scholarly journals Ultrastructural details of mammalian chromosome architecture

2019 ◽  
Author(s):  
Nils Krietenstein ◽  
Sameer Abraham ◽  
Sergey V. Venev ◽  
Nezar Abdennur ◽  
Johan Gibcus ◽  
...  
Keyword(s):  
Dynamic Range ◽  
Embryonic Stem ◽  
Ctcf Binding ◽  
Chromosome Architecture ◽  
Genome Wide ◽  
Domain Boundaries ◽  
Order Of Magnitude ◽  
Cis Interaction

ABSTRACTOver the past decade, 3C-related methods, complemented by increasingly detailed microscopic views of the nucleus, have provided unprecedented insights into chromosome folding in vivo. Here, to overcome the resolution limits inherent to the majority of genome-wide chromosome architecture mapping studies, we extend a recently-developed Hi-C variant, Micro-C, to map chromosome architecture at nucleosome resolution in human embryonic stem cells and fibroblasts. Micro-C maps robustly capture well-described features of mammalian chromosome folding including A/B compartment organization, topologically associating domains (TADs), and cis interaction peaks anchored at CTCF binding sites, while also providing a detailed 1-dimensional map of nucleosome positioning and phasing genome-wide. Compared to high-resolution in situ Hi-C, Micro-C exhibits substantially improved signal-to-noise with an order of magnitude greater dynamic range, enabling not only localization of domain boundaries with single-nucleosome accuracy, but also resolving more than 20,000 additional looping interaction peaks in each cell type. Intriguingly, many of these newly-identified peaks are localized along stripe patterns and form transitive grids, consistent with their anchors being pause sites impeding the process of cohesin-dependent loop extrusion. Together, our analyses provide the highest resolution maps of chromosome folding in human cells to date, and provide a valuable resource for studies of chromosome folding mechanisms.

scholarly journals Genome-Wide Binding Analyses of HOXB1 Revealed a Novel DNA Binding Motif Associated with Gene Repression

2021 ◽  
Vol 9 (1) ◽  
pp. 6
Author(s):  
Narendra Pratap Singh ◽  
Bony De Kumar ◽  
Ariel Paulson ◽  
Mark E. Parrish ◽  
Carrie Scott ◽  
...  
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Embryonic Stem ◽  
Binding Motif ◽  
Binding Assays ◽  
Histone Marks ◽  
Genome Wide ◽  
Hox Cofactors

Knowledge of the diverse DNA binding specificities of transcription factors is important for understanding their specific regulatory functions in animal development and evolution. We have examined the genome-wide binding properties of the mouse HOXB1 protein in embryonic stem cells differentiated into neural fates. Unexpectedly, only a small number of HOXB1 bound regions (7%) correlate with binding of the known HOX cofactors PBX and MEIS. In contrast, 22% of the HOXB1 binding peaks display co-occupancy with the transcriptional repressor REST. Analyses revealed that co-binding of HOXB1 with PBX correlates with active histone marks and high levels of expression, while co-occupancy with REST correlates with repressive histone marks and repression of the target genes. Analysis of HOXB1 bound regions uncovered enrichment of a novel 15 base pair HOXB1 binding motif HB1RE (HOXB1 response element). In vitro template binding assays showed that HOXB1, PBX1, and MEIS can bind to this motif. In vivo, this motif is sufficient for direct expression of a reporter gene and over-expression of HOXB1 selectively represses this activity. Our analyses suggest that HOXB1 has evolved an association with REST in gene regulation and the novel HB1RE motif contributes to HOXB1 function in part through a repressive role in gene expression.

scholarly journals CTCF Interacts with and Recruits the Largest Subunit of RNA Polymerase II to CTCF Target Sites Genome-Wide

2007 ◽  
Vol 27 (5) ◽  
pp. 1631-1648 ◽  
Author(s):  
Igor Chernukhin ◽  
Shaharum Shamsuddin ◽  
Sung Yun Kang ◽  
Rosita Bergström ◽  
Yoo-Wook Kwon ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Gene Activation ◽  
Ctcf Binding ◽  
Pol Ii ◽  
Genome Wide ◽  
Target Sites ◽  
On Chip

ABSTRACT CTCF is a transcription factor with highly versatile functions ranging from gene activation and repression to the regulation of insulator function and imprinting. Although many of these functions rely on CTCF-DNA interactions, it is an emerging realization that CTCF-dependent molecular processes involve CTCF interactions with other proteins. In this study, we report the association of a subpopulation of CTCF with the RNA polymerase II (Pol II) protein complex. We identified the largest subunit of Pol II (LS Pol II) as a protein significantly colocalizing with CTCF in the nucleus and specifically interacting with CTCF in vivo and in vitro. The role of CTCF as a link between DNA and LS Pol II has been reinforced by the observation that the association of LS Pol II with CTCF target sites in vivo depends on intact CTCF binding sequences. “Serial” chromatin immunoprecipitation (ChIP) analysis revealed that both CTCF and LS Pol II were present at the β-globin insulator in proliferating HD3 cells but not in differentiated globin synthesizing HD3 cells. Further, a single wild-type CTCF target site (N-Myc-CTCF), but not the mutant site deficient for CTCF binding, was sufficient to activate the transcription from the promoterless reporter gene in stably transfected cells. Finally, a ChIP-on-ChIP hybridization assay using microarrays of a library of CTCF target sites revealed that many intergenic CTCF target sequences interacted with both CTCF and LS Pol II. We discuss the possible implications of our observations with respect to plausible mechanisms of transcriptional regulation via a CTCF-mediated direct link of LS Pol II to the DNA.

scholarly journals TET-catalyzed 5-carboxylcytosine promotes CTCF binding to suboptimal sequences genome-wide

2018 ◽  
Author(s):  
Kyster K. Nanan ◽  
David M. Sturgill ◽  
Maria F. Prigge ◽  
Morgan Thenoz ◽  
Allissa A. Dillman ◽  
...  
Keyword(s):  
Binding Sites ◽  
Genomic Dna ◽  
Multiple Roles ◽  
Ctcf Binding ◽  
Cellular Model ◽  
Potential Mechanism ◽  
Dynamic Regulation ◽  
Genome Wide

SummaryThe mechanisms supporting dynamic regulation of CTCF binding sites remain poorly understood. Here we describe the TET-catalyzed 5-methylcytosine derivative, 5-carboxylcytosine (5caC) as a factor driving new CTCF binding within genomic DNA. Through a combination of in vivo and in vitro approaches, we reveal that 5caC generally strengthens CTCF association with DNA and facilitates binding to suboptimal sequences. Dramatically, profiling of CTCF binding in a cellular model that accumulates genomic 5caC identified ∼13,000 new CTCF sites. The new sites were enriched for overlapping 5caC and were marked by an overall reduction in CTCF motif strength. As CTCF has multiple roles in gene expression, these findings have wide-reaching implications and point to induced 5caC as a potential mechanism to achieve differential CTCF binding in cells.

scholarly journals BLISS: quantitative and versatile genome-wide profiling of DNA breaks in situ

2016 ◽  
Author(s):  
Winston X. Yan ◽  
Reza Mirzazadeh ◽  
Silvano Garnerone ◽  
David Scott ◽  
Martin W. Schneider ◽  
...  
Keyword(s):  
High Efficiency ◽  
Embryonic Stem ◽  
Dna Breaks ◽  
Drug Induced ◽  
Low Input ◽  
Tissue Sections ◽  
Genome Wide ◽  
Primary Mouse

AbstractWe present a method for genome-wide DNA double-strand Breaks (DSBs) Labeling In Situ and Sequencing (BLISS) which, compared to existing methods, introduces several key features: 1) high efficiency and low input requirement by in situ DSB labeling in cells or tissue sections directly on a solid surface; 2) easy scalability by performing in situ reactions in multi-well plates; 3) high sensitivity by linearly amplifying tagged DSBs using in vitro transcription; and 4) accurate DSB quantification and control of PCR biases by using unique molecular identifiers. We demonstrate the ability to use BLISS to quantify natural and drug-induced DSBs in low-input samples of cancer cells, primary mouse embryonic stem cells, and mouse liver tissue sections. Finally, we applied BLISS to compare the specificity of CRISPR-associated RNA-guided endonucleases Cas9 and Cpf1, and found that Cpf1 has higher specificity than Cas9. These results establish BLISS as a versatile, sensitive, and efficient method for genome-wide DSB mapping in many applications.

scholarly journals Active and poised promoter states drive folding of the extended HoxB locus in mouse embryonic stem cells

2017 ◽  
Author(s):  
Mariano Barbieri ◽  
Sheila Q. Xie ◽  
Elena Torlai Triglia ◽  
Inês de Santiago ◽  
Miguel R. Branco ◽  
...  
Keyword(s):  
Es Cells ◽  
Single Cells ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Genomic Region ◽  
Ctcf Binding ◽  
Test Model ◽  
State Dependent ◽  
Polymer Models

AbstractGene expression states influence the three-dimensional conformation of the genome through poorly understood mechanisms. Here, we investigate the conformation of the murine HoxB locus, a gene-dense genomic region containing closely spaced genes with distinct activation states in mouse embryonic stem (ES) cells. To predict possible folding scenarios, we performed computer simulations of polymer models informed with different chromatin occupancy features, which define promoter activation states or CTCF binding sites. Single cell imaging of the locus folding was performed to test model predictions. While CTCF occupancy alone fails to predict the in vivo folding at genomic length scale of 10 kb, we found that homotypic interactions between active and Polycomb-repressed promoters co-occurring in the same DNA fibre fully explain the HoxB folding patterns imaged in single cells. We identify state-dependent promoter interactions as major drivers of chromatin folding in gene-dense regions.

scholarly journals BAP1 activity regulates PcG occupancy and global chromatin condensation counteracting diffuse PCGF3/5-dependent H2AK119ub1 deposition

2020 ◽  
Author(s):  
Eric Conway ◽  
Federico Rossi ◽  
Simone Tamburri ◽  
Eleonora Ponzo ◽  
Karin Johanna Ferrari ◽  
...  
Keyword(s):  
Transcriptional Repression ◽  
Chromatin Condensation ◽  
Embryonic Stem ◽  
Apparent Contradiction ◽  
Major Function ◽  
Genome Wide ◽  
Cancer Types ◽  
Precise Relationship ◽  
The Stability

AbstractBAP1 is recurrently mutated or deleted in a large number of diverse cancer types, including mesothelioma, uveal melanoma and hepatocellular cholangiocarcinoma. BAP1 is the catalytic subunit of the Polycomb Repressive De-Ubiquitination complex (PR-DUB) which removes PRC1 mediated H2AK119ub1. We and others have shown that H2AK119ub1 is essential for maintaining transcriptional repression and contributes to PRC2 chromatin recruitment. However, the precise relationship between BAP1 and PRC1 remains mechanistically elusive. Using embryonic stem cells, we show that a major function of BAP1 is to restrict H2AK119ub1 deposition to target sites. This increases the stability of PcG complexes with their targets and prevents diffuse accumulation of H2AK119ub1 and H3K27me3 modifications. Loss of BAP1 results in a broad increase in H2AK119ub1 levels that are primarily dependent on PCGF3/5-PRC1 complexes with a mechanism that is reminiscent of X-chromosome inactivation. Increased genome-wide H2AK119ub1 levels titrates away PRC2 from its targets and stimulates diffuse H3K27me3 accumulation across the genome. This decreases the activity of PcG repressive machineries at physiological targets and induces a general compaction of the entire chromatin. Our findings provide evidences for a unifying model that resolves the apparent contradiction between BAP1 catalytic activity and its role in vivo, uncovering molecular vulnerabilities that could be useful for BAP1-related pathologies.

scholarly journals CTCF-dependent chromatin boundaries formed by asymmetric nucleosome arrays with decreased linker length

2019 ◽  
Author(s):  
Christopher T. Clarkson ◽  
Emma A. Deeks ◽  
Ralph Samarista ◽  
Hulkar Mamayusupova ◽  
Victor B. Zhurkin ◽  
...  
Keyword(s):  
Neural Progenitor Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Ctcf Binding ◽  
Embryonic Fibroblasts ◽  
Genome Wide ◽  
Binding Factor ◽  
Chromatin Boundary ◽  
Nucleosome Array ◽  
The Relationship

AbstractThe CCCTC-binding factor (CTCF) organises the genome in 3D through DNA loops and in 1D by setting boundaries isolating different chromatin states, but these processes are not well understood. Here we focus on the relationship between CTCF binding and the decrease of the Nucleosome Repeat Length (NRL) for ∼20 adjacent nucleosomes, affecting up to 10% of the mouse genome. We found that the chromatin boundary near CTCF is created by the nucleosome-depleted region (NDR) asymmetrically located >40 nucleotides 5’-upstream from the centre of CTCF motif. The strength of CTCF binding to DNA is correlated with the decrease of NRL near CTCF and anti-correlated with the level of asymmetry of the nucleosome array. Individual chromatin remodellers have different contributions, with Snf2h having the strongest effect on the NRL decrease near CTCF and Chd4 playing a major role in the symmetry breaking. Upon differentiation of embryonic stem cells to neural progenitor cells and embryonic fibroblasts, a subset of common CTCF sites preserved in all three cell types maintains a relatively small local NRL despite genome-wide NRL increase. The sites which lost CTCF upon differentiation are characterised by nucleosome rearrangement 3’-downstream, but the boundary defined by the NDR 5’-upstream of CTCF motif remains.

Embryonic Stem Cell-Like Features of Testicular Carcinoma in Situ Revealed by Genome-Wide Gene Expression Profiling

2004 ◽  
Vol 64 (14) ◽  
pp. 4736-4743 ◽  
Author(s):  
Kristian Almstrup ◽  
Christina E. Hoei-Hansen ◽  
Ute Wirkner ◽  
Jonathon Blake ◽  
Christian Schwager ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Expression Profiling ◽  
Carcinoma In Situ ◽  
Embryonic Stem ◽  
Genome Wide ◽  
Testicular Carcinoma ◽  
Genome Wide Gene Expression

scholarly journals Synthetic genome readers target clustered binding sites across diverse chromatin states

2016 ◽  
Vol 113 (47) ◽  
pp. E7418-E7427 ◽  
Author(s):  
Graham S. Erwin ◽  
Matthew P. Grieshop ◽  
Devesh Bhimsaria ◽  
Truman J. Do ◽  
José A. Rodríguez-Martínez ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Embryonic Stem ◽  
Live Cells ◽  
Binding Affinities ◽  
Chromatin States ◽  
Synthetic Genome ◽  
Occupancy Patterns ◽  
Genome Wide ◽  
The Impact

Targeting the genome with sequence-specific DNA-binding molecules is a major goal at the interface of chemistry, biology, and precision medicine. Polyamides, composed of N-methylpyrrole and N-methylimidazole monomers, are a class of synthetic molecules that can be rationally designed to “read” specific DNA sequences. However, the impact of different chromatin states on polyamide binding in live cells remains an unresolved question that impedes their deployment in vivo. Here, we use cross-linking of small molecules to isolate chromatin coupled to sequencing to map the binding of two bioactive and structurally distinct polyamides to genomes directly within live H1 human embryonic stem cells. This genome-wide view from live cells reveals that polyamide-based synthetic genome readers bind cognate sites that span a range of binding affinities. Polyamides can access cognate sites within repressive heterochromatin. The occupancy patterns suggest that polyamides could be harnessed to target loci within regions of the genome that are inaccessible to other DNA-targeting molecules.

scholarly journals In vivo dissection of a clustered-CTCF domain boundary reveals developmental principles of regulatory insulation

2021 ◽  
Author(s):  
Chiara Anania ◽  
Rafael D. Acemel ◽  
Johanna Jedamzick ◽  
Adriano Bolondi ◽  
Giulia Cova ◽  
...  
Keyword(s):  
Target Genes ◽  
Domain Boundary ◽  
Boundary Function ◽  
Regulatory Elements ◽  
Ctcf Binding ◽  
Chromatin Interactions ◽  
Genome Wide ◽  
Multiple Levels ◽  
Developmental Principles

Vertebrate genomes organize into topologically associating domains (TADs), delimited by boundaries that insulate regulatory elements from non-target genes. However, how boundary function is established is not well understood. Here, we combine genome-wide analyses and transgenic mouse assays to dissect the regulatory logic of clustered-CTCF boundaries in vivo, interrogating their function at multiple levels: chromatin interactions, transcription and phenotypes. Individual CTCF binding sites (CBS) deletions revealed that the characteristics of specific sites can outweigh other factors like CBS number and orientation. Combined deletions demonstrated that CBS cooperate redundantly and provide boundary robustness. We show that divergent CBS signatures are not strictly required for effective insulation and that chromatin loops formed by non-convergently oriented sites could be mediated by a loop interference mechanism. Further, we observe that insulation strength constitutes a quantitative modulator of gene expression and phenotypes. Our results highlight the modular nature of boundaries and their control over developmental processes.

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