Human INO80/YY1 chromatin remodeling complex transcriptionally regulates the BRCA2- and CDKN1A-interacting protein (BCCIP) in cells

The BCCIP (BRCA2- and CDKN1A-interacting protein) is an important cofactor for BRCA2 in tumor suppression. Although the low expression of BCCIP is observed in multiple clinically diagnosed primary tumor tissues such as ovarian cancer, renal cell carcinoma and colorectal carcinoma, the mechanism of how BCCIP is regulated in cells is still unclear. The human INO80/YY1 chromatin remodeling complex composed of 15 subunits catalyzes ATP-dependent sliding of nucleosomes along DNA. Here, we first report that BCCIP is a novel target gene of the INO80/YY1 complex by presenting a series of experimental evidence. Gene expression studies combined with siRNA knockdown data locked candidate genes including BCCIP of the INO80/YY1 complex. Silencing or over-expressing the subunits of the INO80/YY1 complex regulates the expression level of BCCIP both in mRNA and proteins in cells. Also, the functions of INO80/YY1 complex in regulating the transactivation of BCCIP were confirmed by luciferase reporter assays. Chromatin immunoprecipitation (ChIP) experiments clarify the enrichment of INO80 and YY1 at +0.17 kb downstream of the BCCIP transcriptional start site. However, this enrichment is significantly inhibited by either knocking down INO80 or YY1, suggesting the existence of both INO80 and YY1 is required for recruiting the INO80/YY1 complex to BCCIP promoter region. Our findings strongly indicate that BCCIP is a potential target gene of the INO80/YY1 complex.


INTRODUCTION
The BRCA2-and CDKN1A (Cip1, p21)-interacting protein BCCIP is alternatively spliced α and β isoforms (Liu et al. 2001;Ono et al. 2000). Both isoforms share N-terminal acidic domain (NAD, include 258 amino acids) and the internal conserved domain (ICD) (Liu et al. 2001). As an important cofactor for BRCA2 in tumor suppression, BCCIP has been implicated in many important cellular processes with obvious links to cancer. Knocking down BCCIP with siRNA in cells leads to defective DNA damage repair (Lu et al. 2005), abnormal cell cycle (Meng et al. 2004;Meng et al. 2004) and genomic instability (Meng et al. 2007). Recently, other colleagues and we have reported that BCCIP was down-regulated in several cancer tissues such as renal cell carcinoma, ovarian cancer and colorectal carcinoma (Meng et al. 2003;Liu et al. 2013). Thus, it is important to clarify the role of BCCIP in tumorigenesis, especially to know how the BCCIP is regulated in cells.
Yin Yang 1 (YY1), a member of the GLI-Krüppel class proteins, was first discovered as a DNA binding protein Seto et al. 1991;Park and Atchison 1991;Hariharan et al. 1991). It is a ubiquitously expressed and evolutionarily conserved protein in human cells. Domain research found that YY1 includes not only an activation domain, but also contains a repression domain (Thomas and Seto 1999;Shi et al. 1997). In subsequent studies, YY1 has been clarified as a versatile protein which can either repress or activate gene transcription by recruiting different cofactors such as histone deacetylases (HDACs), methyltransferase enhancer of zeste homolog 2 (Ezh2), CREB-binding protein (CBP), and P300/CBP-associated factor (PCAF) (Yao et al. 2001;Lee et al. 1995).
Using biochemical purification approaches, we previously verified that YY1 is tightly associated with the human INO80 (INO80) chromatin remodeling complex (Jin et al. 2005). All evolutionarily conserved subunits (include actin-related proteins Arp4, Arp5, Arp8, Tip49a and Tip49b AAA + ATPases, and hIes2 and hIes6) assembled on the conserved helicase-SANT-associated/post-HSA (HSA/PTH) and ATPase domains of INO80 protein (Jin et al. 2005). Both HSA/PTH and ATPase domains in INO80 protein are essential for catalyzing the ATP-dependent nucleosome remodeling activity of the INO80 complex. Based on YY1 with Arp4 and Arp8 together participate in assembling helicase-SANT-associated/post-HSA (HSA/PTH) module, suggesting that like other conserved subunits, YY1 is also essential for maintaining the ATP-dependent nucleosome remodeling activity of the INO80 complex (Chen et al. 2011). Experimental evidence indicates that yeast Ino80 is required for proper transcriptional regulation of many target genes (Morrison and Shen 2009), while in Drosophila, INO80 facilitates transcriptional repression of ecdysone-regulated genes during prepupal development (Neuman et al. 2014). Recent experimental results demonstrate that INO80 is also required in ESC self-renewal, somatic cell reprogramming, and blastocyst development (Wang et al. 2014). Although genome-wide studies show that INO80 binds many, but not all, loci in the genome (Moshkin et al. 2012), it is unclear whether there is a direct correlation between INO80 binding and target gene expression levels. One possibility is that YY1 as a transcription factor helps INO80 complex to recognize INO80-target genes and recruits the complex to some target genes such as CDC6 and GRP78 (Cai et al. 2007). In this study, using molecular and cell biology approaches, we first show that the BCCIP might be a novel target gene of the INO80 /YY1 chromatin remodeling complex. Our results will provide a strong theoretical basis to elucidate the BCCIP functions in cells.

RESULTS
BCCIP was selected as a candidate target gene of the INO80/YY1 complex from gene expression profiles Human INO80 complex is one of the most highly conserved chromatin remodelers. Eight core subunits (Arp5, Arp8, TIP49a/b, Ies2, Ies6, Arp4, and YY1) are evolutionary conserved and form an enzyme core including HSA and SNF2 modules. Except for conserved subunits, INO80 complex contains 6 metazoan-specific subunits which all assemble on N-terminus of INO80 protein and form an N-terminal regulatory module (Jin et al. 2005;Chen et al. 2011) (Fig. 1A). Increasing evidence suggests the functions of INO80/YY1 complex in gene transcriptional regulation (Morrison and Shen 2009;Conaway and Conaway 2009), but the precise mechanisms are still unclear. To investigate the target genes of the INO80/YY1 complex, total RNA from 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 DNA microarray. As shown in Fig. 1C, a total of 1932, 1445, 1235, 2707 and 2159 genes were differentially expressed among INO80, Arp8, Arp5, hIes6 or hIes2 and NT siRNA knockdown HeLa cells, respectively. Hundreds of overlapping genes are found to be regulated by INO80, Arp8, Arp5, hIes6 and hIes2, which are components of the HSA and SNF2 module. On the other hand, a total of 602 genes were co-regulated by INO80 and Arp8 which participate in assembling HSA module (data not shown). Selected overlapping co-regulated genes (8 down and 6 up) in INO80/YY1 complex knockdown HeLa cells are shown in Table 1. mRNA from INO80-and Arp8-siRNA knockdown cells (Fig. 1E) was measured with RT-qPCR ( Fig. 1F). Compared to NT siRNA control, selected gene mRNA including BCCIP, TNFRSF21, and BRMS1L was down-regulated with siRNA knockdown of INO80 and Arp8 in HeLa cells. In contrast, RAF1 and PTTG1IP mRNAs was upregulated. However, there was no change of mRNA of RAB24, RAB22A, and ST7L. Table 2 compared the results of microarray illumina and RT-qPCR. In addition, differentially expressed genes (DEGs) in INO80/YY1 complex knocked down cells were used for KEGG annotation by using DAVID web annotation tool. DEG-enriched pathways in INO80 complex knockdown cells were shown previously (Chen et al. 2011). Fig. 1D shows the differentially regulated microarray genes in cancer pathway after knocking down the indicated subunits of INO80/YY1 complex.
The expression of BCCIP was regulated by INO80/YY1 complex in HeLa and 293T cells and annotation (Karolchik et al. 2013). Based on UCSC database (ChIP-Seq) search, we found that YY1 was 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 clarify this observation, we first examined the BCCIP expression level in YY1-siRNA knockdown HeLa cells. As shown in Fig. 2A, dose-dependent down-regulation of BCCIP (lower panel) in YY1-siRNA knockdown (upper panel) HeLa cells was confirmed by qPCR. Furthermore, low protein levels of BCCIP in YY1-siRNA knockdown HeLa cells were also detected by western blot (Fig. 2B) and immunofluorescence staining (Fig. 2C)   (F) Verification of the mRNA of select genes from gene expression profiles. 48 h after siRNA transfection, RT-qPCR was performed to assess the relative mRNA levlel (n = 3). Bar graphs show ratios of RT-qPCR signals to GAPDH (all signals normalized to siNT). **P < 0.01 in comparison with siNT control (Student ttest).

Human INO80 facilitates the transactivation of BCCIP
To further explore whether the INO80/YY1 chromatin remodeling complex affects the transactivation of BCCIP, three designed BCCIP promoter region was sub-cloned into pGL4 luciferase vector (Fig. 3A). Constructed pGL4-BCCIP-Luc plasmids containing different BCCIP promoter region were first tested in dual luciferase assay (Fig. 3B). Then, the impact of INO80 on BCCIP transactivation was estimated by co-transfection of pGL4-BCCIP-Luc with Flag-INO80 plasmids. As shown in Fig. 3C, in contrast to a basal-level luciferase activity, co-transfection of pGL4-BCCIP-Luc and INO80 dose-dependently increased BCCIP-luciferase activity in all cases, indicating the roles of INO80 complex in regulating the transactivation of BCCIP.

Chromatin remodeling activity of the INO80 complex might be associated with regulation of BCCIP expression
To investigate the effects of INO80 complex on BCCIP-mediated transactivation, pGL4-BCCIP-Luc vector containing 391 bp promoter region (−330 ∼+61 bp) was used in subsequent experiments. As shown in Fig. 4A, co-transfection of pGL4-BCCIP-Luc vector with Flag-tagged INO80, Arp5, and Arp8 increased not only luciferase activities (data not shown) but also intracellular BCCIP proteins in a dose-dependent manner. Compared to pcDNA3.1 control group, statistically significant differences were found in INO80 (P < 0.01 at 0.2, 0.4, and 0.8 μg of plasmids, respectively), Arp5 (P < 0.05 at 0.8 μg of plasmids), or Arp8 (P < 0.01 at 0.4 and 0.8 μg of Co-regulated genes selected from different specific subunit of the INO80/YY1 complex knockdown gene expression profiles are tested by quantitative PCR (qPCR). The performed qPCR results were then compared with DNA microarray illumine data.
plasmids, respectively) transfected 293T cells (Fig. 4B), suggesting the importance of enzyme core including HSA and SNF2 modules of the INO80 complex in regulating BCCIP expression. To further confirm this result, point or delete mutations of INO80 protein were designed (Fig. 4C).

INO80/YY1 chromatin remodeling complex is restricted to the BCCIP transcriptional start site proximal region
Data in the previous experiment clearly show that BCCIP level in both mRNA and protein, and the transactivation of BCCIP in cells is regulated by INO80/YY1 chromatin remodeling complex. To further determine whether the BCCIP is transcriptionally controlled by INO80/YY1 complex, indicated primer sets were designed to amplify ChIP DNA (Fig. 5A, upper panel). Amplified ChIP DNA by each specific primer set for BCCIP promoter proximal region as well as the region far away from the transcriptional start site was confirmed by qPCR, and visualized in 2.5% DNA agarose with Ethidium bromide (Fig. 5A, lower panel). Human INO80 and YY1 specific antibodies were then used in ChIP assays. As shown in Fig. 5B, both INO80 (upper) and YY1 (lower) are recruited at +0.17 kb downstream of the BCCIP transcriptional start site, indicating the co-localization of INO80 and YY1 on the BCCIP promoter region. Further analysis of performed qPCR products on 2.5% DNA agarose gel acquired a significant enrichment both in INO80 and YY1 ChIP at +0.17 kb downstream of the BCCIP transcriptional start site (Fig. 5C). However, knockdown INO80 by transfecting pBS-shINO80 (Fig. 6A) in 293T cells significantly blocked the recruitment of INO80 and YY1 at the +0.17 kb downstream of the BCCIP transcriptional start site (P < 0.01 in both cases), confirming the binding of INO80/YY1 complex at BCCIP promoter region (Fig. 6B). Similarly, silencing YY1 by siRNA (Fig. 6C) in 293T cells inhibited the recruitment of INO80 at the +0.17 kb downstream of the BCCIP transcriptional start site (P < 0.01), but not at +2.8 bp far away from the downstream of the BCCIP transcriptional start site, suggesting the requirement of YY1 for binding of INO80 to BCCIP promoter region (Fig. 6D).

DISCUSSION
It has been known that the INO80 complex is highly conserved from yeast to human. Evolutionarily conserved subunits including YY1 form an enzyme core which is composed of two modules (HSA and SNF2) assembled on the conserved HSA/PTH and ATPase domains of the INO80 protein (Fig. 1A). Although the precise functions of those two modules remain to be explored, actin-related protein Arp4 and Arp8 have been reported to bind to DNA and histones, suggesting the contributions of the INO80 complex in recognition of DNA and/or nucleosome substrates (Seeber et al. 2013;Shen et al. 2003). On the other hand, recognition of DNA and/or histones by subunits of INO80 complex is beneficial to the ATP-dependent nucleosome remodeling activity, thereby regulating gene expression through chromatin structure alteration. Based on the analysis of performed gene expression profiles from INO80-, Arp8-, Arp5-, hIes6-and hIes2-knockdown HeLa cells, we not only clarified that over thousands of genes were regulated in each gene expression profile, but also found that hundreds of genes were co-regulated by silencing the subunits which composed of SNF2 (Arp5, Ies6 and Ies2) and HSA (Arp8) modules (Cao et al. 2015). Also, enrichment of differentially expressed genes in multi-KEGG pathways including pathways in cancer was confirmed by KEGG pathway enrichment analysis, indicating the importance of the INO80 complex in cellular biological processes such as cancer pathway (Fig. 1D) (Chen et al. 2011). We previously reported that zinc-finger transcription factor YY1 is tightly associated with human INO80 chromatin remodeling complex, and with actin-related proteins (Arp4 and Arp8) together, YY1 assembles HSA module on the HSA/PTH domain of INO80 protein, demonstrating the roles of YY1 in maintaining the nucleosome remodeling activity of the INO80 complex (Chen et al. 2011;Chen et al. 2013).  More importantly, as an essential co-activator, YY1 can recruit the INO80 complex to some target genes such as CDC6 and GRP78 (Cai et al. 2007). Based on our gene expression profiles and screening experiments, BCCIP was chosen as one of the potential candidates of target genes of the INO80 complex. Based on database search in UCSC Genome Browser, we found that YY1 is restricted at the BCCIP transcriptional start site proximal region in different types of cancer cell lines including A549, HepG2 and HCT116, suggesting the functions of YY1 or INO80/YY1 complex in regulating the expression of BCCIP.
In this report, we first verified that BCCIP is a novel target gene of the INO80 chromatin remodeling complex by presenting a series of experimental evidence. The levels of BCCIP both in mRNA and protein were not only regulated by silencing-or over-expressing-YY1 (Fig. 2), but also were changed by knocking down Arp5,and Arp8 HeLa cells (Figs. 2 and 4A), suggesting the roles of INO80/YY1 complex, but not limited to YY1, in regulating the BCCIP expression. ChIP assays further verified that INO80 and YY1 co-localize the BCCIP promoter region, and the binding site to BCCIP gene was enriched at +0.17 kb downstream of the transcriptional start site (Fig. 5). However, this enrichment was significantly inhibited in either INO80-or YY1-knocking down 293T cells (Fig. 6), suggesting that (i) INO80/YY1 complex binds to the BCCIP promoter region; (ii) binding of YY1 to the BCCIP promoter region requires INO80; (iii) INO80 complex is required for YY1 to gain access to target gene promoters and to activate transcription of target genes.
In summary, we first demonstrate that BCCIP might be a novel target gene of the human INO80/YY1 chromatin remodeling complex. This result will provide a theoretical basis for further understanding and elucidating the functions of BCCIP in tumorigenesis and cellular processes. . PCR products from each primer set were confirmed using 2.5% agarose gel (lower panel). (B) Co-occupying of INO80 and YY1 at the BCCIP promoter region. ChIP assays were performed using INO80 (upper panel) or YY1 (lower panel) antibodies. 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 the standard error of the mean of three independent experiments. (C) Enrichment of INO80/YY1 at the +0.17 kb downstream of the BCCIP transcriptional start site. qPCR product at +0.17 kp downstream of BCCIP transcriptional start site was visualized by ethidium bromide (upper panel) in 2.5% agarose gel. 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).

Statistics
Two-tailed student's t-test was used in this study. Data shown is mean + SD from at least three independent experiments. Statistical probability is expressed as *P < 0.05, **P < 0.01.