High-resolution, genome-wide mapping of positive supercoiling in chromosomes

Supercoiling impacts DNA replication, transcription, protein binding to DNA, and the three-dimensional organization of chromosomes. However, there are currently no methods to directly interrogate or map positive supercoils, so their distribution in genomes remains unknown. Here, we describe a method, GapR-seq, based on the chromatin immunoprecipitation of GapR, a bacterial protein that preferentially recognizes overtwisted DNA, for generating high-resolution maps of positive supercoiling. Applying this method to E. coli and S. cerevisiae, we find that positive supercoiling is widespread, associated with transcription, and particularly enriched between convergently-oriented genes, consistent with the 'twin-domain' model of supercoiling. In yeast, we also find positive supercoils associated with centromeres, cohesin binding sites, autonomously replicating sites, and the borders of R-loops (DNA-RNA hybrids). Our results suggest that GapR-seq is a powerful approach, likely applicable in any organism, to investigate aspects of chromosome structure and organization not accessible by Hi-C or other existing methods.

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High-resolution, genome-wide mapping of positive supercoiling in chromosomes

10.1101/2021.02.25.432897 ◽

2021 ◽

Author(s):

Monica S. Guo ◽

Ryo Kawamura ◽

Megan Littlehale ◽

John F. Marko ◽

Michael T. Laub

Keyword(s):

High Resolution ◽

Chromatin Immunoprecipitation ◽

Binding Sites ◽

Three Dimensional ◽

Domain Model ◽

Bacterial Protein ◽

E Coli ◽

Genome Wide ◽

Powerful Approach ◽

Positive Supercoiling

AbstractSupercoiling impacts DNA replication, transcription, protein binding to DNA, and the three-dimensional organization of chromosomes. However, there are currently no methods to directly interrogate or map positive supercoils, so their distribution in genomes remains unknown. Here, we describe a method, GapR-seq, based on the chromatin immunoprecipitation of GapR, a bacterial protein that preferentially recognizes overtwisted DNA, for generating high-resolution maps of positive supercoiling. Applying this method to E. coli and S. cerevisiae, we find that positive supercoiling is widespread, associated with transcription, and particularly enriched between convergently-oriented genes, consistent with the “twin-domain” model of supercoiling. In yeast, we also find positive supercoils associated with centromeres, cohesin binding sites, autonomously replicating sites, and the borders of R-loops (DNA-RNA hybrids). Our results suggest that GapR-seq is a powerful approach, likely applicable in any organism, to investigate aspects of chromosome structure and organization not accessible by Hi-C or other existing methods.

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Immobilization of Escherichia coli RNA Polymerase and Location of Binding Sites by Use of Chromatin Immunoprecipitation and Microarrays

Journal of Bacteriology ◽

10.1128/jb.187.17.6166-6174.2005 ◽

2005 ◽

Vol 187 (17) ◽

pp. 6166-6174 ◽

Cited By ~ 83

Author(s):

Christopher D. Herring ◽

Marni Raffaelle ◽

Timothy E. Allen ◽

Elenita I. Kanin ◽

Robert Landick ◽

...

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Chromatin Immunoprecipitation ◽

Binding Sites ◽

Reading Frame ◽

E Coli ◽

Genome Wide ◽

Polymerase Binding ◽

Chip Chip ◽

Negative Detection

ABSTRACT The genome-wide location of RNA polymerase binding sites was determined in Escherichia coli using chromatin immunoprecipitation and microarrays (chIP-chip). Cross-linked chromatin was isolated in triplicate from rifampin-treated cells, and DNA bound to RNA polymerase was precipitated with an antibody specific for the β′ subunit. The DNA was amplified and hybridized to “tiled” oligonucleotide microarrays representing the whole genome at 25-bp resolution. A total of 1,139 binding sites were detected and evaluated by comparison to gene expression data from identical conditions and to 961 promoters previously identified by established methods. Of the detected binding sites, 418 were located within 1,000 bp of a known promoter, leaving 721 previously unknown RNA polymerase binding sites. Within 200 bp, we were able to detect 51% (189/368) of the known σ70-specific promoters occurring upstream of an expressed open reading frame and 74% (273/368) within 1,000 bp. Conversely, many known promoters were not detected by chIP-chip, leading to an estimated 26% negative-detection rate. Most of the detected binding sites could be associated with expressed transcription units, but 299 binding sites occurred near inactive transcription units. This map of RNA polymerase binding sites represents a foundation for studies of transcription factors in E. coli and an important evaluation of the chIP-chip technique.

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Genome-Wide Identification of Transcription Start Sites, Promoters and Transcription Factor Binding Sites in E. coli

PLoS ONE ◽

10.1371/journal.pone.0007526 ◽

2009 ◽

Vol 4 (10) ◽

pp. e7526 ◽

Cited By ~ 184

Author(s):

Alfredo Mendoza-Vargas ◽

Leticia Olvera ◽

Maricela Olvera ◽

Ricardo Grande ◽

Leticia Vega-Alvarado ◽

...

Keyword(s):

Transcription Factor ◽

Binding Sites ◽

Transcription Factor Binding Sites ◽

Transcription Factor Binding ◽

Transcription Start ◽

Transcription Start Sites ◽

E Coli ◽

Factor Binding ◽

Genome Wide

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Rapid and Low-Input Profiling of Histone Marks in Plants Using Nucleus CUT&Tag

Frontiers in Plant Science ◽

10.3389/fpls.2021.634679 ◽

2021 ◽

Vol 12 ◽

Author(s):

Weizhi Ouyang ◽

Xiwen Zhang ◽

Yong Peng ◽

Qing Zhang ◽

Zhilin Cao ◽

...

Keyword(s):

Histone Modifications ◽

Chromatin Immunoprecipitation ◽

Binding Sites ◽

Protein A ◽

Transcriptional Factor ◽

Experimental Procedure ◽

Histone Marks ◽

Genome Wide ◽

Efficient Alternative ◽

Tn5 Transposase

Characterizing genome-wide histone posttranscriptional modifications and transcriptional factor occupancy is crucial for deciphering their biological functions. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a powerful method for genome-wide profiling of histone modifications and transcriptional factor-binding sites. However, the current ChIP-seq experimental procedure in plants requires significant material and several days for completion. CUT&Tag is an alternative method of ChIP-seq for low-sample and single-cell epigenomic profiling using protein A-Tn5 transposase fusion proteins (PAT). In this study, we developed a nucleus CUT&Tag (nCUT&Tag) protocol based on the live-cell CUT&Tag technology. Our results indicate that nCUT&Tag could be used for histone modifications profiling in both monocot rice and dicot rapeseed using crosslinked or fresh tissues. In addition, both active and repressive histone marks such as H3K4me3 and H3K9me2 can be identified using our nCUT&Tag. More importantly, all the steps in nCUT&Tag can be finished in only 1 day, and the assay can be performed with as little as 0.01 g of plant tissue as starting materials. Therefore, our results demonstrate that nCUT&Tag is an efficient alternative strategy for plant epigenomic studies.

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The native cistrome and sequence motif families of the maize ear

PLoS Genetics ◽

10.1371/journal.pgen.1009689 ◽

2021 ◽

Vol 17 (8) ◽

pp. e1009689

Author(s):

Savannah D. Savadel ◽

Thomas Hartwig ◽

Zachary M. Turpin ◽

Daniel L. Vera ◽

Pei-Yau Lung ◽

...

Keyword(s):

High Resolution ◽

Binding Sites ◽

Regulatory Networks ◽

Regulatory Elements ◽

Chromatin Interaction ◽

Sequence Motif ◽

Binding Prediction ◽

Interaction Sites ◽

Genome Wide ◽

Dna Regulatory Elements

Elucidating the transcriptional regulatory networks that underlie growth and development requires robust ways to define the complete set of transcription factor (TF) binding sites. Although TF-binding sites are known to be generally located within accessible chromatin regions (ACRs), pinpointing these DNA regulatory elements globally remains challenging. Current approaches primarily identify binding sites for a single TF (e.g. ChIP-seq), or globally detect ACRs but lack the resolution to consistently define TF-binding sites (e.g. DNAse-seq, ATAC-seq). To address this challenge, we developed MNase-defined cistrome-Occupancy Analysis (MOA-seq), a high-resolution (< 30 bp), high-throughput, and genome-wide strategy to globally identify putative TF-binding sites within ACRs. We used MOA-seq on developing maize ears as a proof of concept, able to define a cistrome of 145,000 MOA footprints (MFs). While a substantial majority (76%) of the known ATAC-seq ACRs intersected with the MFs, only a minority of MFs overlapped with the ATAC peaks, indicating that the majority of MFs were novel and not detected by ATAC-seq. MFs were associated with promoters and significantly enriched for TF-binding and long-range chromatin interaction sites, including for the well-characterized FASCIATED EAR4, KNOTTED1, and TEOSINTE BRANCHED1. Importantly, the MOA-seq strategy improved the spatial resolution of TF-binding prediction and allowed us to identify 215 motif families collectively distributed over more than 100,000 non-overlapping, putatively-occupied binding sites across the genome. Our study presents a simple, efficient, and high-resolution approach to identify putative TF footprints and binding motifs genome-wide, to ultimately define a native cistrome atlas.

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RedChIP identifies noncoding RNAs associated with genomic sites occupied by Polycomb and CTCF proteins

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2116222119 ◽

2021 ◽

Vol 119 (1) ◽

pp. e2116222119

Author(s):

Alexey A. Gavrilov ◽

Rinat I. Sultanov ◽

Mikhail D. Magnitov ◽

Aleksandra A. Galitsyna ◽

Erdem B. Dashinimaev ◽

...

Keyword(s):

Chromatin Immunoprecipitation ◽

Binding Sites ◽

Wide Spectrum ◽

Noncoding Rnas ◽

Ctcf Binding ◽

Polycomb Repressive Complex 2 ◽

Proximity Ligation ◽

Chromatin Interactions ◽

Genome Wide ◽

Architectural Protein

Nuclear noncoding RNAs (ncRNAs) are key regulators of gene expression and chromatin organization. The progress in studying nuclear ncRNAs depends on the ability to identify the genome-wide spectrum of contacts of ncRNAs with chromatin. To address this question, a panel of RNA–DNA proximity ligation techniques has been developed. However, neither of these techniques examines proteins involved in RNA–chromatin interactions. Here, we introduce RedChIP, a technique combining RNA–DNA proximity ligation and chromatin immunoprecipitation for identifying RNA–chromatin interactions mediated by a particular protein. Using antibodies against architectural protein CTCF and the EZH2 subunit of the Polycomb repressive complex 2, we identify a spectrum of cis- and trans-acting ncRNAs enriched at Polycomb- and CTCF-binding sites in human cells, which may be involved in Polycomb-mediated gene repression and CTCF-dependent chromatin looping. By providing a protein-centric view of RNA–DNA interactions, RedChIP represents an important tool for studies of nuclear ncRNAs.

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In silico prediction of high-resolution Hi-C interaction matrices

Nature Communications ◽

10.1038/s41467-019-13423-8 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 7

Author(s):

Shilu Zhang ◽

Deborah Chasman ◽

Sara Knaack ◽

Sushmita Roy

Keyword(s):

High Resolution ◽

Genome Organization ◽

Three Dimensional ◽

Predictive Performance ◽

Computational Prediction ◽

3D Genome ◽

Genome Wide ◽

Binding Factor ◽

Sequence Elements ◽

Individual Locus

AbstractThe three-dimensional (3D) organization of the genome plays an important role in gene regulation bringing distal sequence elements in 3D proximity to genes hundreds of kilobases away. Hi-C is a powerful genome-wide technique to study 3D genome organization. Owing to experimental costs, high resolution Hi-C datasets are limited to a few cell lines. Computational prediction of Hi-C counts can offer a scalable and inexpensive approach to examine 3D genome organization across multiple cellular contexts. Here we present HiC-Reg, an approach to predict contact counts from one-dimensional regulatory signals. HiC-Reg predictions identify topologically associating domains and significant interactions that are enriched for CCCTC-binding factor (CTCF) bidirectional motifs and interactions identified from complementary sources. CTCF and chromatin marks, especially repressive and elongation marks, are most important for HiC-Reg’s predictive performance. Taken together, HiC-Reg provides a powerful framework to generate high-resolution profiles of contact counts that can be used to study individual locus level interactions and higher-order organizational units of the genome.

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Structural similarities in the CPC clip motif explain peptide-binding promiscuity between glycosaminoglycans and lipopolysaccharides

Journal of The Royal Society Interface ◽

10.1098/rsif.2017.0423 ◽

2017 ◽

Vol 14 (136) ◽

pp. 20170423 ◽

Cited By ~ 1

Author(s):

David Pulido ◽

Rocío Rebollido-Rios ◽

Javier Valle ◽

David Andreu ◽

Ester Boix ◽

...

Keyword(s):

Binding Sites ◽

Three Dimensional ◽

The Body ◽

Heparin Binding ◽

Structural Determinants ◽

E Coli ◽

High Affinity ◽

Structural Framework ◽

And Control ◽

Proteins And Peptides

Lipopolysaccharides (LPSs) and glycosaminoglycans (GAGs) are polymeric structures containing negatively charged disaccharide units that bind to specialized proteins and peptides in the human body and control fundamental processes such as inflammation and coagulation. Surprisingly, some proteins can bind both LPSs and GAGs with high affinity, suggesting that a cross-communication between these two pathways can occur. Here, we explore whether GAGs and LPSs can share common binding sites in proteins and what are the structural determinants of this binding. We found that the LPS-binding peptide YI12WF, derived from protein FhuA, can bind both heparin and E. coli LPS with high affinity. Most interestingly, mutations decreasing heparin binding in the peptide also reduce LPS affinity. We show that such mutations involve the CPC clip motif in the peptide, a small three-dimensional signature required for heparin binding. Overall, we conclude that negatively charged polysaccharide-containing polymers such as GAGs and LPSs can compete for similar binding sites in proteins, and that the CPC clip motif is essential to bind both ligands. Our results provide a structural framework to explain why these polymers can cross-interact with the same proteins and peptides and thus contribute to the regulation of apparently unrelated processes in the body.

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