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Results for “chromatin accessibility”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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48 records · Page 3
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

An autoinhibited state of 53BP1 revealed by small molecule antagonists and protein engineering

The recruitment of 53BP1 to chromatin, mediated by its recognition of histone H4 dimethylated at lysine 20 (H4K20me2), is important for DNA double-strand break repair. Using a series of small molecule antagonists, we demonstrate a conformational equilibrium between an open and a pre-existing lowly populated closed state of 53BP1 in which the H4K20me2 binding surface is buried at the interface between two interacting 53BP1 molecules. In cells, these antagonists inhibit the chromatin recruitment of wild type 53BP1, but do not affect 53BP1 variants unable to access the closed conformation despite preservation of the H4K20me2 binding site. Thus, this inhibition operates by shifting the conformational equilibrium toward the closed state. Our work therefore identifies an auto-associated form of 53BP1—autoinhibited for chromatin binding—that can be stabilized by small molecule ligands encapsulated between two 53BP1 protomers. Such ligands are valuable research tools to study the function of 53BP1 and have the potential to facilitate the development of new drugs for cancer therapy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Physical models reveal indirect reader protein interactions that facilitate epigenetic crosstalk

The spatial organization of chromatin is governed by epigenetic factors, including epigenetic marks and the reader proteins that bind them. By dictating the accessibility of genomic loci, epigenetic factors contribute to the physical regulation of gene expression, enabling diverse cellular phenotypes to be encoded by a shared genome in an individual. Epigenetic dysregulation can lead to aberrations in chromatin architecture, contributing to diseases such as neurological disorders and cancers. Despite the known importance of chromatin organization for human health, the physical mechanisms governing chromatin folding remain underspecified. In this work, we develop a physical model of chromatin organization based on contributions from multiple epigenetic factors. Using our model, we evaluate how conditions in the nuclear environment and crosstalk between epigenetic marks affect the compartmentalization of chromatin into dense heterochromatin and loose euchromatin. Our results emphasize the role of reader protein binding in chromatin compartmentalization. We show that reader proteins interact through an indirect mechanism facilitated by the shared chromatin “scaffold” to which they bind. Under a scenario where reader proteins compete for binding sites, we find that indirect interactions affect the program adopted by the chromatin fiber. By isolating indirect modes of epigenetic crosstalk, we demonstrate how the interplay between epigenetic patterning and environmental factors influences chromatin architecture.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Binding profiles for 961 Drosophila and C. elegans transcription factors reveal tissue-specific regulatory relationships

A catalog of transcription factor (TF) binding sites in the genome is critical for deciphering regulatory relationships. Here, we present the culmination of the efforts of the modENCODE (model organism Encyclopedia of DNA Elements) and modERN (model organism Encyclopedia of Regulatory Networks) consortia to systematically assay TF binding events in vivo in two major model organisms,Drosophila melanogaster(fly) andCaenorhabditis elegans(worm). These data sets comprise 605 TFs identifying 3.6 M sites in the fly and 356 TFs identifying 0.9 M sites in the worm, and represent the majority of the regulatory space in each genome. We demonstrate that TFs associate with chromatin in clusters termed “metapeaks,” that larger metapeaks have characteristics of high-occupancy target (HOT) regions, and that the importance of consensus sequence motifs bound by TFs depends on metapeak size and complexity. Combining ChIP-seq data with single-cell RNA-seq data in a machine-learning model identifies TFs with a prominent role in promoting target gene expression in specific cell types, even differentiating between parent–daughter cells during embryogenesis. These data are a rich resource for the community that should fuel and guide future investigations into TF function. To facilitate data accessibility and utility, all strains expressing green fluorescent protein (GFP)-tagged TFs are available at the stock centers for each organism. The chromatin immunoprecipitation sequencing data are available through the ENCODE Data Coordinating Center, GEO, and through a direct interface that provides rapid access to processed data sets and summary analyses, as well as widgets to probe the cell-type-specific TF–target relationships.

Biochemistry & Molecular Biology↗
DOE OSTI2021

Transcriptional network orchestrating regional patterning of cortical progenitors

We uncovered a transcription factor (TF) network that regulates cortical regional patterning in radial glial stem cells. Screening the expression of hundreds of TFs in the developing mouse cortex identified 38 TFs that are expressed in gradients in the ventricular zone (VZ). We tested whether their cortical expression was altered in mutant mice with known patterning defects (Emx2, Nr2f1, and Pax6), which enabled us to define a cortical regionalization TF network (CRTFN). To identify genomic programming underlying this network, we performed TF ChIP-seq and chromatin-looping conformation to identify enhancer–gene interactions. To map enhancers involved in regional patterning of cortical progenitors, we performed assays for epigenomic marks and DNA accessibility in VZ cells purified from wild-type and patterning mutant mice. This integrated approach has identified a CRTFN and VZ enhancers involved in cortical regional patterning in the mouse.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

H2A.X promotes endosperm-specific DNA methylation in Arabidopsis thaliana

Background: H2A.X is an H2A variant histone in eukaryotes, unique for its ability to respond to DNA damage, initiating the DNA repair pathway. H2A.X replacement within the histone octamer is mediated by the FAcilitates Chromatin Transactions (FACT) complex, a key chromatin remodeler. FACT is required for DEMETER (DME)-mediated DNA demethylation at certain loci in Arabidopsis thaliana female gametophytes during reproduction. Here, we sought to investigate whether H2A.X is involved in DME- and FACT-mediated DNA demethylation during reproduction. Results: H2A.X is encoded by two genes in Arabidopsis genome, HTA3 and HTA5. We generated h2a.x double mutants, which displayed a normal growth profile, whereby flowering time, seed development, and root tip organization, S-phase progression and proliferation were all normal. However, h2a.x mutants were more sensitive to genotoxic stress, consistent with previous reports. H2A.X fused to Green Fluorescent Protein (GFP) under the H2A.X promoter was highly expressed especially in newly developing Arabidopsis tissues, including in male and female gametophytes, where DME is also expressed. We examined DNA methylation in h2a.x developing seeds and seedlings using whole genome bisulfite sequencing, and found that CG DNA methylation is decreased genome-wide in h2a.x mutant endosperm. Hypomethylation was most striking in transposon bodies, and occurred on both parental alleles in the developing endosperm, but not the embryo or seedling. h2a.x-mediated hypomethylated sites overlapped DME targets, but also included other loci, predominately located in heterochromatic transposons and intergenic DNA. Conclusions: Our genome-wide methylation analyses suggest that H2A.X could function in preventing access of the DME demethylase to non-canonical sites. Overall, our data suggest that H2A.X is required to maintain DNA methylation homeostasis in the unique chromatin environment of the Arabidopsis endosperm.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Genetic variation at transcription factor binding sites largely explains phenotypic heritability in maize

Abstract Comprehensive maps of functional variation at transcription factor (TF) binding sites (cis-elements) are crucial for elucidating how genotype shapes phenotype. Here, we report the construction of a pan-cistrome of the maize leaf under well-watered and drought conditions. We quantified haplotype-specific TF footprints across a pan-genome of 25 maize hybrids and mapped over 200,000 variants, genetic, epigenetic, or both (termed binding quantitative trait loci (bQTL)), linked tocis-element occupancy. Three lines of evidence support the functional significance of bQTL: (1) coincidence with causative loci that regulate traits, includingvgt1,ZmTRE1and the MITE transposon nearZmNAC111under drought; (2) bQTL allelic bias is shared between inbred parents and matches chromatin immunoprecipitation sequencing results; and (3) partitioning genetic variation across genomic regions demonstrates that bQTL capture the majority of heritable trait variation across ~72% of 143 phenotypes. Our study provides an auspicious approach to make functionalcis-variation accessible at scale for genetic studies and targeted engineering of complex traits.

Genetics & Heredity↗
DOE OSTI2024

Chromatin binding by HORMAD proteins regulates meiotic recombination initiation

The meiotic chromosome axis coordinates chromosome organization and interhomolog recombination in meiotic prophase and is essential for fertility. In S. cerevisiae , the HORMAD protein Hop1 mediates the enrichment of axis proteins at nucleosome-rich islands through a central chromatin-binding region (CBR). Here, we use cryoelectron microscopy to show that the Hop1 CBR directly recognizes bent nucleosomal DNA through a composite interface in its PHD and winged helix–turn–helix domains. Targeted disruption of the Hop1 CBR-nucleosome interface causes a localized reduction of axis protein binding and meiotic DNA double-strand breaks (DSBs) in axis islands and leads to defects in chromosome synapsis. Synthetic effects with mutants of the Hop1 regulator Pch2 suggest that nucleosome binding delays a conformational switch in Hop1 from a DSB-promoting, Pch2-inaccessible state to a DSB-inactive, Pch2-accessible state to regulate the extent of meiotic DSB formation. Phylogenetic analyses of meiotic HORMADs reveal an ancient origin of the CBR, suggesting that the mechanisms we uncover are broadly conserved.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Structural basis for $\mathrm{APE1}$ processing $\mathrm{DNA}$ damage in the nucleosome

Genomic DNA is continually exposed to endogenous and exogenous factors that promote DNA damage. Eukaryotic genomic DNA is packaged into nucleosomes, which present a barrier to accessing and effectively repairing DNA damage. The mechanisms by which DNA repair proteins overcome this barrier to repair DNA damage in the nucleosome and protect genomic stability is unknown. Here, we determine how the base excision repair (BER) endonuclease AP-endonuclease 1 (APE1) recognizes and cleaves DNA damage in the nucleosome. Kinetic assays determine that APE1 cleaves solvent-exposed AP sites in the nucleosome with 3-6 orders of magnitude higher efficiency than occluded AP sites. A cryo-electron microscopy structure of APE1 bound to a nucleosome containing a solvent-exposed AP site reveal that APE1 uses a DNA sculpting mechanism for AP site recognition, where APE1 bends the nucleosomal DNA to access the AP site. Notably, additional biochemical and structural characterization of occluded AP sites identify contacts between the nucleosomal DNA and histone octamer that prevent efficient processing of the AP site by APE1. These findings provide a rationale for the position-dependent activity of BER proteins in the nucleosome and suggests the ability of BER proteins to sculpt nucleosomal DNA drives efficient BER in chromatin.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Expanded encyclopaedias of DNA elements in the human and mouse genomes

The human and mouse genomes contain instructions that specify RNAs and proteins and govern the timing, magnitude, and cellular context of their production. To better delineate these elements, phase III of the Encyclopedia of DNA Elements (ENCODE) Project has expanded analysis of the cell and tissue repertoires of RNA transcription, chromatin structure and modification, DNA methylation, chromatin looping, and occupancy by transcription factors and RNA-binding proteins. Here we summarize these efforts, which have produced 5,992 new experimental datasets, including systematic determinations across mouse fetal development. All data are available through the ENCODE data portal (https://www.encodeproject.org), including phase II ENCODE1 and Roadmap Epigenomics2 data. We have developed a registry of 926,535 human and 339,815 mouse candidate cis-regulatory elements, covering 7.9 and 3.4% of their respective genomes, by integrating selected datatypes associated with gene regulation, and constructed a web-based server (SCREEN; http://screen.encodeproject.org) to provide flexible, user-defined access to this resource. Collectively, the ENCODE data and registry provide an expansive resource for the scientific community to build a better understanding of the organization and function of the human and mouse genomes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Characterization of Mammalian In Vivo Enhancers Using Mouse Transgenesis and CRISPR Genome Editing [Book Chapter]

Embryonic morphogenesis is strictly dependent on tight spatiotemporal control of developmental gene expression, which is typically achieved through the concerted activity of multiple enhancers driving cell type-specific expression of a target gene. Mammalian genomes are organized in topologically associated domains, providing a preferred environment and framework for interactions between transcriptional enhancers and gene promoters. While epigenomic profiling and three-dimensional chromatin conformation capture have significantly increased the accuracy of identifying enhancers, assessment of subregional enhancer activities via transgenic reporter assays in mice remains the gold standard for assigning enhancer activity in vivo. Once this activity is defined, the ideal method to explore the functional necessity of a transcriptional enhancer and its contribution to target gene dosage and morphological or physiological processes is deletion of the enhancer sequence from the mouse genome. Here we present detailed protocols for efficient introduction of enhancer-reporter transgenes and CRISPR-mediated genomic deletions into the mouse genome, including a step-by-step guide for pronuclear microinjection of fertilized mouse eggs. We provide instructions for the assembly and genomic integration of enhancer-reporter cassettes that have been used for validation of thousands of putative enhancer sequences accessible through the VISTA enhancer browser, including a recently published method for robust site-directed transgenesis at the H11 safe-harbor locus. Together, these methods enable rapid and large-scale assessment of enhancer activities and sequence variants in mice, which is essential to understand mammalian genome function and genetic diseases.

cis-regulatory elements↗
DOE OSTI2024

An ascomycete H4 variant with an unknown function

Histone variants leading to altered nucleosome structure, dynamics and DNA accessibility occur frequently, albeit rarely for H4. We carried out a comprehensive in silico scrutiny of fungal genomes, which revealed the presence of a novel H4 variant (H4E) in the ascomycetes, throughout the Pezizomycotina, in basal species of the Taphrinomycotina and also in the Glomeromycota. The coding cognate genes show a specific intron/exon organization, different from H4 canonical genes. H4Es diverge from canonical H4s mainly in the N- and C-terminal extensions, showing marked differences in the distribution and number of Lys and Arg residues, which may result in novel post-translational modifications. In Aspergillus nidulans (Pezizomycotina, Eurotiomycetes) the H4E variant protein level is low in mycelia. However, the encoding gene is well expressed at 37°C under nitrogen starvation. H4E localizes to the nucleus and interacts with H3, but its absence or overexpression does not result in any detectable phenotype. Deletion of only one of the of the two canonical H4 genes results in a strikingly impaired growth phenotype, which indicates that H4E cannot replace this canonical histone. Thus, an H4 variant is present throughout a whole subphylum of the ascomycetes, but with hitherto no experimentally detectable function.

59 BASIC BIOLOGICAL SCIENCES↗