In mammalian spermatogenesis, the X and Y chromosomes are transcriptionally silenced during the pachytene stage of meiotic prophase (meiotic sex chromosome inactivation, MSCI), forming a condensed chromatin domain termed the sex or XY body. The nucleosomal core histone H2AX is phosphorylated within the XY chromatin domain just prior to MSCI, and it has been hypothesized that this triggers the chromatin condensation and transcriptional repression. Here, we show that the kinase ATR localizes to XY chromatin at the onset of MSCI and that this localization is disrupted in mice with a mutant form of the tumor suppressor protein BRCA1. In the mutant pachytene cells, ATR is usually present at nonsex chromosomal sites, where it colocalizes with aberrant sites of H2AX phosphorylation; in these cells, there is MSCI failure. In rare pachytene cells, ATR does locate to XY chromatin, H2AX is then phosphorylated, a sex body forms, and MSCI ensues. These observations highlight an important role for BRCA1 in recruiting the kinase ATR to XY chromatin at the onset of MSCI and provide compelling evidence that it is ATR that phosphorylates H2AX and triggers MSCI.

Tumor hypoxia often directly correlates with aggressive phenotype, metastasis progression, and resistance to chemotherapy. Two transcription factors [hypoxia-inducible factor-1A (HIF-1A) and HIF-2A] are dramatically induced in hypoxic areas and regulate the expression of genes necessary for tumor adaptation to the conditions of low oxygen; however, the relative contribution of these factors is controversial. We used RNA interference-mediated inactivation of HIF-1A or HIF-2A followed by microarray analysis to identify genes specifically regulated by either HIF-1 or HIF-2 in hypoxia. We found that, in the MCF7 cell line, the vast majority of hypoxiaresponsive genes (>80%) were dependent on the presence of HIF-1A. However, a small group of genes were preferentially regulated by HIF-2A. Promoter analysis for this group of genes revealed that all of them have putative binding sites for ETS family transcription factors, and 10 of 11 HIF-2A-dependent genes had at least one potential hypoxia-responsive element (HRE) in proximity to an ETS transcription factor binding site. Knockdown of ELK-1, the most often represented member of ETS family, significantly reduced hypoxic induction of the HIF-2A-dependent genes. Physical and functional interaction between ELK-1 and HIF-2A were supported by coimmunoprecipitation of these two proteins, luciferase reporter assay using CITED2 promoter, and binding of ELK-1 protein to the promoters of CITED2 and WISP2 genes in proximity to a HRE. These data suggest that the choice of the target genes by HIF-1 or HIF-2 depends on availability and cooperation of HIFs with other factors recognizing their cognate elements in the promoters. (Cancer Res 2006; 66(11): 5641-7)

Phosphorylation of cyclin-dependent kinases (CDKs) by the CDK-activating kinase is required for the activation of CDK enzymes. Members of two families of CDK inhibitors, p16/p18 and p21/p27, become physically associated with and inhibit the activity of CDKs in response to a variety of growth-modulating signals. Here, we show that the representative members of both families of CDK inhibitors, p21waf1,cip1, p27kip1, and p18, can prevent the phosphorylation of their CDK partners, CDK2 and CDK6, by CDK-activating kinase. No direct interaction between CDK-activating kinase and the CDK inhibitors could be detected, suggesting that binding of these CDK inhibitors to CDK subunits renders CDK inaccessible to the CDK-activating kinase phosphorylation. These findings suggest that a general mechanism of CDK inhibitor function is to block the phosphorylation of CDK enzymes by CDK-activating kinase.

BRCA1 is a susceptibility gene for breast and ovarian cancer with growth-inhibitory activity for which the mechanism of action remains unclear. When introduced into cells, BRCA1 inhibits growth of some but not all cell lines. In an attempt to uncover the mechanism of growth suppression by BRCA1, we examined a panel of cell lines for their ability to reduce colony outgrowth in response to BRCA1 overexpression. Of all variables tested, only those cells with wild-type pRb were sensitive to BRCA1-induced growth suppression. In cells with an intact rb gene, inactivation of pRb by HPV E7 abrogates the growth arrest imposed by BRCA1. In accordance with these observations, we found that BRCA1 could not suppress BrdUrd uptake in primary fibroblasts from rb؊/؊ mice and exhibited an intermediate ability to inhibit DNA synthesis in rb؉/؊ as compared with rb؉/؉ cells. We further found that the BRCA1 protein complexes with the hypophosphorylated form of pRb. This binding is localized to amino acids 304 -394 of BRCA1 protein and requires the ABC domain of pRb. In-frame deletion of BRCA1 fragment involved in interaction with pRb completely abolished the growth-suppressive property of BRCA1. Although it has been reported that BRCA1 interacts with p53, we find the p53 status did not affect the ability of BRCA1 to suppress colony formation. Our data suggest that the growth suppressor function of BRCA1 depends, at least in part, on Rb. G ermline mutations in BRCA1 are found in Ϸ50-60% of hereditary breast and ovarian cancers (1). Despite the unequivocal role of BRCA1 in familial breast cancer susceptibility, the biological function of the BRCA1 protein remains unclear. Experimental data suggest that BRCA1 may be a negative regulator of cell growth. Attenuation of BRCA1 synthesis by antisense oligonucleotide increased the proliferative rate of both benign and malignant mammary epithelial cells in culture (2), and BRCA1 expression decreased the capacity of MCF7 breast cancer cells to form tumors in nude mice (3). However, BRCA1 expression increases as cells progress through the G 1 and S phases of the cell cycle (4, 5). Furthermore, homozygous BRCA1 mutant mice died at day 7.5 of embryogenesis with evidence of abnormal cessation of cellular proliferation accompanied by high levels of the CDK inhibitor p21 and low levels of cyclin E and mdm2 (6, 7). Recently, colocalization and physical interaction of BRCA1 with Rad51 protein has raised the possibility that BRCA1 is involved in DNA repair (8). Moreover, the COOH terminus of the BRCA1 protein can activate transcription in in vitro experiments (9, 10) and coactivate transcription of p53-regulated genes (11,12).The experimental biology of BRCA1 therefore suggests that BRCA1 may have different functions, each best manifested in specific study systems and cell lines. Herein, we show that BRCA1 binds preferentially to the hypophosphorylated form of Rb and that the growth-suppressive phenotype of BRCA1 depends on the presence of a functional Rb protein. These data indicate the complexity of ...