The Mouse INO80 Chromatin-Remodeling Complex Is an Essential Meiotic Factor for Spermatogenesis.

The ability to faithfully transmit genetic information across generations via the germ cells is a critical aspect of mammalian reproduction. The process of germ cell development requires a number of large-scale modulations of chromatin within the nucleus. One such occasion arises during meiotic recombination, when hundreds of DNA double-strand breaks are induced and subsequently repaired, enabling the transfer of genetic information between homologous chromosomes. The inability to properly repair DNA damage is known to lead to an arrest in the developing germ cells and sterility within the animal. Chromatin-remodeling activity, and in particular the BRG1 subunit of the SWI/SNF complex, has been shown to be required for successful completion of meiosis. In contrast, remodeling complexes of the ISWI and CHD families are required for postmeiotic processes. Little is known regarding the contribution of the INO80 family of chromatin-remodeling complexes, which is a particularly interesting candidate due to its well described functions during DNA double-strand break repair. Here we show that INO80 is expressed in developing spermatocytes during the early stages of meiotic prophase I. Based on this information, we used a conditional allele to delete the INO80 core ATPase subunit, thereby eliminating INO80 chromatin-remodeling activity in this lineage. The loss of INO80 resulted in an arrest during meiosis associated with a failure to repair DNA damage during meiotic recombination.

Coexistent ARID1A-PIK3CA mutations promote ovarian clear-cell tumorigenesis through pro-tumorigenic inflammatory cytokine signalling.

Ovarian clear-cell carcinoma (OCCC) is an aggressive form of ovarian cancer with high ARID1A mutation rates. Here we present a mutant mouse model of OCCC. We find that ARID1A inactivation is not sufficient for tumour formation, but requires concurrent activation of the phosphoinositide 3-kinase catalytic subunit, PIK3CA. Remarkably, the mice develop highly penetrant tumours with OCCC-like histopathology, culminating in haemorrhagic ascites and a median survival period of 7.5 weeks. Therapeutic treatment with the pan-PI3K inhibitor, BKM120, prolongs mouse survival by inhibiting the tumour cell growth. Cross-species gene expression comparisons support a role for IL-6 inflammatory cytokine signalling in OCCC pathogenesis. We further show that ARID1A and PIK3CA mutations cooperate to promote tumour growth through sustained IL-6 overproduction. Our findings establish an epistatic relationship between SWI/SNF chromatin remodelling and PI3K pathway mutations in OCCC and demonstrate that these pathways converge on pro-tumorigenic cytokine signalling. We propose that ARID1A protects against inflammation-driven tumorigenesis.

Gene silencing associated with SWI/SNF complex loss during NSCLC development.

UNLABELLED: The SWI/SNF chromatin-remodeling complex regulates gene expression and alters chromatin structures in an ATP-dependent manner. Recent sequencing efforts have shown mutations in BRG1 (SMARCA4), one of two mutually exclusive ATPase subunits in the complex, in a significant number of human lung tumor cell lines and primary non-small cell lung carcinoma (NSCLC) clinical specimens. To determine how BRG1 loss fuels tumor progression in NSCLC, molecular profiling was performed after restoration of BRG1 expression or treatment with a histone deacetylase inhibitor or a DNA methyltransferase (DNMT) inhibitor in a BRG1-deficient NSCLC cells. Importantly, validation studies from multiple cell lines revealed that BRG1 reexpression led to substantial changes in the expression of CDH1, CDH3, EHF, and RRAD that commonly undergo silencing by other epigenetic mechanisms during NSCLC development. Furthermore, treatment with DNMT inhibitors did not restore expression of these transcripts, indicating that this common mechanism of gene silencing did not account for their loss of expression. Collectively, BRG1 loss is an important mechanism for the epigenetic silencing of target genes during NSCLC development. IMPLICATIONS: Inactivation of the SWI/SNF complex provides a novel mechanism to induce gene silencing during NSCLC development.

ARID1a-DNA interactions are required for promoter occupancy by SWI/SNF.

Every known SWI/SNF chromatin-remodeling complex incorporates an ARID DNA binding domain-containing subunit. Despite being a ubiquitous component of the complex, physiological roles for this domain remain undefined. Here, we show that disruption of ARID1a-DNA binding in mice results in embryonic lethality, with mutant embryos manifesting prominent defects in the heart and extraembryonic vasculature. The DNA binding-defective mutant ARID1a subunit is stably expressed and capable of assembling into a SWI/SNF complex with core catalytic properties, but nucleosome substrate binding and promoter occupancy by ARID1a-containing SWI/SNF complexes (BAF-A) are impaired. Depletion of ARID domain-dependent, BAF-A associations at THROMBOSPONDIN 1 (THBS1) led to the concomitant upregulation of this SWI/SNF target gene. Using a THBS1 promoter-reporter gene, we further show that BAF-A directly regulates THBS1 promoter activity in an ARID domain-dependent manner. Our data not only demonstrate that ARID1a-DNA interactions are physiologically relevant in higher eukaryotes but also indicate that these interactions facilitate SWI/SNF binding to target sites in vivo. These findings support the model wherein cooperative interactions among intrinsic subunit-chromatin interaction domains and sequence-specific transcription factors drive SWI/SNF recruitment.

The BRG1 chromatin remodeler protects against ovarian cysts, uterine tumors, and mammary tumors in a lineage-specific manner.

The BRG1 catalytic subunit of SWI/SNF-related complexes is required for mammalian development as exemplified by the early embryonic lethality of Brg1 null homozygous mice. BRG1 is also a tumor suppressor and, in mice, 10% of heterozygous (Brg1(null/+)) females develop mammary tumors. We now demonstrate that BRG1 mRNA and protein are expressed in both the luminal and basal cells of the mammary gland, raising the question of which lineage requires BRG1 to promote mammary homeostasis and prevent oncogenic transformation. To investigate this question, we utilized Wap-Cre to mutate both Brg1 floxed alleles in the luminal cells of the mammary epithelium of pregnant mice where WAP is exclusively expressed within the mammary gland. Interestingly, we found that Brg1(Wap-Cre) conditional homozygotes lactated normally and did not develop mammary tumors even when they were maintained on a Brm-deficient background. However, Brg1(Wap-Cre) mutants did develop ovarian cysts and uterine tumors. Analysis of these latter tissues showed that both, like the mammary gland, contain cells that normally express Brg1 and Wap. Thus, tumor formation in Brg1 mutant mice appears to be confined to particular cell types that require BRG1 and also express Wap. Our results now show that such cells exist both in the ovary and the uterus but not in either the luminal or the basal compartments of the mammary gland. Taken together, these findings indicate that SWI/SNF-related complexes are dispensable in the luminal cells of the mammary gland and therefore argue against the notion that SWI/SNF-related complexes are essential for cell survival. These findings also suggest that the tumor-suppressor activity of BRG1 is restricted to the basal cells of the mammary gland and demonstrate that this function extends to other female reproductive organs, consistent with recent observations of recurrent ARID1A/BAF250a mutations in human ovarian and endometrial tumors.

The Val432Leu polymorphism of the CYP1B1 gene is associated with differences in estrogen metabolism and bone density.

Polymorphisms of the CYP450 genes that encode for the enzymes that metabolize estrogen are linked to hormone-related cancers. We investigated the impact of two polymorphisms of the CYP1B1 gene previously reported to be associated with hormone-related disorders on estrogen metabolism and bone mineral density (BMD), another hormone-dependent condition, in women from different ethnic backgrounds. Four hundred sixty-eight postmenopausal Caucasian women, 220 from St. Louis, MO, USA (mean age=63.5+/-0.53 years) and 248 from Palermo, Italy (mean age=72.9+/-0.44 years) participated in the study. Measurements of urinary estrogen metabolites by enzyme-linked immunoassay, serum estradiol by ultrasensitive radioimmnunoassay, and serum sex hormone-binding globulin by immunoradiometric assay were performed only in the American women, while BMD by dual energy X-ray absorptiometry and genotyping by pyrosequencing were performed in both American and Italian women. Differences in the levels of metabolites, free estradiol index and BMD were analyzed by analysis of covariance. Analysis among the American participants for the Valine432Leucine polymorphism showed that, compared to women with the Val/Val genotype, women with the Leu allele (Val/Leu and Leu/Leu) had significantly higher log-transformed values of total urinary estrogen metabolite (ng/mg-creatinine) levels (1.23+/-0.04, 1.35+/-0.02, and 1.34+/-0.03; p=0.03), and significantly lower BMD (gm/cm(2)) in the lumbar spine (1.009+/-0.02, 0.955+/-0.01 and 0.931+/-0.02; p=0.03) and the femoral neck (0.748+/-0.02, 0.717+/-0.01 and 0.693+/-001, p=0.03) for the Val/Val, Val/Leu and Leu/Leu genotypes respectively. There were no significant differences in the urinary metabolites and BMD in the different genotypes for the Alanine119Serine polymorphism among the American women. Meanwhile, a separate analysis among the Italian women revealed no significant differences in BMD among the different genotypes for the two polymorphisms investigated. In conclusion, women with the Leu allele for the CYP1B1 Val432polymorphism have increased estrogen catabolism, as indicated by higher urinary estrogen metabolites, compared to those with Val/Val genotype. This may lead to relative hypoestrogenism and lower BMD in the lumbar spine and femoral neck in these women. Our data suggest that through its effect on the rate of estrogen catabolism, the Val432Leu polymorphism of the CYP1B1 gene may represent as a possible genetic risk factor for osteoporosis in American women.