Nanoscale chromatin profiling of gastric adenocarcinoma reveals cancer-associated cryptic promoters and somatically acquired regulatory elements

Cleavage Under Targets & Tagmentation (CUT&Tag) is an antibody-directed transposase tethering strategy for in situ chromatin profiling in small samples and single cells. We describe a modified CUT&Tag protocol using a mixture of an antibody to the initiation form of RNA Polymerase II (Pol2 Serine-5 phosphate) and an antibody to repressive Polycomb domains (H3K27me3) followed by computational signal deconvolution to produce high-resolution maps of both the active and repressive regulomes in single cells. The ability to seamlessly map active promoters, enhancers and repressive regulatory elements using a single workflow provides a complete regulome profiling strategy suitable for high-throughput single-cell platforms.

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Rapid In Vivo Mapping of Hematopoietic Cis-Regulatory Elements.

Blood ◽

10.1182/blood.v104.11.4209.4209 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4209-4209

Author(s):

John A. Stamatoyannopouylos ◽

Michael Hawrylycz ◽

Richard Humbert ◽

James C. Wallace ◽

Man Yu ◽

...

Keyword(s):

Chromatin Structure ◽

Regulatory Elements ◽

Regulatory Sequences ◽

Beta Globin ◽

Cis Acting ◽

Human Genomic ◽

Chromatin Profiling ◽

Hematopoietic Cell Lines ◽

Entire Gene

Abstract Cis-acting sequences play defining roles in the control of genes involved in hematopoiesis. However, for the vast majority of such genes, regulatory sequences remain to be defined. We describe a powerful, generic approach to identification of cis-regulatory sequences that may be applied to any gene locus in the context of any cell type. We used densely tiled primers and a novel real-time PCR-based assay to create continuous, high-resolution quantitative profiles of in vivo chromatin structure across entire gene domains. Such profiles can be analyzed using robust statistical algorithms to pinpoint disruptions in chromatin structure that are characteristic of cis-regulatory elements. We analyzed >1Mb of human genomic terrain from diverse gene loci in the context of several hematopoietic cell lines and cleanly delineated a spectrum of classical cis-regulatory activities including enhancers, promoters, insulators, and locus control regions. The approach displayed outstanding sensitivity (100%) and specificity (>99.6%) for known elements and was successful in defining novel elements even in heavily-explored terrain such as the alpha- and beta-globin loci. Since only small quantities of cells are required, the approach can be used readily in the context of hematopoietic progenitors. Systematic application of quantitative chromatin profiling to relevant genes promises to expand dramatically our understanding of the regulation of hematopoiesis.

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SBE6: a novel long-range enhancer involved in driving sonic hedgehog expression in neural progenitor cells

Open Biology ◽

10.1098/rsob.160197 ◽

2016 ◽

Vol 6 (11) ◽

pp. 160197 ◽

Cited By ~ 8

Author(s):

Nezha S. Benabdallah ◽

Philippe Gautier ◽

Betul Hekimoglu-Balkan ◽

Laura A. Lettice ◽

Shipra Bhatia ◽

...

Keyword(s):

Progenitor Cells ◽

Long Range ◽

Sonic Hedgehog ◽

Neural Progenitor Cells ◽

Embryonic Stem ◽

Regulatory Elements ◽

Neural Progenitor ◽

Chromatin Profiling

The expression of genes with key roles in development is under very tight spatial and temporal control, mediated by enhancers. A classic example of this is the sonic hedgehog gene ( Shh ), which plays a pivotal role in the proliferation, differentiation and survival of neural progenitor cells both in vivo and in vitro. Shh expression in the brain is tightly controlled by several known enhancers that have been identified through genetic, genomic and functional assays. Using chromatin profiling during the differentiation of embryonic stem cells to neural progenitor cells, here we report the identification of a novel long-range enhancer for Shh—Shh-brain-enhancer-6 (SBE6)—that is located 100 kb upstream of Shh and that is required for the proper induction of Shh expression during this differentiation programme. This element is capable of driving expression in the vertebrate brain. Our study illustrates how a chromatin-focused approach, coupled to in vivo testing, can be used to identify new cell-type specific cis -regulatory elements, and points to yet further complexity in the control of Shh expression during embryonic brain development.

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Chromatin profiling across the human tumour necrosis factor gene locus reveals a complex, cell type-specific landscape with novel regulatory elements

Nucleic Acids Research ◽

10.1093/nar/gkn444 ◽

2008 ◽

Vol 36 (15) ◽

pp. 4845-4862 ◽

Cited By ~ 18

Author(s):

Jennifer M. Taylor ◽

Kate Wicks ◽

Claire Vandiedonck ◽

Julian C. Knight

Keyword(s):

Tumour Necrosis ◽

Gene Locus ◽

Regulatory Elements ◽

Complex Cell ◽

Cell Type ◽

Human Tumour ◽

Human Tumour Necrosis Factor ◽

Cell Type Specific ◽

Chromatin Profiling ◽

Necrosis Factor

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Three classes of epigenomic regulators converge to hyperactivate the essential maternal gene deadhead within a heterochromatin mini-domain.

10.1101/2021.05.24.445410 ◽

2021 ◽

Author(s):

Daniela Torres-Campana ◽

Béatrice Horard ◽

Sandrine Denaud ◽

Gérard Benoit ◽

Benjamin Loppin ◽

...

Keyword(s):

Regulatory Element ◽

Critical Role ◽

Extreme Case ◽

Regulatory Elements ◽

Chromatin Regulators ◽

Data Analysis Procedure ◽

Chromatin Profiling ◽

Dna Regulatory Elements ◽

Female Germline ◽

Maternal Gene

The formation of a diploid zygote is a highly complex cellular process that is entirely controlled by maternal gene products stored in the egg cytoplasm. This highly specialized transcriptional program is tightly controlled at the chromatin level in the female germline. As an extreme case in point, the massive and specific ovarian expression of the essential thioredoxin Deadhead (DHD) is critically regulated in Drosophila by the histone demethylase Lid and its partner, the histone deacetylase complex scaffold Sin3A, via yet unknown mechanisms. Here, we identified the Brahma chromatin remodeler sub-unit Snr1 and the insulator component Mod(mdg4) as essential for dhd expression and investigated how these epigenomic effectors act with Lid and Sin3A to hyperactivate dhd . Using Cut&Run chromatin profiling with a dedicated data analysis procedure, we found that dhd is intriguingly embedded in an H3K27me3/H3K9me3-enriched mini-domain flanked by DNA regulatory elements, including a dhd promoter-proximal element essential for its expression. Surprisingly, Lid, Sin3A, Snr1 and Mod(mdg4) impact H3K27me3 and this regulatory element in distinct manners. However, we show that these effectors activate dhd independently of H3K27me3/H3K9me3, and that these marks are not required to repress dhd . Together, our study demonstrates an atypical and critical role for chromatin regulators Lid, Sin3A, Snr1 and Mod(mdg4) to trigger tissue-specific hyperactivation within a unique heterochromatin mini-domain.

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Repression of divergent noncoding transcription by a sequence-specific transcription factor

10.1101/314310 ◽

2018 ◽

Author(s):

Andrew CK Wu ◽

Harshil Patel ◽

Minghao Chia ◽

Fabien Moretto ◽

David Frith ◽

...

Keyword(s):

Transcription Factor ◽

Transcription Initiation ◽

Large Fraction ◽

Regulatory Elements ◽

Gene Promoters ◽

Specific Transcription Factor ◽

Highly Expressed Genes ◽

Divergent Transcription ◽

Noncoding Transcription ◽

Cryptic Promoters

SummaryMany active eukaryotic gene promoters exhibit divergent noncoding transcription, but the mechanisms restricting expression of these transcripts are not well understood. Here we demonstrate how a sequence-specific transcription factor represses divergent noncoding transcription at highly expressed genes in yeast. We find that depletion of the transcription factor Rap1 induces noncoding transcription in a large fraction of Rap1 regulated gene promoters. Specifically, Rap1 prevents transcription initiation at cryptic promoters near its binding sites, which is uncoupled from transcription regulation in the protein-coding direction. We further provide evidence that Rap1 acts independently of chromatin-based mechanisms to repress cryptic or divergent transcription. Finally, we show that divergent transcription in the absence of Rap1 is elicited by the RSC chromatin remodeller. We propose that a sequence-specific transcription factor limits access of basal transcription machinery to regulatory elements and adjacent sequences that act as divergent cryptic promoters, thereby providing directionality towards productive transcription.

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Mobilization and Mechanism of Transcription of Integrated Self-Inactivating Lentiviral Vectors

Journal of Virology ◽

10.1128/jvi.79.13.8410-8421.2005 ◽

2005 ◽

Vol 79 (13) ◽

pp. 8410-8421 ◽

Cited By ~ 36

Author(s):

Hideki Hanawa ◽

Derek A. Persons ◽

Arthur W. Nienhuis

Keyword(s):

Lentiviral Vectors ◽

Regulatory Elements ◽

Trap Design ◽

Promoter Sequences ◽

Vector Integration ◽

Vector Genome ◽

Promoter Trap ◽

A Cell ◽

Or Gene ◽

Cryptic Promoters

ABSTRACT Permanent genetic modification of replicating primitive hematopoietic cells by an integrated vector has many potential therapeutic applications. Both oncoretroviral and lentiviral vectors have a predilection for integration into transcriptionally active genes, creating the potential for promoter activation or gene disruption. The use of self-inactivating (SIN) vectors in which a deletion of the enhancer and promoter sequences from the 3′ long terminal repeat (LTR) is copied over into the 5′ LTR during vector integration is designed to improve safety by reducing the risk of mobilization of the vector genome and the influence of the LTR on nearby cellular promoters. Our results indicate that SIN vectors are mobilized in cells expressing lentiviral proteins, with the frequency of mobilization influenced by features of the vector design. The mechanism of transcription of integrated vector genomes was evaluated using a promoter trap design with a vector encoding tat but lacking an upstream promoter in a cell line in which drug resistance depended on tat expression. In six clones studied, all transcripts originated from cryptic promoters either upstream or within the vector genome. We estimate that approximately 1 in 3,000 integrated vector genomes is transcribed, leading to the inference that activation of cryptic promoters must depend on local features of chromatin structure and the constellation of nearby regulatory elements as well as the nature of the regulatory elements within the vector.

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Three classes of epigenomic regulators converge to hyperactivate the essential maternal gene deadhead within a heterochromatin mini-domain

PLoS Genetics ◽

10.1371/journal.pgen.1009615 ◽

2022 ◽

Vol 18 (1) ◽

pp. e1009615

Author(s):

Daniela Torres-Campana ◽

Béatrice Horard ◽

Sandrine Denaud ◽

Gérard Benoit ◽

Benjamin Loppin ◽

...

Keyword(s):

Regulatory Element ◽

Critical Role ◽

Extreme Case ◽

Regulatory Elements ◽

Chromatin Regulators ◽

Data Analysis Procedure ◽

Chromatin Profiling ◽

Dna Regulatory Elements ◽

Female Germline ◽

Maternal Gene

The formation of a diploid zygote is a highly complex cellular process that is entirely controlled by maternal gene products stored in the egg cytoplasm. This highly specialized transcriptional program is tightly controlled at the chromatin level in the female germline. As an extreme case in point, the massive and specific ovarian expression of the essential thioredoxin Deadhead (DHD) is critically regulated in Drosophila by the histone demethylase Lid and its partner, the histone deacetylase complex Sin3A/Rpd3, via yet unknown mechanisms. Here, we identified Snr1 and Mod(mdg4) as essential for dhd expression and investigated how these epigenomic effectors act with Lid and Sin3A to hyperactivate dhd. Using Cut&Run chromatin profiling with a dedicated data analysis procedure, we found that dhd is intriguingly embedded in an H3K27me3/H3K9me3-enriched mini-domain flanked by DNA regulatory elements, including a dhd promoter-proximal element essential for its expression. Surprisingly, Lid, Sin3a, Snr1 and Mod(mdg4) impact H3K27me3 and this regulatory element in distinct manners. However, we show that these effectors activate dhd independently of H3K27me3/H3K9me3, and that dhd remains silent in the absence of these marks. Together, our study demonstrates an atypical and critical role for chromatin regulators Lid, Sin3A, Snr1 and Mod(mdg4) to trigger tissue-specific hyperactivation within a unique heterochromatin mini-domain.

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