In silico characterization of the INO80 subfamily of SWI2/SNF2 chromatin remodeling proteins

Proteins belonging to SNF2 family of DNA dependent ATPases are important members of the chromatin remodeling complexes that are implicated in epigenetic control of gene expression. The yeast Ino80, the catalytic ATPase subunit of the INO80 complex, is the most recently described member of the SNF2 family. Outside the conserved ATPase domain, it has very little similarity with other well-characterized SNF2 proteins hence it is believed to represent a new subfamily. We have identified new members of this subfamily in different organisms and have detected characteristic features of this subfamily. Using various data mining tools we have identified a new, previously undetected domain in all members of this subfamily. This domain designated DBINO is characteristic of the INO80 subfamily and is predicted to have DNA-binding function. The presence of this domain in all the INO80 subfamily proteins from different organisms suggests its conserved function in evolution.     

Biochemical characterisation of the SWI/SNF family member HLTF

HLTF is highly similar in domain organisation to yeast Rad5. We identify PTIP and RPA70, both involved in DNA replication and DNA repair, as HLTF-interacting proteins although cells depleted of HLTF did not show defects in cellular responses to DNA damage. In vitro, HLTF has ATPase activity and E3 ubiquitin ligase activity with a range of E2 ubiquitin conjugating enzymes. HLTF expression is severely reduced in a range of cancer cells, and we suggest that the HLTF antibodies generated in this study could be used for cancer diagnostic purposes.     

SWI/SNF Chromatin-remodeling Factors: Multiscale Analyses and Diverse Functions

Chromatin-remodeling enzymes play essential roles in many biological processes, including gene expression, DNA replication and repair, and cell division. Although one such complex, SWI/SNF, has been extensively studied, new discoveries are still being made. Here, we review SWI/SNF biochemistry; highlight recent genomic and proteomic advances; and address the role of SWI/SNF in human diseases, including cancer and viral infections. These studies have greatly increased our understanding of complex nuclear processes.     

A SWI/SNF-like factor from chicken liver that disrupts nucleosomes and transfers histone octamers in cis and trans

ATP-dependent chromatin remodeling factors have been implicated in nuclear processes involving DNA. Here we report partial purification and characterization of an ATP-dependent chromatin remodeling activity from chicken liver. Nuclear extract from chicken liver was fractionated chromatographically to enrich proteins immunoreacting to antibodies against components of human SWI/SNF, namely BRG1, BAF170, BAF155, and BAF57. Immunoreactivity to these antibodies elutes with a mass of about 2MDa on Sepharose CL-6B gel filtration, suggesting that they constitute a SWI/SNF-like complex (SLC). The SLC displays three chromatin-remodeling activities, viz. nucleosome disruption, octamer transfer, and nucleosome sliding (octamer transfer in cis). We further show that components of SLC, as revealed by immunoreactivity to the above antibodies, display a dynamic nucleocytoplasmic distribution and colocalize with RNA polymerase II in the liver nuclei. This report contributes to the understanding of phylogenetic generality of chromatin remodeling factors in eukaryotes.     

SWI/SNF complex in disorder: SWItching from malignancies to intellectual disability

Heterozygous germline mutations in components of switch/sucrose nonfermenting (SWI/SNF) chromatin remodeling complexes were recently identified in patients with non-syndromic intellectual disability, Coffin-Siris syndrome and Nicolaides-Baraitser syndrome. The common denominator of the phenotype of these patients is severe intellectual disability and speech delay. Somatic and germline mutations in SWI/SNF components were previously implicated in tumor development. This raises the question whether patients with intellectual disability caused by SWI/SNF mutations in the germline are exposed to an increased risk of developing cancer. Here we compare the mutational spectrum of SWI/SNF components in intellectual disability syndromes and cancer, and discuss the implications of the results of this comparison for the patients.     

ATP-dependent chromatin remodeling complex SWI/SNF in cardiogenesis and cardiac progenitor cell development

The recent identification of cardiac progenitor cells (CPCs) provides a new paradigm for studying and treating heart disease.To realize the full potential of CPCs for therapeutic purposes,it is essenti...     

Coronary development is regulated by ATP-dependent SWI/SNF chromatin remodeling component BAF180

Dissecting the molecular mechanisms that guide the proper development of epicardial cell lineages is critical for understanding the etiology of both congenital and adult forms of human cardiovascular disease. In this study, we describe the function of BAF180, a polybromo protein in ATP-dependent SWI/SNF chromatin remodeling complexes, in coronary development. Ablation of BAF180 leads to impaired epithelial-to-mesenchymal-transition (EMT) and arrested maturation of epicardium around E11.5. Three-dimensional collagen gel assays revealed that the BAF180 mutant epicardial cells indeed possess significantly compromised migrating and EMT potentials. Consequently, the mutant hearts form abnormal surface nodules and fail to develop the fine and continuous plexus of coronary vessels that cover the entire ventricle around E14. PECAM and α-SMA staining assays indicate that these nodules are defective structures resulting from the failure of endothelial and smooth muscle cells within them to form coronary vessels. PECAM staining also reveal that there are very few coronary vessels inside the myocardium of mutant hearts. Consistent with this, quantitative RT-PCR analysis indicate that the expression of genes involved in FGF, TGF, and VEGF pathways essential for coronary development are down-regulated in mutant hearts. Together, these data reveal for the first time that BAF180 is critical for coronary vessel formation.     

Essential role of ARID2 protein-containing SWI/SNF complex in tissue-specific gene expression

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.     

Ubiquitin-dependent and Ubiquitin-independent Control of Subunit Stoichiometry in the SWI/SNF Complex

The mammalian SWI/SNF chromatin remodeling complex is a key player in multiple chromatin transactions. Core subunits of this complex, including the ATPase, Brg-1, and various Brg-1-associated factors (BAFs), work in concert to maintain a functional remodeling complex. This intra-complex regulation is supervised by protein-protein interactions, as stoichiometric levels of BAF proteins are maintained by proteasomal degradation. We show that the mechanism of BAF155-mediated stabilization of BAF57 involves blocking its ubiquitination by preventing interaction with TRIP12, an E3 ubiquitin ligase. Consequently, as opposed to complexed BAF57, whose principal lysines are unavailable for ubiquitination, uncomplexed BAF57 can be freely ubiquitinated and degraded by the proteasome. Additionally, a BAF57 mutant, which contains no lysine residues, was found to retain its ability to be stabilized by interaction with BAF155, suggesting that in addition to the ubiquitin-dependent mechanism of BAF57 degradation, there exists a ubiquitin-independent mechanism that may involve the direct interaction of BAF57 with the proteasome. We propose that this regulatory mechanism exists to ensure functional fidelity of the complex and prevent the accumulation of uncomplexed proteins, which may disrupt the normal activity of the complex.     

Mutations in two non‐canonical Arabidopsis SWI2/SNF2 chromatin remodeling ATPases cause embryogenesis and stem cell maintenance defects

SWI2/SNF2 chromatin remodeling ATPases play important roles in plant and metazoan development. Whereas metazoans generally encode one or two SWI2/SNF2 ATPase genes, Arabidopsis encodes four such chromatin regulators: the well‐studied BRAHMA and SPLAYED ATPases, as well as two closely related non‐canonical SWI2/SNF2 ATPases, CHR12 and CHR23. No developmental role has as yet been described for CHR12 and CHR23. Here, we show that although strong single chr12 or chr23 mutants are morphologically indistinguishable from the wild type, chr12 chr23 double mutants cause embryonic lethality. The double mutant embryos fail to initiate root and shoot meristems, and display few and aberrant cell divisions. Weak double mutant embryos give rise to viable seedlings with dramatic defects in the maintenance of both the shoot and the root stem cell populations. Paradoxically, the stem cell defects are correlated with increased expression of the stem cell markers WUSCHEL and WOX5. During subsequent development, the meristem defects are partially overcome to allow for the formation of very small, bushy adult plants. Based on the observed morphological defects, we named the two chromatin remodelers MINUSCULE 1 and 2. Possible links between minu1 minu2 defects and defects in hormone signaling and replication‐coupled chromatin assembly are discussed.     

Next-generation bromodomain inhibitors of the SWI/SNF complex enhance DNA damage and cell death in glioblastoma

Glioblastoma (GBM) is an aggressive brain cancer with a poor prognosis. While surgical resection is the primary treatment, adjuvant temozolomide (TMZ) chemotherapy and radiotherapy only provide slight improvement in disease course and outcome. Unfortunately, most treated patients experience recurrence of highly aggressive, therapy-resistant tumours and eventually succumb to the disease. To increase chemosensitivity and overcome therapy resistance, we have modified the chemical structure of the PFI-3 bromodomain inhibitor of the BRG1 and BRM catalytic subunits of the SWI/SNF chromatin remodelling complex. Our modifications resulted in compounds that sensitized GBM to the DNA alkylating agent TMZ and the radiomimetic bleomycin. We screened these chemical analogues using a cell death ELISA with GBM cell lines and a cellular thermal shift assay using epitope tagged BRG1 or BRM bromodomains expressed in GBM cells. An active analogue, IV-129, was then identified and further modified, resulting in new generation of bromodomain inhibitors with distinct properties. IV-255 and IV-275 had higher bioactivity than IV-129, with IV-255 selectively binding to the bromodomain of BRG1 and not BRM, while IV-275 bound well to both BRG1 and BRM bromodomains. In contrast, IV-191 did not bind to either bromodomain or alter GBM chemosensitivity. Importantly, both IV-255 and IV-275 markedly increased the extent of DNA damage induced by TMZ and bleomycin as determined by nuclear γH2AX staining. Our results demonstrate that these next-generation inhibitors selectively bind to the bromodomains of catalytic subunits of the SWI/SNF complex and sensitize GBM to the anticancer effects of TMZ and bleomycin. This approach holds promise for improving the treatment of GBM.