Strong replicators associated with open chromatin are sufficient to establish an early replicating domain

AbstractVertebrate genomes replicate according to a precise temporal program strongly correlated with their organization into topologically associating domains. However, the molecular mechanisms underlying the establishment of early-replicating domains remain largely unknown. We defined two minimal cis-element modules containing a strong replication origin and chromatin modifier binding sites capable of shifting a targeted mid-late replicating region for earlier replication. When inserted side-by-side, these modules acted in cooperation, with similar effects on two late-replicating regions. Targeted insertions of these two modules at two chromosomal sites separated by 30 kb brought these two modules into close physical proximity and induced the formation of an early-replicating domain. Thus, combinations of strong origins and cis-elements capable of opening the chromatin structure are the basic units of early-replicating domains, and are absent from late-replicated regions. These findings are consistent with those of genome-wide studies mapping strong initiation sites and open chromatin marks in vertebrate genomes.

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The role of epigenetic modifications, long-range contacts, enhancers and topologically associating domains in the regulation of glioma grade-specific genes

Scientific Reports ◽

10.1038/s41598-021-95009-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ilona E. Grabowicz ◽

Bartek Wilczyński ◽

Bożena Kamińska ◽

Adria-Jaume Roura ◽

Bartosz Wojtaś ◽

...

Keyword(s):

Gene Expression ◽

Long Range ◽

Genetic Alterations ◽

Chromatin Organization ◽

Open Chromatin ◽

Low Grade ◽

Strong Impact ◽

Cell Matrix ◽

Topologically Associating Domains ◽

Genome Wide

AbstractGenome-wide studies have uncovered specific genetic alterations, transcriptomic patterns and epigenetic profiles associated with different glioma types. We have recently created a unique atlas encompassing genome-wide profiles of open chromatin, histone H3K27ac and H3Kme3 modifications, DNA methylation and transcriptomes of 33 glioma samples of different grades. Here, we intersected genome-wide atlas data with topologically associating domains (TADs) and demonstrated that the chromatin organization and epigenetic landscape of enhancers have a strong impact on genes differentially expressed in WHO low grade versus high grade gliomas. We identified TADs enriched in glioma grade-specific genes and/or epigenetic marks. We found the set of transcription factors, including REST, E2F1 and NFKB1, that are most likely to regulate gene expression in multiple TADs, containing specific glioma-related genes. Moreover, many genes associated with the cell–matrix adhesion Gene Ontology group, in particular 14 PROTOCADHERINs, were found to be regulated by long-range contacts with enhancers. Presented results demonstrate the existence of epigenetic differences associated with chromatin organization driving differential gene expression in gliomas of different malignancy.

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Investigation of the Interaction of Human Origin Recognition Complex Subunit 1 with G-Quadruplex DNAs of Human c-myc Promoter and Telomere Regions

International Journal of Molecular Sciences ◽

10.3390/ijms22073481 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3481

Author(s):

Afaf Eladl ◽

Yudai Yamaoki ◽

Shoko Hoshina ◽

Haruka Horinouchi ◽

Keiko Kondo ◽

...

Keyword(s):

Fluorescence Anisotropy ◽

Binding Sites ◽

Replication Origin ◽

Origin Recognition Complex ◽

Chemical Shift Perturbation ◽

Human Origin ◽

Replication Origins ◽

Genome Wide ◽

G Quadruplex ◽

Origin Recognition

Origin recognition complex (ORC) binds to replication origins in eukaryotic DNAs and plays an important role in replication. Although yeast ORC is known to sequence-specifically bind to a replication origin, how human ORC recognizes a replication origin remains unknown. Previous genome-wide studies revealed that guanine (G)-rich sequences, potentially forming G-quadruplex (G4) structures, are present in most replication origins in human cells. We previously suggested that the region comprising residues 413–511 of human ORC subunit 1, hORC1413–511, binds preferentially to G-rich DNAs, which form a G4 structure in the absence of hORC1413–511. Here, we investigated the interaction of hORC1413-511 with various G-rich DNAs derived from human c-myc promoter and telomere regions. Fluorescence anisotropy revealed that hORC1413–511 binds preferentially to DNAs that have G4 structures over ones having double-stranded structures. Importantly, circular dichroism (CD) and nuclear magnetic resonance (NMR) showed that those G-rich DNAs retain the G4 structures even after binding with hORC1413–511. NMR chemical shift perturbation analyses revealed that the external G-tetrad planes of the G4 structures are the primary binding sites for hORC1413–511. The present study suggests that human ORC1 may recognize replication origins through the G4 structure.

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methyl-ATAC-seq measures DNA methylation at accessible chromatin

10.1101/445486 ◽

2018 ◽

Cited By ~ 1

Author(s):

R Spektor ◽

ND Tippens ◽

CA Mimoso ◽

PD Soloway

Keyword(s):

Dna Methylation ◽

Binding Sites ◽

Open Chromatin ◽

Regulatory Sequences ◽

Protein Binding Sites ◽

3 Dimensional ◽

Genome Wide ◽

Gene Regulatory ◽

Nucleosome Location ◽

Accessible Chromatin

ABSTRACTChromatin features are characterized by genome-wide assays for nucleosome location, protein binding sites, 3-dimensional interactions, and modifications to histones and DNA. For example, Assay for Transposase Accessible Chromatin sequencing (ATAC-seq) identifies nucleosome-depleted (open) chromatin, which harbors potentially active gene regulatory sequences; and bisulfite sequencing (BS-seq) quantifies DNA methylation. When two distinct chromatin features like these are assayed separately in populations of cells, it is impossible to determine, with certainty, where the features are coincident in the genome by simply overlaying datasets. Here we describe methyl-ATAC-seq (mATAC-seq), which implements modifications to ATAC-seq, including subjecting the output to BS-seq. Merging these assays into a single protocol identifies the locations of open chromatin, and reveals, unambiguously, the DNA methylation state of the underlying DNA. Such combinatorial methods eliminate the need to perform assays independently and infer where features are coincident.

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Genomic Study of Replication Initiation in Human Chromosomes Reveals the Influence of Transcription Regulation and Chromatin Structure on Origin Selection

Molecular Biology of the Cell ◽

10.1091/mbc.e09-08-0707 ◽

2010 ◽

Vol 21 (3) ◽

pp. 393-404 ◽

Cited By ~ 94

Author(s):

Neerja Karnani ◽

Christopher M. Taylor ◽

Ankit Malhotra ◽

Anindya Dutta

Keyword(s):

Rna Polymerase Ii ◽

Chromatin Structure ◽

Binding Sites ◽

Transcription Initiation ◽

Origin Recognition Complex ◽

Replication Initiation ◽

Open Chromatin ◽

Lambda Exonuclease ◽

Genomic Study ◽

Exonuclease Digestion

DNA replication in metazoans initiates from multiple chromosomal loci called origins. Currently, there are two methods to purify origin-centered nascent strands: lambda exonuclease digestion and anti-bromodeoxyuridine immunoprecipitation. Because both methods have unique strengths and limitations, we purified nascent strands by both methods, hybridized them independently to tiling arrays (1% genome) and compared the data to have an accurate view of genome-wide origin distribution. By this criterion, we identified 150 new origins that were reproducible across the methods. Examination of a subset of these origins by chromatin immunoprecipitation against origin recognition complex (ORC) subunits 2 and 3 showed 93% of initiation peaks to localize at/within 1 kb of ORC binding sites. Correlation of origins with functional elements of the genome revealed origin activity to be significantly enriched around transcription start sites (TSSs). Consistent with proximity to TSSs, we found a third of initiation events to occur at or near the RNA polymerase II binding sites. Interestingly, ∼50% of the early origin activity was localized within 5 kb of transcription regulatory factor binding region clusters. The chromatin signatures around the origins were enriched in H3K4-(di- and tri)-methylation and H3 acetylation modifications on histones. Affinity of origins for open chromatin was also reiterated by their proximity to DNAse I-hypersensitive sites. Replication initiation peaks were AT rich, and >50% of the origins mapped to evolutionarily conserved regions of the genome. In summary, these findings indicate that replication initiation is influenced by transcription initiation and regulation as well as chromatin structure.

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Mutational bias in spermatogonia impacts the anatomy of regulatory sites in the human genome

10.1101/2021.06.10.447556 ◽

2021 ◽

Author(s):

Vera B Kaiser ◽

Lana Talmane ◽

Yatendra Kumar ◽

Fiona Semple ◽

Marie MacLennan ◽

...

Keyword(s):

Chromatin Structure ◽

Meiotic Recombination ◽

Binding Sites ◽

Evolutionary Process ◽

Mutational Bias ◽

Open Chromatin ◽

Structural Variant ◽

Regulatory Architecture ◽

Mutational Processes ◽

Regulatory Sites

Mutation in the germline is the ultimate source of genetic variation, but little is known about the influence of germline chromatin structure on mutational processes. Using ATAC-seq, we profile the open chromatin landscape of human spermatogonia, the most proliferative cell-type of the germline, identifying transcription factor binding sites (TFBSs) and PRDM9-binding sites, a subset of which will initiate meiotic recombination. We observe an increase in rare structural variant (SV) breakpoints at PRDM9-bound sites, implicating meiotic recombination in the generation of structural variation. Many germline TFBSs, such as NRF, are also associated with increased rates of SV breakpoints, apparently independent of recombination. Singleton short insertions (>=5 bp) are highly enriched at TFBSs, particularly at sites bound by testis active TFs, and their rates correlate with those of structural variant breakpoints. Short insertions often duplicate the TFBS motif, leading to clustering of motif sites near regulatory regions in this male-driven evolutionary process. Increased mutation loads at germline TFBSs disproportionately affect neural enhancers with activity in spermatogonia, potentially altering neurodevelopmental regulatory architecture. Local chromatin structure in spermatogonia is thus pervasive in shaping both evolution and disease.

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Mutational bias in spermatogonia impacts the anatomy of regulatory sites in the human genome

Genome Research ◽

10.1101/gr.275407.121 ◽

2021 ◽

Author(s):

Vera B. Kaiser ◽

Lana Talmane ◽

Yatendra Kumar ◽

Fiona Semple ◽

Marie MacLennan ◽

...

Keyword(s):

Chromatin Structure ◽

Meiotic Recombination ◽

Binding Sites ◽

Evolutionary Process ◽

Mutational Bias ◽

Open Chromatin ◽

Structural Variant ◽

Regulatory Architecture ◽

Mutational Processes ◽

Regulatory Sites

Mutation in the germline is the ultimate source of genetic variation, but little is known about the influence of germline chromatin structure on mutational processes. Using ATAC-seq, we profile the open chromatin landscape of human spermatogonia, the most proliferative cell type of the germline, identifying transcription factor binding sites (TFBSs) and PRDM9 binding sites, a subset of which will initiate meiotic recombination. We observe an increase in rare structural variant (SV) breakpoints at PRDM9-bound sites, implicating meiotic recombination in the generation of structural variation. Many germline TFBSs, such as NRF1, are also associated with increased rates of SV breakpoints, apparently independent of recombination. Singleton short insertions (≥5 bp) are highly enriched at TFBSs, particularly at sites bound by testis active TFs, and their rates correlate with those of structural variant breakpoints. Short insertions often duplicate the TFBS motif, leading to clustering of motif sites near regulatory regions in this male-driven evolutionary process. Increased mutation loads at germline TFBSs disproportionately affect neural enhancers with activity in spermatogonia, potentially altering neurodevelopmental regulatory architecture. Local chromatin structure in spermatogonia is thus pervasive in shaping both evolution and disease.

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B1 SINE-binding ZFP266 impedes reprogramming through suppression of chromatin opening by pioneering factors

10.1101/2022.01.04.474927 ◽

2022 ◽

Author(s):

Daniel F Kaemena ◽

Masahito Yoshihara ◽

James Ashmore ◽

Meryam Beniazza ◽

Suling Zhao ◽

...

Keyword(s):

Binding Sites ◽

Molecular Mechanisms ◽

Zinc Finger Protein ◽

Chromatin Accessibility ◽

Finger Protein ◽

Genome Wide ◽

Ipsc Generation ◽

Cellular Identity ◽

Induced Pluripotent ◽

Chromatin Opening

Successful generation of induced pluripotent stem cells (iPSCs) via the overexpression of Oct4 (Pou5f1), Sox2, Klf4 and c-Myc (OSKM) highlights the power of transcription factor (TF)-mediated cellular conversions. Nevertheless, iPSC reprogramming is inherently inefficient and understanding the molecular mechanisms underlying this inefficiency holds the key to control cellular identity successfully. Here, we report 16 novel reprogramming roadblock genes identified by CRISPR/Cas9-mediated genome-wide knockout (KO) screening. Of these, disruption of KRAB zinc finger protein (KRAB-ZFP) Zfp266 strongly and consistently enhanced iPSC generation in several iPSC reprogramming settings, emerging as the most robust roadblock. Further analyses revealed that ZFP266 bound Short Interspersed Nuclear Elements (SINEs) adjacent to OSK binding sites and impedes chromatin opening. This work serves as a resource for better understanding reprogramming mechanisms and proposes SINEs as a critical genetic element that regulates chromatin accessibility at enhancers for efficient pluripotency induction.

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C/EBPδ-induced epigenetic changes control the dynamic gene transcription of S100A8 and S100A9

10.1101/2021.12.05.471309 ◽

2021 ◽

Author(s):

Saskia-Larissa Jauch-Speer ◽

Jonas Wolf ◽

Marisol Herrera-Rivero ◽

Leonie Martens ◽

Achmet Imam Chasan ◽

...

Keyword(s):

Binding Sites ◽

Molecular Mechanisms ◽

Epigenetic Changes ◽

Promoter Regions ◽

Inflammatory Conditions ◽

Genome Wide ◽

Promising Target ◽

Cardiovascular Patients ◽

Factor C ◽

S100a9 Expression

The proinflammatory alarmins S100A8 and S100A9 are among the most abundant proteins in neutrophils and monocytes but completely silenced after differentiation to macrophages. The molecular mechanisms of the extraordinarily dynamic transcriptional regulation of s100a8 and s100a9 genes, however, are only barely understood. Using an unbiased genome-wide CRISPR/Cas9 knockout based screening approach in immortalized murine monocytes we identified the transcription factor C/EBPδ as a central regulator of S100A8 and S100A9 expression. S100a8 and S100a9 expression was further controlled by the C/EBPδ-antagonists ATF3 and FBXW7. We confirmed the clinical relevance of this regulatory network in subpopulations of human monocytes in a clinical cohort of cardiovascular patients. Moreover, we identified specific C/EBPδ-binding sites within s100a8 and s100a9 promoter regions, and demonstrated that C/EBPδ-dependent JMJD3-mediated demethylation of H3K27me3 is indispensable for their expression. Overall, our work uncovered C/EBPδ as a novel regulator of S100A8 and S100A9 expression. Therefore, C/EBPδ represents a promising target for modulation of inflammatory conditions that are characterised by S100A8 and S100A9 overexpression.

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Research Resource: Genome-Wide Profiling of Progesterone Receptor Binding in the Mouse Uterus

Molecular Endocrinology ◽

10.1210/me.2011-1355 ◽

2012 ◽

Vol 26 (8) ◽

pp. 1428-1442 ◽

Cited By ~ 89

Author(s):

Cory A. Rubel ◽

Rainer B. Lanz ◽

Ramakrishna Kommagani ◽

Heather L. Franco ◽

John P. Lydon ◽

...

Keyword(s):

Transcription Factor ◽

Progesterone Receptor ◽

Binding Sites ◽

Molecular Mechanisms ◽

Target Genes ◽

Massively Parallel Sequencing ◽

Mouse Uterus ◽

Ovariectomized Mice ◽

Genome Wide ◽

Uterine Function

Progesterone (P4) signaling through its nuclear transcription factor, the progesterone receptor (PR), is essential for normal uterine function. Although deregulation of PR-mediated signaling is known to underscore uterine dysfunction and a number of endometrial pathologies, the early molecular mechanisms of this deregulation are unclear. To address this issue, we have defined the genome-wide PR cistrome in the murine uterus using chromatin immunoprecipitation (ChIP) followed by massively parallel sequencing (ChIP-seq). In uteri of ovariectomized mice, we identified 6367 PR-binding sites in the absence of P4 ligand; however, this number increased at nearly 3-fold (18,432) after acute P4 exposure. Sequence analysis revealed that approximately 73% of these binding sites contain a progesterone response element or a half-site motif recognized by the PR. Many previously identified P4 target genes known to regulate uterine function were found to contain PR-binding sites, confirming the validity of our methodology. Interestingly, when the ChIP-seq data were coupled with our microarray expression data, we identified a novel regulatory role for uterine P4 in circadian rhythm gene expression, thereby uncovering a hitherto unexpected new circadian biology for P4 in this tissue. Further mining of the ChIP-seq data revealed Sox17 as a direct transcriptional PR target gene in the uterus. As a member of the Sox transcription factor family, Sox17 represents a potentially novel mediator of PR action in the murine uterus. Collectively, our first line of analysis of the uterine PR cistrome provides the first insights into the early molecular mechanisms that underpin normal uterine responsiveness to acute P4 exposure. Future analysis promises to reveal the PR interactome and, in turn, potential therapeutic targets for the diagnosis and/or treatment of endometrial dysfunction.

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An Integrative Approach for Fine-Mapping Chromatin Interactions

10.1101/605576 ◽

2019 ◽

Author(s):

Artur Jaroszewicz ◽

Jason Ernst

Keyword(s):

High Resolution ◽

Binding Sites ◽

Biological Significance ◽

Computational Method ◽

Integrative Approach ◽

Genome Architecture ◽

Open Chromatin ◽

Chromatin Interactions ◽

Genome Wide ◽

Evolutionarily Conserved

AbstractChromatin interactions play an important role in genome architecture and regulation. The Hi-C assay generates such interactions maps genome-wide, but at relatively low resolutions (e.g., 5-25kb), which is substantially larger than the resolution of transcription factor binding sites or open chromatin sites that are potential sources of such interactions. To predict the sources of Hi-C identified interactions at a high resolution (e.g., 100bp), we developed a computational method that integrates ChIP-seq data of transcription factors and histone marks and DNase-seq data. Our method,χ-SCNN, uses this data to first train a Siamese Convolutional Neural Network (SCNN) to discriminate between called Hi-C interactions and non-interactions.χ-SCNN then predicts the high-resolution source of each Hi-C interaction using a feature attribution method. We show these predictions recover original Hi-C peaks after extending them to be coarser. We also showχ-SCNN predictions enrich for evolutionarily conserved bases, eQTLs, and CTCF motifs, supporting their biological significance.χ-SCNN provides an approach for analyzing important aspects of genome architecture and regulation at a higher resolution than previously possible.χ-SCNN software is available on GitHub (https://github.com/ernstlab/X-SCNN).

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