bims-crepig 2022-06-19 papers

bims-crepig

Biomed News

on Chromatin regulation and epigenetics in cell fate and cancer

Issue of 2022‒06‒19
twenty-six papers selected by
Connor Rogerson
University of Cambridge

The chromatin remodeller ATRX facilitates diverse nuclear processes, in a stochastic manner, in both heterochromatin and euchromatin.
Context-dependent enhancer function revealed by targeted inter-TAD relocation.
Ep300 sequestration to functionally distinct glucocorticoid receptor binding loci underlie rapid gene activation and repression.
EnhFFL: A database of enhancer mediated feed-forward loops for human and mouse.
Transcriptional regulation and chromatin architecture maintenance are decoupled functions at the Sox2 locus.
Building regulatory landscapes reveals that an enhancer can recruit cohesin to create contact domains, engage CTCF sites and activate distant genes.
DLX1 and the NuRD complex cooperate in enhancer decommissioning and transcriptional repression.
EagleC: A deep-learning framework for detecting a full range of structural variations from bulk and single-cell contact maps.
The cohesin acetylation cycle controls chromatin loop length through a PDS5A brake mechanism.
Ranking reprogramming factors for cell differentiation.
YAP1 maintains active chromatin state in head and neck squamous cell carcinomas that promotes tumorigenesis through cooperation with BRD4.
SCADIE: simultaneous estimation of cell type proportions and cell type-specific gene expressions using SCAD-based iterative estimating procedure.
Genomic features underlie the co-option of SVA transposons as cis-regulatory elements in human pluripotent stem cells.
Oncofusion-driven de novo enhancer assembly promotes malignancy in Ewing sarcoma via aberrant expression of the stereociliary protein LOXHD1.
Chromatin interaction maps identify Wnt responsive cis-regulatory elements coordinating Paupar-Pax6 expression in neuronal cells.
Allelic imbalance of chromatin accessibility in cancer identifies candidate causal risk variants and their mechanisms.
Integrated multi-omics reveal polycomb repressive complex 2 restricts human trophoblast induction.
ARID1A loss derepresses a group of human endogenous retrovirus-H loci to modulate BRD4-dependent transcription.
Structural basis of nucleosome retention during transcription elongation.
SpyChIP identifies cell type-specific transcription factor occupancy from complex tissues.
Nucleome programming is required for the foundation of totipotency in mammalian germline development.
A PDX1 cistrome and single-cell transcriptome resource of the developing pancreas.
Analysis of estrogen-regulated enhancer RNAs identifies a functional motif required for enhancer assembly and gene expression.
Remodeling of gene regulatory networks underlying thermogenic stimuli-induced adipose beiging.
RIViT-seq enables systematic identification of regulons of transcriptional machineries.
Transcriptional and functional motifs defining renal function revealed by single-nucleus RNA sequencing.

Nat Commun. 2022 Jun 17. 13(1): 3485

The chromatin remodeller ATRX facilitates diverse nuclear processes, in a stochastic manner, in both heterochromatin and euchromatin.

Julia Truch, Damien J Downes, Caroline Scott, E Ravza Gür, Jelena M Telenius, Emmanouela Repapi, Ron Schwessinger, Matthew Gosden, Jill M Brown, Stephen Taylor, Pak Leng Cheong, Jim R Hughes, Douglas R Higgs, Richard J Gibbons.

The chromatin remodeller ATRX interacts with the histone chaperone DAXX to deposit the histone variant H3.3 at sites of nucleosome turnover. ATRX is known to bind repetitive, heterochromatic regions of the genome including telomeres, ribosomal DNA and pericentric repeats, many of which are putative G-quadruplex forming sequences (PQS). At these sites ATRX plays an ancillary role in a wide range of nuclear processes facilitating replication, chromatin modification and transcription. Here, using an improved protocol for chromatin immunoprecipitation, we show that ATRX also binds active regulatory elements in euchromatin. Mutations in ATRX lead to perturbation of gene expression associated with a reduction in chromatin accessibility, histone modification, transcription factor binding and deposition of H3.3 at the sequences to which it normally binds. In erythroid cells where downregulation of α-globin expression is a hallmark of ATR-X syndrome, perturbation of chromatin accessibility and gene expression occurs in only a subset of cells. The stochastic nature of this process suggests that ATRX acts as a general facilitator of cell specific transcriptional and epigenetic programmes, both in heterochromatin and euchromatin.

DOI: https://doi.org/10.1038/s41467-022-31194-7
Nat Commun. 2022 Jun 17. 13(1): 3488

Context-dependent enhancer function revealed by targeted inter-TAD relocation.

Christopher Chase Bolt, Lucille Lopez-Delisle, Aurélie Hintermann, Bénédicte Mascrez, Antonella Rauseo, Guillaume Andrey, Denis Duboule.

The expression of some genes depends on large, adjacent regions of the genome that contain multiple enhancers. These regulatory landscapes frequently align with Topologically Associating Domains (TADs), where they integrate the function of multiple similar enhancers to produce a global, TAD-specific regulation. We asked if an individual enhancer could overcome the influence of one of these landscapes, to drive gene transcription. To test this, we transferred an enhancer from its native location, into a nearby TAD with a related yet different functional specificity. We used the biphasic regulation of Hoxd genes during limb development as a paradigm. These genes are first activated in proximal limb cells by enhancers located in one TAD, which is then silenced when the neighboring TAD activates its enhancers in distal limb cells. We transferred a distal limb enhancer into the proximal limb TAD and found that its new context suppresses its normal distal specificity, even though it is bound by HOX13 transcription factors, which are responsible for the distal activity. This activity can be rescued only when a large portion of the surrounding environment is removed. These results indicate that, at least in some cases, the functioning of enhancer elements is subordinated to the host chromatin context, which can exert a dominant control over its activity.

DOI: https://doi.org/10.1038/s41467-022-31241-3
Nucleic Acids Res. 2022 Jun 17. pii: gkac488. [Epub ahead of print]

Ep300 sequestration to functionally distinct glucocorticoid receptor binding loci underlie rapid gene activation and repression.

Avital Sarusi Portuguez, Ivana Grbesa, Moran Tal, Rachel Deitch, Dana Raz, Limor Kliker, Ran Weismann, Michal Schwartz, Olga Loza, Leslie Cohen, Libi Marchenkov-Flam, Myong-Hee Sung, Tommy Kaplan, Ofir Hakim.

The rapid transcriptional response to the transcription factor, glucocorticoid receptor (GR), including gene activation or repression, is mediated by the spatial association of genes with multiple GR binding sites (GBSs) over large genomic distances. However, only a minority of the GBSs have independent GR-mediated activating capacity, and GBSs with independent repressive activity were rarely reported. To understand the positive and negative effects of GR we mapped the regulatory environment of its gene targets. We show that the chromatin interaction networks of GR-activated and repressed genes are spatially separated and vary in the features and configuration of their GBS and other non-GBS regulatory elements. The convergence of the KLF4 pathway in GR-activated domains and the STAT6 pathway in GR-repressed domains, impose opposite transcriptional effects to GR, independent of hormone application. Moreover, the ROR and Rev-erb transcription factors serve as positive and negative regulators, respectively, of GR-mediated gene activation. We found that the spatial crosstalk between GBSs and non-GBSs provides a physical platform for sequestering the Ep300 co-activator from non-GR regulatory loci in both GR-activated and -repressed gene compartments. While this allows rapid gene repression, Ep300 recruitment to GBSs is productive specifically in the activated compartments, thus providing the basis for gene induction.

DOI: https://doi.org/10.1093/nar/gkac488
Precis Clin Med. 2021 Jun;4(2): 129-135

EnhFFL: A database of enhancer mediated feed-forward loops for human and mouse.

Ran Kang, Zhengtang Tan, Mei Lang, Linqi Jin, Yin Zhang, Yiming Zhang, Tailin Guo, Zhiyun Guo.

  Feed-forward loops (FFLs) are thought to be one of the most common and important classes of transcriptional network motifs involved in various diseases. Enhancers are cis-regulatory elements that positively regulate protein-coding genes or microRNAs (miRNAs) by recruiting DNA-binding transcription factors (TFs). However, a comprehensive resource to identify, store, and analyze the FFLs of typical enhancer and super-enhancer FFLs is not currently available. Here, we present EnhFFL, an online database to provide a data resource for users to browse and search typical enhancer and super-enhancer FFLs. The current database covers 46 280/7000 TF-enhancer-miRNA FFLs, 9997/236 enhancer-miRNA-gene FFLs, 3 561 164/3 193 182 TF-enhancer-gene FFLs, and 1259/235 TF-enhancer feed-back loops (FBLs) across 91 tissues/cell lines of human and mouse, respectively. Users can browse loops by selecting species, types of tissue/cell line, and types of FFLs. EnhFFL supports searching elements including name/ID, genomic location, and the conservation of miRNA target genes. We also developed tools for users to screen customized FFLs using the threshold of q value as well as the confidence score of miRNA target genes. Disease and functional enrichment analysis showed that master miRNAs that are widely engaged in FFLs including TF-enhancer-miRNAs and enhancer-miRNA-genes are significantly involved in tumorigenesis. Database URL:http://lcbb.swjtu.edu.cn/EnhFFL/.

Keywords:  database; enhancer; feed-forward loop; miRNA; transcription factor

DOI:  https://doi.org/10.1093/pcmedi/pbab006
Genes Dev. 2022 Jun 16.

Transcriptional regulation and chromatin architecture maintenance are decoupled functions at the Sox2 locus.

Tiegh Taylor, Natalia Sikorska, Virlana M Shchuka, Sanjay Chahar, Chenfan Ji, Neil N Macpherson, Sakthi D Moorthy, Marit A C de Kort, Shanelle Mullany, Nawrah Khader, Zoe E Gillespie, Lida Langroudi, Ian C Tobias, Tineke L Lenstra, Jennifer A Mitchell, Tom Sexton.

  How distal regulatory elements control gene transcription and chromatin topology is not clearly defined, yet these processes are closely linked in lineage specification during development. Through allele-specific genome editing and chromatin interaction analyses of the Sox2 locus in mouse embryonic stem cells, we found a striking disconnection between transcriptional control and chromatin architecture. We traced nearly all Sox2 transcriptional activation to a small number of key transcription factor binding sites, whose deletions have no effect on promoter-enhancer interaction frequencies or topological domain organization. Local chromatin architecture maintenance, including at the topologically associating domain (TAD) boundary downstream from the Sox2 enhancer, is widely distributed over multiple transcription factor-bound regions and maintained in a CTCF-independent manner. Furthermore, partial disruption of promoter-enhancer interactions by ectopic chromatin loop formation has no effect on Sox2 transcription. These findings indicate that many transcription factors are involved in modulating chromatin architecture independently of CTCF.

Keywords:  CTCF; TAD; allele-specific; chromatin loop; enhancer; genome engineering; transcription

DOI:  https://doi.org/10.1101/gad.349489.122
Nat Struct Mol Biol. 2022 Jun;29(6): 563-574

Building regulatory landscapes reveals that an enhancer can recruit cohesin to create contact domains, engage CTCF sites and activate distant genes.

Niels J Rinzema, Konstantinos Sofiadis, Sjoerd J D Tjalsma, Marjon J A M Verstegen, Yuva Oz, Christian Valdes-Quezada, Anna-Karina Felder, Teodora Filipovska, Stefan van der Elst, Zaria de Andrade Dos Ramos, Ruiqi Han, Peter H L Krijger, Wouter de Laat.

Developmental gene expression is often controlled by distal regulatory DNA elements called enhancers. Distant enhancer action is restricted to structural chromosomal domains that are flanked by CTCF-associated boundaries and formed through cohesin chromatin loop extrusion. To better understand how enhancers, genes and CTCF boundaries together form structural domains and control expression, we used a bottom-up approach, building series of active regulatory landscapes in inactive chromatin. We demonstrate here that gene transcription levels and activity over time reduce with increased enhancer distance. The enhancer recruits cohesin to stimulate domain formation and engage flanking CTCF sites in loop formation. It requires cohesin exclusively for the activation of distant genes, not of proximal genes, with nearby CTCF boundaries supporting efficient long-range enhancer action. Our work supports a dual activity model for enhancers: its classic role of stimulating transcription initiation and elongation from target gene promoters and a role of recruiting cohesin for the creation of chromosomal domains, the engagement of CTCF sites in chromatin looping and the activation of distal target genes.

DOI: https://doi.org/10.1038/s41594-022-00787-7
Development. 2022 Jun 01. pii: dev199508. [Epub ahead of print]149(11):

DLX1 and the NuRD complex cooperate in enhancer decommissioning and transcriptional repression.

James D Price, Susan Lindtner, Athena Ypsilanti, Fadya Binyameen, Jeffrey R Johnson, Billy W Newton, Nevan J Krogan, John L R Rubenstein.

  In the developing subpallium, the fate decision between neurons and glia is driven by expression of Dlx1/2 or Olig1/2, respectively, two sets of transcription factors with a mutually repressive relationship. The mechanism by which Dlx1/2 repress progenitor and oligodendrocyte fate, while promoting transcription of genes needed for differentiation, is not fully understood. We identified a motif within DLX1 that binds RBBP4, a NuRD complex subunit. ChIP-seq studies of genomic occupancy of DLX1 and six different members of the NuRD complex show that DLX1 and NuRD colocalize to putative regulatory elements enriched near other transcription factor genes. Loss of Dlx1/2 leads to dysregulation of genome accessibility at putative regulatory elements near genes repressed by Dlx1/2, including Olig2. Consequently, heterozygosity of Dlx1/2 and Rbbp4 leads to an increase in the production of OLIG2+ cells. These findings highlight the importance of the interplay between transcription factors and chromatin remodelers in regulating cell-fate decisions.

Keywords:  Chromatin; DLX; Enhancer; Mouse; NuRD complex; Subpallium; Transcription factor

DOI:  https://doi.org/10.1242/dev.199508
Sci Adv. 2022 Jun 17. 8(24): eabn9215

EagleC: A deep-learning framework for detecting a full range of structural variations from bulk and single-cell contact maps.

Xiaotao Wang, Yu Luan, Feng Yue.

The Hi-C technique has been shown to be a promising method to detect structural variations (SVs) in human genomes. However, algorithms that can use Hi-C data for a full-range SV detection have been severely lacking. Current methods can only identify interchromosomal translocations and long-range intrachromosomal SVs (>1 Mb) at less-than-optimal resolution. Therefore, we develop EagleC, a framework that combines deep-learning and ensemble-learning strategies to predict a full range of SVs at high resolution. We show that EagleC can uniquely capture a set of fusion genes that are missed by whole-genome sequencing or nanopore. Furthermore, EagleC also effectively captures SVs in other chromatin interaction platforms, such as HiChIP, Chromatin interaction analysis with paired-end tag sequencing (ChIA-PET), and capture Hi-C. We apply EagleC in more than 100 cancer cell lines and primary tumors and identify a valuable set of high-quality SVs. Last, we demonstrate that EagleC can be applied to single-cell Hi-C and used to study the SV heterogeneity in primary tumors.

DOI: https://doi.org/10.1126/sciadv.abn9215
Nat Struct Mol Biol. 2022 Jun;29(6): 586-591

The cohesin acetylation cycle controls chromatin loop length through a PDS5A brake mechanism.

Marjon S van Ruiten, Démi van Gent, Ángela Sedeño Cacciatore, Astrid Fauster, Laureen Willems, Maarten L Hekkelman, Liesbeth Hoekman, Maarten Altelaar, Judith H I Haarhuis, Thijn R Brummelkamp, Elzo de Wit, Benjamin D Rowland.

Cohesin structures the genome through the formation of chromatin loops and by holding together the sister chromatids. The acetylation of cohesin's SMC3 subunit is a dynamic process that involves the acetyltransferase ESCO1 and deacetylase HDAC8. Here we show that this cohesin acetylation cycle controls the three-dimensional genome in human cells. ESCO1 restricts the length of chromatin loops, and of architectural stripes emanating from CTCF sites. HDAC8 conversely promotes the extension of such loops and stripes. This role in controlling loop length turns out to be distinct from the canonical role of cohesin acetylation that protects against WAPL-mediated DNA release. We reveal that acetylation controls the interaction of cohesin with PDS5A to restrict chromatin loop length. Our data support a model in which this PDS5A-bound state acts as a brake that enables the pausing and restart of loop enlargement. The cohesin acetylation cycle hereby provides punctuation in the process of genome folding.

DOI: https://doi.org/10.1038/s41594-022-00773-z
Nat Methods. 2022 Jun 16.

Ranking reprogramming factors for cell differentiation.

Jennifer Hammelman, Tulsi Patel, Michael Closser, Hynek Wichterle, David Gifford.

Transcription factor over-expression is a proven method for reprogramming cells to a desired cell type for regenerative medicine and therapeutic discovery. However, a general method for the identification of reprogramming factors to create an arbitrary cell type is an open problem. Here we examine the success rate of methods and data for differentiation by testing the ability of nine computational methods (CellNet, GarNet, EBseq, AME, DREME, HOMER, KMAC, diffTF and DeepAccess) to discover and rank candidate factors for eight target cell types with known reprogramming solutions. We compare methods that use gene expression, biological networks and chromatin accessibility data, and comprehensively test parameter and preprocessing of input data to optimize performance. We find the best factor identification methods can identify an average of 50-60% of reprogramming factors within the top ten candidates, and methods that use chromatin accessibility perform the best. Among the chromatin accessibility methods, complex methods DeepAccess and diffTF have higher correlation with the ranked significance of transcription factor candidates within reprogramming protocols for differentiation. We provide evidence that AME and diffTF are optimal methods for transcription factor recovery that will allow for systematic prioritization of transcription factor candidates to aid in the design of new reprogramming protocols.

DOI: https://doi.org/10.1038/s41592-022-01522-2
Cell Rep. 2022 Jun 14. pii: S2211-1247(22)00756-2. [Epub ahead of print]39(11): 110970

YAP1 maintains active chromatin state in head and neck squamous cell carcinomas that promotes tumorigenesis through cooperation with BRD4.

Nana Chen, Gabriel Golczer, Subhoshree Ghose, Brian Lin, Adam Langenbucher, Jason Webb, Haymanti Bhanot, Nicholas B Abt, Derrick Lin, Mark Varvares, Martin Sattler, Ann Marie Egloff, Richard Joh, Ravindra Uppaluri, Kevin S Emerick, Michael S Lawrence, Srinivas Vinod Saladi.

  Analysis of The Cancer Genome Atlas and other published data of head and neck squamous cell carcinoma (HNSCC) reveals somatic alterations of the Hippo-YAP pathway in approximately 50% of HNSCC. Better strategies to target the YAP1 transcriptional complex are sought. Here, we show that FAT1, an upstream inhibitor of YAP1, is mutated either by missense or by truncating mutation in 29% of HNSCC. Comprehensive proteomic and drug-screening studies across pan-cancer models confirm that FAT1-mutant HNSCC exhibits selective and higher sensitivity to BRD4 inhibition by JQ1. Epigenomic analysis reveals an active chromatin state in FAT1-mutant HNSCC cells that is driven by the YAP/TAZ transcriptional complex through recruitment of BRD4 to deposit active histone marks, thereby maintaining an oncogenic transcriptional state. This study reveals a detailed cooperative mechanism between YAP1 and BRD4 in HNSCC and suggests a specific therapeutic opportunity for the treatment of this subset of head and neck cancer patients.

Keywords:  ATAD2; BRD4; CP: Cancer; CP: Molecular biology; FAT1 mutation; HNSCC; JQ1; NRG1; OTX-015; PD-L1; YAP1; hippo pathway

DOI:  https://doi.org/10.1016/j.celrep.2022.110970
Genome Biol. 2022 Jun 15. 23(1): 129

SCADIE: simultaneous estimation of cell type proportions and cell type-specific gene expressions using SCAD-based iterative estimating procedure.

Daiwei Tang, Seyoung Park, Hongyu Zhao.

  A challenge in bulk gene differential expression analysis is to differentiate changes due to cell type-specific gene expression and cell type proportions. SCADIE is an iterative algorithm that simultaneously estimates cell type-specific gene expression profiles and cell type proportions, and performs cell type-specific differential expression analysis at the group level. Through its unique penalty and objective function, SCADIE more accurately identifies cell type-specific differentially expressed genes than existing methods, including those that may be missed from single cell RNA-Seq data. SCADIE has robust performance with respect to the choice of deconvolution methods and the sources and quality of input data.

Keywords:  Cell type-specific differential expression; Deconvolution; RNA-Seq; SCAD; scRNA-seq

DOI:  https://doi.org/10.1186/s13059-022-02688-w
PLoS Genet. 2022 Jun 15. 18(6): e1010225

Genomic features underlie the co-option of SVA transposons as cis-regulatory elements in human pluripotent stem cells.

Samantha M Barnada, Andrew Isopi, Daniela Tejada-Martinez, Clément Goubert, Sruti Patoori, Luca Pagliaroli, Mason Tracewell, Marco Trizzino.

Domestication of transposable elements (TEs) into functional cis-regulatory elements is a widespread phenomenon. However, the mechanisms behind why some TEs are co-opted as functional enhancers while others are not are underappreciated. SINE-VNTR-Alus (SVAs) are the youngest group of transposons in the human genome, where ~3,700 copies are annotated, nearly half of which are human-specific. Many studies indicate that SVAs are among the most frequently co-opted TEs in human gene regulation, but the mechanisms underlying such processes have not yet been thoroughly investigated. Here, we leveraged CRISPR-interference (CRISPRi), computational and functional genomics to elucidate the genomic features that underlie SVA domestication into human stem-cell gene regulation. We found that ~750 SVAs are co-opted as functional cis-regulatory elements in human induced pluripotent stem cells. These SVAs are significantly closer to genes and harbor more transcription factor binding sites than non-co-opted SVAs. We show that a long DNA motif composed of flanking YY1/2 and OCT4 binding sites is enriched in the co-opted SVAs and that these two transcription factors bind consecutively on the TE sequence. We used CRISPRi to epigenetically repress active SVAs in stem cell-like NCCIT cells. Epigenetic perturbation of active SVAs strongly attenuated YY1/OCT4 binding and influenced neighboring gene expression. Ultimately, SVA repression resulted in ~3,000 differentially expressed genes, 131 of which were the nearest gene to an annotated SVA. In summary, we demonstrated that SVAs modulate human gene expression, and uncovered that location and sequence composition contribute to SVA domestication into gene regulatory networks.

DOI: https://doi.org/10.1371/journal.pgen.1010225
Cell Rep. 2022 Jun 14. pii: S2211-1247(22)00757-4. [Epub ahead of print]39(11): 110971

Oncofusion-driven de novo enhancer assembly promotes malignancy in Ewing sarcoma via aberrant expression of the stereociliary protein LOXHD1.

Qu Deng, Ramakrishnan Natesan, Florencia Cidre-Aranaz, Shehbeel Arif, Ying Liu, Reyaz Ur Rasool, Pei Wang, Erick Mitchell-Velasquez, Chandan Kanta Das, Endrit Vinca, Zvi Cramer, Patrick J Grohar, Margaret Chou, Chandan Kumar-Sinha, Kristy Weber, T S Karin Eisinger-Mathason, Nicolas Grillet, Thomas Grünewald, Irfan A Asangani.

  Ewing sarcoma (EwS) is a highly aggressive tumor of bone and soft tissues that mostly affects children and adolescents. The pathognomonic oncofusion EWSR1::FLI1 transcription factor drives EwS by orchestrating an oncogenic transcription program through de novo enhancers. By integrative analysis of thousands of transcriptomes representing pan-cancer cell lines, primary cancers, metastasis, and normal tissues, we identify a 32-gene signature (ESS32 [Ewing Sarcoma Specific 32]) that stratifies EwS from pan-cancer. Among the ESS32, LOXHD1, encoding a stereociliary protein, is the most highly expressed gene through an alternative transcription start site. Deletion or silencing of EWSR1::FLI1 bound upstream de novo enhancer results in loss of the LOXHD1 short isoform, altering EWSR1::FLI1 and HIF1α pathway genes and resulting in decreased proliferation/invasion of EwS cells. These observations implicate LOXHD1 as a biomarker and a determinant of EwS metastasis and suggest new avenues for developing LOXHD1-targeted drugs or cellular therapies for this deadly disease.

Keywords:  CP: Cancer; CP: Molecular biology; EWSR1::FLI1; Ewing sarcoma; LOXHD1; biomarker; enhancer; hypoxia; integrative genomics; metastasis; transcription

DOI:  https://doi.org/10.1016/j.celrep.2022.110971
PLoS Genet. 2022 Jun;18(6): e1010230

Chromatin interaction maps identify Wnt responsive cis-regulatory elements coordinating Paupar-Pax6 expression in neuronal cells.

Ioanna Pavlaki, Michael Shapiro, Giuseppina Pisignano, Stephanie M E Jones, Jelena Telenius, Silvia Muñoz-Descalzo, Robert J Williams, Jim R Hughes, Keith W Vance.

Central nervous system-expressed long non-coding RNAs (lncRNAs) are often located in the genome close to protein coding genes involved in transcriptional control. Such lncRNA-protein coding gene pairs are frequently temporally and spatially co-expressed in the nervous system and are predicted to act together to regulate neuronal development and function. Although some of these lncRNAs also bind and modulate the activity of the encoded transcription factors, the regulatory mechanisms controlling co-expression of neighbouring lncRNA-protein coding genes remain unclear. Here, we used high resolution NG Capture-C to map the cis-regulatory interaction landscape of the key neuro-developmental Paupar-Pax6 lncRNA-mRNA locus. The results define chromatin architecture changes associated with high Paupar-Pax6 expression in neurons and identify both promoter selective as well as shared cis-regulatory-promoter interactions involved in regulating Paupar-Pax6 co-expression. We discovered that the TCF7L2 transcription factor, a regulator of chromatin architecture and major effector of the Wnt signalling pathway, binds to a subset of these candidate cis-regulatory elements to coordinate Paupar and Pax6 co-expression. We describe distinct roles for Paupar in Pax6 expression control and show that the Paupar DNA locus contains a TCF7L2 bound transcriptional silencer whilst the Paupar transcript can act as an activator of Pax6. Our work provides important insights into the chromatin interactions, signalling pathways and transcription factors controlling co-expression of adjacent lncRNAs and protein coding genes in the brain.

DOI: https://doi.org/10.1371/journal.pgen.1010230
Nat Genet. 2022 Jun;54(6): 837-849

Allelic imbalance of chromatin accessibility in cancer identifies candidate causal risk variants and their mechanisms.

Dennis Grishin, Alexander Gusev.

While many germline cancer risk variants have been identified through genome-wide association studies (GWAS), the mechanisms by which these variants operate remain largely unknown. Here we used 406 cancer ATAC-Seq samples across 23 cancer types to identify 7,262 germline allele-specific accessibility QTLs (as-aQTLs). Cancer as-aQTLs had stronger enrichment for cancer risk heritability (up to 145 fold) than any other functional annotation across seven cancer GWAS. Most cancer as-aQTLs directly altered transcription factor (TF) motifs and exhibited differential TF binding and gene expression in functional screens. To connect as-aQTLs to putative risk mechanisms, we introduced the regulome-wide associations study (RWAS). RWAS identified genetically associated accessible peaks at >70% of known breast and prostate loci and discovered new risk loci in all examined cancer types. Integrating as-aQTL discovery, motif analysis and RWAS identified candidate causal regulatory elements and their probable upstream regulators. Our work establishes cancer as-aQTLs and RWAS analysis as powerful tools to study the genetic architecture of cancer risk.

DOI: https://doi.org/10.1038/s41588-022-01075-2
Nat Cell Biol. 2022 Jun;24(6): 858-871

Integrated multi-omics reveal polycomb repressive complex 2 restricts human trophoblast induction.

Dick W Zijlmans, Irene Talon, Sigrid Verhelst, Adam Bendall, Karlien Van Nerum, Alok Javali, Andrew A Malcolm, Sam S F A van Knippenberg, Laura Biggins, San Kit To, Adrian Janiszewski, Danielle Admiraal, Ruth Knops, Nikky Corthout, Bradley P Balaton, Grigorios Georgolopoulos, Amitesh Panda, Natarajan V Bhanu, Amanda J Collier, Charlene Fabian, Ryan N Allsop, Joel Chappell, Thi Xuan Ai Pham, Michael Oberhuemer, Cankat Ertekin, Lotte Vanheer, Paraskevi Athanasouli, Frederic Lluis, Dieter Deforce, Joop H Jansen, Benjamin A Garcia, Michiel Vermeulen, Nicolas Rivron, Maarten Dhaenens, Hendrik Marks, Peter J Rugg-Gunn, Vincent Pasque.

Human naive pluripotent stem cells have unrestricted lineage potential. Underpinning this property, naive cells are thought to lack chromatin-based lineage barriers. However, this assumption has not been tested. Here we define the chromatin-associated proteome, histone post-translational modifications and transcriptome of human naive and primed pluripotent stem cells. Our integrated analysis reveals differences in the relative abundance and activities of distinct chromatin modules. We identify a strong enrichment of polycomb repressive complex 2 (PRC2)-associated H3K27me3 in the chromatin of naive pluripotent stem cells and H3K27me3 enrichment at promoters of lineage-determining genes, including trophoblast regulators. PRC2 activity acts as a chromatin barrier restricting the differentiation of naive cells towards the trophoblast lineage, whereas inhibition of PRC2 promotes trophoblast-fate induction and cavity formation in human blastoids. Together, our results establish that human naive pluripotent stem cells are not epigenetically unrestricted, but instead possess chromatin mechanisms that oppose the induction of alternative cell fates.

DOI: https://doi.org/10.1038/s41556-022-00932-w
Nat Commun. 2022 Jun 17. 13(1): 3501

ARID1A loss derepresses a group of human endogenous retrovirus-H loci to modulate BRD4-dependent transcription.

Chunhong Yu, Xiaoyun Lei, Fang Chen, Song Mao, Lu Lv, Honglu Liu, Xueying Hu, Runhan Wang, Licong Shen, Na Zhang, Yang Meng, Yunfan Shen, Jiale Chen, Pishun Li, Shi Huang, Changwei Lin, Zhuohua Zhang, Kai Yuan.

Transposable elements (TEs) through evolutionary exaptation have become an integral part of the human genome, offering ample regulatory sequences and shaping chromatin 3D architecture. While the functional impacts of TE-derived sequences on early embryogenesis have been recognized, their roles in malignancy are only starting to emerge. Here we show that many TEs, especially the pluripotency-related human endogenous retrovirus H (HERVH), are abnormally activated in colorectal cancer (CRC) samples. Transcriptional upregulation of HERVH is associated with mutations of several tumor suppressors, particularly ARID1A. Knockout of ARID1A in CRC cells leads to increased transcription at several HERVH loci, which involves compensatory contribution by ARID1B. Suppression of HERVH in CRC cells and patient-derived organoids impairs tumor growth. Mechanistically, HERVH transcripts colocalize with nuclear BRD4 foci, modulating their dynamics and co-regulating many target genes. Altogether, we uncover a critical role for ARID1A in restraining HERVH, whose abnormal activation can promote tumorigenesis by stimulating BRD4-dependent transcription.

DOI: https://doi.org/10.1038/s41467-022-31197-4
Science. 2022 Jun 17. 376(6599): 1313-1316

Structural basis of nucleosome retention during transcription elongation.

Martin Filipovski, Jelly H M Soffers, Seychelle M Vos, Lucas Farnung.

In eukaryotes, RNA polymerase (Pol) II transcribes chromatin and must move past nucleosomes, often resulting in nucleosome displacement. How Pol II unwraps the DNA from nucleosomes to allow transcription and how DNA rewraps to retain nucleosomes has been unclear. Here, we report the 3.0-angstrom cryo-electron microscopy structure of a mammalian Pol II-DSIF-SPT6-PAF1c-TFIIS-nucleosome complex stalled 54 base pairs within the nucleosome. The structure provides a mechanistic basis for nucleosome retention during transcription elongation where upstream DNA emerging from the Pol II cleft has rewrapped the proximal side of the nucleosome. The structure uncovers a direct role for Pol II and transcription elongation factors in nucleosome retention and explains how nucleosomes are retained to prevent the disruption of chromatin structure across actively transcribed genes.

DOI: https://doi.org/10.1126/science.abo3851
Proc Natl Acad Sci U S A. 2022 Jun 21. 119(25): e2122900119

SpyChIP identifies cell type-specific transcription factor occupancy from complex tissues.

Siqian Feng, Richard S Mann.

  Chromatin immunoprecipitation (ChIP) is an important technique for characterizing protein-DNA binding in vivo. One drawback of ChIP-based techniques is the lack of cell type-specificity when profiling complex tissues. To overcome this limitation, we developed SpyChIP to identify cell type-specific transcription factor (TF) binding sites in native physiological contexts without tissue dissociation or nuclei sorting. SpyChIP takes advantage of a specific covalent isopeptide bond that rapidly forms between the 15-amino acid SpyTag and the 17-kDa protein SpyCatcher. In SpyChIP, the target TF is fused with SpyTag by genome engineering, and an epitope tagged SpyCatcher is expressed in cell populations of interest, where it covalently binds to SpyTag-TF. Cell type-specific ChIP is obtained by immunoprecipitating chromatin prepared from whole tissues using antibodies directed against the epitope-tagged SpyCatcher. Using SpyChIP, we identified the genome-wide binding profiles of the Hox protein Ultrabithorax (Ubx) in two distinct cell types of the Drosophila haltere imaginal disc. Our results revealed extensive region-specific Ubx-DNA binding events, highlighting the significance of cell type-specific ChIP and the limitations of whole-tissue ChIP approaches. Analysis of Ubx::SpyChIP results provided insights into the relationship between chromatin accessibility and Ubx-DNA binding, as well as different mechanisms Ubx employs to regulate its downstream cis-regulatory modules. In addition to SpyChIP, we suggest that SpyTag-SpyCatcher technology, as well as other protein pairs that form covalent isopeptide bonds, will facilitate many additional in vivo applications that were previously impractical.

Keywords:  ChIP; Drosophila; SpyTag; Ubx; transcription factor

DOI:  https://doi.org/10.1073/pnas.2122900119
EMBO J. 2022 Jun 15. e110600

Nucleome programming is required for the foundation of totipotency in mammalian germline development.

Masahiro Nagano, Bo Hu, Shihori Yokobayashi, Akitoshi Yamamura, Fumiya Umemura, Mariel Coradin, Hiroshi Ohta, Yukihiro Yabuta, Yukiko Ishikura, Ikuhiro Okamoto, Hiroki Ikeda, Naofumi Kawahira, Yoshiaki Nosaka, Sakura Shimizu, Yoji Kojima, Ken Mizuta, Tomoko Kasahara, Yusuke Imoto, Killian Meehan, Roman Stocsits, Gordana Wutz, Yasuaki Hiraoka, Yasuhiro Murakawa, Takuya Yamamoto, Kikue Tachibana, Jan-Michel Peters, Leonid A Mirny, Benjamin A Garcia, Jacek Majewski, Mitinori Saitou.

  Germ cells are unique in engendering totipotency, yet the mechanisms underlying this capacity remain elusive. Here, we perform comprehensive and in-depth nucleome analysis of mouse germ-cell development in vitro, encompassing pluripotent precursors, primordial germ cells (PGCs) before and after epigenetic reprogramming, and spermatogonia/spermatogonial stem cells (SSCs). Although epigenetic reprogramming, including genome-wide DNA de-methylation, creates broadly open chromatin with abundant enhancer-like signatures, the augmented chromatin insulation safeguards transcriptional fidelity. These insulatory constraints are then erased en masse for spermatogonial development. Notably, despite distinguishing epigenetic programming, including global DNA re-methylation, the PGCs-to-spermatogonia/SSCs development entails further euchromatization. This accompanies substantial erasure of lamina-associated domains, generating spermatogonia/SSCs with a minimal peripheral attachment of chromatin except for pericentromeres-an architecture conserved in primates. Accordingly, faulty nucleome maturation, including persistent insulation and improper euchromatization, leads to impaired spermatogenic potential. Given that PGCs after epigenetic reprogramming serve as oogenic progenitors as well, our findings elucidate a principle for the nucleome programming that creates gametogenic progenitors in both sexes, defining a basis for nuclear totipotency.

Keywords:  3D genome organization; epigenetic reprogramming; germ cells; lamina-associated domains; nucleome

DOI:  https://doi.org/10.15252/embj.2022110600
Development. 2022 Jun 16. pii: dev.200432. [Epub ahead of print]

A PDX1 cistrome and single-cell transcriptome resource of the developing pancreas.

Xiaodun Yang, Jeffrey C Raum, Junil Kim, Reynold Yu, Juxiang Yang, Gabriella Rice, Changhong Li, Kyoung-Jae Won, Diana E Stanescu, Doris A Stoffers.

  Pancreatic and duodenal homeobox 1 (PDX1) is crucial for pancreas organogenesis, yet the dynamic changes in PDX1 binding in human or mouse developing pancreas have not been examined. To address this knowledge gap, we performed PDX1 ChIP-seq and single-cell RNA-seq using fetal human pancreata. We integrated our datasets with published datasets and revealed the dynamics of PDX1 binding and potential cell-lineage-specific PDX1 bound genes in the pancreas from fetal to adult stages. We identified a core set of developmentally conserved PDX1 bound genes that reveal the broad multifaceted role of PDX1 in pancreas development. Despite the well-known, dramatic changes in PDX1 function and expression, we found that PDX1 bound genes are largely conserved from embryonic to adult stages. This points towards a dual role of PDX1 in regulating the expression of its targets at different ages, dependent on other functionally-congruent or directly-interacting partners. We also showed that PDX1 binding is largely conserved in mouse pancreas. Together, our study reveals PDX1 targets in the developing pancreas in vivo and provides an essential resource for future studies on pancreas development.

Keywords:  ChIP-seq; Human; Mouse; PDX1 cistrome; Pancreas development; Single-cell RNA-seq

DOI:  https://doi.org/10.1242/dev.200432
Cell Rep. 2022 Jun 14. pii: S2211-1247(22)00726-4. [Epub ahead of print]39(11): 110944

Analysis of estrogen-regulated enhancer RNAs identifies a functional motif required for enhancer assembly and gene expression.

Tim Y Hou, W Lee Kraus.

  To better understand the functions of non-coding enhancer RNAs (eRNAs), we annotated the estrogen-regulated eRNA transcriptome in estrogen receptor α (ERα)-positive breast cancer cells using PRO-cap and RNA sequencing. We then cloned a subset of the eRNAs identified, fused them to single guide RNAs, and targeted them to their ERα enhancers of origin using CRISPR/dCas9. Some of the eRNAs tested modulated the expression of cognate, but not heterologous, target genes after estrogen treatment by increasing ERα recruitment and stimulating p300-catalyzed H3K27 acetylation at the enhancer. We identified a ∼40 nucleotide functional eRNA regulatory motif (FERM) present in many eRNAs that was necessary and sufficient to modulate gene expression, but not the specificity of activation, after estrogen treatment. The FERM interacted with BCAS2, an RNA-binding protein amplified in breast cancers. The ectopic expression of a targeted eRNA controlling the expression of an oncogene resulted in increased cell proliferation, demonstrating the regulatory potential of eRNAs in breast cancer.

Keywords:  Breast cancer; CP: Molecular biology; H3K27 acetylation; coregulator; enhancer; enhancer RNA (eRNA); estrogen receptor alpha (ERα); noncoding RNA; p300/CBP; pre-mRNA-splicing factor (SPF27/BCAS2); transcription

DOI:  https://doi.org/10.1016/j.celrep.2022.110944
Commun Biol. 2022 Jun 14. 5(1): 584

Remodeling of gene regulatory networks underlying thermogenic stimuli-induced adipose beiging.

Seoyeon Lee, Abigail M Benvie, Hui Gyu Park, Roman Spektor, Blaine Harlan, J Thomas Brenna, Daniel C Berry, Paul D Soloway.

Beige adipocytes are induced by cold temperatures or β3-adrenergic receptor (Adrb3) agonists. They create heat through glucose and fatty acid (FA) oxidation, conferring metabolic benefits. The distinct and shared mechanisms by which these treatments induce beiging are unknown. Here, we perform single-nucleus assay for transposase-accessible chromatin sequencing (snATAC-seq) on adipose tissue from mice exposed to cold or an Adrb3 agonist to identify cellular and chromatin accessibility dynamics during beiging. Both stimuli induce chromatin remodeling that influence vascularization and inflammation in adipose. Beige adipocytes from cold-exposed mice have increased accessibility at genes regulating glycolytic processes, whereas Adrb3 activation increases cAMP responses. While both thermogenic stimuli increase accessibility at genes regulating thermogenesis, lipogenesis, and beige adipocyte development, the kinetics and magnitudes of the changes are distinct for the stimuli. Accessibility changes at lipogenic genes are linked to functional changes in lipid composition of adipose. Both stimuli tend to decrease the proportion of palmitic acids, a saturated FA in adipose. However, Adrb3 activation increases the proportion of monounsaturated FAs, whereas cold increases the proportion of polyunsaturated FAs. These findings reveal common and distinct mechanisms of cold and Adrb3 induced beige adipocyte biogenesis, and identify unique functional consequences of manipulating these pathways in vivo.

DOI: https://doi.org/10.1038/s42003-022-03531-5
Nat Commun. 2022 Jun 17. 13(1): 3502

RIViT-seq enables systematic identification of regulons of transcriptional machineries.

Hiroshi Otani, Nigel J Mouncey.

Transcriptional regulation is a critical process to ensure expression of genes necessary for growth and survival in diverse environments. Transcription is mediated by multiple transcription factors including activators, repressors and sigma factors. Accurate computational prediction of the regulon of target genes for transcription factors is difficult and experimental identification is laborious and not scalable. Here, we demonstrate regulon identification by in vitro transcription-sequencing (RIViT-seq) that enables systematic identification of regulons of transcription factors by combining an in vitro transcription assay and RNA-sequencing. Using this technology, target genes of 11 sigma factors were identified in Streptomyces coelicolor A3(2). The RIViT-seq data expands the transcriptional regulatory network in this bacterium, discovering regulatory cascades and crosstalk between sigma factors. Implementation of RIViT-seq with other transcription factors and in other organisms will improve our understanding of transcriptional regulatory networks across biology.

DOI: https://doi.org/10.1038/s41467-022-31191-w
Proc Natl Acad Sci U S A. 2022 Jun 21. 119(25): e2203179119

Transcriptional and functional motifs defining renal function revealed by single-nucleus RNA sequencing.

Jun Xu, Yifang Liu, Hongjie Li, Alexander J Tarashansky, Colin H Kalicki, Ruei-Jiun Hung, Yanhui Hu, Aram Comjean, Sai Saroja Kolluru, Bo Wang, Stephen R Quake, Liqun Luo, Andrew P McMahon, Julian A T Dow, Norbert Perrimon.

  Recent advances in single-cell sequencing provide a unique opportunity to gain novel insights into the diversity, lineage, and functions of cell types constituting a tissue/organ. Here, we performed a single-nucleus study of the adult Drosophila renal system, consisting of Malpighian tubules and nephrocytes, which shares similarities with the mammalian kidney. We identified 11 distinct clusters representing renal stem cells, stellate cells, regionally specific principal cells, garland nephrocyte cells, and pericardial nephrocytes. Characterization of the transcription factors specific to each cluster identified fruitless (fru) as playing a role in stem cell regeneration and Hepatocyte nuclear factor 4 (Hnf4) in regulating glycogen and triglyceride metabolism. In addition, we identified a number of genes, including Rho guanine nucleotide exchange factor at 64C (RhoGEF64c), Frequenin 2 (Frq2), Prip, and CG1093 that are involved in regulating the unusual star shape of stellate cells. Importantly, the single-nucleus dataset allows visualization of the expression at the organ level of genes involved in ion transport and junctional permeability, providing a systems-level view of the organization and physiological roles of the tubules. Finally, a cross-species analysis allowed us to match the fly kidney cell types to mouse kidney cell types and planarian protonephridia, knowledge that will help the generation of kidney disease models. Altogether, our study provides a comprehensive resource for studying the fly kidney.

Keywords:  Malpighian tubules; cross-species; kidney disease; nephrocytes; snRNA-seq

DOI:  https://doi.org/10.1073/pnas.2203179119