Oncogene (2007) 26, 2543–2553 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE Regulation of clustered gene expression by cofactor of BRCA1 (COBRA1) in breast cancer cells SE Aiyar1, AL Blair1, DA Hopkinson, S Bekiranov and R Li Department of Biochemistry and Molecular Genetics, School of Medicine, University of Virginia, Charlottesville, VA, USA Eucaryotic genes that are coordinately expressed tend to the gene organization patterns within a genome. In be clustered. Furthermore, gene clusters across chromo- contrast to the long-held presumption that genes are somal regions are often upregulated in various tumors. randomly distributed throughout the eucaryotic ge- However, relatively little is known about how gene nomes, there is compelling evidence for clustering of clusters are coordinately expressed in physiological or co-expressed genes in every eucaryotic genome exam- pathological conditions. Cofactor of BRCA1 (COBRA1), ined so far (Hurst et al., 2004). For example, 25% of a subunit of the human negative elongation factor, has genes in budding yeast that are transcribed in the same been shown to repress estrogen-stimulated transcription cell-cycle stage are adjacently located (Cho et al., 1998). of trefoil factor 1 (TFF1 or pS2)by stalling RNA The gene-clustering phenomenon is perhaps even more polymerase II. Here, we carried out a genome-wide study prominent in multicellular organisms. It is reported that to identify additional physiological target genes of 20% of genes in the entire fly genome are organized into COBRA1 in breast cancer cells. The study identified a clusters with similar expression patterns (Spellman and total of 134 genes that were either activated or repressed Rubin, 2002). Likewise, a large number of housekeeping upon small hairpin RNA-mediated reduction of COBRA1. genes and highly expressed genes in the human genome Interestingly, many COBRA1-regulated genes reside as are clustered (Caron et al., 2001; Lercher et al., 2002). clusters on the chromosomes and have been previously Co-expression of most gene clusters is unlikely owing to implicated in cancer development. Detailed examination a ‘bystander effect’ wherein genes might be activated of two such clusters on chromosome 21 (21q22)and simply because they are adjacent to other actively chromosome X (Xp11)reveals that COBRA1 is physically transcribed genes, as syntenic analyses of mouse and associated with a subset of its regulated genes in each human genomes indicate that the order of co-expressed cluster. In addition, COBRA1 was shown to regulate genes in a cluster is conserved by natural selection both estrogen-dependent and -independent transcription of (Singer et al., 2005). Therefore, clustering of co- the gene cluster at 21q22, which encompasses the pre- expressed genes may have a functional advantage in viously identified COBRA1-regulated TFF1 (pS2)locus. coordinating gene expression during cell proliferation, Thus, COBRA1 plays a critical role in the regulation of differentiation and development. clustered gene expression at preferred chromosomal In addition to their potential roles in normal domains in breast cancer cells. physiology, gene clusters across chromosomal regions Oncogene (2007) 26, 2543–2553. doi:10.1038/sj.onc.1210047; are often upregulated in various tumors (Zhou et al., published online 16 October 2006 2003; Koon et al., 2004). For example, members of the trefoil factor family (TFF1–3) are tandem clustered Keywords: COBRA1; clustered gene expression; estrogen; within a 55 kb genomic region at chromosome 21q22.3 transcription; NELF (Chinery et al., 1995; Schmitt et al., 1996). Clinical studies suggest a strong association of TFF1/TFF3 expression with breast cancer (Poulsom et al., 1997; Mikhitarian et al., 2005; Xu et al., 2005; Smid et al., 2006). In a recent study of primary breast tumor Introduction samples, overexpression of TFF1 (pS2) and TFF3 was most significantly correlated with bone metastasis in Recent rigorous examination of complete eucaryotic breast cancer (Smid et al., 2006). In addition, it has been genomes, combined with gene expression profiling shown that plasma levels of TFF1, TFF2 and TFF3 are analyses, has shed exciting and unexpected insight into increased in patients with advanced prostate cancer (Vestergaard et al., 2006). Therefore, coordinate expres- Correspondence: Dr R Li, Department of Biochemistry and Molecular sion of the TFF gene cluster may represent a significant Genetics, School of Medicine, University of Virginia, 1300 Jefferson marker in breast and prostate cancer progression. Park Avenue, Charlottesville, VA 22908, USA. Although part of the upregulated clustered gene E-mail: [email protected] 1These authors contributed equally to this work. expression in tumor samples could be attributed to gene Received 22 May 2006; revised 24 August 2006; accepted 6 September amplification, epigenetic changes in high-order chro- 2006; published online 16 October 2006 matin structure may also lead to dysregulation of COBRA1 regulates clustered gene expression SE Aiyar et al 2544 coordinated gene expression at the transcription level in human cancers. Despite the abundant evidence for clustering of co-expressed genes in eucaryotic genomes, the mechanistic basis and protein regulators that dictate the coordinated expression of clustered genes are poorly understood. Cofactor of BRCA1 (COBRA1) was first identified as a BRCA1-interacting protein capable of inducing large- scale chromatin reorganization (Ye et al., 2001). COBRA1 was subsequently found to be one of the four subunits of the negative elongation factor complex NELF (NELF-B) (Narita et al., 2003). The NELF complex was biochemically purified based on its ability to inhibit transcription elongation by stalling RNA polymerase II (RNAPII) within the promoter-proximal region (Wada et al., 1998; Yamaguchi et al., 1999). Recent in vivo work shows that COBRA1 interacts with the ligand-binding domain of estrogen receptor a (ERa) (Aiyar et al., 2004). In response to estrogen, COBRA1 and the rest of the NELF complex are recruited to a Figure 1 Gene ontology analysis of COBRA1-regulated genes as identified by microarray. (a) Immunoblots showing the decreased subset of ERa-regulated promoters such as TFF1 (pS2), expression of COBRA1 protein in the COBRA1 knockdown and inhibit the ligand-dependent gene activation by T47D-derived cells (sh2A and shGA). Parental T47D and interfering with the RNAPII movement (Aiyar et al., shEGFP-expressing cells were used as controls. (b) The Ingenuity 2004). To better understand the pattern of gene Gene Ontology Analysis tool is used to identify several biological pathways that are relatively enriched with COBRA1-regulated regulation by COBRA1, we conducted a genome-wide genes. The P-value for each category is shown on the top of the search for COBRA1 target genes by comparing gene bar. expression profiles in control and COBRA1 knockdown breast cancer cells. Our work led to the key discovery that COBRA1 regulates both ligand-dependent and - regulated genes. Of note, 31 out of the 134 COBRA1- independent expression of cancer-associated genes that regulated genes are either membrane-associated or tend to cluster on the chromosomes, including the TFF secretory (solid squares in Supplementary Table 1). gene cluster. Thus, this finding suggests a link between Using the Ingenuity Gene Ontology Analysis software regulation of transcription elongation and higher order program (www.ingenuity.com), we found that the list of chromatin structure in breast cancer cells. COBRA1-regulated genes is enriched with those in- volved in cell cycle control, antigen presentation and amino-acid synthesis pathways (Figure 1b). A total of 21 COBRA1-regulated genes have been Results reported to be estrogen-responsive (http://defiant.i2r. a-star.edu.sg/projects/Ergdb-v2/index.htm) (solid trian- Whole-genome analysis of COBRA1-regulated genes gles in Supplementary Table 1), and 17 of them are To conduct a genome-wide search for genes regulated by repressed by COBRA1 (upregulated in the COBRA1 COBRA1, ERa-positive breast cancer cell line T47D knockdown cells). This extends our previous observa- was infected with a retrovirus expressing either a control tion of the COBRA1-mediated repression of estrogen- small hairpin RNA (shRNA) against enhanced green responsive transcription at the TFF1 (pS2) and C3 loci fluorescent protein (EGFP) (shEGFP) or shRNA (Aiyar et al., 2004). In addition, a large number of against COBRA1 (sh2A). shRNA-mediated knockdown COBRA1-regulated genes have been previously impli- of COBRA1 was verified by Western blotting cated in cancer development. These include CD74 in (Figure 1a). Total RNA isolated from duplicates of myeloma (Burton et al., 2004), Claudin 1 in colon cancer each shRNA-expressing cell line was subjected to (Dhawan et al., 2005), AZGP1 in prostate cancer microarray analyses using the Affymetrix microarray (Lapointe et al., 2004) and the cancer/testis-specific G- chips (HG-U133A). Examination of the microarray antigens (GAGEs) (Bodey, 2002). In particular, multiple data revealed a total of 134 genes with a P-value of COBRA1-regulated genes have been associated with less than or equal to 0.05 and a fold change in breast cancer. These include APOD (Lopez-Boado signal intensity greater than 1.5 (Supplementary et al., 1994; Blais et al., 1995; Rassart et al., 2000), Table 1). Among these genes, 84 were stimulated (i.e. CLDN1 (Kramer et al., 2000; Hoevel et al., 2002; COBRA1-repressed)