bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2022–11–13
24 papers selected by
Connor Rogerson, University of Cambridge
  1. Hi-TrAC reveals division of labor of transcription factors in organizing chromatin loops.
  2. Mcm2 promotes stem cell differentiation via its ability to bind H3-H4.
  3. Transcription factor antagonism regulates heterogeneity in embryonic stem cell states.
  4. Systematic discovery and functional dissection of enhancers needed for cancer cell fitness and proliferation.
  5. CTCF blocks antisense transcription initiation at divergent promoters.
  6. A global high-density chromatin interaction network reveals functional long-range and trans-chromosomal relationships.
  7. The assembly of mammalian SWI/SNF chromatin remodeling complexes is regulated by lysine-methylation dependent proteolysis.
  8. Endocrine resistance and breast cancer plasticity are controlled by CoREST.
  9. Learning the histone codes with large genomic windows and three-dimensional chromatin interactions using transformer.
  10. FRET-FISH probes chromatin compaction at individual genomic loci in single cells.
  11. Histone H3 proline 16 hydroxylation regulates mammalian gene expression.
  12. dcHiC detects differential compartments across multiple Hi-C datasets.
  13. Reversible Myc hypomorphism identifies a key Myc-dependency in early cancer evolution.
  14. Pharmacological blockade of TEAD-YAP reveals its therapeutic limitation in cancer cells.
  15. Loss of neuronal Tet2 enhances hippocampal-dependent cognitive function.
  16. The bromodomain protein TRIM28 controls the balance between growth and invasiveness in melanoma.
  17. A new cut&run low volume-urea (LoV-U) protocol optimized for transcriptional co-factors uncovers Wnt/b-catenin tissue-specific genomic targets.
  18. Nucleoporins facilitate ORC loading onto chromatin.
  19. Deep learning-assisted genome-wide characterization of massively parallel reporter assays.
  20. Increased chromatin accessibility facilitates intron retention in specific cell differentiation states.
  21. Paraspeckles interact with SWI/SNF subunit ARID1B to regulate transcription and splicing.
  22. ZBTB2 links p53 deficiency to HIF-1-mediated hypoxia signaling to promote cancer aggressiveness.
  23. EpiFactors 2022: expansion and enhancement of a curated database of human epigenetic factors and complexes.
  24. Stromal FOXF2 suppresses prostate cancer progression and metastasis by enhancing antitumor immunity.
  1. Nat Commun. 2022 Nov 05. 13(1): 6679
    Hi-TrAC reveals division of labor of transcription factors in organizing chromatin loops.
    Shuai Liu, Yaqiang Cao, Kairong Cui, Qingsong Tang, Keji Zhao.
      The three-dimensional genomic structure plays a critical role in gene expression, cellular differentiation, and pathological conditions. It is pivotal to elucidate fine-scale chromatin architectures, especially interactions of regulatory elements, to understand the temporospatial regulation of gene expression. In this study, we report Hi-TrAC as a proximity ligation-free, robust, and sensitive technique to profile genome-wide chromatin interactions at high-resolution among regulatory elements. Hi-TrAC detects chromatin looping among accessible regions at single nucleosome resolution. With almost half-million identified loops, we reveal a comprehensive interaction network of regulatory elements across the genome. After integrating chromatin binding profiles of transcription factors, we discover that cohesin complex and CTCF are responsible for organizing long-range chromatin loops, related to domain formation; whereas ZNF143 and HCFC1 are involved in structuring short-range chromatin loops between regulatory elements, which directly regulate gene expression. Thus, we introduce a methodology to identify a delicate and comprehensive network of cis-regulatory elements, revealing the complexity and a division of labor of transcription factors in organizing chromatin loops for genome organization and gene expression.
    DOI:  https://doi.org/10.1038/s41467-022-34276-8
  2. Elife. 2022 Nov 10. pii: e80917. [Epub ahead of print]11
    Mcm2 promotes stem cell differentiation via its ability to bind H3-H4.
    Xiaowei Xu, Xu Hua, Kyle Brown, Xiaojun Ren, Zhiguo Zhang.
      Mcm2, a subunit of the Mcm2-7 helicase best known for its role in DNA replication, contains a histone binding motif that facilitates the transfer of parental histones following DNA replication. Here we show that Mcm2 is important for the differentiation of mouse embryonic stem (ES) cells. The Mcm2-2A mutation defective in histone binding shows defects in silencing of pluripotent genes and the induction of lineage specific genes. The defects in the induction of lineage specific genes in the mutant cells are likely, at least in part, due to reduced binding to Asf1a, a histone chaperone that binds Mcm2 and is important for nucleosome disassembly at bivalent chromatin domains containing repressive H3K27me3 and active H3K4me3 modifications during differentiation. Mcm2 localizes at transcription starting sites and the binding of Mcm2 at gene promoters is disrupted in both Mcm2-2A ES cells and neuro-precursor cells (NPCs). Reduced Mcm2 binding at bivalent chromatin domains in Mcm2-2A ES cells correlates with decreased chromatin accessibility at corresponding sites in NPCs. Together, our studies reveal a novel function of Mcm2 in ES cell differentiation, likely through manipulating chromatin landscapes at bivalent chromatin domains.
    Keywords:  chromosomes; gene expression; mouse
    DOI:  https://doi.org/10.7554/eLife.80917
  3. Mol Cell. 2022 Nov 04. pii: S1097-2765(22)01022-X. [Epub ahead of print]
    Transcription factor antagonism regulates heterogeneity in embryonic stem cell states.
    Sofia Hu, Emily Metcalf, Dig Bijay Mahat, Lynette Chan, Noor Sohal, Meenakshi Chakraborty, Maxwell Hamilton, Arundeep Singh, Abhyudai Singh, Jacqueline A Lees, Phillip A Sharp, Salil Garg.
      Gene expression heterogeneity underlies cell states and contributes to developmental robustness. While heterogeneity can arise from stochastic transcriptional processes, the extent to which it is regulated is unclear. Here, we characterize the regulatory program underlying heterogeneity in murine embryonic stem cell (mESC) states. We identify differentially active and transcribed enhancers (DATEs) across states. DATEs regulate differentially expressed genes and are distinguished by co-binding of transcription factors Klf4 and Zfp281. In contrast to other factors that interact in a positive feedback network stabilizing mESC cell-type identity, Klf4 and Zfp281 drive opposing transcriptional and chromatin programs. Abrogation of factor binding to DATEs dampens variation in gene expression, and factor loss alters kinetics of switching between states. These results show antagonism between factors at enhancers results in gene expression heterogeneity and formation of cell states, with implications for the generation of diverse cell types during development.
    Keywords:  Klf4; Zfp281; cell-to-cell variation; embryonic stem cell state; enhancer RNA; gene expression variation; transcription factors
    DOI:  https://doi.org/10.1016/j.molcel.2022.10.022
  4. Cell Rep. 2022 Nov 08. pii: S2211-1247(22)01501-7. [Epub ahead of print]41(6): 111630
    Systematic discovery and functional dissection of enhancers needed for cancer cell fitness and proliferation.
    Poshen B Chen, Patrick C Fiaux, Kai Zhang, Bin Li, Naoki Kubo, Shan Jiang, Rong Hu, Emma Rooholfada, Sihan Wu, Mengchi Wang, Wei Wang, Graham McVicker, Paul S Mischel, Bing Ren.
      A scarcity of functionally validated enhancers in the human genome presents a significant hurdle to understanding how these cis-regulatory elements contribute to human diseases. We carry out highly multiplexed CRISPR-based perturbation and sequencing to identify enhancers required for cell proliferation and fitness in 10 human cancer cell lines. Our results suggest that the cell fitness enhancers, unlike their target genes, display high cell-type specificity of chromatin features. They typically adopt a modular structure, comprised of activating elements enriched for motifs of oncogenic transcription factors, surrounded by repressive elements enriched for motifs recognized by transcription factors with tumor suppressor functions. We further identify cell fitness enhancers that are selectively accessible in clinical tumor samples, and the levels of chromatin accessibility are associated with patient survival. These results reveal functional enhancers across multiple cancer cell lines, characterize their context-dependent chromatin organization, and yield insights into altered transcription programs in cancer cells.
    Keywords:  CP: Cancer; CP: Molecular biology; cell proliferation; functional enhancer; gene regulation; oncogene
    DOI:  https://doi.org/10.1016/j.celrep.2022.111630
  5. Nat Struct Mol Biol. 2022 Nov 11.
    CTCF blocks antisense transcription initiation at divergent promoters.
    Jing Luan, Marit W Vermunt, Camille M Syrett, Allison Coté, Jacob M Tome, Haoyue Zhang, Anran Huang, Jennifer M Luppino, Cheryl A Keller, Belinda M Giardine, Shiping Zhang, Margaret C Dunagin, Zhe Zhang, Eric F Joyce, John T Lis, Arjun Raj, Ross C Hardison, Gerd A Blobel.
      Transcription at most promoters is divergent, initiating at closely spaced oppositely oriented core promoters to produce sense transcripts along with often unstable upstream antisense transcripts (uasTrx). How antisense transcription is regulated and to what extent it is coordinated with sense transcription is not well understood. Here, by combining acute degradation of the multi-functional transcription factor CTCF and nascent transcription measurements, we find that CTCF specifically suppresses antisense but not sense transcription at hundreds of divergent promoters. Primary transcript RNA-FISH shows that CTCF lowers burst fraction but not burst intensity of uasTrx and that co-bursting of sense and antisense transcripts is disfavored. Genome editing, chromatin conformation studies and high-resolution transcript mapping revealed that precisely positioned CTCF directly suppresses the initiation of uasTrx, in a manner independent of its architectural function. In sum, CTCF shapes the transcriptional landscape in part by suppressing upstream antisense transcription.
    DOI:  https://doi.org/10.1038/s41594-022-00855-y
  6. Genome Biol. 2022 Nov 09. 23(1): 238
    A global high-density chromatin interaction network reveals functional long-range and trans-chromosomal relationships.
    Ruchi Lohia, Nathan Fox, Jesse Gillis.
       BACKGROUND: Chromatin contacts are essential for gene-expression regulation; however, obtaining a high-resolution genome-wide chromatin contact map is still prohibitively expensive owing to large genome sizes and the quadratic scale of pairwise data. Chromosome conformation capture (3C)-based methods such as Hi-C have been extensively used to obtain chromatin contacts. However, since the sparsity of these maps increases with an increase in genomic distance between contacts, long-range or trans-chromatin contacts are especially challenging to sample.
    RESULTS: Here, we create a high-density reference genome-wide chromatin contact map using a meta-analytic approach. We integrate 3600 human, 6700 mouse, and 500 fly Hi-C experiments to create species-specific meta-Hi-C chromatin contact maps with 304 billion, 193 billion, and 19 billion contacts in respective species. We validate that meta-Hi-C contact maps are uniquely powered to capture functional chromatin contacts in both cis and trans. We find that while individual dataset Hi-C networks are largely unable to predict any long-range coexpression (median 0.54 AUC), meta-Hi-C networks perform comparably in both cis and trans (0.65 AUC vs 0.64 AUC). Similarly, for long-range expression quantitative trait loci (eQTL), meta-Hi-C contacts outperform all individual Hi-C experiments, providing an improvement over the conventionally used linear genomic distance-based association. Assessing between species, we find patterns of chromatin contact conservation in both cis and trans and strong associations with coexpression even in species for which Hi-C data is lacking.
    CONCLUSIONS: We have generated an integrated chromatin interaction network which complements a large number of methodological and analytic approaches focused on improved specificity or interpretation. This high-depth "super-experiment" is surprisingly powerful in capturing long-range functional relationships of chromatin interactions, which are now able to predict coexpression, eQTLs, and cross-species relationships. The meta-Hi-C networks are available at https://labshare.cshl.edu/shares/gillislab/resource/HiC/ .
    Keywords:  Coexpression; Hi-C; Meta-analysis; eQTL
    DOI:  https://doi.org/10.1186/s13059-022-02790-z
  7. Nat Commun. 2022 Nov 05. 13(1): 6696
    The assembly of mammalian SWI/SNF chromatin remodeling complexes is regulated by lysine-methylation dependent proteolysis.
    Pengfei Guo, Nam Hoang, Joseph Sanchez, Elaine H Zhang, Keshari Rajawasam, Kristiana Trinidad, Hong Sun, Hui Zhang.
      The assembly of mammalian SWI/SNF chromatin remodeling complexes is developmentally programed, and loss/mutations of SWI/SNF subunits alter the levels of other components through proteolysis, causing cancers. Here, we show that mouse Lsd1/Kdm1a deletion causes dramatic dissolution of SWI/SNF complexes and that LSD1 demethylates the methylated lysine residues in SMARCC1 and SMARCC2 to preserve the structural integrity of SWI/SNF complexes. The methylated SMARCC1/SMARCC2 are targeted for proteolysis by L3MBTL3 and the CRL4DCAF5 ubiquitin ligase complex. We identify SMARCC1 as the critical target of LSD1 and L3MBTL3 to maintain the pluripotency and self-renewal of embryonic stem cells. L3MBTL3 also regulates SMARCC1/SMARCC2 proteolysis induced by the loss of SWI/SNF subunits. Consistently, mouse L3mbtl3 deletion causes striking accumulation of SWI/SNF components, associated with embryonic lethality. Our studies reveal that the assembly/disassembly of SWI/SNF complexes is dynamically controlled by a lysine-methylation dependent proteolytic mechanism to maintain the integrity of the SWI/SNF complexes.
    DOI:  https://doi.org/10.1038/s41467-022-34348-9
  8. Nat Struct Mol Biol. 2022 Nov 07.
    Endocrine resistance and breast cancer plasticity are controlled by CoREST.
    Liliana Garcia-Martinez, Andrew M Adams, Ho Lam Chan, Yuichiro Nakata, Natalia Weich, Stephanie Stransky, Zhao Zhang, Mohamed Alshalalfa, Leonor Sarria, Brandon A Mahal, Susan B Kesmodel, Toni Celià-Terrassa, Zhijie Liu, Saverio Minucci, Daniel Bilbao, Simone Sidoli, Ramiro E Verdun, Lluis Morey.
      Resistance to cancer treatment remains a major clinical hurdle. Here, we demonstrate that the CoREST complex is a key determinant of endocrine resistance and ER+ breast cancer plasticity. In endocrine-sensitive cells, CoREST is recruited to regulatory regions co-bound to ERα and FOXA1 to regulate the estrogen pathway. In contrast, during temporal reprogramming towards a resistant state, CoREST is recruited to AP-1 sites. In reprogrammed cells, CoREST favors chromatin opening, cJUN binding to chromatin, and gene activation by controlling SWI/SNF recruitment independently of the demethylase activity of the CoREST subunit LSD1. Genetic and pharmacological CoREST inhibition reduces tumorigenesis and metastasis of endocrine-sensitive and endocrine-resistant xenograft models. Consistently, CoREST controls a gene signature involved in invasiveness in clinical breast tumors resistant to endocrine therapies. Our studies reveal CoREST functions that are co-opted to drive cellular plasticity and resistance to endocrine therapies and tumorigenesis, thus establishing CoREST as a potential therapeutic target for the treatment of advanced breast cancer.
    DOI:  https://doi.org/10.1038/s41594-022-00856-x
  9. Nat Commun. 2022 Nov 05. 13(1): 6678
    Learning the histone codes with large genomic windows and three-dimensional chromatin interactions using transformer.
    Dohoon Lee, Jeewon Yang, Sun Kim.
      The quantitative characterization of the transcriptional control by histone modifications has been challenged by many computational studies, but most of them only focus on narrow and linear genomic regions around promoters, leaving a room for improvement. We present Chromoformer, a transformer-based, three-dimensional chromatin conformation-aware deep learning architecture that achieves the state-of-the-art performance in the quantitative deciphering of the histone codes in gene regulation. The core essence of Chromoformer architecture lies in the three variants of attention operation, each specialized to model individual hierarchy of transcriptional regulation involving from core promoters to distal elements in contact with promoters through three-dimensional chromatin interactions. In-depth interpretation of Chromoformer reveals that it adaptively utilizes the long-range dependencies between histone modifications associated with transcription initiation and elongation. We also show that the quantitative kinetics of transcription factories and Polycomb group bodies can be captured by Chromoformer. Together, our study highlights the great advantage of attention-based deep modeling of complex interactions in epigenomes.
    DOI:  https://doi.org/10.1038/s41467-022-34152-5
  10. Nat Commun. 2022 Nov 05. 13(1): 6680
    FRET-FISH probes chromatin compaction at individual genomic loci in single cells.
    Ana Mota, Szymon Berezicki, Erik Wernersson, Luuk Harbers, Xiaoze Li-Wang, Katarina Gradin, Christiane Peuckert, Nicola Crosetto, Magda Bienko.
      Chromatin compaction is a key biophysical property that influences multiple DNA transactions. Lack of chromatin accessibility is frequently used as proxy for chromatin compaction. However, we currently lack tools for directly probing chromatin compaction at individual genomic loci. To fill this gap, here we present FRET-FISH, a method combining fluorescence resonance energy transfer (FRET) with DNA fluorescence in situ hybridization (FISH) to probe chromatin compaction at select loci in single cells. We first validate FRET-FISH by comparing it with ATAC-seq, demonstrating that local compaction and accessibility are strongly correlated. FRET-FISH also detects expected differences in compaction upon treatment with drugs perturbing global chromatin condensation. We then leverage FRET-FISH to study local chromatin compaction on the active and inactive X chromosome, along the nuclear radius, in different cell cycle phases, and during increasing passage number. FRET-FISH is a robust tool for probing local chromatin compaction in single cells.
    DOI:  https://doi.org/10.1038/s41467-022-34183-y
  11. Nat Genet. 2022 Nov;54(11): 1721-1735
    Histone H3 proline 16 hydroxylation regulates mammalian gene expression.
    Xijuan Liu, Jun Wang, Joshua A Boyer, Weida Gong, Shuai Zhao, Ling Xie, Qiong Wu, Cheng Zhang, Kanishk Jain, Yiran Guo, Javier Rodriguez, Mingjie Li, Hidetaka Uryu, Chengheng Liao, Lianxin Hu, Jin Zhou, Xiaobing Shi, Yi-Hsuan Tsai, Qin Yan, Weibo Luo, Xian Chen, Brian D Strahl, Alex von Kriegsheim, Qi Zhang, Gang Greg Wang, Albert S Baldwin, Qing Zhang.
      Histone post-translational modifications (PTMs) are important for regulating various DNA-templated processes. Here, we report the existence of a histone PTM in mammalian cells, namely histone H3 with hydroxylation of proline at residue 16 (H3P16oh), which is catalyzed by the proline hydroxylase EGLN2. We show that H3P16oh enhances direct binding of KDM5A to its substrate, histone H3 with trimethylation at the fourth lysine residue (H3K4me3), resulting in enhanced chromatin recruitment of KDM5A and a corresponding decrease of H3K4me3 at target genes. Genome- and transcriptome-wide analyses show that the EGLN2-KDM5A axis regulates target gene expression in mammalian cells. Specifically, our data demonstrate repression of the WNT pathway negative regulator DKK1 through the EGLN2-H3P16oh-KDM5A pathway to promote WNT/β-catenin signaling in triple-negative breast cancer (TNBC). This study characterizes a regulatory mark in the histone code and reveals a role for H3P16oh in regulating mammalian gene expression.
    DOI:  https://doi.org/10.1038/s41588-022-01212-x
  12. Nat Commun. 2022 Nov 11. 13(1): 6827
    dcHiC detects differential compartments across multiple Hi-C datasets.
    Abhijit Chakraborty, Jeffrey G Wang, Ferhat Ay.
      The compartmental organization of mammalian genomes and its changes play important roles in distinct biological processes. Here, we introduce dcHiC, which utilizes a multivariate distance measure to identify significant changes in compartmentalization among multiple contact maps. Evaluating dcHiC on four collections of bulk and single-cell contact maps from in vitro mouse neural differentiation (n = 3), mouse hematopoiesis (n = 10), human LCLs (n = 20) and post-natal mouse brain development (n = 3 stages), we show its effectiveness and sensitivity in detecting biologically relevant changes, including those orthogonally validated. dcHiC reported regions with dynamically regulated genes associated with cell identity, along with correlated changes in chromatin states, subcompartments, replication timing and lamin association. With its efficient implementation, dcHiC enables high-resolution compartment analysis as well as standalone browser visualization, differential interaction identification and time-series clustering. dcHiC is an essential addition to the Hi-C analysis toolbox for the ever-growing number of bulk and single-cell contact maps. Available at: https://github.com/ay-lab/dcHiC .
    DOI:  https://doi.org/10.1038/s41467-022-34626-6
  13. Nat Commun. 2022 Nov 09. 13(1): 6782
    Reversible Myc hypomorphism identifies a key Myc-dependency in early cancer evolution.
    Nicole M Sodir, Luca Pellegrinet, Roderik M Kortlever, Tania Campos, Yong-Won Kwon, Shinseog Kim, Daniel Garcia, Alessandra Perfetto, Panayiotis Anastasiou, Lamorna Brown Swigart, Mark J Arends, Trevor D Littlewood, Gerard I Evan.
      Germ-line hypomorphism of the pleiotropic transcription factor Myc in mice, either through Myc gene haploinsufficiency or deletion of Myc enhancers, delays onset of various cancers while mice remain viable and exhibit only relatively mild pathologies. Using a genetically engineered mouse model in which Myc expression may be systemically and reversibly hypomorphed at will, we asked whether this resistance to tumour progression is also emplaced when Myc hypomorphism is acutely imposed in adult mice. Indeed, adult Myc hypomorphism profoundly blocked KRasG12D-driven lung and pancreatic cancers, arresting their evolution at the early transition from indolent pre-tumour to invasive cancer. We show that such arrest is due to the incapacity of hypomorphic levels of Myc to drive release of signals that instruct the microenvironmental remodelling necessary to support invasive cancer. The cancer protection afforded by long-term adult imposition of Myc hypomorphism is accompanied by only mild collateral side effects, principally in haematopoiesis, but even these are circumvented if Myc hypomorphism is imposed metronomically whereas potent cancer protection is retained.
    DOI:  https://doi.org/10.1038/s41467-022-34079-x
  14. Nat Commun. 2022 Nov 08. 13(1): 6744
    Pharmacological blockade of TEAD-YAP reveals its therapeutic limitation in cancer cells.
    Yang Sun, Lu Hu, Zhipeng Tao, Gopala K Jarugumilli, Hannah Erb, Alka Singh, Qi Li, Jennifer L Cotton, Patricia Greninger, Regina K Egan, Y Tony Ip, Cyril H Benes, Jianwei Che, Junhao Mao, Xu Wu.
      Targeting TEAD autopalmitoylation has been proposed as a therapeutic approach for YAP-dependent cancers. Here we show that TEAD palmitoylation inhibitor MGH-CP1 and analogues block cancer cell "stemness", organ overgrowth and tumor initiation in vitro and in vivo. MGH-CP1 sensitivity correlates significantly with YAP-dependency in a large panel of cancer cell lines. However, TEAD inhibition or YAP/TAZ knockdown leads to transient inhibition of cell cycle progression without inducing cell death, undermining their potential therapeutic utilities. We further reveal that TEAD inhibition or YAP/TAZ silencing leads to VGLL3-mediated transcriptional activation of SOX4/PI3K/AKT signaling axis, which contributes to cancer cell survival and confers therapeutic resistance to TEAD inhibitors. Consistently, combination of TEAD and AKT inhibitors exhibits strong synergy in inducing cancer cell death. Our work characterizes the therapeutic opportunities and limitations of TEAD palmitoylation inhibitors in cancers, and uncovers an intrinsic molecular mechanism, which confers potential therapeutic resistance.
    DOI:  https://doi.org/10.1038/s41467-022-34559-0
  15. Cell Rep. 2022 Nov 08. pii: S2211-1247(22)01481-4. [Epub ahead of print]41(6): 111612
    Loss of neuronal Tet2 enhances hippocampal-dependent cognitive function.
    Karishma J B Pratt, Jeremy M Shea, Laura Remesal-Gomez, Gregor Bieri, Lucas K Smith, Julien Couthouis, Christopher P Chen, Irena J Roy, Geraldine Gontier, Saul A Villeda.
      DNA methylation has emerged as a critical modulator of neuronal plasticity and cognitive function. Notwithstanding, the role of enzymes that demethylate DNA remain to be fully explored. Here, we report that loss of ten-eleven translocation methylcytosine dioxygenase 2 (Tet2), which catalyzes oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), in adult neurons enhances cognitive function. In the adult mouse hippocampus, we detected an enrichment of Tet2 in neurons. Viral-mediated neuronal overexpression and RNA interference of Tet2 altered dendritic complexity and synaptic-plasticity-related gene expression in vitro. Overexpression of neuronal Tet2 in adult hippocampus, and loss of Tet2 in adult glutamatergic neurons, resulted in differential hydroxymethylation associated with genes involved in synaptic transmission. Functionally, overexpression of neuronal Tet2 impaired hippocampal-dependent memory, while loss of neuronal Tet2 enhanced memory. Ultimately, these data identify neuronal Tet2 as a molecular target to boost cognitive function.
    Keywords:  CP: Molecular biology; CP: Neuroscience; TET2; chromatin; cognition; epigenetics; hippocampus; hydroxymethylation; memory; methylation; neurons
    DOI:  https://doi.org/10.1016/j.celrep.2022.111612
  16. EMBO Rep. 2022 Nov 07. e54944
    The bromodomain protein TRIM28 controls the balance between growth and invasiveness in melanoma.
    William A Nyberg, Diego A Velasquez-Pulgarin, Tianlin He, Maria Sjöstrand, Lucia Pellé, Ruxandra Covacu, Alexander Espinosa.
      Melanoma tumors are highly metastatic partly due to the ability of melanoma cells to transition between invasive and proliferative states. However, the mechanisms underlying this plasticity are still not fully understood. To identify new epigenetic regulators of melanoma plasticity, we combined data mining, tumor models, proximity proteomics, and CUT&RUN sequencing. We focus on the druggable family of bromodomain epigenetic readers and identify TRIM28 as a new regulator of melanoma plasticity. We find that TRIM28 promotes the expression of pro-invasive genes and that TRIM28 controls the balance between invasiveness and growth of melanoma cells. We demonstrate that TRIM28 acts via the transcription factor JUNB that directly regulates the expression of pro-invasive and pro-growth genes. Mechanistically, TRIM28 controls the expression of JUNB by negatively regulating its transcriptional elongation by RNA polymerase II. In conclusion, our results demonstrate that a TRIM28-JUNB axis controls the balance between invasiveness and growth in melanoma tumors and suggest that the bromodomain protein TRIM28 could be targeted to reduce tumor spread.
    Keywords:  Invasiveness; JUNB; Melanoma growth; Tripartite motif-containing 28
    DOI:  https://doi.org/10.15252/embr.202254944
  17. Development. 2022 Nov 10. pii: dev.201124. [Epub ahead of print]
    A new cut&run low volume-urea (LoV-U) protocol optimized for transcriptional co-factors uncovers Wnt/b-catenin tissue-specific genomic targets.
    Gianluca Zambanini, Anna Nordin, Mattias Jonasson, Pierfrancesco Pagella, Claudio Cantù.
      Upon WNT/b-catenin pathway activation, stabilized b-catenin travels to the nucleus where it associates with the TCF/LEF transcription factors, constitutively bound to genomic Wnt Responsive Elements (WREs), to activate target gene transcription. Discovering the binding profile of b-catenin is therefore required to unambiguously assign direct targets of WNT signaling. Cleavage Under Targets and Release Using Nuclease (CUT&RUN) has emerged as prime technique for mapping the binding profile of DNA-interacting proteins. Here we present a modified version of CUT&RUN, named LoV-U (Low Volume and Urea), that enables the robust and reproducible generation of b-catenin binding profiles, uncovering direct WNT/β-catenin target genes in human cells, as well as in cells isolated from developing mouse tissues. CUT&RUN-LoV-U outperforms original CUT&RUN when targeting co-factors that do not bind the DNA, can profile all classes of chromatin regulators, and is well suited for simultaneous processing of several samples. We submit that the application of our protocol will allow the detection of the complex system of tissue-specific WNT/β-catenin target genes, together with other non-DNA-binding transcriptional regulators that act downstream of ontogenetically fundamental signaling cascades.
    Keywords:  Beta-catenin; CUT&RUN; Chromatin; Transcription; Transcriptional regulation; WNT signalling
    DOI:  https://doi.org/10.1242/dev.201124
  18. Cell Rep. 2022 Nov 08. pii: S2211-1247(22)01455-3. [Epub ahead of print]41(6): 111590
    Nucleoporins facilitate ORC loading onto chromatin.
    Logan Richards, Christopher L Lord, Mary Lauren Benton, John A Capra, Jared T Nordman.
      The origin recognition complex (ORC) binds throughout the genome to initiate DNA replication. In metazoans, it is still unclear how ORC is targeted to specific loci to facilitate helicase loading and replication initiation. Here, we perform immunoprecipitations coupled with mass spectrometry for ORC2 in Drosophila embryos. Surprisingly, we find that ORC2 associates with multiple subunits of the Nup107-160 subcomplex of the nuclear pore. Bioinformatic analysis reveals that, relative to all modENCODE factors, nucleoporins are among the most enriched factors at ORC2 binding sites. Critically, depletion of the nucleoporin Elys, a member of the Nup107-160 complex, decreases ORC2 loading onto chromatin. Depleting Elys also sensitizes cells to replication fork stalling, which could reflect a defect in establishing dormant replication origins. Our work reveals a connection between ORC, replication initiation, and nucleoporins, suggesting a function for nucleoporins in metazoan replication initiation.
    Keywords:  CP: Molecular biology; DNA replication; drosophila; genome stability; nuclear pore
    DOI:  https://doi.org/10.1016/j.celrep.2022.111590
  19. Nucleic Acids Res. 2022 Nov 09. pii: gkac990. [Epub ahead of print]
    Deep learning-assisted genome-wide characterization of massively parallel reporter assays.
    Fred Lu, Aaron Sossin, Nathan Abell, Stephen B Montgomery, Zihuai He.
      Massively parallel reporter assay (MPRA) is a high-throughput method that enables the study of the regulatory activities of tens of thousands of DNA oligonucleotides in a single experiment. While MPRA experiments have grown in popularity, their small sample sizes compared to the scale of the human genome limits our understanding of the regulatory effects they detect. To address this, we develop a deep learning model, MpraNet, to distinguish potential MPRA targets from the background genome. This model achieves high discriminative performance (AUROC = 0.85) at differentiating MPRA positives from a set of control variants that mimic the background genome when applied to the lymphoblastoid cell line. We observe that existing functional scores represent very distinct functional effects, and most of them fail to characterize the regulatory effect that MPRA detects. Using MpraNet, we predict potential MPRA functional variants across the genome and identify the distributions of MPRA effect relative to other characteristics of genetic variation, including allele frequency, alternative functional annotations specified by FAVOR, and phenome-wide associations. We also observed that the predicted MPRA positives are not uniformly distributed across the genome; instead, they are clumped together in active regions comprising 9.95% of the genome and inactive regions comprising 89.07% of the genome. Furthermore, we propose our model as a screen to filter MPRA experiment candidates at genome-wide scale, enabling future experiments to be more cost-efficient by increasing precision relative to that observed from previous MPRAs.
    DOI:  https://doi.org/10.1093/nar/gkac990
  20. Nucleic Acids Res. 2022 Nov 10. pii: gkac994. [Epub ahead of print]
    Increased chromatin accessibility facilitates intron retention in specific cell differentiation states.
    DEEP Consortium
      Dynamic intron retention (IR) in vertebrate cells is of widespread biological importance. Aberrant IR is associated with numerous human diseases including several cancers. Despite consistent reports demonstrating that intrinsic sequence features can help introns evade splicing, conflicting findings about cell type- or condition-specific IR regulation by trans-regulatory and epigenetic mechanisms demand an unbiased and systematic analysis of IR in a controlled experimental setting. We integrated matched mRNA sequencing (mRNA-Seq), whole-genome bisulfite sequencing (WGBS), nucleosome occupancy methylome sequencing (NOMe-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq) data from primary human myeloid and lymphoid cells. Using these multi-omics data and machine learning, we trained two complementary models to determine the role of epigenetic factors in the regulation of IR in cells of the innate immune system. We show that increased chromatin accessibility, as revealed by nucleosome-free regions, contributes substantially to the retention of introns in a cell-specific manner. We also confirm that intrinsic characteristics of introns are key for them to evade splicing. This study suggests an important role for chromatin architecture in IR regulation. With an increasing appreciation that pathogenic alterations are linked to RNA processing, our findings may provide useful insights for the development of novel therapeutic approaches that target aberrant splicing.
    DOI:  https://doi.org/10.1093/nar/gkac994
  21. EMBO Rep. 2022 Nov 10. e55345
    Paraspeckles interact with SWI/SNF subunit ARID1B to regulate transcription and splicing.
    Divya Reddy, Saikat Bhattacharya, Michaella Levy, Ying Zhang, Madelaine Gogol, Hua Li, Laurence Florens, Jerry L Workman.
      Paraspeckles are subnuclear RNA-protein structures that are implicated in important processes including cellular stress response, differentiation, and cancer progression. However, it is unclear how paraspeckles impart their physiological effect at the molecular level. Through biochemical analyses, we show that paraspeckles interact with the SWI/SNF chromatin-remodeling complex. This is specifically mediated by the direct interaction of the long-non-coding RNA NEAT1 of the paraspeckles with ARID1B of the cBAF-type SWI/SNF complex. Strikingly, ARID1B depletion, in addition to resulting in loss of interaction with the SWI/SNF complex, decreases the binding of paraspeckle proteins to chromatin modifiers, transcription factors, and histones. Functionally, the loss of ARID1B and NEAT1 influences the transcription and the alternative splicing of a common set of genes. Our findings reveal that dynamic granules such as the paraspeckles may leverage the specificity of epigenetic modifiers to impart their regulatory effect, thus providing a molecular basis for their function.
    Keywords:  chromatin; lncRNA; paralogs; phase separation; transcription
    DOI:  https://doi.org/10.15252/embr.202255345
  22. EMBO Rep. 2022 Nov 07. e202154042
    ZBTB2 links p53 deficiency to HIF-1-mediated hypoxia signaling to promote cancer aggressiveness.
    Sho Koyasu, Shoichiro Horita, Keisuke Saito, Minoru Kobayashi, Hiroshi Ishikita, Christalle Ct Chow, Gouki Kambe, Shigeto Nishikawa, Toshi Menju, Akiyo Morinibu, Yasushi Okochi, Yoshiaki Tabuchi, Yasuhito Onodera, Norihiko Takeda, Hiroshi Date, Gregg L Semenza, Ester M Hammond, Hiroshi Harada.
      Aberrant activation of the hypoxia-inducible transcription factor HIF-1 and dysfunction of the tumor suppressor p53 have been reported to induce malignant phenotypes and therapy resistance of cancers. However, their mechanistic and functional relationship remains largely unknown. Here, we reveal a mechanism by which p53 deficiency triggers the activation of HIF-1-dependent hypoxia signaling and identify zinc finger and BTB domain-containing protein 2 (ZBTB2) as an important mediator. ZBTB2 forms homodimers via its N-terminus region and increases the transactivation activity of HIF-1 only when functional p53 is absent. The ZBTB2 homodimer facilitates invasion, distant metastasis, and growth of p53-deficient, but not p53-proficient, cancers. The intratumoral expression levels of ZBTB2 are associated with poor prognosis in lung cancer patients. ZBTB2 N-terminus-mimetic polypeptides competitively inhibit ZBTB2 homodimerization and significantly suppress the ZBTB2-HIF-1 axis, leading to antitumor effects. Our data reveal an important link between aberrant activation of hypoxia signaling and loss of a tumor suppressor and provide a rationale for targeting a key mediator, ZBTB2, to suppress cancer aggressiveness.
    Keywords:  HIF-1; ZBTB2; cancer; hypoxia; p53
    DOI:  https://doi.org/10.15252/embr.202154042
  23. Nucleic Acids Res. 2022 Nov 09. pii: gkac989. [Epub ahead of print]
    EpiFactors 2022: expansion and enhancement of a curated database of human epigenetic factors and complexes.
    Daria Marakulina, Ilya E Vorontsov, Ivan V Kulakovskiy, Andreas Lennartsson, Finn Drabløs, Yulia A Medvedeva.
      We present an update of EpiFactors, a manually curated database providing information about epigenetic regulators, their complexes, targets, and products which is openly accessible at http://epifactors.autosome.org. An updated version of the EpiFactors contains information on 902 proteins, including 101 histones and protamines, and, as a main update, a newly curated collection of 124 lncRNAs involved in epigenetic regulation. The amount of publications concerning the role of lncRNA in epigenetics is rapidly growing. Yet, the resource that compiles, integrates, organizes, and presents curated information on lncRNAs in epigenetics is missing. EpiFactors fills this gap and provides data on epigenetic regulators in an accessible and user-friendly form. For 820 of the genes in EpiFactors, we include expression estimates across multiple cell types assessed by CAGE-Seq in the FANTOM5 project. In addition, the updated EpiFactors contains information on 73 protein complexes involved in epigenetic regulation. Our resource is practical for a wide range of users, including biologists, bioinformaticians and molecular/systems biologists.
    DOI:  https://doi.org/10.1093/nar/gkac989
  24. Nat Commun. 2022 Nov 11. 13(1): 6828
    Stromal FOXF2 suppresses prostate cancer progression and metastasis by enhancing antitumor immunity.
    Deyong Jia, Zhicheng Zhou, Oh-Joon Kwon, Li Zhang, Xing Wei, Yiqun Zhang, Mingyang Yi, Martine P Roudier, Mary C Regier, Ruth Dumpit, Peter S Nelson, Mark Headley, Lawrence True, Daniel W Lin, Colm Morrissey, Chad J Creighton, Li Xin.
      Cancer-associated fibroblasts (CAFs) mediate an immunosuppressive effect, but the underlying mechanism remains incompletely defined. Here we show that increasing prostatic stromal Foxf2 suppresses the growth and progression of both syngeneic and autochthonous mouse prostate cancer models in an immunocompetent context. Mechanistically, Foxf2 moderately attenuates the CAF phenotype and transcriptionally downregulates Cxcl5, which diminish the immunosuppressive myeloid cells and enhance T cell cytotoxicity. Increasing prostatic stromal Foxf2 sensitizes prostate cancer to the immune checkpoint blockade therapies. Augmenting lung stromal Foxf2 also mediates an immunosuppressive milieu and inhibits lung colonization of prostate cancer. FOXF2 is expressed higher in the stroma of human transition zone (TZ) than peripheral zone (PZ) prostate. The stromal FOXF2 expression level in primary prostate cancers inversely correlates with the Gleason grade. Our study establishes Foxf2 as a stromal transcription factor modulating the tumor immune microenvironment and potentially explains why cancers are relatively rare and indolent in the TZ prostate.
    DOI:  https://doi.org/10.1038/s41467-022-34665-z
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