Ontology highlight
ABSTRACT:
PROVIDER: PRJNA590635 | ENA |
REPOSITORIES: ENA
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Project description:Nuclear receptors function as ligand-regulated transcription factors whose ability to regulate diverse physiological processes is closely linked with conformational changes induced upon ligand binding. Understanding how conformational populations of nuclear receptors are shifted by various ligands could illuminate strategies for the design of synthetic modulators to regulate specific transcriptional programs. Here, we investigate ligand-induced conformational changes using a reconstructed, ancestral nuclear receptor. By making substitutions at a key position, we engineer receptor variants with altered ligand specificities. We use atomistic molecular dynamics (MD) simulations with enhanced sampling to generate ensembles of wildtype and engineered receptors in combination with multiple ligands, followed by conformational analysis and prediction of ligand activity. We combine cellular and biophysical experiments to allow correlation of MD-based predictions with functional ligand profiles, as well as elucidation of mechanisms underlying altered transcription in receptor variants. We determine that conformational ensembles accurately predict ligand responses based on observed population shifts, even within engineered receptors that were constitutively active or transcriptionally unresponsive in experiments. These studies provide a platform which will allow structural characterization of physiologically-relevant conformational ensembles, as well as provide the ability to design and predict transcriptional responses in novel ligands.
2022-10-12 | PXD036076 | Pride
Project description:A key limitation of the commonly-used CRISPR enzyme S. pyogenes Cas9 is the strict requirement of an NGG protospacer-adjacent motif (PAM) at the target site, which reduces the number of accessible genomic loci. This constraint can be limiting for genome editing applications that require precise Cas9 positioning. Recently, two Cas9 variants with a relaxed PAM requirement (NG) have been developed (xCas9 and Cas9-NG) but their activity has been measured at only a small number of endogenous sites. Here we devised a high-throughput Cas9 pooled competition screen to compare the performance of both PAM-flexible Cas9 variants and wild-type Cas9 at thousands of genomic loci and across 3 modalities (gene knock-out, transcriptional activation and suppression). We show that PAM flexibility comes at a substantial cost of decreased DNA targeting and cutting. Of the PAM-flexible variants, we found that Cas9-NG outperforms xCas9 regardless of genome engineering modality or PAM. Finally, we combined xCas9 mutations with those of Cas9-NG, creating a stronger transcriptional modulator than existing PAM-flexible Cas9 variants.
2020-01-26 | GSE143892 | GEO
Project description:Human somatic cells may contain up to seven members of the histone H1 family contributing to chromatin compaction and regulation of nuclear processes, apparently with certain subtype specificities. Previous studies in T47D breast cancer cells determined that H1 variants are distributed in a variant-specific manner throughout the genome, although only H1.2 and H1X endogenous variants were mapped. Now, we performed ChIP-seq of five endogenous H1 variants (H1.0, H1.2, H1.4, H1.5, H1X) in T47D cells.
2021-08-16 | GSE166645 | GEO
Project description:CRISPR-Cas transcriptional tools have been widely applied for programmable regulation of complex biological networks. In comparison to eukaryotic systems, bacterial CRISPR activation (CRISPRa) has stringent target site requirements for effective gene activation. While genes may not always have an NGG protospacer adjacent motif (PAM) at the appropriate position, PAM-flexible dCas9 variants can expand the range of targetable sites. Here we systematically evaluate a panel of PAM-flexible dCas9 variants for their ability to activate bacterial genes. We observe that dxCas9-NG provides a high dynamic range of gene activation for sites with NGN PAMs while dSpRY permits modest activity across almost any PAM. Similar trends were observed for heterologous and endogenous promoters. For all variants tested, improved PAM-flexibility comes with the tradeoff that CRISPRi-mediated gene repression becomes less effective. Weaker CRISPR interference (CRISPRi) gene repression can be partially rescued by expressing multiple sgRNAs to target many sites in the gene of interest. Our work provides a framework to choose the most effective dCas9 variant for a given set of gene targets, which will further expand the utility of CRISPRa/i gene regulation in bacterial systems.
2022-11-15 | GSE217559 | GEO
Project description:PamcKO/cKO mice, with loxp sites flanking exons previously deleted in the global Pam knockout, were created, viable and equivalent to wildtype mice in every test. Eliminating Pam expression in excitatory forebrain neurons reduced anxiety-like behavior, increased locomotor responsiveness to cocaine and improved thermoregulation in the cold. Eliminating Pam expression in cardiomyocytes increased anxiety-like behavior, improved thermoregulation and markedly diminished atrial levels of pro-atrial natriuretic peptide. RNASeq analysis of atria revealed a set of PAM-regulated transcripts related to cardiac function.
2019-08-13 | GSE132180 | GEO
Project description:Human somatic cells may contain up to seven members of the histone H1 family contributing to chromatin compaction and regulation of nuclear processes, apparently with certain subtype specificities. Our previous studies of H1.0, H1.2, H1.4, H1.5, and H1X genome-wide distribution determined that H1 variants are distributed in a variant-specific manner throughout the genome. We have been able to extend our study of the genomic distribution of the H1 family from five to six proteins thanks to the acquisition and validation of a new ChIP-grade endogenous antibody against histone H1.3.
2024-01-09 | GSE236878 | GEO
Project description:To study target sequence specificity, selectivity, and reaction kinetics of Streptococcus pyogenes Cas9 activity, we challenged libraries of random variant targets with purified Cas9::guide RNA complexes in vitro. Cleavage kinetics were nonlinear, with a burst of initial activity followed by slower sustained cleavage. Consistent with other recent analyses of Cas9 sequence specificity, we observe considerable (albeit incomplete) impairment of cleavage for targets mutated in the PAM sequence or in "seed" sequences matching the proximal 8 bp of the guide. A second target region requiring close homology was located at the other end of the guide::target duplex (positions 13-18 relative to the PAM). Strikingly, a subset of variants which broke homology in the intervening region consistently increased the capacity of Cas9 to cleave in extended reactions. Sequences flanking the guide+PAM region had measurable (albeit modest) effects on cleavage. Taken together, these studies provide both a basis for predicting effective cleavage targets and a basis for potential optimization of guide RNAs to yield efficiency beyond that of the simple perfect-match guides.
2014-11-14 | GSE58426 | GEO
Project description:Fusion of active protein domains to the nuclease-deficient clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9 (dCas9) has been widely used for epigenome editing, but the specificities of these engineered proteins have still not been fully investigated. Targeted methylation of specific gene loci offers a direct approach to perturb DNA methylation-associated biological processes. In this study, we generated and validated the global off-target characteristics of CRISPR-guided DNA methyltransferases (CRISPRme) by fusing the catalytic domain of DNMT3A or DNMT3B to the C terminus of the dCas9 protein from S. pyogenes. Using targeted quantitative bisulfite pyrosequencing and whole genome bisulfite sequencing (WGBS), we prove that CRISPRme can efficiently methylate the CpG dinucleotides flanking its target sites in genomic loci (uPA and TGFBR3) in human cells (HEK293T) with CpG-methylation levels exceeding 70% for some target sites. Using qPCR, fluorescent reporter cells, and RNA sequencing, we found that CRISPRme can mediate transient inhibition of gene expression which appears to result from Cas9-mediated interference with transcription rather than de novo DNA methylation. Analyses of whole genome methylation did not identify global methylation changes, however a substantial number of CRISPRme off-target differentially methylated regions (DMR, over 6000) were still identified. The majority of these DMRs were hypermethylated both in cells expressing CRISPRme alone and cells expressing CRISPRme together with gRNAs. These off-target hypermethylated sites were enriched in gene bodies, introns, 5’UTR, CGI shores, Alu sequences, open chromatin and PAM rich regions, but not correlated with off-target binding sites predicted by ChIP-seq. Our results prove that CRISPRme allows for efficient RNA-guided methylation of endogenous CpGs, however with high frequencies of off-target methylation indicating that further improvements of the specificity of CRISPR-dCas9 based DNA methylation modifiers are still required.
2016-12-14 | GSE92261 | GEO