of the hINO80 chromatin remodeling complex using in vitro biological experiments combined knockdown/over-expression approaches. The expression of BCCIP both in mRNA and protein levels were significantly decreased after knocking down hINO80, Arp8 or YY1 in HeLa cells 335 (Fig .2C and D). In contrast, over-expression of hINO80, Arp5 or Arp8 significantly enhanced luciferase activity of pGL4-BCCIP (Fig 3). In addition, ChIP assay with hINO80- and YY1-specific antibodies verified that hINO80 and YY1 co-localize the BCCIP promoter region, and the binding site to BCCIP gene was mainly restricted at +0.25 kb downstream of the transcriptional start site (Fig 4B and C). Interestingly, either hINO80 or YY1 binding to BCCIP 340 promoter region was significantly reduced in siINO80 knockdown 293T cells (Fig. 5C and D), suggesting that (i) binding of YY1 to the BCCIP transcriptional start site proximal region requires hINO80; (ii) the nucleosome remodeling activity of hINO80 complex is required for YY1 to gain access to target gene promoters and to activate transcription of target genes. In this report, although we did not show the direct evidence that INO80 complex is recruited to the BCCIP 345 promoter by YY1, however, based on previous reports, we prefer to believe that this a possibility. In summary, we first demonstrate that BCCIP is a novel target gene of the human INO80 chromatin remodeling complex. This result provides a theoretical basis for further understanding and elucidating the functions of BCCIP in tumorigenesis and cellular processes. 4 Competing interests 350 The authors declare that they have no competing interests. 5 Authors Contributions Performed the experiments: JS, YS, JD, YY and SS. Designed the experiments: JS, and JJ. Wrote the paper: JS and JJ. All authors read and approved the final manuscript.

355 References

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425 染色质重塑酶 INO80 复合物的潜在靶 基因-BCCIP 苏家明，隋毅，丁健，杨洋，沈双，金景姬

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（吉林大学 生命科学学院） 摘要：目前认为 BRCA2/CDKN1A-相关蛋白 BCCIP 在肿瘤抑制过程中作为 BRCA2 的辅助因 430 子发挥重要的作用。已知 BCCIP 在多种肿瘤中包括卵巢癌、肾癌和大肠癌中呈现低表达。本 研究结合生物化学和分子生物学技术，利用染色质免疫沉淀、细胞染色、报告基因、基因沉 默、qPCR 等方法，发现 BCCIP 在细胞内的表达受染色质重塑酶的调控。用 siRNA 沉默复合 物中的催化亚基 INO80 或其它亚基如 Arp5 和 YY1 都明显降低了 BCCIP 的 mRNA 和蛋白的表达 水平。相反，顺时转染 INO80 或其它亚基可以明显提高 pGL4-BCCIP 的荧光素酶活性。进一 435 步研究还证实，INO80 复合物募集于 BCCIP 基因启动子区（+0.25kb）。本研究首次证实 BCCIP 可能是染色质重塑酶 INO80 复合物的潜在靶基因。本研究结果不仅为后续研究 INO80 和 BCCIP 之间的相互关系提供了理论基础，同时也为进一步研究 BCCIP 与 BRCA2 在肿瘤抑制中 的作用机制提供了实验依据。

440 关键词：BCCIP；INO80；染色质重塑酶

中图分类号：Q 生物科学

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