bims-crepig 2023-04-16 papers

bims-crepig

Biomed News

on Chromatin regulation and epigenetics in cell fate and cancer

Issue of 2023–04–16
twenty-one papers selected by
Connor Rogerson, University of Cambridge

Understanding cell fate acquisition in stem-cell-derived pancreatic islets using single-cell multiome-inferred regulomes.
Broad compatibility between yeast UAS elements and core promoters and identification of promoter elements that determine cofactor specificity.
PRC2.1- and PRC2.2-specific accessory proteins drive recruitment of different forms of canonical PRC1.
The esBAF and ISWI nucleosome remodeling complexes influence occupancy of overlapping dinucleosomes and fragile nucleosomes in murine embryonic stem cells.
DisP-seq reveals the genome-wide functional organization of DNA-associated disordered proteins.
Modulation of transcription burst amplitude underpins dosage compensation in the Drosophila embryo.
Elevated enhancer-oncogene contacts and higher oncogene expression levels by recurrent CTCF inactivating mutations in acute T cell leukemia.
Stalled oligodendrocyte differentiation in IDH-mutant gliomas.
Cell-to-cell variability in Myc dynamics drives transcriptional heterogeneity in cancer cells.
Hemato-vascular specification requires arnt1 and arnt2 genes in zebrafish embryos.
The epigenetic reader, BRD2, mediates cholangiocyte senescence via interaction with ETS1.
Landscape of prostate-specific membrane antigen heterogeneity and regulation in AR-positive and AR-negative metastatic prostate cancer.
ABL kinases regulate the stabilization of HIF-1α and MYC through CPSF1.
A multi-omics integrative analysis based on CRISPR screens re-defines the pluripotency regulatory network in ESCs.
High-resolution Nanopore methylome-maps reveal random hyper-methylation at CpG-poor regions as driver of chemoresistance in leukemias.
Fob1-dependent condensin recruitment and loop extrusion on yeast chromosome III.
PASK links cellular energy metabolism with a mitotic self-renewal network to establish differentiation competence.
Endothelial FAT1 inhibits angiogenesis by controlling YAP/TAZ protein degradation via E3 ligase MIB2.
Histone 4 lysine 5/12 acetylation enables developmental plasticity of Pristionchus mouth form.
Super-enhancer-driven TOX2 mediates oncogenesis in Natural Killer/T Cell Lymphoma.
Extrachromosomal DNA in the cancerous transformation of Barrett's oesophagus.

Dev Cell. 2023 Apr 03. pii: S1534-5807(23)00107-7. [Epub ahead of print]

Understanding cell fate acquisition in stem-cell-derived pancreatic islets using single-cell multiome-inferred regulomes.

Han Zhu, Gaowei Wang, Kim-Vy Nguyen-Ngoc, Dongsu Kim, Michael Miller, Georgina Goss, Jenna Kovsky, Austin R Harrington, Diane C Saunders, Alexander L Hopkirk, Rebecca Melton, Alvin C Powers, Sebastian Preissl, Francesca M Spagnoli, Kyle J Gaulton, Maike Sander.

  Pancreatic islet cells derived from human pluripotent stem cells hold great promise for modeling and treating diabetes. Differences between stem-cell-derived and primary islets remain, but molecular insights to inform improvements are limited. Here, we acquire single-cell transcriptomes and accessible chromatin profiles during in vitro islet differentiation and pancreas from childhood and adult donors for comparison. We delineate major cell types, define their regulomes, and describe spatiotemporal gene regulatory relationships between transcription factors. CDX2 emerged as a regulator of enterochromaffin-like cells, which we show resemble a transient, previously unrecognized, serotonin-producing pre-β cell population in fetal pancreas, arguing against a proposed non-pancreatic origin. Furthermore, we observe insufficient activation of signal-dependent transcriptional programs during in vitro β cell maturation and identify sex hormones as drivers of β cell proliferation in childhood. Altogether, our analysis provides a comprehensive understanding of cell fate acquisition in stem-cell-derived islets and a framework for manipulating cell identities and maturity.

Keywords:  ATAC-seq; CDX2; RNA-seq; development; fetal pancreas; gene regulatory network; human pluripotent stem cells; islets; pancreas; serotonin; signals; single-cell genomics; transcription factors; β cell

DOI:  https://doi.org/10.1016/j.devcel.2023.03.011
Cell Rep. 2023 Apr 12. pii: S2211-1247(23)00398-4. [Epub ahead of print]42(4): 112387

Broad compatibility between yeast UAS elements and core promoters and identification of promoter elements that determine cofactor specificity.

Jeremy A Schofield, Steven Hahn.

  Three classes of yeast protein-coding genes are distinguished by their dependence on the transcription cofactors TFIID, SAGA, and Mediator (MED) Tail, but whether this dependence is determined by the core promoter, upstream activating sequences (UASs), or other gene features is unclear. Also unclear is whether UASs can broadly activate transcription from the different promoter classes. Here, we measure transcription and cofactor specificity for thousands of UAS-core promoter combinations and find that most UASs broadly activate promoters regardless of regulatory class, while few display strong promoter specificity. However, matching UASs and promoters from the same gene class is generally important for optimal expression. We find that sensitivity to rapid depletion of MED Tail or SAGA is dependent on the identity of both UAS and core promoter, while dependence on TFIID localizes to only the promoter. Finally, our results suggest the role of TATA and TATA-like promoter sequences in MED Tail function.

Keywords:  CP: Molecular biology; SAGA; TFIID; core promoters; enhancers; large-scale reporter; mediator; transcriptional coactivators; upstream activating sequences (UASs)

DOI:  https://doi.org/10.1016/j.celrep.2023.112387
Mol Cell. 2023 Apr 03. pii: S1097-2765(23)00208-3. [Epub ahead of print]

PRC2.1- and PRC2.2-specific accessory proteins drive recruitment of different forms of canonical PRC1.

Eleanor Glancy, Cheng Wang, Ellen Tuck, Evan Healy, Simona Amato, Hannah K Neikes, Andrea Mariani, Marlena Mucha, Michiel Vermeulen, Diego Pasini, Adrian P Bracken.

  Polycomb repressive complex 2 (PRC2) mediates H3K27me3 deposition, which is thought to recruit canonical PRC1 (cPRC1) via chromodomain-containing CBX proteins to promote stable repression of developmental genes. PRC2 forms two major subcomplexes, PRC2.1 and PRC2.2, but their specific roles remain unclear. Through genetic knockout (KO) and replacement of PRC2 subcomplex-specific subunits in naïve and primed pluripotent cells, we uncover distinct roles for PRC2.1 and PRC2.2 in mediating the recruitment of different forms of cPRC1. PRC2.1 catalyzes the majority of H3K27me3 at Polycomb target genes and is sufficient to promote recruitment of CBX2/4-cPRC1 but not CBX7-cPRC1. Conversely, while PRC2.2 is poor at catalyzing H3K27me3, we find that its accessory protein JARID2 is essential for recruitment of CBX7-cPRC1 and the consequent 3D chromatin interactions at Polycomb target genes. We therefore define distinct contributions of PRC2.1- and PRC2.2-specific accessory proteins to Polycomb-mediated repression and uncover a new mechanism for cPRC1 recruitment.

Keywords:  CBX2; CBX4; CBX7; H3K27me3; JARID2; PRC1; PRC2.1; PRC2.2; Polycomb; Polycomb-like protein

DOI:  https://doi.org/10.1016/j.molcel.2023.03.018
BMC Genomics. 2023 Apr 13. 24(1): 201

The esBAF and ISWI nucleosome remodeling complexes influence occupancy of overlapping dinucleosomes and fragile nucleosomes in murine embryonic stem cells.

David C Klein, Kris Troy, Sarah A Tripplehorn, Sarah J Hainer.

   BACKGROUND: Nucleosome remodeling factors regulate the occupancy and positioning of nucleosomes genome-wide through ATP-driven DNA translocation. While many nucleosomes are consistently well-positioned, some nucleosomes and alternative nucleosome structures are more sensitive to nuclease digestion or are transitory. Fragile nucleosomes are nucleosome structures that are sensitive to nuclease digestion and may be composed of either six or eight histone proteins, making these either hexasomes or octasomes. Overlapping dinucleosomes are composed of two merged nucleosomes, lacking one H2A:H2B dimer, creating a 14-mer wrapped by ~ 250 bp of DNA. In vitro studies of nucleosome remodeling suggest that the collision of adjacent nucleosomes by sliding stimulates formation of overlapping dinucleosomes.
RESULTS: To better understand how nucleosome remodeling factors regulate alternative nucleosome structures, we depleted murine embryonic stem cells of the transcripts encoding remodeler ATPases BRG1 or SNF2H, then performed MNase-seq. We used high- and low-MNase digestion to assess the effects of nucleosome remodeling factors on nuclease-sensitive or "fragile" nucleosome occupancy. In parallel we gel-extracted MNase-digested fragments to enrich for overlapping dinucleosomes. We recapitulate prior identification of fragile nucleosomes and overlapping dinucleosomes near transcription start sites, and identify enrichment of these features around gene-distal DNaseI hypersensitive sites, CTCF binding sites, and pluripotency factor binding sites. We find that BRG1 stimulates occupancy of fragile nucleosomes but restricts occupancy of overlapping dinucleosomes.
CONCLUSIONS: Overlapping dinucleosomes and fragile nucleosomes are prevalent within the ES cell genome, occurring at hotspots of gene regulation beyond their characterized existence at promoters. Although neither structure is fully dependent on either nucleosome remodeling factor, both fragile nucleosomes and overlapping dinucleosomes are affected by knockdown of BRG1, suggesting a role for the complex in creating or removing these structures.

Keywords:  Chromatin; Fragile nucleosomes; Nucleosomes; Overlapping dinucleosomes; Remodeling; Stem cells; Subnucleosomes

DOI:  https://doi.org/10.1186/s12864-023-09287-4
Nat Biotechnol. 2023 Apr 10.

DisP-seq reveals the genome-wide functional organization of DNA-associated disordered proteins.

Yu-Hang Xing, Rui Dong, Lukuo Lee, Shruthi Rengarajan, Nicolò Riggi, Gaylor Boulay, Miguel N Rivera.

Intrinsically disordered regions (IDRs) in DNA-associated proteins are known to influence gene regulation, but their distribution and cooperative functions in genome-wide regulatory programs remain poorly understood. Here we describe DisP-seq (disordered protein precipitation followed by DNA sequencing), an antibody-independent chemical precipitation assay that can simultaneously map endogenous DNA-associated disordered proteins genome-wide through a combination of biotinylated isoxazole precipitation and next-generation sequencing. DisP-seq profiles are composed of thousands of peaks that are associated with diverse chromatin states, are enriched for disordered transcription factors (TFs) and are often arranged in large lineage-specific clusters with high local concentrations of disordered proteins and different combinations of histone modifications linked to regulatory potential. We use DisP-seq to analyze cancer cells and reveal how disordered protein-associated islands enable IDR-dependent mechanisms that control the binding and function of disordered TFs, including oncogene-dependent sequestration of TFs through long-range interactions and the reactivation of differentiation pathways upon loss of oncogenic stimuli in Ewing sarcoma.

DOI: https://doi.org/10.1038/s41587-023-01737-4
Cell Rep. 2023 Apr 13. pii: S2211-1247(23)00393-5. [Epub ahead of print]42(4): 112382

Modulation of transcription burst amplitude underpins dosage compensation in the Drosophila embryo.

Lauren Forbes Beadle, Hongpeng Zhou, Magnus Rattray, Hilary L Ashe.

  Dosage compensation, the balancing of X-linked gene expression between sexes and to the autosomes, is critical to an organism's fitness and survival. In Drosophila, dosage compensation involves hypertranscription of the male X chromosome. Here, we use quantitative live imaging and modeling at single-cell resolution to study X chromosome dosage compensation in Drosophila. We show that the four X chromosome genes studied undergo transcriptional bursting in male and female embryos. Mechanistically, our data reveal that transcriptional upregulation of male X chromosome genes is primarily mediated by a higher RNA polymerase II initiation rate and burst amplitude across the expression domain. In contrast, burst frequency is spatially modulated in nuclei within the expression domain in response to different transcription factor concentrations to tune the transcriptional response. Together, these data show how the local and global regulation of distinct burst parameters can establish the complex transcriptional outputs underpinning developmental patterning.

Keywords:  CP: Molecular biology; Drosophila embryo; MS2; RNA polymerase II; X chromosome; burst amplitude; dosage compensation; live imaging; transcriptional bursting

DOI:  https://doi.org/10.1016/j.celrep.2023.112382
Cell Rep. 2023 Apr 13. pii: S2211-1247(23)00384-4. [Epub ahead of print]42(4): 112373

Elevated enhancer-oncogene contacts and higher oncogene expression levels by recurrent CTCF inactivating mutations in acute T cell leukemia.

Willem K Smits, Carlo Vermeulen, Rico Hagelaar, Shunsuke Kimura, Eric M Vroegindeweij, Jessica G C A M Buijs-Gladdines, Ellen van de Geer, Marjon J A M Verstegen, Erik Splinter, Simon V van Reijmersdal, Arjan Buijs, Niels Galjart, Winfried van Eyndhoven, Max van Min, Roland Kuiper, Patrick Kemmeren, Charles G Mullighan, Wouter de Laat, Jules P P Meijerink.

  Monoallelic inactivation of CCCTC-binding factor (CTCF) in human cancer drives altered methylated genomic states, altered CTCF occupancy at promoter and enhancer regions, and deregulated global gene expression. In patients with T cell acute lymphoblastic leukemia (T-ALL), we find that acquired monoallelic CTCF-inactivating events drive subtle and local genomic effects in nearly half of t(5; 14) (q35; q32.2) rearranged patients, especially when CTCF-binding sites are preserved in between the BCL11B enhancer and the TLX3 oncogene. These solitary intervening sites insulate TLX3 from the enhancer by inducing competitive looping to multiple binding sites near the TLX3 promoter. Reduced CTCF levels or deletion of the intervening CTCF site abrogates enhancer insulation by weakening competitive looping while favoring TLX3 promoter to BCL11B enhancer looping, which elevates oncogene expression levels and leukemia burden.

Keywords:  BCL11B; CP: Cancer; CTCF mutation; T cell acute lymphoblastic leukemia; TLX3; competitive chromatin looping; enhancer insulation; oncogene regulation

DOI:  https://doi.org/10.1016/j.celrep.2023.112373
Genome Med. 2023 Apr 13. 15(1): 24

Stalled oligodendrocyte differentiation in IDH-mutant gliomas.

Yanfei Wei, Guanzhang Li, Jing Feng, Fan Wu, Zheng Zhao, Zhaoshi Bao, Wei Zhang, Xiaodong Su, Jiuyi Li, Xueling Qi, Zejun Duan, Yunqiu Zhang, Sandra Ferreyra Vega, Asgeir Store Jakola, Yingyu Sun, Helena Carén, Tao Jiang, Xiaolong Fan.

BACKGROUND: Roughly 50% of adult gliomas harbor isocitrate dehydrogenase (IDH) mutations. According to the 2021 WHO classification guideline, these gliomas are diagnosed as astrocytomas, harboring no 1p19q co-deletion, or oligodendrogliomas, harboring 1p19q co-deletion. Recent studies report that IDH-mutant gliomas share a common developmental hierarchy. However, the neural lineages and differentiation stages in IDH-mutant gliomas remain inadequately characterized.
METHODS: Using bulk transcriptomes and single-cell transcriptomes, we identified genes enriched in IDH-mutant gliomas with or without 1p19q co-deletion, we also assessed the expression pattern of stage-specific signatures and key regulators of oligodendrocyte lineage differentiation. We compared the expression of oligodendrocyte lineage stage-specific markers between quiescent and proliferating malignant single cells. The gene expression profiles were validated using RNAscope analysis and myelin staining and were further substantiated using data of DNA methylation and single-cell ATAC-seq. As a control, we assessed the expression pattern of astrocyte lineage markers.
RESULTS: Genes concordantly enriched in both subtypes of IDH-mutant gliomas are upregulated in oligodendrocyte progenitor cells (OPC). Signatures of early stages of oligodendrocyte lineage and key regulators of OPC specification and maintenance are enriched in all IDH-mutant gliomas. In contrast, signature of myelin-forming oligodendrocytes, myelination regulators, and myelin components are significantly down-regulated or absent in IDH-mutant gliomas. Further, single-cell transcriptomes of IDH-mutant gliomas are similar to OPC and differentiation-committed oligodendrocyte progenitors, but not to myelinating oligodendrocyte. Most IDH-mutant glioma cells are quiescent; quiescent cells and proliferating cells resemble the same differentiation stage of oligodendrocyte lineage. Mirroring the gene expression profiles along the oligodendrocyte lineage, analyses of DNA methylation and single-cell ATAC-seq data demonstrate that genes of myelination regulators and myelin components are hypermethylated and show inaccessible chromatin status, whereas regulators of OPC specification and maintenance are hypomethylated and show open chromatin status. Markers of astrocyte precursors are not enriched in IDH-mutant gliomas.
CONCLUSIONS: Our studies show that despite differences in clinical manifestation and genomic alterations, all IDH-mutant gliomas resemble early stages of oligodendrocyte lineage and are stalled in oligodendrocyte differentiation due to blocked myelination program. These findings provide a framework to accommodate biological features and therapy development for IDH-mutant gliomas.

DOI: https://doi.org/10.1186/s13073-023-01175-6
Cell Rep. 2023 Apr 13. pii: S2211-1247(23)00412-6. [Epub ahead of print]42(4): 112401

Cell-to-cell variability in Myc dynamics drives transcriptional heterogeneity in cancer cells.

Chad Liu, Takamasa Kudo, Xin Ye, Karen Gascoigne.

  Cell-to-cell heterogeneity is vital for tumor evolution and survival. How cancer cells achieve and exploit this heterogeneity remains an active area of research. Here, we identify c-Myc as a highly heterogeneously expressed transcription factor and an orchestrator of transcriptional and phenotypic diversity in cancer cells. By monitoring endogenous c-Myc protein in individual living cells, we report the surprising pulsatile nature of c-Myc expression and the extensive cell-to-cell variability in its dynamics. We further show that heterogeneity in c-Myc dynamics leads to variable target gene transcription and that timing of c-Myc expression predicts cell-cycle progression rates and drug sensitivities. Together, our data advocate for a model in which cancer cells increase the heterogeneity of functionally diverse transcription factors such as c-Myc to rapidly survey transcriptional landscapes and survive stress.

Keywords:  CP: Cancer; CP: Cell biology; Myc; cancer; cell-to-cell heterogeneity; gene expression; transcription

DOI:  https://doi.org/10.1016/j.celrep.2023.112401
Development. 2023 Apr 11. pii: dev.200500. [Epub ahead of print]

Hemato-vascular specification requires arnt1 and arnt2 genes in zebrafish embryos.

Hailey E Edwards, Mary Jane Edgington, Jaclyn P Souder, Daniel A Gorelick.

  During embryonic development, a subset of cells in the mesoderm germ layer are specified as hemato-vascular progenitor cells, which then differentiate into endothelial cells and hematopoietic stem and progenitor cells. In zebrafish, the transcription factor npas4l (cloche) is required for the specification of hemato-vascular progenitor cells. However, it is unclear if npas4l is the sole factor at the top of the hemato-vascular specification cascade. Here we show that arnt1 and arnt2 genes are required for hemato-vascular specification. We found that arnt1;arnt2 double mutant zebrafish embryos, but not arnt1 or arnt2 single mutants, lack blood cells and most endothelial cells. arnt1/2 mutants have reduced or absent expression of etsrp and tal1, the earliest known endothelial and hematopoietic transcription factor genes. We found that Npas4l binds both Arnt1 and Arnt2 proteins in vitro, consistent with the idea that PAS domain-containing bHLH transcription factors act in a multimeric complex to regulate gene expression. Our results demonstrate that npas4l, arnt1 and arnt2 act together to regulate endothelial and hematopoietic cell fate, where each gene is necessary, but not sufficient, to drive hemato-vascular specification.

Keywords:  Arnt; Hematopoiesis; Hematovascular specification; Zebrafish; npas4l

DOI:  https://doi.org/10.1242/dev.200500
Gastroenterology. 2023 Apr 12. pii: S0016-5085(23)00592-9. [Epub ahead of print]

The epigenetic reader, BRD2, mediates cholangiocyte senescence via interaction with ETS1.

Jeong-Han Kang, Patrick L Splinter, Christy E Trussoni, Nicholas E Pirius, Gregory J Gores, Nicholas F LaRusso, Steven P O'Hara.

   BACKGROUND & AIMS: We reported that cholangiocyte senescence, regulated by the transcription factor, ETS1, is a pathogenic feature of Primary Sclerosing Cholangitis (PSC). Furthermore, Histone 3 Lysine 27 is acetylated at senescence-associated loci. The epigenetic readers, bromodomain and extra-terminal domain (BET) proteins, bind acetylated histones, recruit transcription factors, and drive gene expression. Thus, we tested the hypothesis that BET proteins interact with ETS1 to drive gene expression and cholangiocyte senescence.
METHODS: We performed immunofluorescence for BET proteins (BRD2 and 4) in PSC patient and mouse model liver tissue. Using Normal Human Cholangiocytes (NHC), NHC experimentally induced to senescence (NHCsen), and PSC patient-derived cholangiocytes (PSCDCs), we assessed senescence, fibroinflammatory secretome, and apoptosis following BET inhibition or RNAi depletion. We assessed BET interaction with ETS1 in NHCsen and PSC patient tissues, and the effects of BET inhibitors on liver fibrosis, senescence, and inflammatory gene expression in mouse models.
RESULTS: PSC patient and mouse model tissue exhibited increased cholangiocyte BRD2 and 4 protein (∼5x) compared to non-disease controls. NHCsen exhibited increased BRD2 and 4 (∼2x), while PSCDCs exhibited increased BRD2 protein (∼2x) relative to NHC. BET inhibition in NHCsen and PSCDCs reduced senescence markers and inhibited the fibroinflammatory secretome. ETS1 interacted with BRD2 in NHCsen and BRD2 depletion diminished NHCsen p21 expression. BET inhibitors reduced senescence, fibroinflammatory gene expression, and fibrosis in the DDC-fed and Mdr2-/- mouse models.
CONCLUSION: Our data suggest that BRD2 is an essential mediator of the senescent cholangiocyte phenotype and is a potential therapeutic target for patients with PSC.

Keywords:  BET proteins; Epigenetics; Primary sclerosing cholangitis (PSC); Senescence

DOI:  https://doi.org/10.1053/j.gastro.2023.03.235
Nat Cancer. 2023 Apr 10.

Landscape of prostate-specific membrane antigen heterogeneity and regulation in AR-positive and AR-negative metastatic prostate cancer.

Martin K Bakht, Yasutaka Yamada, Sheng-Yu Ku, Varadha Balaji Venkadakrishnan, Joshua A Korsen, Teja M Kalidindi, Kei Mizuno, Shin Hye Ahn, Ji-Heui Seo, Maria Mica Garcia, Francesca Khani, Olivier Elemento, Henry W Long, Alain Chaglassian, Nagavarakishore Pillarsetty, Jason S Lewis, Matthew Freedman, Anthony P Belanger, Quang-De Nguyen, Himisha Beltran.

Tumor expression of prostate-specific membrane antigen (PSMA) is lost in 15-20% of men with castration-resistant prostate cancer (CRPC), yet the underlying mechanisms remain poorly defined. In androgen receptor (AR)-positive CRPC, we observed lower PSMA expression in liver lesions versus other sites, suggesting a role of the microenvironment in modulating PSMA. PSMA suppression was associated with promoter histone 3 lysine 27 methylation and higher levels of neutral amino acid transporters, correlating with 18F-fluciclovine uptake on positron emission tomography imaging. While PSMA is regulated by AR, we identified a subset of AR-negative CRPC with high PSMA. HOXB13 and AR co-occupancy at the PSMA enhancer and knockout models point to HOXB13 as an upstream regulator of PSMA in AR-positive and AR-negative prostate cancer. These data demonstrate how PSMA expression is differentially regulated across metastatic lesions and in the context of the AR, which may inform selection for PSMA-targeted therapies and development of complementary biomarkers.

DOI: https://doi.org/10.1038/s43018-023-00539-6
Proc Natl Acad Sci U S A. 2023 Apr 18. 120(16): e2210418120

ABL kinases regulate the stabilization of HIF-1α and MYC through CPSF1.

Benjamin Mayro, Jacob P Hoj, Christian G Cerda-Smith, Haley M Hutchinson, Michael W Caminear, Hannah L Thrash, Peter S Winter, Suzanne E Wardell, Donald P McDonnell, Colleen Wu, Kris C Wood, Ann Marie Pendergast.

  The hypoxia-inducible factor 1-α (HIF-1α) enables cells to adapt and respond to hypoxia (Hx), and the activity of this transcription factor is regulated by several oncogenic signals and cellular stressors. While the pathways controlling normoxic degradation of HIF-1α are well understood, the mechanisms supporting the sustained stabilization and activity of HIF-1α under Hx are less clear. We report that ABL kinase activity protects HIF-1α from proteasomal degradation during Hx. Using a fluorescence-activated cell sorting (FACS)-based CRISPR/Cas9 screen, we identified HIF-1α as a substrate of the cleavage and polyadenylation specificity factor-1 (CPSF1), an E3-ligase which targets HIF-1α for degradation in the presence of an ABL kinase inhibitor in Hx. We show that ABL kinases phosphorylate and interact with CUL4A, a cullin ring ligase adaptor, and compete with CPSF1 for CUL4A binding, leading to increased HIF-1α protein levels. Further, we identified the MYC proto-oncogene protein as a second CPSF1 substrate and show that active ABL kinase protects MYC from CPSF1-mediated degradation. These studies uncover a role for CPSF1 in cancer pathobiology as an E3-ligase antagonizing the expression of the oncogenic transcription factors, HIF-1α and MYC.

Keywords:  ABL kinases; CPSF1; E3-ligase; HIF-1α; MYC

DOI:  https://doi.org/10.1073/pnas.2210418120
Commun Biol. 2023 Apr 14. 6(1): 410

A multi-omics integrative analysis based on CRISPR screens re-defines the pluripotency regulatory network in ESCs.

Yan Ruan, Jiaqi Wang, Meng Yu, Fengsheng Wang, Jiangjun Wang, Yixiao Xu, Lianlian Liu, Yuda Cheng, Ran Yang, Chen Zhang, Yi Yang, JiaLi Wang, Wei Wu, Yi Huang, Yanping Tian, Guangxing Chen, Junlei Zhang, Rui Jian.

A comprehensive and precise definition of the pluripotency gene regulatory network (PGRN) is crucial for clarifying the regulatory mechanisms in embryonic stem cells (ESCs). Here, after a CRISPR/Cas9-based functional genomics screen and integrative analysis with other functional genomes, transcriptomes, proteomes and epigenome data, an expanded pluripotency-associated gene set is obtained, and a new PGRN with nine sub-classes is constructed. By integrating the DNA binding, epigenetic modification, chromatin conformation, and RNA expression profiles, the PGRN is resolved to six functionally independent transcriptional modules (CORE, MYC, PAF, PRC, PCGF and TBX). Spatiotemporal transcriptomics reveal activated CORE/MYC/PAF module activity and repressed PRC/PCGF/TBX module activity in both mouse ESCs (mESCs) and pluripotent cells of early embryos. Moreover, this module activity pattern is found to be shared by human ESCs (hESCs) and cancers. Thus, our results provide novel insights into elucidating the molecular basis of ESC pluripotency.

DOI: https://doi.org/10.1038/s42003-023-04700-w
Commun Biol. 2023 04 08. 6(1): 382

High-resolution Nanopore methylome-maps reveal random hyper-methylation at CpG-poor regions as driver of chemoresistance in leukemias.

Alberto Magi, Gianluca Mattei, Alessandra Mingrino, Chiara Caprioli, Chiara Ronchini, GianMaria Frigè, Roberto Semeraro, Davide Bolognini, Alessandro Rambaldi, Anna Candoni, Emanuela Colombo, Luca Mazzarella, Pier Giuseppe Pelicci.

Aberrant DNA methylation at CpG dinucleotides is a cancer hallmark that is associated with the emergence of resistance to anti cancer treatment, though molecular mechanisms and biological significance remain elusive. Genome scale methylation maps by currently used methods are based on chemical modification of DNA and are best suited for analyses of methylation at CpG rich regions (CpG islands). We report the first high coverage whole-genome map in cancer using the long read nanopore technology, which allows simultaneous DNA-sequence and -methylation analyses on native DNA. We analyzed clonal epigenomic/genomic evolution in Acute Myeloid Leukemias (AMLs) at diagnosis and relapse, after chemotherapy. Long read sequencing coupled to a novel computational method allowed definition of differential methylation at unprecedented resolution, and showed that the relapse methylome is characterized by hypermethylation at both CpG islands and sparse CpGs regions. Most differentially methylated genes, however, were not differentially expressed nor enriched for chemoresistance genes. A small fraction of under-expressed and hyper-methylated genes at sparse CpGs, in the gene body, was significantly enriched in transcription factors (TFs). Remarkably, these few TFs supported large gene-regulatory networks including 50% of all differentially expressed genes in the relapsed AMLs and highly-enriched in chemoresistance genes. Notably, hypermethylated regions at sparse CpGs were poorly conserved in the relapsed AMLs, under-represented at their genomic positions and showed higher methylation entropy, as compared to CpG islands. Analyses of available datasets confirmed TF binding to their target genes and conservation of the same gene-regulatory networks in large patient cohorts. Relapsed AMLs carried few patient specific structural variants and DNA mutations, apparently not involved in drug resistance. Thus, drug resistance in AMLs can be mainly ascribed to the selection of random epigenetic alterations at sparse CpGs of a few transcription factors, which then induce reprogramming of the relapsing phenotype, independently of clonal genomic evolution.

DOI: https://doi.org/10.1038/s42003-023-04756-8
PLoS Genet. 2023 Apr 14. 19(4): e1010705

Fob1-dependent condensin recruitment and loop extrusion on yeast chromosome III.

Manikarna Dinda, Ryan D Fine, Shekhar Saha, Zhenjia Wang, Chongzhi Zang, Mingguang Li, Jeffrey S Smith.

Despite recent advances in single-molecule and structural analysis of condensin activity in vitro, mechanisms of functional condensin loading and loop extrusion that lead to specific chromosomal organization remain unclear. In Saccharomyces cerevisiae, the most prominent condensin loading site is the rDNA locus on chromosome XII, but its repetitiveness deters rigorous analysis of individual genes. An equally prominent non-rDNA condensin site is located on chromosome III (chrIII). It lies in the promoter of a putative non-coding RNA gene called RDT1, which is in a segment of the recombination enhancer (RE) that dictates MATa-specific chrIII organization. Here, we unexpectedly find that condensin is recruited to the RDT1 promoter in MATa cells through hierarchical interactions with Fob1, Tof2, and cohibin (Lrs4/Csm1), a set of nucleolar factors that also recruit condensin to the rDNA. Fob1 directly binds to this locus in vitro, while its binding in vivo depends on an adjacent Mcm1/α2 binding site that provides MATa cell specificity. We also uncover evidence for condensin-driven loop extrusion anchored by Fob1 and cohibin at RDT1 that unidirectionally extends toward MATa on the right arm of chrIII, supporting donor preference during mating-type switching. S. cerevisiae chrIII therefore provides a new platform for the study of programmed condensin-mediated chromosome conformation.

DOI: https://doi.org/10.1371/journal.pgen.1010705
Elife. 2023 Apr 13. pii: e81717. [Epub ahead of print]12

PASK links cellular energy metabolism with a mitotic self-renewal network to establish differentiation competence.

Michael Xiao, Chia-Hua Wu, Graham Meek, Brian Kelly, Dara Buendia Castillo, Lyndsay E A Young, Sara Martire, Sajina Dhungel, Elizabeth McCauley, Purbita Saha, Altair L Dube, Matthew S Gentry, Laura A Banaszynski, Ramon C Sun, Chintan K Kikani.

  Quiescent stem cells are activated in response to a mechanical or chemical injury to their tissue niche. Activated cells rapidly generate a heterogeneous progenitor population that regenerates the damaged tissues. While the transcriptional cadence that generates heterogeneity is known, the metabolic pathways influencing the transcriptional machinery to establish a heterogeneous progenitor population remains unclear. Here, we describe a novel pathway downstream of mitochondrial glutamine metabolism that confers stem cell heterogeneity and establishes differentiation competence by countering post-mitotic self-renewal machinery. We discovered that mitochondrial glutamine metabolism induces CBP/EP300-dependent acetylation of stem cell-specific kinase, PASK, resulting in its release from cytoplasmic granules and subsequent nuclear migration. In the nucleus, PASK catalytically outcompetes mitotic WDR5-anaphase-promoting complex/cyclosome (APC/C) interaction resulting in the loss of post-mitotic Pax7 expression and exit from self-renewal. In concordance with these findings, genetic or pharmacological inhibition of PASK or glutamine metabolism upregulated Pax7 expression, reduced stem cell heterogeneity, and blocked myogenesis in vitro and muscle regeneration in mice. These results explain a mechanism whereby stem cells co-opt the proliferative functions of glutamine metabolism to generate transcriptional heterogeneity and establish differentiation competence by countering the mitotic self-renewal network via nuclear PASK.

Keywords:  cell biology; human; mouse

DOI:  https://doi.org/10.7554/eLife.81717
Nat Commun. 2023 Apr 08. 14(1): 1980

Endothelial FAT1 inhibits angiogenesis by controlling YAP/TAZ protein degradation via E3 ligase MIB2.

Rui Li, Jingchen Shao, Young-June Jin, Haruya Kawase, Yu Ting Ong, Kerstin Troidl, Qi Quan, Lei Wang, Remy Bonnavion, Astrid Wietelmann, Francoise Helmbacher, Michael Potente, Johannes Graumann, Nina Wettschureck, Stefan Offermanns.

Activation of endothelial YAP/TAZ signaling is crucial for physiological and pathological angiogenesis. The mechanisms of endothelial YAP/TAZ regulation are, however, incompletely understood. Here we report that the protocadherin FAT1 acts as a critical upstream regulator of endothelial YAP/TAZ which limits the activity of these transcriptional cofactors during developmental and tumor angiogenesis by promoting their degradation. We show that loss of endothelial FAT1 results in increased endothelial cell proliferation in vitro and in various angiogenesis models in vivo. This effect is due to perturbed YAP/TAZ protein degradation, leading to increased YAP/TAZ protein levels and expression of canonical YAP/TAZ target genes. We identify the E3 ubiquitin ligase Mind Bomb-2 (MIB2) as a FAT1-interacting protein mediating FAT1-induced YAP/TAZ ubiquitination and degradation. Loss of MIB2 expression in endothelial cells in vitro and in vivo recapitulates the effects of FAT1 depletion and causes decreased YAP/TAZ degradation and increased YAP/TAZ signaling. Our data identify a pivotal mechanism of YAP/TAZ regulation involving FAT1 and its associated E3 ligase MIB2, which is essential for YAP/TAZ-dependent angiogenesis.

DOI: https://doi.org/10.1038/s41467-023-37671-x
Nat Commun. 2023 Apr 13. 14(1): 2095

Histone 4 lysine 5/12 acetylation enables developmental plasticity of Pristionchus mouth form.

Michael S Werner, Tobias Loschko, Thomas King, Shelley Reich, Tobias Theska, Mirita Franz-Wachtel, Boris Macek, Ralf J Sommer.

Development can be altered to match phenotypes with the environment, and the genetic mechanisms that direct such alternative phenotypes are beginning to be elucidated. Yet, the rules that govern environmental sensitivity vs. invariant development, and potential epigenetic memory, remain unknown. Here, we show that plasticity of nematode mouth forms is determined by histone 4 lysine 5 and 12 acetylation (H4K5/12ac). Acetylation in early larval stages provides a permissive chromatin state, which is susceptible to induction during the critical window of environmental sensitivity. As development proceeds deacetylation shuts off switch gene expression to end the critical period. Inhibiting deacetylase enzymes leads to fixation of prior developmental trajectories, demonstrating that histone modifications in juveniles can carry environmental information to adults. Finally, we provide evidence that this regulation was derived from an ancient mechanism of licensing developmental speed. Altogether, our results show that H4K5/12ac enables epigenetic regulation of developmental plasticity that can be stored and erased by acetylation and deacetylation, respectively.

DOI: https://doi.org/10.1038/s41467-023-37734-z
Mol Cancer. 2023 04 10. 22(1): 69

Super-enhancer-driven TOX2 mediates oncogenesis in Natural Killer/T Cell Lymphoma.

Jianbiao Zhou, Sabrina Hui-Min Toh, Tze King Tan, Kalpnaa Balan, Jing Quan Lim, Tuan Zea Tan, Sinan Xiong, Yunlu Jia, Siok-Bian Ng, Yanfen Peng, Anand D Jeyasekharan, Shuangyi Fan, Soon Thye Lim, Chin-Ann Johnny Ong, Choon Kiat Ong, Takaomi Sanda, Wee-Joo Chng.

   BACKGROUND: Extranodal natural killer/T-cell lymphoma (NKTL) is an aggressive type of non-Hodgkin lymphoma with dismal outcome. A better understanding of disease biology and key oncogenic process is necessary for the development of targeted therapy. Super-enhancers (SEs) have been shown to drive pivotal oncogenes in various malignancies. However, the landscape of SEs and SE-associated oncogenes remain elusive in NKTL.
METHODS: We used Nano-ChIP-seq of the active enhancer marker histone H3 lysine 27 acetylation (H3K27ac) to profile unique SEs NKTL primary tumor samples. Integrative analysis of RNA-seq and survival data further pinned down high value, novel SE oncogenes. We utilized shRNA knockdown, CRISPR-dCas9, luciferase reporter assay, ChIP-PCR to investigate the regulation of transcription factor (TF) on SE oncogenes. Multi-color immunofluorescence (mIF) staining was performed on an independent cohort of clinical samples. Various function experiments were performed to evaluate the effects of TOX2 on the malignancy of NKTL in vitro and in vivo.
RESULTS: SE landscape was substantially different in NKTL samples in comparison with normal tonsils. Several SEs at key transcriptional factor (TF) genes, including TOX2, TBX21(T-bet), EOMES, RUNX2, and ID2, were identified. We confirmed that TOX2 was aberrantly overexpressed in NKTL relative to normal NK cells and high expression of TOX2 was associated with worse survival. Modulation of TOX2 expression by shRNA, CRISPR-dCas9 interference of SE function impacted on cell proliferation, survival and colony formation ability of NKTL cells. Mechanistically, we found that RUNX3 regulates TOX2 transcription by binding to the active elements of its SE. Silencing TOX2 also impaired tumor formation of NKTL cells in vivo. Metastasis-associated phosphatase PRL-3 has been identified and validated as a key downstream effector of TOX2-mediated oncogenesis.
CONCLUSIONS: Our integrative SE profiling strategy revealed the landscape of SEs, novel targets and insights into molecular pathogenesis of NKTL. The RUNX3-TOX2-SE-TOX2-PRL-3 regulatory pathway may represent a hallmark of NKTL biology. Targeting TOX2 could be a valuable therapeutic intervene for NKTL patients and warrants further study in clinic.

Keywords:  Epigenetics; Natural Killer/T Cell Lymphoma; PRL-3; RUNX3; Super-enhancer; TOX2; Therapeutic targets

DOI:  https://doi.org/10.1186/s12943-023-01767-1
Nature. 2023 Apr 12.

Extrachromosomal DNA in the cancerous transformation of Barrett's oesophagus.

Jens Luebeck, Alvin Wei Tian Ng, Patricia C Galipeau, Xiaohong Li, Carissa A Sanchez, Annalise C Katz-Summercorn, Hoon Kim, Sriganesh Jammula, Yudou He, Scott M Lippman, Roel G W Verhaak, Carlo C Maley, Ludmil B Alexandrov, Brian J Reid, Rebecca C Fitzgerald, Thomas G Paulson, Howard Y Chang, Sihan Wu, Vineet Bafna, Paul S Mischel.

Oncogene amplification on extrachromosomal DNA (ecDNA) drives the evolution of tumours and their resistance to treatment, and is associated with poor outcomes for patients with cancer1-6. At present, it is unclear whether ecDNA is a later manifestation of genomic instability, or whether it can be an early event in the transition from dysplasia to cancer. Here, to better understand the development of ecDNA, we analysed whole-genome sequencing (WGS) data from patients with oesophageal ademocarcinoma (EAC) or Barrett's oesophagus. These data included 206 biopsies in Barrett's oesophagus surveillance and EAC cohorts from Cambridge University. We also analysed WGS and histology data from biopsies that were collected across multiple regions at 2 time points from 80 patients in a case-control study at the Fred Hutchinson Cancer Center. In the Cambridge cohorts, the frequency of ecDNA increased between Barrett's-oesophagus-associated early-stage (24%) and late-stage (43%) EAC, suggesting that ecDNA is formed during cancer progression. In the cohort from the Fred Hutchinson Cancer Center, 33% of patients who developed EAC had at least one oesophageal biopsy with ecDNA before or at the diagnosis of EAC. In biopsies that were collected before cancer diagnosis, higher levels of ecDNA were present in samples from patients who later developed EAC than in samples from those who did not. We found that ecDNAs contained diverse collections of oncogenes and immunomodulatory genes. Furthermore, ecDNAs showed increases in copy number and structural complexity at more advanced stages of disease. Our findings show that ecDNA can develop early in the transition from high-grade dysplasia to cancer, and that ecDNAs progressively form and evolve under positive selection.

DOI: https://doi.org/10.1038/s41586-023-05937-5