Identification of multiple distinct Snf2 subfamilies with conserved structural motifs

The Snf2 family of helicase-related proteins includes the catalytic subunits of ATP-dependent chromatin remodelling complexes found in all eukaryotes. These act to regulate the structure and dynamic properties of chromatin and so influence a broad range of nuclear processes. We have exploited progress in genome sequencing to assemble a comprehensive catalogue of over 1300 Snf2 family members. Multiple sequence alignment of the helicase-related regions enables 24 distinct subfamilies to be identified, a considerable expansion over earlier surveys. Where information is known, there is a good correlation between biological or biochemical function and these assignments, suggesting Snf2 family motor domains are tuned for specific tasks. Scanning of complete genomes reveals all eukaryotes contain members of multiple subfamilies, whereas they are less common and not ubiquitous in eubacteria or archaea. The large sample of Snf2 proteins enables additional distinguishing conserved sequence blocks within the helicase-like motor to be identified. The establishment of a phylogeny for Snf2 proteins provides an opportunity to make informed assignments of function, and the identification of conserved motifs provides a framework for understanding the mechanisms by which these proteins function

Histone H2AX Y142 phosphorylation is a low abundance modification

We employ targeted mass spectrometry to compare the levels of H2AX S139 phosphorylation (γH2AX) and Y142 phosphorylation. We use synthetic peptides to facilitate MS optimisation and estimate relative detection efficiencies for the different modifications. Despite phosphopeptide enrichment from large amounts of starting material, we are unable to detect endogenous H2AX Y142 phosphorylation, indicating that it is present in low abundance (<1%). We also calculate the relative levels of H2AX compared to other H2A isoforms and quantify the proportion of H2AX that is phosphorylated on S139 (γH2AX) after ionising radiation

Analysis of individual remodeled nucleosomes reveals decreased histone–DNA contacts created by hSWI/SNF

Chromatin remodeling enzymes use the energy of ATP hydrolysis to alter histone–DNA contacts and regulate DNA-based processes in eukaryotes. Whether different subfamilies of remodeling complexes generate distinct products remains uncertain. We have developed a protocol to analyze nucleosome remodeling on individual products formed in vitro. We used a DNA methyltransferase to examine DNA accessibility throughout nucleosomes that had been remodeled by the ISWI and SWI/SNF families of enzymes. We confirmed that ISWI-family enzymes mainly created patterns of accessibility consistent with canonical nucleosomes. In contrast, SWI/SNF-family enzymes generated widespread DNA accessibility. The protection patterns created by these enzymes were usually located at the extreme ends of the DNA and showed no evidence for stable loop formation on individual molecules. Instead, SWI/SNF family proteins created extensive accessibility by generating heterogeneous products that had fewer histone–DNA contacts than a canonical nucleosome, consistent with models in which a canonical histone octamer has been ‘pushed’ off of the end of the DNA

Quantitative test of the barrier nucleosome model for statistical positioning of nucleosomes up- and downstream of transcription start sites

The positions of nucleosomes in eukaryotic genomes determine which parts of the DNA sequence are readily accessible for regulatory proteins and which are not. Genome-wide maps of nucleosome positions have revealed a salient pattern around transcription start sites, involving a nucleosome-free region (NFR) flanked by a pronounced periodic pattern in the average nucleosome density. While the periodic pattern clearly reflects well-positioned nucleosomes, the positioning mechanism is less clear. A recent experimental study by Mavrich et al. argued that the pattern observed in S. cerevisiae is qualitatively consistent with a `barrier nucleosome model', in which the oscillatory pattern is created by the statistical positioning mechanism of Kornberg and Stryer. On the other hand, there is clear evidence for intrinsic sequence preferences of nucleosomes, and it is unclear to what extent these sequence preferences affect the observed pattern. To test the barrier nucleosome model, we quantitatively analyze yeast nucleosome positioning data both up- and downstream from NFRs. Our analysis is based on the Tonks model of statistical physics which quantifies the interplay between the excluded-volume interaction of nucleosomes and their positional entropy. We find that although the typical patterns on the two sides of the NFR are different, they are both quantitatively described by the same physical model, with the same parameters, but different boundary conditions. The inferred boundary conditions suggest that the first nucleosome downstream from the NFR (the +1 nucleosome) is typically directly positioned while the first nucleosome upstream is statistically positioned via a nucleosome-repelling DNA region. These boundary conditions, which can be locally encoded into the genome sequence, significantly shape the statistical distribution of nucleosomes over a range of up to ~1000 bp to each side.Comment: includes supporting materia

Deep-learning predicted PET can be subtracted from the true clinical fluorodeoxyglucose PET co-registered to MRI to identify the epileptogenic zone in focal epilepsy

Objective Normal interictal [18F]FDG-PET can be predicted from the corresponding T1w MRI with Generative Adversarial Networks (GANs). A technique we call SIPCOM (Subtraction Interictal PET Co-registered to MRI) can then be used to compare epilepsy patients' predicted and clinical PET. We assessed the ability of SIPCOM to identify the Resection Zone (RZ) in patients with drug-resistant epilepsy (DRE) with reference to visual and statistical parametric mapping (SPM) analysis. Methods Patients with complete presurgical work-up and subsequent SEEG and cortectomy were included. RZ localisation, the reference region, was assigned to one of eighteen anatomical brain regions. SIPCOM was implemented using healthy controls to train a GAN. To compare, the clinical PET coregistered to MRI was visually assessed by two trained readers, and a standard SPM analysis was performed. Results Twenty patients aged 17-50 (32 ± 7.8) years were included, 14 (70%) with temporal lobe epilepsy (TLE). Eight (40%) were MRI-negative. After surgery, 14 patients (70%) had a good outcome (Engel I-II). RZ localisation rate was 60% with SIPCOM vs 35% using SPM (P = 0.015) and vs 85% using visual analysis (P = 0.54). Results were similar for Engel I-II patients, the RZ localisation rate was 64% with SIPCOM vs 36% with SPM. With SIPCOM localisation was correct in 67% in MRI-positive vs 50% in MRI-negative patients, and 64% in TLE vs 43% in extra-TLE. The average number of false-positive clusters was 2.2 ± 1.3 using SIPCOM vs 2.3 ± 3.1 using SPM. All RZs localized with SPM were correctly localized with SIPCOM. In one case, PET and MRI were visually reported as negative, but both SIPCOM and SPM localized the RZ. Significance SIPCOM performed better than the reference computer-assisted method (SPM) for RZ detection in a group of operated DRE patients. SIPCOM's impact on epilepsy management needs to be prospectively validated. Key points AI can predict PET from T1w magnetic resonance imaging [MRI] with good accuracy. Subtraction of the clinical from the predicted PET (SIPCOM) in analogy to SISCOM. SIPCOM performed better than SPM to localize the resection zone in [18F]FDG-PET. SIPCOM performance advantages were relatively similar in each sub-group (MRI positive/negative patients, temporal lobe epilepsy [TLE] vs extra-TLE patients). Visual analysis performed better than SIPCOM except in one case

Long-range effects of histone point mutations on DNA remodeling revealed from computational analyses of SIN-mutant nucleosome structures

The packaging of DNA into nucleosomes impedes the binding and access of molecules involved in its processing. The SWI/SNF multi-protein assembly, found in yeast, is one of many regulatory factors that stimulate the remodeling of DNA required for its transcription. Amino-acid point mutations in histones H3 or H4 partially bypass the requirement of the SWI/SNF complex in this system. The mechanisms underlying the observed remodeling, however, are difficult to discern from the crystal structures of nucleosomes bearing these so-called SIN (SWI/SNF INdependent) mutations. Here, we report detailed analyses of the conformations and interactions of the histones and DNA in these assemblies. We find that the loss of direct protein–DNA contacts near point-mutation sites, reported previously, is coupled to unexpected additional long-range effects, i.e. loss of intermolecular contacts and accompanying DNA conformational changes at sequentially and spatially distant sites. The SIN mutations seemingly transmit information relevant to DNA binding across the nucleosome. The energetic cost of deforming the DNA to the states found in the SIN-mutant structures helps to distinguish the mutants that show phenotypes in yeast from those that do not. Models incorporating these deformed dimer steps suggest ways that nucleosomal DNA may be remodeled during its biological processing

Strong physical constraints on sequence-specific target location by proteins on DNA molecules

Sequence-specific binding to DNA in the presence of competing non-sequence-specific ligands is a problem faced by proteins in all organisms. It is akin to the problem of parking a truck at a loading bay by the side of a road in the presence of cars parked at random along the road. Cars even partially covering the loading bay prevent correct parking of the truck. Similarly on DNA, non-specific ligands interfere with the binding and function of sequence-specific proteins. We derive a formula for the probability that the loading bay is free from parked cars. The probability depends on the size of the loading bay and allows an estimation of the size of the footprint on the DNA of the sequence-specific protein by assaying protein binding or function in the presence of increasing concentrations of non-specific ligand. Assaying for function gives an ‘activity footprint’; the minimum length of DNA required for function rather than the more commonly measured physical footprint. Assaying the complex type I restriction enzyme, EcoKI, gives an activity footprint of ∼66 bp for ATP hydrolysis and 300 bp for the DNA cleavage function which is intimately linked with translocation of DNA by EcoKI. Furthermore, considering the coverage of chromosomal DNA by proteins in vivo, our theory shows that the search for a specific DNA sequence is very difficult; most sites are obscured by parked cars. This effectively rules out any significant role in target location for mechanisms invoking one-dimensional, linear diffusion along DNA

Escherichia coli RNA polymerase-associated SWI/SNF protein RapA: evidence for RNA-directed binding and remodeling activity

Helicase-like SWI/SNF proteins are present in organisms belonging to distant kingdoms from bacteria to humans, indicating that they perform a very basic and ubiquitous form of nucleic acid management; current studies associate the activity of SWI/SNF proteins with remodeling of DNA and DNA–protein complexes. The bacterial SWI/SNF homolog RapA—an integral part of the Escherichia coli RNA polymerase complex—has been implicated in remodeling post-termination DNA–RNA polymerase–RNA ternary complexes (PTC), however its explicit nucleic acid substrates and mechanism remain elusive. Our work presents evidence indicating that RNA is a key substrate of RapA. Specifically, the formation of stable RapA–RNA intermediates in transcription and other, independent lines of evidence presented herein indicate that RapA binds and remodels RNA during transcription. Our results are consistent with RapA promoting RNA release from DNA–RNA polymerase–RNA ternary complexes; this process may be accompanied by the destabilization of non-canonical DNA–RNA complexes (putative DNA–RNA triplexes). Taken together, our data indicate a novel RNA remodeling activity for RapA, a representative of the SWI/SNF protein superfamily

Rad51 Polymerization Reveals a New Chromatin Remodeling Mechanism

Rad51 protein is a well known protagonist of homologous recombination in eukaryotic cells. Rad51 polymerization on single-stranded DNA and its role in presynaptic filament formation have been extensively documented. Rad51 polymerizes also on double-stranded DNA but the significance of this filament formation remains unclear. We explored the behavior of Saccharomyces cerevisiae Rad51 on dsDNA and the influence of nucleosomes on Rad51 polymerization mechanism to investigate its putative role in chromatin accessibility to recombination machinery. We combined biochemical approaches, transmission electron microscopy (TEM) and atomic force microscopy (AFM) for analysis of the effects of the Rad51 filament on chromatinized templates. Quantitative analyses clearly demonstrated the occurrence of chromatin remodeling during nucleoprotein filament formation. During Rad51 polymerization, recombinase proteins moved all the nucleosomal arrays in front of the progressing filament. This polymerization process had a powerful remodeling effect, as Rad51 destabilized the nucleosomes along considerable stretches of DNA. Similar behavior was observed with RecA. Thus, recombinase polymerization is a powerful mechanism of chromatin remodeling. These remarkable features open up new possibilities for understanding DNA recombination and reveal new types of ATP-dependent chromatin dynamics