A systems understanding of nuclear organization and events is critical for determining how cells divide, differentiate, and respond to stimuli and for identifying the causes of diseases. Chromatin remodeling complexes such as SWI/SNF have been implicated in a wide variety of cellular processes including gene expression, nuclear organization, centromere function, and chromosomal stability, and mutations in SWI/SNF components have been linked to several types of cancer. To better understand the biological processes in which chromatin remodeling proteins participate, we globally mapped binding regions for several components of the SWI/SNF complex throughout the human genome using ChIP-Seq. SWI/SNF components were found to lie near regulatory elements integral to transcription (e.g. 5′ ends, RNA Polymerases II and III, and enhancers) as well as regions critical for chromosome organization (e.g. CTCF, lamins, and DNA replication origins). Interestingly we also find that certain configurations of SWI/SNF subunits are associated with transcripts that have higher levels of expression, whereas other configurations of SWI/SNF factors are associated with transcripts that have lower levels of expression. To further elucidate the association of SWI/SNF subunits with each other as well as with other nuclear proteins, we also analyzed SWI/SNF immunoprecipitated complexes by mass spectrometry. Individual SWI/SNF factors are associated with their own family members, as well as with cellular constituents such as nuclear matrix proteins, key transcription factors, and centromere components, implying a ubiquitous role in gene regulation and nuclear function. We find an overrepresentation of both SWI/SNF-associated regions and proteins in cell cycle and chromosome organization. Taken together the results from our ChIP and immunoprecipitation experiments suggest that SWI/SNF facilitates gene regulation and genome function more broadly and through a greater diversity of interactions than previously appreciated.
Genetic information and programming are not entirely contained in DNA sequence but are also governed by chromatin structure. Gaining a greater understanding of chromatin remodeling complexes can bridge gaps between processes in the genome and the epigenome and can offer insights into diseases such as cancer. We identified targets of the chromatin remodeling complex, SWI/SNF, on a genome-wide scale using ChIP-Seq. We also identify proteins that co-purify with its various components via immunoprecipitation combined with mass spectrometry. By integrating these newly-identified regions with a combination of novel and published data sources, we identify pathways and cellular compartments in which SWI/SNF plays a major role as well as discern general characteristics of SWI/SNF target sites. Our parallel evaluations of multiple SWI/SNF factors indicate that these subunits are found in highly dynamic and combinatorial assemblies. Our study presents the first genome-wide and unified view of multiple SWI/SNF components and also provides a valuable resource to the scientific community as an important data source to be integrated with future genomic and epigenomic studies.
The yeast SNF-SWI complex is required for transcriptional activation of diverse genes and has been shown to alter chromatin structure. The complex has at least 10 components, including SNF2/SWI2, SNF5, SNF6, SWI1/ADR6, and SWI3, and has been widely conserved in eukaryotes. Here we report the characterization of a new component. We identified proteins that interact in the two-hybrid system with the N-terminal region of SNF2, preceding the ATPase domain. In addition to SWI3, we recovered a new 19-kDa protein, designated SNF11. Like other SNF/SWI proteins, SNF11 functions as a transcriptional activator in genetic assays. SNF11 interacts with SNF2 in vitro and copurifies with the SNF-SWI complex from yeast cells. Using a specific antibody, we showed that SNF11 coimmunoprecipitates with members of the SNF-SWI complex and that SNF11 is tightly and stoichiometrically associated with the complex. Furthermore, SNF11 was detected in purified SNF-SWI complex by staining with Coomassie blue dye; its presence previously went unrecognized because it does not stain with silver. SNF11 interacts with a 40-residue sequence of SNF2 that is highly conserved, suggesting that SNF11 homologs exist in other organisms.
Yeast (Saccharomyces cerevisiae) SWI/SNF is a prototype for a large family of ATP-dependent chromatin-remodeling enzymes that facilitate numerous DNA-mediated processes. Swi2/Snf2 is the catalytic subunit of SWI/SNF, and it is the founding member of a novel subfamily of the SF2 superfamily of DNA helicase/ATPases. Here we present a functional analysis of the diagnostic set of helicase/ATPase sequence motifs found within all Swi2p/Snf2p family members. Whereas many of these motifs play key roles in ATP binding and/or hydrolysis, we identify residues within conserved motif V that are specifically required to couple ATP hydrolysis to chromatin-remodeling activity. Interestingly, motif V of the human Swi2p/Snf2p homolog, Brg1p, has been shown to be a possible hot spot for mutational alterations associated with cancers.
The Saccharomyces cerevisiae SNF2 gene affects the expression of many diversely regulated genes and has been implicated in transcriptional activation. We report here the cloning and characterization of STH1, a gene that is homologous to SNF2. STH1 is essential for mitotic growth and is functionally distinct from SNF2. A bifunctional STH1-beta-galactosidase protein is located in the nucleus. The predicted 155,914-Da STH1 protein is 72% identical to SNF2 over 661 amino acids and 46% identical over another stretch of 66 amino acids. Both STH1 and SNF2 contain a putative nucleoside triphosphate-binding site and sequences resembling the consensus helicase motifs. The large region of homology shared by STH1 and SNF2 is conserved among other eukaryotic proteins, and STH1 and SNF2 appear to define a novel family of proteins related to helicases.
As part of large protein complexes, Snf2 family ATPases are responsible for energy supply during chromatin remodeling, but the precise mechanism of action of many of these proteins is largely unknown. They influence many processes in plants, such as the response to environmental stress. This analysis is the first comprehensive study of Snf2 family ATPases in plants. We here present a comparative analysis of 1159 candidate plant Snf2 genes in 33 complete and annotated plant genomes, including two green algae. The number of Snf2 ATPases shows considerable variation across plant genomes (17-63 genes). The DRD1, Rad5/16 and Snf2 subfamily members occur most often. Detailed analysis of the plant-specific DRD1 subfamily in related plant genomes shows the occurrence of a complex series of evolutionary events. Notably tomato carries unexpected gene expansions of DRD1 gene members. Most of these genes are expressed in tomato, although at low levels and with distinct tissue or organ specificity. In contrast, the Snf2 subfamily genes tend to be expressed constitutively in tomato. The results underpin and extend the Snf2 subfamily classification, which could help to determine the various functional roles of Snf2 ATPases and to target environmental stress tolerance and yield in future breeding.
The Saccharomyces cerevisiae Swi/Snf complex has a role in remodeling chromatin structure to facilitate transcriptional activation. The complex has 11 components, including Swi1/Adr6, Swi2/Snf2, Swi3, Snf5, Snf6, Snf11, Swp73/Snf12, and Tfg3. Mammalian homologs of these proteins have been shown to form multiple Swi/Snf-related complexes. Here we characterize an S. cerevisiae Swi3 homolog (Swh3) and present evidence that it associates in a complex with a Snf2 homolog, Sthl. We identified Swh3 as a protein that interacts with the N terminus of Snf2 in the two-hybrid system. Swh3 and Swi3 are functionally distinct, and overexpression of one does not compensate for loss of the other. Swh3 is essential for viability and does not activate transcription of reporters. The Snf2 sequence that interacts with Swh3 was mapped to a region conserved in Sth1. We show that Swh3 and Sth1 fusion proteins interact in the two-hybrid system and coimmunoprecipitate from yeast cell extracts. We also map interactions between Swh3 and Sth1 and examine the role of a leucine zipper motif in self-association of Swh3. These findings, together with previous analysis of Sth1, indicate that Swh3 and Sth1 are associated in a complex that is functionally distinct from the Swi/Snf complex and essential for viability.
Like other DNA viruses that replicate in the nucleus, herpes simplex virus 1 (HSV-1) regulates the association of histones with its genome to promote viral replication and gene expression. We previously demonstrated that SNF2H, a member of the ISWI family of chromatin-remodeling factors, is concentrated in HSV-1 replication compartments in the nuclei of infected cells, suggesting that this cellular enzyme plays a role in viral replication. We show here that small interfering RNA (siRNA)-mediated knockdown of SNF2H in HEp-2 cells resulted in an approximately 20-fold decrease in HSV-1 replication, arguing that SNF2H promotes efficient HSV-1 replication. Decreases in HSV-1 replication were observed with multiple SNF2H-specific siRNAs, and the extent of the replication decrease correlated with the amount of SNF2H knockdown, indicating that the phenotype resulted from decreased SNF2H levels rather than off-target effects of the siRNAs. We also observed a decrease in the accumulation of immediate-early (IE) gene products in HSV-1-infected cells in which SNF2H was knocked down. Histone H3 occupancy on viral promoters was increased in HSV-1-infected cells that were transfected with SNF2H-specific siRNAs, suggesting that SNF2H promotes removal of histones from viral promoters during infection. Furthermore, chromatin immunoprecipitation (ChIP) studies showed that SNF2H associated with the HSV-1 genome during infection, which suggests that SNF2H may directly remodel viral chromatin. We hypothesize that SNF2H is recruited to viral promoters during HSV-1 infection, where it can remodel the chromatin state of the viral genome, facilitate the transcription of immediate-early genes, and enhance viral replication.
It is becoming increasingly appreciated that regulation of the state of chromatin is a major determinant in control of gene expression. It has also become clear that the state of chromatin of the herpes simplex virus type 1 (HSV-1) genome is dynamically regulated during both productive and latent stages of infection. In addition, multiple viral gene products have been reported to play roles in regulating the viral chromatin state. However, the cellular chromatin-remodeling factors involved in altering nucleosome occupancy at viral genes remain largely unknown. The results in this report represent the first evidence that cellular chromatin-remodeling proteins, and SNF2H in particular, can play important roles in regulating the chromatin state of the HSV-1 genome during infection. This work also further establishes HSV-1 infection as a useful model to study chromatin control of gene expression and suggests that disrupting the regulation of viral chromatin states can possibly be exploited as a novel antiviral therapeutic target.
The yeast SWI2/SNF2 polypeptide is a subunit of the SWI/SNF protein complex that is required for many transcriptional activators to function in a chromatin context. SWI2 is believed to be the founding member of a new subfamily of DNA-stimulated ATPases/DNA helicases that includes proteins that function in DNA repair (RAD5, RAD16, ERCC6), recombination (RAD54), transcription (MOT1, ISWI, brm, BRG1, hBRM) and cell cycle control (STH1). We have created a set of 16 mutations within the SWI2 ATPase domain and have analyzed the functional consequences of these mutations in vivo. We have identified residues within each of the seven ATPase motifs that are required for SWI2 function. We have also identified crucial residues that are interspersed between the known ATPase motifs. In contrast, we identify other highly conserved residues that appear to be dispensable for SWI2 function. We also find that single amino acid changes in ATPase motifs IV and VI lead to a dominant negative phenotype. None of the 12 SWI2 mutations that disrupt SWI2 activity in vivo alter the assembly of the SWI/SNF complex. These studies provide an invaluable framework for biochemical analysis of the SWI2 ATPase and for functional analysis of other SWI2 family members.
Function prediction by homology is widely used to provide preliminary functional annotations for genes for which experimental evidence of function is unavailable or limited. This approach has been shown to be prone to systematic error, including percolation of annotation errors through sequence databases. Phylogenomic analysis avoids these errors in function prediction but has been difficult to automate for high-throughput application. To address this limitation, we present a computationally efficient pipeline for phylogenomic classification of proteins. This pipeline uses the SCI-PHY (Subfamily Classification in Phylogenomics) algorithm for automatic subfamily identification, followed by subfamily hidden Markov model (HMM) construction. A simple and computationally efficient scoring scheme using family and subfamily HMMs enables classification of novel sequences to protein families and subfamilies. Sequences representing entirely novel subfamilies are differentiated from those that can be classified to subfamilies in the input training set using logistic regression. Subfamily HMM parameters are estimated using an information-sharing protocol, enabling subfamilies containing even a single sequence to benefit from conservation patterns defining the family as a whole or in related subfamilies. SCI-PHY subfamilies correspond closely to functional subtypes defined by experts and to conserved clades found by phylogenetic analysis. Extensive comparisons of subfamily and family HMM performances show that subfamily HMMs dramatically improve the separation between homologous and non-homologous proteins in sequence database searches. Subfamily HMMs also provide extremely high specificity of classification and can be used to predict entirely novel subtypes. The SCI-PHY Web server at http://phylogenomics.berkeley.edu/SCI-PHY/ allows users to upload a multiple sequence alignment for subfamily identification and subfamily HMM construction. Biologists wishing to provide their own subfamily definitions can do so. Source code is available on the Web page. The Berkeley Phylogenomics Group PhyloFacts resource contains pre-calculated subfamily predictions and subfamily HMMs for more than 40,000 protein families and domains at http://phylogenomics.berkeley.edu/phylofacts/.
Predicting the function of a gene or protein (gene product) from its primary sequence is a major focus of many bioinformatics methods. In this paper, the authors present a three-stage computational pipeline for gene functional annotation in an evolutionary framework to reduce the systematic errors associated with the standard protocol (annotation transfer from predicted homologs). In the first stage, a functional hierarchy is estimated for each protein family and subfamilies are identified. In the second stage, hidden Markov models (HMMs) (a type of statistical model) are constructed for each subfamily to model both the family-defining and subfamily-specific signatures. In the third stage, subfamily HMMs are used to assign novel sequences to functional subtypes. Extensive experimental validation of these methods shows that predicted subfamilies correspond closely to functional subtypes identified by experts and to conserved clades in phylogenetic trees; that subfamily HMMs increase the separation between homologs and non-homologs in sequence database discrimination tests relative to the use of a single HMM for the family; and that specificity of classification of novel sequences to subfamilies using subfamily HMMs is near perfect (1.5% error rate when sequences are assigned to the top-scoring subfamily, and <0.5% error rate when logistic regression of scores is employed).
Helicase-like proteins play a crucial role in nucleic acid- and chromatin-mediated reactions. In this study, we identified 134 helicase-like proteins in the nematode Caenorhabditis elegans and classified the proteins into 10 known subfamilies and a group of orphan genes on the basis of sequence similarity. We characterized loss-of-function phenotypes in RNA interference (RNAi)-treated animals for helicase family members, using the RNAi feeding method, and found several previously unreported phenotypes. Fifty-one (39.5%) of 129 genes tested showed development- or growth-defect phenotypes, and many of these genes were putative nematode homologs of essential genes in a unicellular eukaryote, budding yeast, suggesting conservation of these essential proteins in both species. Comparative analyses between these species identified evolutionarily diverged nematode proteins as well as conserved family members. Chromosome mapping of the nematode genes revealed 10 pairs of putative duplicated genes and clusters of C. elegans-specific SNF2-like genes and Helitrons. Analyses of transcriptional profile data revealed a predominantly oogenesis- and germline-enriched expression of many helicase-like genes. Finally, we identified the D2005.5(drh-3) gene in an RNAi-based screen for genes involved in resistance to X-ray irradiation. Analysis of DRH-3 will clarify the potentially novel mechanism by which it protects against X-ray-induced damage in C. elegans.
C. elegans; comparative genomics; drh-3; helicase family; RNAi-based screen
Helicases are motor proteins that catalyze the unwinding of duplex nucleic acids in an ATP-dependent manner. They are involved in almost all the nucleic acid transactions. In the present study, we report a comprehensive analysis of helicase gene family in human and its comparison with homologs in model organisms. The human genome encodes for 95 non-redundant helicase proteins, of which 64 are RNA helicases and 31 are DNA helicases. 57 RNA helicases are validated based on annotations and occurrence of conserved helicase signature motifs. These include 14 DExH and 37 DExD subfamily members, six other members such as U5.snRNP, ATR-X, Suv3, FANCJ, and two of superkiller viralicidic activity 2-like helicases. 31 DNA helicases are also identified, which include RecQ, MCM and RuvB-like helicases. Finding a set of helicases in human and almost similar sequences in model organisms suggests that the “core” members of helicase gene family are highly conserved throughout evolution. The present study gives an overview of members of RNA and DNA helicases encoded by the human genome along with their conserved motifs, phylogeny and homologs in model organisms. The study on comparing these homologs will spread light on the organization and complexity of helicase gene family in model organisms. The comprehensive analysis of human helicases presented in this study will further provide an invaluable resource for elaborate biological research on these helicases.
DEAD-box; DHX and DDX helicases; human helicases; MCM proteins; RecQ helicases; RNA helicases
The SWI/SNF family of chromatin-remodeling complexes facilitates gene activation by assisting transcription machinery to gain access to targets in chromatin. This family includes BAF (also called hSWI/SNF-A) and PBAF (hSWI/SNF-B) from humans and SWI/SNF and Rsc from Saccharomyces cerevisiae. However, the relationship between the human and yeast complexes is unclear because all human subunits published to date are similar to those of both yeast SWI/SNF and Rsc. Also, the two human complexes have many identical subunits, making it difficult to distinguish their structures or functions. Here we describe the cloning and characterization of BAF250, a subunit present in human BAF but not PBAF. BAF250 contains structural motifs conserved in yeast SWI1 but not in any Rsc components, suggesting that BAF is related to SWI/SNF. BAF250 is also a homolog of the Drosophila melanogaster Osa protein, which has been shown to interact with a SWI/SNF-like complex in flies. BAF250 possesses at least two conserved domains that could be important for its function. First, it has an AT-rich DNA interaction-type DNA-binding domain, which can specifically bind a DNA sequence known to be recognized by a SWI/SNF family-related complex at the β-globin locus. Second, BAF250 stimulates glucocorticoid receptor-dependent transcriptional activation, and the stimulation is sharply reduced when the C-terminal region of BAF250 is deleted. This region of BAF250 is capable of interacting directly with the glucocorticoid receptor in vitro. Our data suggest that BAF250 confers specificity to the human BAF complex and may recruit the complex to its targets through either protein-DNA or protein-protein interactions.
Chromatin remodelling is the ATP-dependent change in nucleosome organisation driven by Snf2 family ATPases. The biochemistry of this process depends on the behaviours of ATP dependent motor proteins and their dynamic nucleosome substrates, which brings significant technical and conceptual challenges. Steady progress has been made in characterizing the polypeptides that these enzymes are comprised of. Divergence in the sequences of different subfamilies of Snf2 related proteins suggests that the motors are adapted for different functions. Recently structural insights have suggested that the Snf2 ATPase acts as a context-sensitive DNA translocase. This may have arisen as a means to enable efficient access to DNA in the high density of the eukaryotic nucleus. How the enzymes engage nucleosomes and how the network of non-covalent interactions within the nucleosome respond to the force applied remains unclear, and it remains prudent to recognise the potential for both DNA distortions and dynamics within the underlying histone octamer structure.
The Snf1 protein kinase plays a central role in the response to glucose starvation in the yeast Saccharomyces cerevisiae. Previously, we showed that two-hybrid interaction between Snf1 and its activating subunit, Snf4, is inhibited by high levels of glucose. These findings, together with biochemical evidence that Snf1 and Snf4 remain associated in cells grown in glucose, suggested that another protein (or proteins) anchors Snf1 and Snf4 into a complex. Here, we examine the possibility that a family of proteins, comprising Sip1, Sip2, and Gal83, serves this purpose. We first show that the fraction of cellular Snf4 protein that is complexed with Snf1 is reduced in a sip1delta sip2delta gal83delta triple mutant. We then present evidence that Sip1, Sip2, and Gal83 each interact independently with both Snf1 and Snf4 via distinct domains. A conserved internal region binds to the Snf1 regulatory domain, and the conserved C-terminal ASC domain binds to Snf4. Interactions were mapped by using the two-hybrid system and were confirmed by in vitro binding studies. These findings indicate that the Sip1/Sip2/Gal83 family anchors Snf1 and Snf4 into a complex. Finally, the interaction of the yeast Sip2 protein with a plant Snf1 homolog suggests that this function is conserved in plants.
Regulation of surface antigenic variation in Giardia lamblia is controlled post-transcriptionally by an RNA-interference (RNAi) pathway that includes a Dicer-like bidentate RNase III (gDicer). This enzyme, however, lacks the RNA helicase domain present in Dicer enzymes from higher eukaryotes. The participation of several RNA helicases in practically all organisms in which RNAi was studied suggests that RNA helicases are potentially involved in antigenic variation, as well as during Giardia differentiation into cysts.
An extensive in silico analysis of the Giardia genome identified 32 putative Super Family 2 RNA helicases that contain almost all the conserved RNA helicase motifs. Phylogenetic studies and sequence analysis separated them into 22 DEAD-box, 6 DEAH-box and 4 Ski2p-box RNA helicases, some of which are homologs of well-characterized helicases from higher organisms. No Giardia putative helicase was found to have significant homology to the RNA helicase domain of Dicer enzymes. Additionally a series of up- and down-regulated putative RNA helicases were found during encystation and antigenic variation by qPCR experiments. Finally, we were able to recognize 14 additional putative helicases from three different families (RecQ family, Swi2/Snf2 and Rad3 family) that could be considered DNA helicases.
This is the first comprehensive analysis of the Super Family 2 helicases from the human intestinal parasite G. lamblia. The relative and variable expression of particular RNA helicases during both antigenic variation and encystation agrees with the proposed participation of these enzymes during both adaptive processes. The putatives RNA and DNA helicases identified in this early-branching eukaryote provide initial information regarding the biological role of these enzymes in cell adaptation and differentiation.
RNA/DNA helicases; Giardia lamblia; Encystation; Antigenic variation; Cell differentiation; Gene expression; RNAi; Dicer
The Saccharomyces cerevisiae SNF1 protein kinase, a member of the SNF1/AMP-activated protein kinase (AMPK) family, is activated by three kinases, Sak1, Tos3, and Elm1, which phosphorylate the Snf1 catalytic subunit on Thr-210 in response to glucose limitation and other stresses. Sak1 is the primary Snf1-activating kinase and is associated with Snf1 in a complex. Here we examine the interaction of Sak1 with SNF1. We report that Sak1 coimmunopurifies with the Snf1 catalytic subunit from extracts of both glucose-replete and glucose-limited cultures and that interaction occurs independently of the phosphorylation state of Snf1 Thr-210, Snf1 catalytic activity, and other SNF1 subunits. Sak1 interacts with the Snf1 kinase domain, and nonconserved sequences C terminal to the Sak1 kinase domain mediate interaction with Snf1 and augment the phosphorylation and activation of Snf1. The Sak1 C terminus is modified in response to glucose depletion, dependent on SNF1 activity. Replacement of the C terminus of Elm1 (or Tos3) with that of Sak1 enhanced the ability of the Elm1 kinase domain to interact with and phosphorylate Snf1. These findings indicate that the C terminus of Sak1 confers its function as the primary Snf1-activating kinase and suggest that the physical association of Sak1 with SNF1 facilitates responses to environmental change.
Chromatin regulates many key processes in the nucleus by controlling access to the underlying DNA. SNF2-like factors are ATP-driven enzymes that play key roles in the dynamics of chromatin by remodelling nucleosomes and other nucleoprotein complexes. Even simple eukaryotes such as yeast contain members of several subfamilies of SNF2-like factors. The FUN30/ETL1 subfamily of SNF2 remodellers is conserved from yeasts to humans, but is poorly characterized. We show that the deletion of FUN30 leads to sensitivity to the topoisomerase I poison camptothecin and to severe cell cycle progression defects when the Orc5 subunit is mutated. We demonstrate a role of FUN30 in promoting silencing in the heterochromatin-like mating type locus HMR, telomeres and the rDNA repeats. Chromatin immunoprecipitation experiments demonstrate that Fun30 binds at the boundary element of the silent HMR and within the silent HMR. Mapping of nucleosomes in vivo using micrococcal nuclease demonstrates that deletion of FUN30 leads to changes of the chromatin structure at the boundary element. A point mutation in the ATP-binding site abrogates the silencing function of Fun30 as well as its toxicity upon overexpression, indicating that the ATPase activity is essential for these roles of Fun30. We identify by amino acid sequence analysis a putative CUE motif as a feature of FUN30/ETL1 factors and show that this motif assists Fun30 activity. Our work suggests that Fun30 is directly involved in silencing by regulating the chromatin structure within or around silent loci.
Mutations in genes whose products modify chromatin structure have been recognized as a cause of X-linked mental retardation (XLMR). These genes encode proteins that regulate DNA methylation (MeCP2), modify histones (RSK2 and JARID1C), and remodel nucleosomes through ATP hydrolysis (ATRX). Thus, genes encoding other chromatin modifying proteins should also be considered as disease candidate genes. In this work, we have characterized the SNF2L gene, encoding an ATP-dependent chromatin remodeling protein of the ISWI family, and sequenced the gene in patients from 12 XLMR families linked to Xq25-26.
We used an in silico and RT-PCR approach to fully characterize specific SNF2L isoforms. Mutation screening was performed in 12 patients from individual families with syndromic or non-syndromic XLMR. We sequenced each of the 25 exons encompassing the entire coding region, complete 5' and 3' untranslated regions, and consensus splice-sites.
The SNF2L gene spans 77 kb and is encoded by 25 exons that undergo alternate splicing to generate several distinct transcripts. Specific isoforms are generated through the alternate use of exons 1 and 13, and by the use of alternate donor splice sites within exon 24. Alternate splicing within exon 24 removes a NLS sequence and alters the subcellular distribution of the SNF2L protein. We identified 3 single nucleotide polymorphisms but no mutations in our 12 patients.
Our results demonstrate that there are numerous splice variants of SNF2L that are expressed in multiple cell types and which alter subcellular localization and function. SNF2L mutations are not a cause of XLMR in our cohort of patients, although we cannot exclude the possibility that regulatory mutations might exist. Nonetheless, SNF2L remains a candidate for XLMR localized to Xq25-26, including the Shashi XLMR syndrome.
The SNF2 family of proteins includes representatives from a variety of species with roles in cellular processes such as transcriptional regulation (e.g. MOT1, SNF2 and BRM), maintenance of chromosome stability during mitosis (e.g. lodestar) and various aspects of processing of DNA damage, including nucleotide excision repair (e.g. RAD16 and ERCC6), recombinational pathways (e.g. RAD54) and post-replication daughter strand gap repair (e.g. RAD5). This family also includes many proteins with no known function. To better characterize this family of proteins we have used molecular phylogenetic techniques to infer evolutionary relationships among the family members. We have divided the SNF2 family into multiple subfamilies, each of which represents what we propose to be a functionally and evolutionarily distinct group. We have then used the subfamily structure to predict the functions of some of the uncharacterized proteins in the SNF2 family. We discuss possible implications of this evolutionary analysis on the general properties and evolution of the SNF2 family.
Epstein-Barr nuclear antigen 2 (EBNA2), one of the six viral nuclear proteins expressed in latently infected B lymphocytes, is essential to the immortalization of B cells by Epstein-Barr virus (EBV). EBNA2 promotes transcriptional transactivation of viral and cellular genes by acting as an adapter molecule that binds to cellular sequence-specific DNA-binding proteins, JK recombination signal-binding protein (RBP-JK), and PU.1 and engages multiple members of the RNA polymerase II transcription complex. In the present study, we show that EBNA2 also interacts with hSNF5/Ini1, the human homolog of the yeast transcription factor SNF5. Gel filtration fractionation of partially purified EBV-positive lymphocyte nuclear extracts shows that a fraction of EBNA2 coelutes with both hSNF5/Ini1 and BRG1, a human homolog of SWI/SNF2, in the high-molecular-mass region (1.5 to 2.0 MDa) of a Superose 6 chromatogram. An affinity-purified rabbit antibody directed against hSNF5/Ini1 coimmunoprecipitates EBNA2 from this high-molecular-mass nuclear protein fraction, demonstrating that EBNA2 and hSNF5/Ini1 interact in vivo. This interaction is restricted to a subpopulation of phosphorylated viral EBNA2. Deletion mutation analysis of EBNA2 shows that the proline-rich aminoterminal end and a domain within the divergent region of EBNA2 mediate EBNA2-hSNF5/Ini1 interaction. Since the SNF-SWI complex participates in gene regulation through the alteration of nucleosome configuration and may be a component of the RNA polymerase II holoenzyme, the EBNA2-hSNF5/Ini1 interaction supports the hypothesis that EBNA2 facilitates transcriptional transactivation by acting as a transcription adapter molecule. We postulate that EBNA2 engages the hSNF-SWI complex to generate an open chromatin conformation at the EBNA2-responsive target genes, thereby potentiating the function of the RBP-JK-EBNA2-polymerase II transcription complex.
The SWI/SNF family of ATP-dependent chromatin-remodeling factors plays a central role in eukaryotic transcriptional regulation. In yeast and human cells, two subclasses have been recognized: one comprises yeast SWI/SNF and human BAF, and the other includes yeast RSC and human PBAF. Therefore, it was puzzling that Drosophila appeared to contain only a single SWI/SNF-type remodeler, the Brahma (BRM) complex. Here, we report the identification of two novel BRM complex-associated proteins: Drosophila Polybromo and BAP170, a conserved protein not described previously. Biochemical analysis established that Drosophila contains two distinct BRM complexes: (i) the BAP complex, defined by the presence of OSA and the absence of Polybromo and BAP170, and (ii) the PBAP complex, containing Polybromo and BAP170 but lacking OSA. Determination of the genome-wide distributions of OSA and Polybromo on larval salivary gland polytene chromosomes revealed that BAP and PBAP display overlapping but distinct distribution patterns. Both complexes associate predominantly with regions of open, hyperacetylated chromatin but are largely excluded from Polycomb-bound repressive chromatin. We conclude that, like yeast and human cells, Drosophila cells express two distinct subclasses of the SWI/SNF family. Our results support a close reciprocity of chromatin regulation by ATP-dependent remodelers and histone-modifying enzymes.
Chromatin facilitates the housing of eukaryotic DNA within the nucleus and restricts access to the underlying sequences. Thus, the regulation of chromatin structure provides an excellent platform for regulating processes that require information stored within genomic DNA. Snf2 proteins are a family of helicase-like proteins that direct energy derived from ATP hydrolysis into the mechanical remodelling of chromatin structure. Here, we highlight some of the recent discoveries regarding this family of proteins and show Snf2 proteins have roles in many aspects of genetic metabolism. Recent developments include new insights into the mechanism for nucleosome spacing and histone dimer exchange; together with growing evidence for the involvement of Snf2 proteins in DNA repair.
Emerging evidence demonstrates that subunits of the SWI/SNF chromatin remodeling complex are specifically mutated at high frequency in a variety of human cancer types. SNF5 (SMARCB1/INI1/BAF47), a core subunit of the SWI/SNF complex, is inactivated in the vast majority of rhabdoid tumors (RT), an aggressive type of pediatric cancer. SNF5-deficient cancers are diploid and genomically stable, suggesting that epigenetically based changes in transcription are key drivers of tumor formation caused by SNF5 loss. However, there is limited understanding of the target genes that drive cancer formation following SNF5 loss. Here we performed comparative expression analyses upon three independent SNF5-deficient cancer data sets from both human and mouse and identify downregulation of the BIN1 tumor suppressor as a conserved event in primary SNF5-deficient cancers. We show that SNF5 recruits the SWI/SNF complex to the BIN1 promoter, and that the marked reduction of BIN1 expression in RT correlates with decreased SWI/SNF occupancy. Functionally, we demonstrate that re-expression of BIN1 specifically compromises the proliferation of SNF5-deficient RT cell lines. Identification of BIN1 as a SNF5 target gene reveals a novel tumor suppressive regulatory mechanism whose disruption can drive cancer formation.
BAF47; BIN1; INI1; rhabdoid tumor; SMARCB1; SNF5
In the yeast Saccharomyces cerevisiae, the Snf1 protein kinase of the Snf1/AMP-activated protein kinase (AMPK) family regulates a wide range of responses to stress caused by glucose deprivation. The stress signal is relayed via upregulation of Snf1, which depends on phosphorylation of its activation loop Thr210 residue by upstream kinases. Although Snf1 is also required for coping with various stresses unrelated to glucose deprivation, some evidence suggests a role for low-level basal activity of unphosphorylated Snf1, rather than a specific signaling function. We previously found that Snf1 is required for diploid pseudohyphal differentiation, a developmental response to nitrogen limitation. Here, we present evidence that Snf1 is directly involved in nitrogen signaling. First, genetic analyses suggest that pseudohyphal differentiation depends on the stimulatory phosphorylation of Snf1 at Thr210. Second, immunochemical data indicate that nitrogen limitation improves Thr210 phosphorylation. Analyses of pseudohyphal differentiation in cells with catalytically inactive and hyperactive Snf1 support the role of Snf1 activity. Finally, we show that Snf1 is negatively regulated by the rapamycin-sensitive TOR kinase which plays essential roles in signaling nitrogen and amino acid availability. This and other evidence implicate Snf1 in the integration of signals regarding nitrogen and carbon stress. TOR and Snf1/AMPK are highly conserved in evolution, and their novel functional interaction in yeast suggests similar mechanisms in other eukaryotes.
Sans-fille (SNF) is the Drosophila homologue of mammalian general splicing factors U1A and U2B″, and it is essential in Drosophila sex determination. We found that, besides its ability to bind U1 snRNA, SNF can also bind polyuridine RNA tracts flanking the male-specific exon of the master switch gene Sex-lethal (Sxl) pre-mRNA specifically, similar to Sex-lethal protein (SXL). The polyuridine RNA binding enables SNF directly inhibit Sxl exon 3 splicing, as the dominant negative mutant SNF1621 binds U1 snRNA but not polyuridine RNA. Unlike U1A, both RNA recognition motifs (RRMs) of SNF can recognize polyuridine RNA tracts independently, even though SNF and U1A share very high sequence identity and overall structure similarity. As SNF RRM1 tends to self-associate on the opposite side of the RNA binding surface, it is possible for SNF to bridge the formation of super-complexes between two introns flanking Sxl exon 3 or between a intron and U1 snRNP, which serves the molecular basis for SNF to directly regulate Sxl splicing. Taken together, a new functional model for SNF in Drosophila sex determination is proposed. The key of the new model is that SXL and SNF function similarly in promoting Sxl male-specific exon skipping with SNF being an auxiliary or backup to SXL, and it is the combined dose of SXL and SNF governs Drosophila sex determination.