bims-crepig 2022-02-06 papers

bims-crepig

Biomed News

on Chromatin regulation and epigenetics in cell fate and cancer

Issue of 2022‒02‒06
twenty-nine papers selected by
Connor Rogerson
University of Cambridge, MRC Cancer Unit

MYC overexpression leads to increased chromatin interactions at superenhancers and MYC binding sites.
SIX1 reprograms myogenic transcription factors to maintain the rhabdomyosarcoma undifferentiated state.
High-Resolution ATAC-Seq Analysis of Frozen Clinical Tissues.
MethReg: estimating the regulatory potential of DNA methylation in gene transcription.
Spt5 histone binding activity preserves chromatin during transcription by RNA polymerase II.
SETD2 loss perturbs the kidney cancer epigenetic landscape to promote metastasis and engenders actionable dependencies on histone chaperone complexes.
Genome organization controls transcriptional dynamics during development.
Eco1-dependent cohesin acetylation anchors chromatin loops and cohesion to define functional meiotic chromosome domains.
spatzie: an R package for identifying significant transcription factor motif co-enrichment from enhancer-promoter interactions.
The H3.3 chaperone Hira complex orchestrates oocyte developmental competence.
An oncogenic enhancer encodes selective selenium dependency in AML.
CT-FOCS: a novel method for inferring cell type-specific enhancer-promoter maps.
Super interactive promoters provide insight into cell type-specific regulatory networks in blood lineage cell types.
Epigenomic priming of immune genes implicates oligodendroglia in multiple sclerosis susceptibility.
Loss of TET reprograms Wnt signaling through impaired demethylation to promote lung cancer development.
Capture-C: a modular and flexible approach for high-resolution chromosome conformation capture.
Noncoding genetic variation in GATA3 increases acute lymphoblastic leukemia risk through local and global changes in chromatin conformation.
Heat shock induces premature transcript termination and reconfigures the human transcriptome.
The transcriptional corepressor CTBP-1 acts with the SOX family transcription factor EGL-13 to maintain AIA interneuron cell identity in Caenorhabditis elegans.
A pan-CRISPR analysis of mammalian cell specificity identifies ultra-compact sgRNA subsets for genome-scale experiments.
Mutant Kras co-opts a proto-oncogenic enhancer network in inflammation-induced metaplastic progenitor cells to initiate pancreatic cancer.
Histone chaperone FACT complex coordinates with HIF to mediate an expeditious transcription program to adapt to poorly oxygenated cancers.
Downregulation of the FTO m6A RNA demethylase promotes EMT-mediated progression of epithelial tumors and sensitivity to Wnt inhibitors.
SoxD genes are required for adult neural stem cell activation.
Novel classes and evolutionary turnover of histone H2B variants in the mammalian germline.
Waves of sumoylation support transcription dynamics during adipocyte differentiation.
Zeb2 drives invasive and microbiota-dependent colon carcinoma.
TNF-α-producing macrophages determine subtype identity and prognosis via AP1 enhancer reprogramming in pancreatic cancer.
Multi-CUT&Tag to simultaneously profile multiple chromatin factors.

Genome Res. 2022 Feb 03. pii: gr.276313.121. [Epub ahead of print]

MYC overexpression leads to increased chromatin interactions at superenhancers and MYC binding sites.

Yi Xiang See, Kaijing Chen, Melissa J Fullwood.

The MYC oncogene encodes for the MYC protein and is frequently dysregulated across multiple cancer cell types, making it an attractive target for cancer therapy. MYC overexpression leads to MYC binding at active enhancers, resulting in a global transcriptional amplification of active genes. Since superenhancers are frequently dysregulated in cancer, we hypothesized that MYC preferentially invades into superenhancers and alters the cancer genome organization. To that end, we performed ChIP-seq, RNA-seq, 4C-seq and SIQHiC (Spike-in Quantitative Hi-C) on the U2OS osteosarcoma cell line with tetracycline-inducible MYC MYC overexpression in U2OS cells modulated histone acetylation and increased MYC binding at superenhancers. SIQHiC analysis revealed increased global chromatin contact frequency, particularly at chromatin interactions connecting MYC binding sites at promoters and enhancers. Immunofluorescence staining showed that MYC molecules formed punctate foci at these transcriptionally active domains after MYC overexpression. These results demonstrate the accumulation of overexpressed MYC at promoter-enhancer hubs and suggest that MYC invades into enhancers through spatial proximity. At the same time, the increased protein-protein interactions may strengthen these chromatin interactions to increase chromatin contact frequency. CTCF siRNA knockdown in MYC overexpressed U2OS cells demonstrated that removal of architectural proteins can disperse MYC and abrogate the increase in chromatin contacts. By elucidating the chromatin landscape of MYC driven cancers, we can potentially target MYC associated chromatin interactions for cancer therapy.

DOI: https://doi.org/10.1101/gr.276313.121
Cell Rep. 2022 Feb 01. pii: S2211-1247(22)00034-1. [Epub ahead of print]38(5): 110323

SIX1 reprograms myogenic transcription factors to maintain the rhabdomyosarcoma undifferentiated state.

Jessica Y Hsu, Etienne P Danis, Stephanie Nance, Jenean H O'Brien, Annika L Gustafson, Veronica M Wessells, Andrew E Goodspeed, Jared C Talbot, Sharon L Amacher, Paul Jedlicka, Joshua C Black, James C Costello, Adam D Durbin, Kristin B Artinger, Heide L Ford.

  Rhabdomyosarcoma (RMS) is a pediatric muscle sarcoma characterized by expression of the myogenic lineage transcription factors (TFs) MYOD1 and MYOG. Despite high expression of these TFs, RMS cells fail to terminally differentiate, suggesting the presence of factors that alter their functions. Here, we demonstrate that the developmental TF SIX1 is highly expressed in RMS and critical for maintaining a muscle progenitor-like state. SIX1 loss induces differentiation of RMS cells into myotube-like cells and impedes tumor growth in vivo. We show that SIX1 maintains the RMS undifferentiated state by controlling enhancer activity and MYOD1 occupancy at loci more permissive to tumor growth over muscle differentiation. Finally, we demonstrate that a gene signature derived from SIX1 loss correlates with differentiation status and predicts RMS progression in human disease. Our findings demonstrate a master regulatory role of SIX1 in repression of RMS differentiation via genome-wide alterations in MYOD1 and MYOG-mediated transcription.

Keywords:  CUT&RUN; MYOD1; SIX1; chromatin; mouse xenograft; muscle differentiation; muscle progenitor; rhabdomyosarcoma; transcriptional control; zebrafish

DOI:  https://doi.org/10.1016/j.celrep.2022.110323
Methods Mol Biol. 2022 ;2458 259-267

High-Resolution ATAC-Seq Analysis of Frozen Clinical Tissues.

Paloma Cejas, Henry W Long.

  The ATAC-seq method enables the genome-wide analysis of accessible chromatin revealing transcriptionally active and poised regulatory elements. The ATAC-seq analysis of clinical specimens at a single-cell resolution reveals the cellular composition of the tissue contributing to the understanding of intra-tissue heterogeneity. Here we describe our method for nuclei isolation from frozen specimens with wide applicability across tissue types, producing nuclei suitable for a number of molecular profiling methods including ATAC-seq in bulk and at a single-cell resolution.

Keywords:  ATAC-seq; ChIP-seq; Chromatin state; Clinical frozen tissues; Nuclei isolation; Single-cell analysis

DOI:  https://doi.org/10.1007/978-1-0716-2140-0_14
Nucleic Acids Res. 2022 Jan 31. pii: gkac030. [Epub ahead of print]

MethReg: estimating the regulatory potential of DNA methylation in gene transcription.

Tiago C Silva, Juan I Young, Eden R Martin, X Steven Chen, Lily Wang.

Epigenome-wide association studies often detect many differentially methylated sites, and many are located in distal regulatory regions. To further prioritize these significant sites, there is a critical need to better understand the functional impact of CpG methylation. Recent studies demonstrated that CpG methylation-dependent transcriptional regulation is a widespread phenomenon. Here, we present MethReg, an R/Bioconductor package that analyzes matched DNA methylation and gene expression data, along with external transcription factor (TF) binding information, to evaluate, prioritize and annotate CpG sites with high regulatory potential. At these CpG sites, TF-target gene associations are often only present in a subset of samples with high (or low) methylation levels, so they can be missed by analyses that use all samples. Using colorectal cancer and Alzheimer's disease datasets, we show MethReg significantly enhances our understanding of the regulatory roles of DNA methylation in complex diseases.

DOI: https://doi.org/10.1093/nar/gkac030
EMBO J. 2022 Feb 01. e109783

Spt5 histone binding activity preserves chromatin during transcription by RNA polymerase II.

Cecile Evrin, Albert Serra-Cardona, Shoufu Duan, Progya P Mukherjee, Zhiguo Zhang, Karim P M Labib.

  Nucleosomes are disrupted transiently during eukaryotic transcription, yet the displaced histones must be retained and redeposited onto DNA, to preserve nucleosome density and associated histone modifications. Here, we show that the essential Spt5 processivity factor of RNA polymerase II (Pol II) plays a direct role in this process in budding yeast. Functional orthologues of eukaryotic Spt5 are present in archaea and bacteria, reflecting its universal role in RNA polymerase processivity. However, eukaryotic Spt5 is unique in having an acidic amino terminal tail (Spt5N) that is sandwiched between the downstream nucleosome and the upstream DNA that emerges from Pol II. We show that Spt5N contains a histone-binding motif that is required for viability in yeast cells and prevents loss of nucleosomal histones within actively transcribed regions. These findings indicate that eukaryotic Spt5 combines two essential activities, which together couple processive transcription to the efficient capture and re-deposition of nucleosomal histones.

Keywords:  RNA polymerase II; Spt5; chromatin; histone; transcription

DOI:  https://doi.org/10.15252/embj.2021109783
Nat Cancer. 2022 Feb 03.

SETD2 loss perturbs the kidney cancer epigenetic landscape to promote metastasis and engenders actionable dependencies on histone chaperone complexes.

Yuchen Xie, Merve Sahin, Sonali Sinha, Yufeng Wang, Amrita M Nargund, Yang Lyu, Song Han, Yiyu Dong, James J Hsieh, Christina S Leslie, Emily H Cheng.

SETD2 is a histone H3 lysine 36 (H3K36) trimethyltransferase that is mutated with high prevalence (13%) in clear cell renal cell carcinoma (ccRCC). Genomic profiling of primary ccRCC tumors reveals a positive correlation between SETD2 mutations and metastasis. However, whether and how SETD2 loss promotes metastasis remains unclear. In this study, we used a SETD2-mutant (SETD2MT) metastatic ccRCC human-derived cell line and xenograft models and showed that H3K36me3 restoration greatly reduced distant metastases of ccRCC in mice in a matrix metalloproteinase 1 (MMP1)-dependent manner. An integrated multiomics analysis using assay for transposase-accessible chromatin using sequencing (ATAC-seq), chromatin immunoprecipitation-sequencing (ChIP-seq) and RNA sequencing (RNA-seq) established a tumor suppressor model in which loss of SETD2-mediated H3K36me3 activates enhancers to drive oncogenic transcriptional output through regulation of chromatin accessibility. Furthermore, we uncovered mechanism-based therapeutic strategies for SETD2-deficient cancer through the targeting of specific histone chaperone complexes, including ASF1A/ASF1B and SPT16. Overall, SETD2 loss creates a permissive epigenetic landscape for cooperating oncogenic drivers to amplify transcriptional output, providing unique therapeutic opportunities.

DOI: https://doi.org/10.1038/s43018-021-00316-3
Science. 2022 Feb 04. 375(6580): 566-570

Genome organization controls transcriptional dynamics during development.

Philippe J Batut, Xin Yang Bing, Zachary Sisco, João Raimundo, Michal Levo, Michael S Levine.

Past studies offer contradictory claims for the role of genome organization in the regulation of gene activity. Here, we show through high-resolution chromosome conformation analysis that the Drosophila genome is organized by two independent classes of regulatory sequences, tethering elements and insulators. Quantitative live imaging and targeted genome editing demonstrate that this two-tiered organization is critical for the precise temporal dynamics of Hox gene transcription during development. Tethering elements mediate long-range enhancer-promoter interactions and foster fast activation kinetics. Conversely, the boundaries of topologically associating domains (TADs) prevent spurious interactions with enhancers and silencers located in neighboring TADs. These two levels of genome organization operate independently of one another to ensure precision of transcriptional dynamics and the reliability of complex patterning processes.

DOI: https://doi.org/10.1126/science.abi7178
Elife. 2022 Feb 01. pii: e74447. [Epub ahead of print]11

Eco1-dependent cohesin acetylation anchors chromatin loops and cohesion to define functional meiotic chromosome domains.

Rachael E Barton, Lucia F Massari, Daniel Robertson, Adèle L Marston.

  Cohesin organizes the genome by forming intra-chromosomal loops and inter-sister chromatid linkages. During gamete formation by meiosis, chromosomes are reshaped to support crossover recombination and two consecutive rounds of chromosome segregation. Here we show that meiotic chromosomes are organised into functional domains by Eco1 acetyltransferase-dependent positioning of both chromatin loops and sister chromatid cohesion in budding yeast. Eco1 acetylates the Smc3 cohesin subunit in meiotic S phase to establish chromatin boundaries, independently of DNA replication. Boundary formation by Eco1 is critical for prophase exit and for the maintenance of cohesion until meiosis II, but is independent of the ability of Eco1 to antagonize the cohesin-release factor, Wpl1. Conversely, prevention of cohesin release by Wpl1 is essential for centromeric cohesion, kinetochore monoorientation and co-segregation of sister chromatids in meiosis I. Our findings establish Eco1 as a key determinant of chromatin boundaries and cohesion positioning, revealing how local chromosome structuring directs genome transmission into gametes.

Keywords:  S. cerevisiae; cell biology; chromosomes; gene expression

DOI:  https://doi.org/10.7554/eLife.74447
Nucleic Acids Res. 2022 Jan 31. pii: gkac036. [Epub ahead of print]

spatzie: an R package for identifying significant transcription factor motif co-enrichment from enhancer-promoter interactions.

Jennifer Hammelman, Konstantin Krismer, David K Gifford.

Genomic interactions provide important context to our understanding of the state of the genome. One question is whether specific transcription factor interactions give rise to genome organization. We introduce spatzie, an R package and a website that implements statistical tests for significant transcription factor motif cooperativity between enhancer-promoter interactions. We conducted controlled experiments under realistic simulated data from ChIP-seq to confirm spatzie is capable of discovering co-enriched motif interactions even in noisy conditions. We then use spatzie to investigate cell type specific transcription factor cooperativity within recent human ChIA-PET enhancer-promoter interaction data. The method is available online at https://spatzie.mit.edu.

DOI: https://doi.org/10.1093/nar/gkac036
Development. 2022 Feb 03. pii: dev.200044. [Epub ahead of print]

The H3.3 chaperone Hira complex orchestrates oocyte developmental competence.

Rowena Smith, Andrej Susor, Hao Ming, Janet Tait, Marco Conti, Zongliang Jiang, Chih-Jen Lin.

  Successful reproduction requires an oocyte competent to sustaining early embryo development. By the end of oogenesis, the oocyte has entered a transcriptionally silenced state, the mechanisms and significance of which remain poorly understood. Histone H3.3, an H3 variant, has unique cell cycle-independent functions in chromatin structure and gene expression. Here we characterised H3.3 chaperone Hira/Cabin1/Ubn1 complex showing that loss-of-function of any of these subunits causes early embryogenesis failure. Transcriptome and nascent RNA analyses revealed that transcription is aberrantly silenced in mutant oocytes. Histone marks including H3K4me3 and H3K9me3 are reduced and chromatin accessibility is impaired in Hira/Cabin1 mutants. Misregulated genes in mutant oocytes include Zscan4, a 2-cell specific gene involved in zygote genome activation. Overexpression of Zscan4 in the oocyte partially recapitulates the phenotypes of Hira mutants and Zscan4 knockdown in Cabin1 mutant oocytes partially restored their developmental potential, illustrating that temporal and spatial expression of Zscan4 is fine-tuned at the oocyte-to-embryo transition. Thus, the H3.3 chaperone Hira complex has a maternal effect function in oocyte developmental competence and embryogenesis by modulating chromatin condensation and transcriptional quiescence.

Keywords:  Hira complex; Histone H3.3; Oocyte-to-embryo transition; Zygotic genome activation; competent oocyte

DOI:  https://doi.org/10.1242/dev.200044
Cell Stem Cell. 2022 Jan 27. pii: S1934-5909(22)00003-0. [Epub ahead of print]

An oncogenic enhancer encodes selective selenium dependency in AML.

Kenneth Eagle, Yajian Jiang, Xiangguo Shi, Minhua Li, Nikolaus P Obholzer, Tianyuan Hu, Monika W Perez, Jošt Vrabič Koren, Ayumi Kitano, Joanna S Yi, Charles Y Lin, Daisuke Nakada.

  Deregulation of transcription is a hallmark of acute myeloid leukemia (AML) that drives oncogenic expression programs and presents opportunities for therapeutic targeting. By integrating comprehensive pan-cancer enhancer landscapes with genetic dependency mapping, we find that AML-enriched enhancers encode for more selective tumor dependencies. We hypothesized that this approach could identify actionable dependencies downstream of oncogenic driver events and discovered a MYB-regulated AML-enriched enhancer regulating SEPHS2, a key component of the selenoprotein production pathway. Using a combination of patient samples and mouse models, we show that this enhancer upregulates SEPHS2, promoting selenoprotein production and antioxidant function required for AML survival. SEPHS2 and other selenoprotein pathway genes are required for AML growth in vitro. SEPHS2 knockout and selenium dietary restriction significantly delay leukemogenesis in vivo with little effect on normal hematopoiesis. These data validate the utility of enhancer mapping in target identification and suggest that selenoprotein production is an actionable target in AML.

Keywords:  AML; MYB; SEPHS2; enhancer; hematopoiesis; hematopoietic stem cell; leukemia stem cell; selenium; selenocysteine

DOI:  https://doi.org/10.1016/j.stem.2022.01.003
Nucleic Acids Res. 2022 Jan 31. pii: gkac048. [Epub ahead of print]

CT-FOCS: a novel method for inferring cell type-specific enhancer-promoter maps.

Tom Aharon Hait, Ran Elkon, Ron Shamir.

Spatiotemporal gene expression patterns are governed to a large extent by the activity of enhancer elements, which engage in physical contacts with their target genes. Identification of enhancer-promoter (EP) links that are functional only in a specific subset of cell types is a key challenge in understanding gene regulation. We introduce CT-FOCS (cell type FOCS), a statistical inference method that uses linear mixed effect models to infer EP links that show marked activity only in a single or a small subset of cell types out of a large panel of probed cell types. Analyzing 808 samples from FANTOM5, covering 472 cell lines, primary cells and tissues, CT-FOCS inferred such EP links more accurately than recent state-of-the-art methods. Furthermore, we show that strictly cell type-specific EP links are very uncommon in the human genome.

DOI: https://doi.org/10.1093/nar/gkac048
PLoS Genet. 2022 Jan 31. 18(1): e1009984

Super interactive promoters provide insight into cell type-specific regulatory networks in blood lineage cell types.

Jia Wen, Taylor M Lagler, Quan Sun, Yuchen Yang, Jiawen Chen, Yuriko Harigaya, Vijay G Sankaran, Ming Hu, Alexander P Reiner, Laura M Raffield, Yun Li.

Existing studies of chromatin conformation have primarily focused on potential enhancers interacting with gene promoters. By contrast, the interactivity of promoters per se, while equally critical to understanding transcriptional control, has been largely unexplored, particularly in a cell type-specific manner for blood lineage cell types. In this study, we leverage promoter capture Hi-C data across a compendium of blood lineage cell types to identify and characterize cell type-specific super-interactive promoters (SIPs). Notably, promoter-interacting regions (PIRs) of SIPs are more likely to overlap with cell type-specific ATAC-seq peaks and GWAS variants for relevant blood cell traits than PIRs of non-SIPs. Moreover, PIRs of cell-type-specific SIPs show enriched heritability of relevant blood cell trait (s), and are more enriched with GWAS variants associated with blood cell traits compared to PIRs of non-SIPs. Further, SIP genes tend to express at a higher level in the corresponding cell type. Importantly, SIP subnetworks incorporating cell-type-specific SIPs and ATAC-seq peaks help interpret GWAS variants. Examples include GWAS variants associated with platelet count near the megakaryocyte SIP gene EPHB3 and variants associated lymphocyte count near the native CD4 T-Cell SIP gene ETS1. Interestingly, around 25.7% ~ 39.6% blood cell traits GWAS variants residing in SIP PIR regions disrupt transcription factor binding motifs. Importantly, our analysis shows the potential of using promoter-centric analyses of chromatin spatial organization data to identify biologically important genes and their regulatory regions.

DOI: https://doi.org/10.1371/journal.pgen.1009984
Neuron. 2022 Jan 28. pii: S0896-6273(21)01089-8. [Epub ahead of print]

Epigenomic priming of immune genes implicates oligodendroglia in multiple sclerosis susceptibility.

Mandy Meijer, Eneritz Agirre, Mukund Kabbe, Cassandra A van Tuijn, Abeer Heskol, Chao Zheng, Ana Mendanha Falcão, Marek Bartosovic, Leslie Kirby, Daniela Calini, Michael R Johnson, M Ryan Corces, Thomas J Montine, Xingqi Chen, Howard Y Chang, Dheeraj Malhotra, Gonçalo Castelo-Branco.

  Multiple sclerosis (MS) is characterized by a targeted attack on oligodendroglia (OLG) and myelin by immune cells, which are thought to be the main drivers of MS susceptibility. We found that immune genes exhibit a primed chromatin state in single mouse and human OLG in a non-disease context, compatible with transitions to immune-competent states in MS. We identified BACH1 and STAT1 as transcription factors involved in immune gene regulation in oligodendrocyte precursor cells (OPCs). A subset of immune genes presents bivalency of H3K4me3/H3K27me3 in OPCs, with Polycomb inhibition leading to their increased activation upon interferon gamma (IFN-γ) treatment. Some MS susceptibility single-nucleotide polymorphisms (SNPs) overlap with these regulatory regions in mouse and human OLG. Treatment of mouse OPCs with IFN-γ leads to chromatin architecture remodeling at these loci and altered expression of interacting genes. Thus, the susceptibility for MS may involve OLG, which therefore constitutes novel targets for immunological-based therapies for MS.

Keywords:  Polycomb; chromatin; genome-wide association studies; histone modifications; major histocompatibility complex; multiple sclerosis; myelin; neuroimmunology; oligodendrocyte; single-nucleotide polymorphisms

DOI:  https://doi.org/10.1016/j.neuron.2021.12.034
Proc Natl Acad Sci U S A. 2022 Feb 08. pii: e2107599119. [Epub ahead of print]119(6):

Loss of TET reprograms Wnt signaling through impaired demethylation to promote lung cancer development.

Qin Xu, Chao Wang, Jia-Xin Zhou, Zhi-Mei Xu, Juan Gao, Pengfei Sui, Colum P Walsh, Hongbin Ji, Guo-Liang Xu.

  Oncogenic imbalance of DNA methylation is well recognized in cancer development. The ten-eleven translocation (TET) family of dioxygenases, which facilitates DNA demethylation, is frequently dysregulated in cancers. How such dysregulation contributes to tumorigenesis remains poorly understood, especially in solid tumors which present infrequent mutational incidence of TET genes. Here, we identify loss-of-function mutations of TET in 7.4% of human lung adenocarcinoma (LUAD), which frequently co-occur with oncogenic KRAS mutations, and this co-occurrence is predictive of poor survival in LUAD patients. Using an autochthonous mouse model of KrasG12D -driven LUAD, we show that individual or combinational loss of Tet genes markedly promotes tumor development. In this Kras-mutant and Tet-deficient model, the premalignant lung epithelium undergoes neoplastic reprogramming of DNA methylation and transcription, with a particular impact on Wnt signaling. Among the Wnt-associated components that undergo reprogramming, multiple canonical Wnt antagonizing genes present impaired expression arising from elevated DNA methylation, triggering aberrant activation of Wnt signaling. These impairments can be largely reversed upon the restoration of TET activity. Correspondingly, genetic depletion of β-catenin, the transcriptional effector of Wnt signaling, substantially reverts the malignant progression of Tet-deficient LUAD. These findings reveal TET enzymes as critical epigenetic barriers against lung tumorigenesis and highlight the therapeutic vulnerability of TET-mutant lung cancer through targeting Wnt signaling.

Keywords:  DNA dioxygenases; Wnt antagonizing genes; epigenetic barriers; lung adenocarcinoma; mouse models

DOI:  https://doi.org/10.1073/pnas.2107599119
Nat Protoc. 2022 Feb 04.

Capture-C: a modular and flexible approach for high-resolution chromosome conformation capture.

Damien J Downes, Alastair L Smith, Magdalena A Karpinska, Taras Velychko, Kevin Rue-Albrecht, David Sims, Thomas A Milne, James O J Davies, A Marieke Oudelaar, Jim R Hughes.

Chromosome conformation capture (3C) methods measure the spatial proximity between DNA elements in the cell nucleus. Many methods have been developed to sample 3C material, including the Capture-C family of protocols. Capture-C methods use oligonucleotides to enrich for interactions of interest from sequencing-ready 3C libraries. This approach is modular and has been adapted and optimized to work for sampling of disperse DNA elements (NuTi Capture-C), including from low cell inputs (LI Capture-C), as well as to generate Hi-C like maps for specific regions of interest (Tiled-C) and to interrogate multiway interactions (Tri-C). We present the design, experimental protocol and analysis pipeline for NuTi Capture-C in addition to the variations for generation of LI Capture-C, Tiled-C and Tri-C data. The entire procedure can be performed in 3 weeks and requires standard molecular biology skills and equipment, access to a next-generation sequencing platform, and basic bioinformatic skills. Implemented with other sequencing technologies, these methods can be used to identify regulatory interactions and to compare the structural organization of the genome in different cell types and genetic models.

DOI: https://doi.org/10.1038/s41596-021-00651-w
Nat Genet. 2022 Feb 03.

Noncoding genetic variation in GATA3 increases acute lymphoblastic leukemia risk through local and global changes in chromatin conformation.

Hongbo Yang, Hui Zhang, Yu Luan, Tingting Liu, Wentao Yang, Kathryn G Roberts, Mao-Xiang Qian, Bo Zhang, Wenjian Yang, Virginia Perez-Andreu, Jie Xu, Sriranga Iyyanki, Da Kuang, Lena A Stasiak, Shalini C Reshmi, Julie Gastier-Foster, Colton Smith, Ching-Hon Pui, William E Evans, Stephen P Hunger, Leonidas C Platanias, Mary V Relling, Charles G Mullighan, Mignon L Loh, Feng Yue, Jun J Yang.

Inherited noncoding genetic variants confer significant disease susceptibility to childhood acute lymphoblastic leukemia (ALL) but the molecular processes linking germline polymorphisms with somatic lesions in this cancer are poorly understood. Through targeted sequencing in 5,008 patients, we identified a key regulatory germline variant in GATA3 associated with Philadelphia chromosome-like ALL (Ph-like ALL). Using CRISPR-Cas9 editing and samples from patients with Ph-like ALL, we showed that this variant activated a strong enhancer that upregulated GATA3 transcription. This, in turn, reshaped global chromatin accessibility and three-dimensional genome organization, including regions proximal to the ALL oncogene CRLF2. Finally, we showed that GATA3 directly regulated CRLF2 and potentiated the JAK-STAT oncogenic effects during leukemogenesis. Taken together, we provide evidence for a distinct mechanism by which a germline noncoding variant contributes to oncogene activation, epigenetic regulation and three-dimensional genome reprogramming.

DOI: https://doi.org/10.1038/s41588-021-00993-x
Mol Cell. 2022 Jan 29. pii: S1097-2765(22)00007-7. [Epub ahead of print]

Heat shock induces premature transcript termination and reconfigures the human transcriptome.

Simona Cugusi, Richard Mitter, Gavin P Kelly, Jane Walker, Zhong Han, Paola Pisano, Michael Wierer, Aengus Stewart, Jesper Q Svejstrup.

  The heat shock (HS) response involves rapid induction of HS genes, whereas transcriptional repression is established more slowly at most other genes. Previous data suggested that such repression results from inhibition of RNA polymerase II (RNAPII) pause release, but here, we show that HS strongly affects other phases of the transcription cycle. Intriguingly, while elongation rates increase upon HS, processivity markedly decreases, so that RNAPII frequently fails to reach the end of genes. Indeed, HS results in widespread premature transcript termination at cryptic, intronic polyadenylation (IPA) sites near gene 5'-ends, likely via inhibition of U1 telescripting. This results in dramatic reconfiguration of the human transcriptome with production of new, previously unannotated, short mRNAs that accumulate in the nucleus. Together, these results shed new light on the basic transcription mechanisms induced by growth at elevated temperature and show that a genome-wide shift toward usage of IPA sites can occur under physiological conditions.

Keywords:  CDK9; CPSF3; SCAF4; SCAF8; TT-seq; U1 snRNA; alternative polyadenylation; cryptic polyadenylation sites; elongation; heat shock; pTEFb; pause release; premature termination; telescripting; transcriptional repression

DOI:  https://doi.org/10.1016/j.molcel.2022.01.007
Elife. 2022 Feb 04. pii: e74557. [Epub ahead of print]11

The transcriptional corepressor CTBP-1 acts with the SOX family transcription factor EGL-13 to maintain AIA interneuron cell identity in Caenorhabditis elegans.

Josh Saul, Takashi Hirose, H Robert Horvitz.

  Cell identity is characterized by a distinct combination of gene expression, cell morphology, and cellular function established as progenitor cells divide and differentiate. Following establishment, cell identities can be unstable and require active and continuous maintenance throughout the remaining life of a cell. Mechanisms underlying the maintenance of cell identities are incompletely understood. Here, we show that the gene ctbp-1, which encodes the transcriptional corepressor C-terminal binding protein-1 (CTBP-1), is essential for the maintenance of the identities of the two AIA interneurons in the nematode Caenorhabditis elegans. ctbp-1 is not required for the establishment of the AIA cell fate but rather functions cell-autonomously and can act in later larval stage and adult worms to maintain proper AIA gene expression, morphology and function. From a screen for suppressors of the ctbp-1 mutant phenotype, we identified the gene egl-13, which encodes a SOX family transcription factor. We found that egl-13 regulates AIA function and aspects of AIA gene expression, but not AIA morphology. We conclude that the CTBP-1 protein maintains AIA cell identity in part by utilizing EGL-13 to repress transcriptional activity in the AIAs. More generally, we propose that transcriptional corepressors like CTBP-1 might be critical factors in the maintenance of cell identities, harnessing the DNA-binding specificity of transcription factors like EGL-13 to selectively regulate gene expression in a cell-specific manner.

Keywords:  C. elegans; cell fate; cell-identity maintenance; developmental biology; genetics; genomics; transcriptional corepressor

DOI:  https://doi.org/10.7554/eLife.74557
Nat Commun. 2022 Feb 02. 13(1): 625

A pan-CRISPR analysis of mammalian cell specificity identifies ultra-compact sgRNA subsets for genome-scale experiments.

Boyang Zhao, Yiyun Rao, Scott Leighow, Edward P O'Brien, Luke Gilbert, Justin R Pritchard.

A genetic knockout can be lethal to one human cell type while increasing growth rate in another. This context specificity confounds genetic analysis and prevents reproducible genome engineering. Genome-wide CRISPR compendia across most common human cell lines offer the largest opportunity to understand the biology of cell specificity. The prevailing viewpoint, synthetic lethality, occurs when a genetic alteration creates a unique CRISPR dependency. Here, we use machine learning for an unbiased investigation of cell type specificity. Quantifying model accuracy, we find that most cell type specific phenotypes are predicted by the function of related genes of wild-type sequence, not synthetic lethal relationships. These models then identify unexpected sets of 100-300 genes where reduced CRISPR measurements can produce genome-scale loss-of-function predictions across >18,000 genes. Thus, it is possible to reduce in vitro CRISPR libraries by orders of magnitude-with some information loss-when we remove redundant genes and not redundant sgRNAs.

DOI: https://doi.org/10.1038/s41467-022-28045-w
Nat Cancer. 2021 Jan;2(1): 49-65

Mutant Kras co-opts a proto-oncogenic enhancer network in inflammation-induced metaplastic progenitor cells to initiate pancreatic cancer.

Yong Li, Yi He, Junya Peng, Zhendong Su, Zeyao Li, Bingjie Zhang, Jing Ma, Meilian Zhuo, Di Zou, Xinde Liu, Xinhong Liu, Wenze Wang, Dan Huang, Mengyue Xu, Jianbin Wang, Haiteng Deng, Jing Xue, Wei Xie, Xun Lan, Mo Chen, Yupei Zhao, Wenming Wu, Charles J David.

Kras-activating mutations display the highest incidence in pancreatic ductal adenocarcinoma. Pancreatic inflammation accelerates mutant Kras-driven tumorigenesis in mice, suggesting high selectivity in the cells that oncogenic Kras transforms, although the mechanisms dictating this specificity are poorly understood. Here we show that pancreatic inflammation is coupled to the emergence of a transient progenitor cell population that is readily transformed in the presence of mutant KrasG12D. These progenitors harbor a proto-oncogenic transcriptional program driven by a transient enhancer network. KrasG12D mutations lock this enhancer network in place, providing a sustained Kras-dependent oncogenic program that drives tumors throughout progression. Enhancer co-option occurs through functional interactions between the Kras-activated transcription factors Junb and Fosl1 and pancreatic lineage transcription factors, potentially accounting for inter-tissue specificity of oncogene transformation. The pancreatic ductal adenocarcinoma cell of origin thus provides an oncogenic transcriptional program that fuels tumor progression beyond initiation, accounting for the intra-tissue selectivity of Kras transformation.

DOI: https://doi.org/10.1038/s43018-020-00134-z
Cell Rep. 2022 Feb 01. pii: S2211-1247(22)00016-X. [Epub ahead of print]38(5): 110304

Histone chaperone FACT complex coordinates with HIF to mediate an expeditious transcription program to adapt to poorly oxygenated cancers.

Jialing Shen, Chunxue Yang, Misty Shuo Zhang, Don Wai-Ching Chin, For-Fan Chan, Cheuk-Ting Law, Gengchao Wang, Carol Lai-Hung Cheng, Mengnuo Chen, Rebecca Ting-Chi Wan, Mengjie Wu, Zhijian Kuang, Rakesh Sharma, Terence Kin Wah Lee, Irene Oi-Lin Ng, Carmen Chak-Lui Wong, Chun-Ming Wong.

  Cancer cells adapt to hypoxia through HIFs (hypoxia-inducible factors), which initiate the transcription of numerous genes for cancer cell survival in the hypoxia microenvironment. In this study, we find that the FACT (facilitates chromatin transcription) complex works cooperatively with HIFs to facilitate the expeditious expression of HIF targets for hypoxia adaptation. Knockout (KO) of the FACT complex abolishes HIF-mediated transcription by impeding transcription elongation in hypoxic cancer cells. Interestingly, the FACT complex is post-translationally regulated by PHD/VHL-mediated hydroxylation and proteasomal degradation, in similar fashion to HIF-1/2α. Metabolic tracing confirms that FACT KO suppresses glycolytic flux and impairs lactate extrusion, leading to intracellular acidification and apoptosis in cancer cells. Therapeutically, hepatic artery ligation and anti-angiogenic inhibitors adversely induce intratumoral hypoxia, while co-treatment with FACT inhibitor curaxin remarkably hinders the growth of hypoxic tumors. In summary, our findings suggest that the FACT complex is a critical component of hypoxia adaptation and a therapeutic target for hypoxic tumors.

Keywords:  FACT complex; HIF; anti-angiogenic inhibitors; cancer; glycolysis; hepatic artery ligation; histone chaperone; hypoxia; intracellular acidification; lactate production

DOI:  https://doi.org/10.1016/j.celrep.2022.110304
Nat Cancer. 2021 Jun;2(6): 611-628

Downregulation of the FTO m6A RNA demethylase promotes EMT-mediated progression of epithelial tumors and sensitivity to Wnt inhibitors.

Jana Jeschke, Evelyne Collignon, Clémence Al Wardi, Mohammad Krayem, Martin Bizet, Yan Jia, Soizic Garaud, Zéna Wimana, Emilie Calonne, Bouchra Hassabi, Renato Morandini, Rachel Deplus, Pascale Putmans, Gaurav Dube, Nitesh Kumar Singh, Alexander Koch, Kateryna Shostak, Lara Rizzotto, Robert L Ross, Christine Desmedt, Yacine Bareche, Françoise Rothé, Jacqueline Lehmann-Che, Martine Duterque-Coquillaud, Xavier Leroy, Gerben Menschaert, Luis Teixeira, Mingzhou Guo, Patrick A Limbach, Pierre Close, Alain Chariot, Eleonora Leucci, Ghanem Ghanem, Bi-Feng Yuan, Karen Willard-Gallo, Christos Sotiriou, Jean-Christophe Marine, François Fuks.

Post-transcriptional modifications of RNA constitute an emerging regulatory layer of gene expression. The demethylase fat mass- and obesity-associated protein (FTO), an eraser of N6-methyladenosine (m6A), has been shown to play a role in cancer, but its contribution to tumor progression and the underlying mechanisms remain unclear. Here, we report widespread FTO downregulation in epithelial cancers associated with increased invasion, metastasis and worse clinical outcome. Both in vitro and in vivo, FTO silencing promotes cancer growth, cell motility and invasion. In human-derived tumor xenografts (PDXs), FTO pharmacological inhibition favors tumorigenesis. Mechanistically, we demonstrate that FTO depletion elicits an epithelial-to-mesenchymal transition (EMT) program through increased m6A and altered 3'-end processing of key mRNAs along the Wnt signaling cascade. Accordingly, FTO knockdown acts via EMT to sensitize mouse xenografts to Wnt inhibition. We thus identify FTO as a key regulator, across epithelial cancers, of Wnt-triggered EMT and tumor progression and reveal a therapeutically exploitable vulnerability of FTO-low tumors.

DOI: https://doi.org/10.1038/s43018-021-00223-7
Cell Rep. 2022 Feb 01. pii: S2211-1247(22)00025-0. [Epub ahead of print]38(5): 110313

SoxD genes are required for adult neural stem cell activation.

Lingling Li, Cristina Medina-Menéndez, Laura García-Corzo, Carmen M Córdoba-Beldad, Alejandra C Quiroga, Elena Calleja Barca, Valeriya Zinchuk, Sara Muñoz-López, Pilar Rodríguez-Martín, Maria Ciorraga, Inés Colmena, Silvia Fernández, Carlos Vicario, Silvia K Nicolis, Véronique Lefebvre, Helena Mira, Aixa V Morales.

  The adult neurogenic niche in the hippocampus is maintained through activation of reversibly quiescent neural stem cells (NSCs) with radial glia-like morphology (RGLs). Here, we show that the expression of SoxD transcription factors Sox5 and Sox6 is enriched in activated RGLs. Using inducible deletion of Sox5 or Sox6 in the adult mouse brain, we show that both genes are required for RGL activation and the generation of new neurons. Conversely, Sox5 overexpression in cultured NSCs interferes with entry in quiescence. Mechanistically, expression of the proneural protein Ascl1 (a key RGL regulator) is severely downregulated in SoxD-deficient RGLs, and Ascl1 transcription relies on conserved Sox motifs. Additionally, loss of Sox5 hinders the RGL activation driven by neurogenic stimuli such as environmental enrichment. Altogether, our data suggest that SoxD genes are key mediators in the transition of adult RGLs from quiescence to an activated mitotic state under physiological situations.

Keywords:  Lamb-Shaffer syndrome; SOXopathy; Sox5; Sox6; Tolchin-Le Caignec syndrome; adult neurogenesis; hippocampus; neural stem cells; quiescence

DOI:  https://doi.org/10.1016/j.celrep.2022.110313
Mol Biol Evol. 2022 Jan 31. pii: msac019. [Epub ahead of print]

Novel classes and evolutionary turnover of histone H2B variants in the mammalian germline.

Pravrutha Raman, Mary C Rominger, Janet M Young, Antoine Molaro, Toshio Tsukiyama, Harmit S Malik.

  Histones and their post-translational modifications facilitate diverse chromatin functions in eukaryotes. Core histones (H2A, H2B, H3, and H4) package genomes after DNA replication. In contrast, variant histones promote specialized chromatin functions, including DNA repair, genome stability, and epigenetic inheritance. Previous studies have identified only a few H2B variants in animals; their roles and evolutionary origins remain largely unknown. Here, using phylogenomic analyses, we reveal the presence of five H2B variants broadly present in mammalian genomes. Three of these variants have been previously described: H2B.1, H2B.L (also called subH2B), and H2B.W. In addition, we identify and describe two new variants: H2B.K and H2B.N. Four of these variants originated in mammals, whereas H2B.K arose prior to the last common ancestor of bony vertebrates. We find that though H2B variants are subject to high gene turnover, most are broadly retained in mammals, including humans. Despite an overall signature of purifying selection, H2B variants evolve more rapidly than core H2B with considerable divergence in sequence and length. All five H2B variants are expressed in the germline. H2B.K and H2B.N are predominantly expressed in oocytes, an atypical expression site for mammalian histone variants. Our findings suggest that H2B variants likely encode potentially redundant but vital functions via unusual chromatin packaging or non-chromatin functions in mammalian germline cells. Our discovery of novel histone variants highlights the advantages of comprehensive phylogenomic analyses and provides unique opportunities to study how innovations in chromatin function evolve.

Keywords:  gene duplication; histone variants; oogenesis; positive selection; pseudogenes; spermatogenesis

DOI:  https://doi.org/10.1093/molbev/msac019
Nucleic Acids Res. 2022 Jan 31. pii: gkac027. [Epub ahead of print]

Waves of sumoylation support transcription dynamics during adipocyte differentiation.

Xu Zhao, Ivo A Hendriks, Stéphanie Le Gras, Tao Ye, Lucía Ramos-Alonso, Aurélie Nguéa P, Guro Flor Lien, Fatemeh Ghasemi, Arne Klungland, Bernard Jost, Jorrit M Enserink, Michael L Nielsen, Pierre Chymkowitch.

Tight control of gene expression networks required for adipose tissue formation and plasticity is essential for adaptation to energy needs and environmental cues. However, the mechanisms that orchestrate the global and dramatic transcriptional changes leading to adipocyte differentiation remain to be fully unraveled. We investigated the regulation of nascent transcription by the sumoylation pathway during adipocyte differentiation using SLAMseq and ChIPseq. We discovered that the sumoylation pathway has a dual function in differentiation; it supports the initial downregulation of pre-adipocyte-specific genes, while it promotes the establishment of the mature adipocyte transcriptional program. By characterizing endogenous sumoylome dynamics in differentiating adipocytes by mass spectrometry, we found that sumoylation of specific transcription factors like PPARγ/RXR and their co-factors are associated with the transcription of adipogenic genes. Finally, using RXR as a model, we found that sumoylation may regulate adipogenic transcription by supporting the chromatin occurrence of transcription factors. Our data demonstrate that the sumoylation pathway supports the rewiring of transcriptional networks required for formation of functional adipocytes. This study also provides the scientists in the field of cellular differentiation and development with an in-depth resource of the dynamics of the SUMO-chromatin landscape, SUMO-regulated transcription and endogenous sumoylation sites during adipocyte differentiation.

DOI: https://doi.org/10.1093/nar/gkac027
Nat Cancer. 2020 Jun;1(6): 620-634

Zeb2 drives invasive and microbiota-dependent colon carcinoma.

Karolina Slowicka, Ioanna Petta, Gillian Blancke, Esther Hoste, Emilie Dumas, Mozes Sze, Hanna Vikkula, Enrico Radaelli, Jody J Haigh, Sven Jonckheere, Joachim Taminau, Niels Vandamme, Andy Wullaert, Eugene Tulchinsky, David Nittner, Pieter Van Vlierberghe, Gert De Hertogh, Pamela Baldin, Emre Etlioglu, Pratyaksha Wirapati, Louis Boon, Bart N Lambrecht, Chris Callewaert, Sabine Tejpar, Steven Goossens, Geert Berx, Lars Vereecke, Geert van Loo.

Colorectal cancer (CRC) is highly prevalent in Western society, and increasing evidence indicates strong contributions of environmental factors and the intestinal microbiota to CRC initiation, progression and even metastasis. We have identified a synergistic inflammatory tumor-promoting mechanism through which the resident intestinal microbiota boosts invasive CRC development in an epithelial-to-mesenchymal transition-prone tissue environment. Intestinal epithelial cell (IEC)-specific transgenic expression of the epithelial-to-mesenchymal transition regulator Zeb2 in mice (Zeb2IEC-Tg/+) leads to increased intestinal permeability, myeloid cell-driven inflammation and spontaneous invasive CRC development. Zeb2IEC-Tg/+ mice develop a dysplastic colonic epithelium, which progresses to severely inflamed neoplastic lesions while the small intestinal epithelium remains normal. Zeb2IEC-Tg/+ mice are characterized by intestinal dysbiosis, and microbiota depletion with broad-spectrum antibiotics or germ-free rederivation completely prevents cancer development. Zeb2IEC-Tg/+ mice represent the first mouse model of spontaneous microbiota-dependent invasive CRC and will help us to better understand host-microbiome interactions driving CRC development in humans.

DOI: https://doi.org/10.1038/s43018-020-0070-2
Nat Cancer. 2021 Nov;2(11): 1185-1203

TNF-α-producing macrophages determine subtype identity and prognosis via AP1 enhancer reprogramming in pancreatic cancer.

Mengyu Tu, Lukas Klein, Elisa Espinet, Theodoros Georgomanolis, Florian Wegwitz, Xiaojuan Li, Laura Urbach, Adi Danieli-Mackay, Stefan Küffer, Kamil Bojarczuk, Athanasia Mizi, Ufuk Günesdogan, Björn Chapuy, Zuguang Gu, Albrecht Neesse, Uday Kishore, Philipp Ströbel, Elisabeth Hessmann, Stephan A Hahn, Andreas Trumpp, Argyris Papantonis, Volker Ellenrieder, Shiv K Singh.

Large-scale genomic profiling of pancreatic cancer (PDAC) has revealed two distinct subtypes: 'classical' and 'basal-like'. Their variable coexistence within the stromal immune microenvironment is linked to differential prognosis; however, the extent to which these neoplastic subtypes shape the stromal immune landscape and impact clinical outcome remains unclear. By combining preclinical models, patient-derived xenografts, as well as FACS-sorted PDAC patient biopsies, we show that the basal-like neoplastic state is sustained via BRD4-mediated cJUN/AP1 expression, which induces CCL2 to recruit tumor necrosis factor (TNF)-α-secreting macrophages. TNF-α+ macrophages force classical neoplastic cells into an aggressive phenotypic state via lineage reprogramming. Integration of ATAC-, ChIP- and RNA-seq data revealed distinct JUNB/AP1 (classical) and cJUN/AP1 (basal-like)-driven regulation of PDAC subtype identity. Pharmacological inhibition of BRD4 led to suppression of the BRD4-cJUN-CCL2-TNF-α axis, restoration of classical subtype identity and a favorable prognosis. Hence, patient-tailored therapy for a cJUNhigh/TNF-αhigh subtype is paramount in overcoming highly inflamed and aggressive PDAC states.

DOI: https://doi.org/10.1038/s43018-021-00258-w
STAR Protoc. 2022 Mar 18. 3(1): 101100

Multi-CUT&Tag to simultaneously profile multiple chromatin factors.

Sneha Gopalan, Thomas G Fazzio.

  Genome-wide chromatin mapping approaches typically focus on one protein at a time. We recently developed multi-CUT&Tag, which enables simultaneous mapping of multiple chromatin proteins in the same single cells or pools of cells. Using barcoded adapters loaded onto antibody-protein A-Tn5 transposase complexes, multi-CUT&Tag marks the locations of each chromatin protein and directly detects colocalization of different proteins in the same cell(s). Although slightly more laborious than CUT&Tag, multi-CUT&Tag provides a powerful option for generating multi-factor maps for epigenomic profiling. For complete details on the use and execution of this protocol, please refer to Gopalan et al. (2021).

Keywords:  Antibody; Genomics; Molecular Biology; Molecular/Chemical Probes; Protein Biochemistry; Protein expression and purification; Sequence analysis; Sequencing; Single Cell

DOI:  https://doi.org/10.1016/j.xpro.2021.101100