Protect-seq: genome-wide profiling of nuclease inaccessible domains reveals physical properties of chromatin

Spracklin, George; Pradhan, Sriharsa

doi:10.1093/nar/gkz1150

Cited by 11 publications

(20 citation statements)

References 46 publications

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“…For instance, HCT116 colon cancer cells appear to harbor compartmentalization signatures in Hi-C that are distinct from other cell lines (Nichols & Corces, 2021; Xiong & Ma, 2019). Furthermore, using Protect-seq, which measures peripheral heterochromatin, we have previously shown that in HCT116, H3K27me3 and H3K9me3 distinguish between two structurally distinct types of heterochromatin (Spracklin & Pradhan, 2020). Therefore, we sought to identify groups of loci through similarities in long-range interaction profiles (i.e.…”

Section: Resultsmentioning

confidence: 99%

“…Prompted by these observations, we sought out perturbation systems that disrupt H3K9me3-HP1α/β chromatin. First, the DNMT1 hypomorph and DNMT3b knockout cell line (termed DKO) (Rhee et al, 2002), is a DNA methylation-deficient line derived from HCT116 that has been reported to have defects in H3K9me3 (Lay et al 2015) and HP1α/β deposition (Spracklin & Pradhan, 2020). To investigate acute effects of DNA methylation dysfunction on compartmentalization and chromatin state, we also examined HCT116 cells treated with 5-Azacytidine for 48h (hereafter referred to as 5Aza) ( Figure 5A ).…”

Section: Resultsmentioning

confidence: 99%

“…To address this in more detail, we examined the chromatin state at disrupted domains in DKO cells. We used available data to examine histone modifications H3K27me3, H3K9me2, H3K9me3, as well as H2A.Z in DKO cells (Lay et al, 2015; Spracklin & Pradhan, 2020). Indeed, persistent domains maintain a H3K9me3-HP1α/β chromatin state as expected.…”

Section: Resultsmentioning

confidence: 99%

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Heterochromatin diversity modulates genome compartmentalization and loop extrusion barriers

Spracklin

Abdennur

Imakaev

et al. 2021

Preprint

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Two dominant processes organizing chromosomes are loop extrusion and the compartmental segregation of active and inactive chromatin. The molecular players involved in loop extrusion during interphase, cohesin and CTCF, have been extensively studied and experimentally validated. However, neither the molecular determinants nor the functional roles of compartmentalization are well understood. Here, we distinguish three inactive chromatin states using contact frequency profiling, comprising two types of heterochromatin and a previously uncharacterized inactive state exhibiting a neutral interaction preference. We find that heterochromatin marked by long continuous stretches of H3K9me3, HP1α and HP1β correlates with a conserved signature of strong compartmentalization and is abundant in HCT116 colon cancer cells. We demonstrate that disruption of DNA methyltransferase activity dramatically remodels genome compartmentalization as a consequence of the loss of H3K9me3 and HP1 binding. Interestingly, H3K9me3-HP1α/β is replaced by the neutral inactive state and retains late replication timing. Furthermore, we show that H3K9me3-HP1α/β heterochromatin is permissive to loop extrusion by cohesin but refractory to CTCF, explaining a paucity of visible loop extrusion-associated patterns in Hi-C. Accordingly, CTCF loop extrusion barriers are reactivated upon loss of H3K9me3-HP1α/β, not as a result of canonical demethylation of the CTCF binding motif but due to an intrinsic resistance of H3K9me3-HP1α/β heterochromatin to CTCF binding. Together, our work reveals a dynamic structural and organizational diversity of the inactive portion of the genome and establishes new connections between the regulation of chromatin state and chromosome organization, including an interplay between DNA methylation, compartmentalization and loop extrusion.HighlightsThree inactive chromatin states are distinguishable by long-range contact frequencies in HCT116, respectively associated with H3K9me3, H3K27me3 and a H3K9me2 state with neutral contact preferences.H3K9me3-HP1α/β heterochromatin has a high degree of homotypic affinity and is permissive to loop extrusion but depleted in extrusion barriers.Disrupting DNA methylation causes widespread loss of H3K9me3-HP1α/β and dramatic remodeling of genome compartmentalization.H3K9me3-HP1α/β is replaced by the neutral inactive state, which gains CTCF loop extrusion barriers and associated contact frequency patterns.DNA methylation suppresses CTCF binding via two distinct mechanisms.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Heterochromatin diversity modulates genome compartmentalization and loop extrusion barriers

Spracklin

Abdennur

Imakaev

et al. 2021

Preprint

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“…Instead, ChIP-seq relies on crosslinking and antibodies, which are sources of technical biases and potential issues of sensitivity or cross-reactivity, respectively. More recently proposed high-throughput sequencing methods for genome-wide mapping of LADs and heterochromatin, such as gradient-seq 40,41 and protect-seq 42 , also rely on crosslinking.…”

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confidence: 99%

SAMMY-seq reveals early alteration of heterochromatin and deregulation of bivalent genes in Hutchinson-Gilford Progeria Syndrome

et al. 2020

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Hutchinson-Gilford progeria syndrome is a genetic disease caused by an aberrant form of Lamin A resulting in chromatin structure disruption, in particular by interfering with lamina associated domains. Early molecular alterations involved in chromatin remodeling have not been identified thus far. Here, we present SAMMY-seq, a high-throughput sequencing-based method for genome-wide characterization of heterochromatin dynamics. Using SAMMY-seq, we detect early stage alterations of heterochromatin structure in progeria primary fibroblasts. These structural changes do not disrupt the distribution of H3K9me3 in early passage cells, thus suggesting that chromatin rearrangements precede H3K9me3 alterations described at later passages. On the other hand, we observe an interplay between changes in chromatin accessibility and Polycomb regulation, with site-specific H3K27me3 variations and transcriptional dysregulation of bivalent genes. We conclude that the correct assembly of lamina associated domains is functionally connected to the Polycomb repression and rapidly lost in early molecular events of progeria pathogenesis.

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“…Other chromatin features are correlated with inaccessible chromatin. Constitutive heterochromatin domains marked by H3K9me3 and H3K27me3 are reported to be largely inaccessible in higher eukaryotes [38], but chromatin inaccessibility within these regions has not yet been directly measured in filamentous fungi. In Saccharomyces cerevisiae, methylation of H3K36 is mediated by a single histone methyltransferase, SET2, which associates with elongating RNA polymerase to methylate H3K36 in coding sequences [39][40][41][42].…”

Section: Introductionmentioning

confidence: 99%

Chromatin accessibility profiling in Neurospora crassa reveals molecular features associated with accessible and inaccessible chromatin

Ferraro

Ameri

et al. 2021

BMC Genomics

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Background Regulation of chromatin accessibility and transcription are tightly coordinated processes. Studies in yeast and higher eukaryotes have described accessible chromatin regions, but little work has been done in filamentous fungi. Results Here we present a genome-scale characterization of accessible chromatin regions in Neurospora crassa, which revealed characteristic molecular features of accessible and inaccessible chromatin. We present experimental evidence of inaccessibility within heterochromatin regions in Neurospora, and we examine features of both accessible and inaccessible chromatin, including the presence of histone modifications, types of transcription, transcription factor binding, and relative nucleosome turnover rates. Chromatin accessibility is not strictly correlated with expression level. Accessible chromatin regions in the model filamentous fungus Neurospora are characterized the presence of H3K27 acetylation and commonly associated with pervasive non-coding transcription. Conversely, methylation of H3 lysine-36 catalyzed by ASH1 is correlated with inaccessible chromatin within promoter regions. Conclusions: In N. crassa, H3K27 acetylation is the most predictive histone modification for open chromatin. Conversely, our data show that H3K36 methylation is a key marker of inaccessible chromatin in gene-rich regions of the genome. Our data are consistent with an expanded role for H3K36 methylation in intergenic regions of filamentous fungi compared to the model yeasts, S. cerevisiae and S. pombe, which lack homologs of the ASH1 methyltransferase.

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Protect-seq: genome-wide profiling of nuclease inaccessible domains reveals physical properties of chromatin

Cited by 11 publications

References 46 publications

Heterochromatin diversity modulates genome compartmentalization and loop extrusion barriers

Heterochromatin diversity modulates genome compartmentalization and loop extrusion barriers

SAMMY-seq reveals early alteration of heterochromatin and deregulation of bivalent genes in Hutchinson-Gilford Progeria Syndrome

Chromatin accessibility profiling in Neurospora crassa reveals molecular features associated with accessible and inaccessible chromatin

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