04-080 Sigma-AldrichAnti-BAF250a/ARID1a Antibody, clone PSG3

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.

Hepatocellular carcinoma (HCC) is a common malignancy worldwide, which is especially prevalent in Asia. Elucidating the molecular basis of HCC is crucial to develop targeted diagnostic tools and novel therapies. Recent studies have identified AT-rich interactive domain-containing protein 1A (ARID1A) as a broad-spectrum tumor suppressor. We evaluated the clinical implications of decreased ARID1A expression in HCC, and investigated the mechanisms of ARID1A-mediated tumor suppression.Quantitative PCR, western blotting, immunohistochemical analysis of ARID1A mRNA and protein expression was conducted in 64 paired HCC and adjacent non-tumorous tissues. ARID1A function was evaluated in vitro in MHCC-97H and Huh7 HCC cell lines, and in vivo in a xenografted HCC tumor model.ARID1A mRNA and protein expression were significantly decreased in HCC tissues, and decreased expression was significantly associated with overall metastasis, including local lymph node and distant metastasis, and poor prognosis. ARID1A knockdown promoted HCC cell migration and invasion in vitro, whereas overexpression of ARID1A inhibited migration and invasion. E-cadherin levels were closely correlated with ARID1A expression, suggesting a role in migration and invasion. In addition, ARID1A and E-cadherin (CDH1) expression were found to be regulated in a coordinated fashion in HCC samples. Furthermore, ARID1A knockdown significantly increased HCC tumor growth and lung metastasis in vivo.ARID1A served as an important tumor suppressor. Decreased expression of ARID1A was associated with tumor progression, metastasis, and reduced overall survival in mice and humans. ARID1A could represent a promising candidate therapeutic target for HCC.

While the importance of gene enhancers in transcriptional regulation is well established, the mechanisms and the protein factors that determine enhancers activity have only recently begun to be unravelled. Recent studies have shown that progesterone receptor (PR) binds regions that display typical features of gene enhancers. Here, we show by ChIP-seq experiments that the chromatin remodeler CHD8 mostly binds promoters under proliferation conditions. However, upon progestin stimulation, CHD8 re-localizes to PR enhancers also enriched in p300 and H3K4me1. Consistently, CHD8 depletion severely impairs progestin-dependent gene regulation. CHD8 binding is PR-dependent but independent of the pioneering factor FOXA1. The SWI/SNF chromatin-remodelling complex is required for PR-dependent gene activation. Interestingly, we show that CHD8 interacts with the SWI/SNF complex and that depletion of BRG1 and BRM, the ATPases of SWI/SNF complex, impairs CHD8 recruitment. We also show that CHD8 is not required for H3K27 acetylation, but contributes to increase accessibility of the enhancer to DNaseI. Furthermore, CHD8 was required for RNAPII recruiting to the enhancers and for transcription of enhancer-derived RNAs (eRNAs). Taken together our data demonstrate that CHD8 is involved in late stages of PR enhancers activation.

Unfolding of the gene expression program that converts precursor cells to their terminally differentiated counterparts is critically dependent on the nucleosome-remodeling activity of the mammalian SWI/SNF complex. The complex can be powered by either of two alternative ATPases, BRM or BRG1. BRG1 is critical for development and the activation of tissue specific genes and is found in two major stable configurations. The complex of BRG1-associated factors termed BAF is the originally characterized form of mammalian SWI/SNF. A more recently recognized configuration shares many of the same subunits but is termed PBAF in recognition of a unique subunit, the polybromo protein (PBRM1). Two other unique subunits, BRD7 and ARID2, are also diagnostic of PBAF. PBAF plays an essential role in development, apparent from the embryonic lethality of Pbmr1-null mice, but very little is known about the role of PBAF, or its signature subunits, in tissue-specific gene expression in individual differentiation programs. Osteoblast differentiation is an attractive model for tissue-specific gene expression because the process is highly regulated and remains tightly synchronized over a period of several weeks. This model was used here, with a stable shRNA-mediated depletion approach, to examine the role of the signature PBAF subunit, ARID2, during differentiation. This analysis identifies a critical role for ARID2-containing complexes in promoting osteoblast differentiation and supports a view that the PBAF subset of SWI/SNF contributes importantly to maintaining cellular identity and activating tissue-specific gene expression.

Steroid hormones regulate gene expression by interaction of their receptors with hormone responsive elements (HREs) and recruitment of kinases, chromatin remodeling complexes, and coregulators to their target promoters. Here we show that in breast cancer cells the BAF, but not the closely related PBAF complex, is required for progesterone induction of several target genes including MMTV, where it catalyzes localized displacement of histones H2A and H2B and subsequent NF1 binding. PCAF is also needed for induction of progesterone target genes and acetylates histone H3 at K14, an epigenetic mark that interacts with the BAF subunits by anchoring the complex to chromatin. In the absence of PCAF, full loading of target promoters with hormone receptors and BAF is precluded, and induction is compromised. Thus, activation of hormone-responsive promoters requires cooperation of at least two chromatin remodeling activities, BAF and PCAF.

Steroid hormones induce transcription of their responsive genes by complex mechanisms including synergism between the hormone receptors and other transcription factors. On the mouse mammary tumor virus (MMTV) promoter progesterone induction is mediated by the reciprocal synergism between progesterone receptor (PR) and the ubiquitous transcription factor NF1. PR binding mediates ATP-dependent displacement of histone H2A and H2B enabling NF1 access to its target site. In minichromosomes assembled in vitro NF1 binding facilitates access of PR to the hormone responsive elements (HRE) by precluding reforming of the histone octamer, but the function of NF1 in living cells remains unclear. Here we show, that depleting NF1 by small interfering RNAs or mutating the NF1 binding site significantly compromises transcription of the MMTV promoter. The central HREs 2 and 3 are not needed for ATP-dependent H2A/H2B displacement or NF1 binding but are critical for full PR binding and MMTV transactivation. We found that NF1 binding to the MMTV promoter on a H3/H4 histone tetramer particle exposes the central HREs and facilitates their binding by PR, suggesting a possible mechanism for the reciprocal synergism between PR and NF1. transactivation. We found that NF1 binding to the MMTV promoter on a H3/H4 histone tetramer particle exposes the central HREs and facilitates their binding by PR, suggesting a possible mechanism for the reciprocal synergism between PR and NF1.

p270 (ARID1A) is a member of the ARID family of DNA-binding proteins and a subunit of human SWI/SNF-related complexes, which use the energy generated by an integral ATPase subunit to remodel chromatin. ARID1B is an independent gene product with an open reading frame that is more than 60% identical with p270. We have generated monoclonal antibodies specific for either p270 or ARID1B to facilitate the investigation of ARID1B and its potential interaction with human SWI/SNF complexes in vivo. Immunocomplex analysis provides direct evidence that endogenous ARID1B is associated with SWI/SNF-related complexes and indicates that p270 and ARID1B, similar to the ATPase subunits BRG1 and hBRM, are alternative, mutually exclusive subunits of the complexes. The ARID-containing subunits are not specific to the ATPases. Each associates with both BRG1 and hBRM, thus increasing the number of distinct subunit combinations known to be present in cells. Analysis of the panels of cell lines indicates that ARID1B, similar to p270, has a broad tissue distribution. The ratio of p270/ARID1B in typical cells is approx. 3.5:1, and BRG1 is distributed proportionally between the two ARID subunits. Analysis of DNA-binding behaviour indicates that ARID1B binds DNA in a non-sequence-specific manner similar to p270.

The SWI/SNF family of chromatin-remodeling complexes has been discovered in many species and has been shown to regulate gene expression by assisting transcriptional machinery to gain access to their sites in chromatin. Several complexes of this family have been reported for humans. In this study, two additional complexes are described that belong to the same SWI/SNF family. These new complexes contain as many as eight subunits identical to those found in other SWI/SNF complexes, and they possess a similar ATP-dependent nucleosome disruption activity. But unlike known SWI/SNFs, the new complexes are low in abundance and contain an extra subunit conserved between human and yeast SWI/SNF complexes. This subunit, ENL, is a homolog of the yeast SWI/SNF subunit, ANC1/TFG3. Moreover, ENL is a fusion partner for the gene product of MLL that is a common target for chromosomal translocations in human acute leukemia. The resultant MLL-ENL fusion protein associates and cooperates with SWI/SNF complexes to activate transcription of the promoter of HoxA7, a downstream target essential for oncogenic activity of MLL-ENL. Our data suggest that human SWI/SNF complexes show considerable heterogeneity, and one or more may be involved in the etiology of leukemia by cooperating with MLL fusion proteins.

The mammalian SWI/SNF-related complexes facilitate gene transcription by remodeling chromatin using the energy of ATP hydrolysis. The recruitment of these complexes to promoters remains poorly understood and may involve histone modifications or direct interactions with site-specific transcription factors or other cofactors. Here we report the isolation of two related but distinct cDNA clones, hOsa1 and hOsa2, that encode the largest subunits of human SWI/SNF. hOsa1 is identical to previously reported BAF250, and hOsa2 shares a high degree of sequence similarity with hOsa1. Mass spectrometric analysis, and immunoblotting with antibodies specific to hOsa1 or hOsa2 demonstrate the presence of both proteins in SWI/SNF-A but not in the related polybromo-BRG1-associated factors complex purified from HeLa cells. Co-precipitation studies indicate that hOsa1 and hOsa2 associate with BRG1 and hBRM through the C-terminal domain of hOsa. We define multiple domains within hBRM and BRG1 that interact with the hOsa C terminus. In cultured mammalian cells, hOsa1 and hOsa2 stimulate transcription by the glucocorticoid, estrogen, and androgen receptors. The glucocorticoid receptor-mediated activation is not observed with the C-terminal domain or with the hOsa2 polypeptide lacking the ARID DNA binding domain. These results suggest that hOsa1 and hOsa2 participate in promoting transcriptional activation by the steroid hormone receptors.