Genes coding for polysomal 9S RNA of sea urchins: conservation and divergence.
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Mapping of protein-DNA interactions at single-cell resolution remains a central challenge in epigenomics, particularly for transcription factors (TFs), whose sparse binding limits reliable detection. Here, we establish DeChIC-seq (DNA Deaminase-based Chromatin Immuno-Conversion sequencing), a conversion-based strategy that uses a protein A-DddAtox fusion to directly record protein-DNA interactions by inducing localized C-to-U conversions near antibody-bound chromatin. Retaining genome-wide background sequence information without immunoprecipitation, DeChIC-seq enables profiling of histone modifications and sensitive detection of TF binding. Integration with single-cell whole-genome amplification extends DeChIC-seq to single-cell applications (scDeChIC-seq), enabling chromatin profiling of individual cells. Applied to mouse embryogenesis, scDeChIC-seq resolves lineage-specific chromatin states through profiling of H3K4me3, CTCF, and RAD21 and sensitively detects TF binding, including that of NR5A2, TFAP2C, and KLF5, from extremely limited blastomere inputs. This underscores its strong potential for detecting TF-binding sites in scarce biological samples. DeChIC-seq establishes a conversion-based framework for chromatin profiling that enables mechanistic dissection of TF-driven gene regulation across rare cells, developmental systems, and disease contexts.
Spike-in normalization enables quantitative analysis of chromatin immunoprecipitation sequencing (ChIP-seq) signal. Here we introduce a robust dual spike-in normalization approach for ChIP-seq (ChIP-wrangler), optimize parameters and verify its accuracy in quantifying changes in ChIP-seq signal and detecting technical artifacts. We use ChIP-wrangler to revisit recent claims that active histone marks depend on transcription. We show that acute depletion of RNA polymerase II (RNAPII) has a modest impact on H3K27ac levels, with only 6% of peaks significantly changing after RNAPII depletion, indicating that histone acetylation maintenance is not entirely dependent on ongoing transcription. Promoters and enhancers are differentially affected, with 82% of decreasing acetylation peaks located at promoter-distal elements with enhancer-related motifs. ChIP-wrangler provides increased rigor and 'guardrails' for successful spike-in normalization and, as applied here, refines the understanding of crosstalk between RNAPII activity and transcription-associated histone marks.
DNA methylation and histone modifications encode epigenetic information. Recently, major progress was made to measure either mark at a single-cell resolution; however, a method for simultaneous detection is lacking, preventing study of their interactions. Here, to bridge this gap, we developed scEpi2-seq. Our technique provides a readout of histone modifications and DNA methylation at the single-cell and single-molecule level. Application in a cell line with the FUCCI cell cycle reporter system reveals how DNA methylation maintenance is influenced by the local chromatin context. In addition, profiling of H3K27me3 and DNA methylation in the mouse intestine yields insights into epigenetic interactions during cell type specification. Differentially methylated regions also demonstrated independent cell-type regulation in addition to H3K27me3 regulation, which reinforces that CpG methylation acts as an additional layer of control in facultative heterochromatin.
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The males and females of the brine shrimp Artemia franciscana are highly dimorphic, and this dimorphism is associated with substantial sex-biased gene expression in heads and gonads. How these sex-specific patterns of expression are regulated at the molecular level is unknown. A. franciscana also has differentiated ZW sex chromosomes, with complete dosage compensation, but the molecular mechanism through which compensation is achieved is unknown. Here, we conducted CUT&TAG assays targeting 7 post-translational histone modifications (H3K27me3, H3K9me2, H3K9me3, H3K36me3, H3K27ac, H3K4me3, and H4K16ac) in heads and gonads of A. franciscana, allowing us to divide the genome into 12 chromatin states. We further defined functional chromatin signatures for all genes, which were correlated with transcript level abundances. Differences in the occupancy of the profiled epigenetic marks between sexes were associated with differential gene expression between males and females. Finally, we found a significant enrichment of the permissive H4K16ac histone mark in the Z-specific region in both tissues of females but not males, supporting the role of this histone mark in mediating dosage compensation of the Z chromosome.
Cytoplasmic messenger RNAs of eukaryotic cells are distributed between polysomal and post-polysomal fractions (free) as protein-bound complexes. These studies were designed to determine whether a specific mRNA isolated from different subcellular compartments is complexed with the same family of polypeptides. As a first approach we have examined the proteins associated with mRNA which codes for histone H4. To perform these experiments HeLa cells were exposed to ultraviolet light to cross-link in vivo polypeptides which are closely associated with nucleic acid. To identify the polypeptides associated with mRNA specific for histones a genomic probe for histone H4 mRNA was immobilized on epoxy-cellulose. By hybrid selection specific mRNPs containing histone mRNA were isolated. Our results reveal the existence of a number of polypeptides associated with both polysomal and post-polysomal histone mRNAs. In polysomal histone mRNA two polypeptides of Mr = 49 000 and 52 500 were the major components. In contrast polypeptides of Mr = 43 000 and 57 000 were the major polypeptide components of post-polysomal (or free) histone mRNA. Furthermore, these results also suggest that the polypeptides associated with either polysomal or free H4 histone mRNA represent a subset of proteins found in poly(A)-free fractions or poly(A)-rich mRNA fractions.
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