bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2024‒11‒10
24 papers selected by
Connor Rogerson, University of Cambridge
  1. Decoding the epigenetics and chromatin loop dynamics of androgen receptor-mediated transcription.
  2. ChromaFold predicts the 3D contact map from single-cell chromatin accessibility.
  3. DNA-directed termination of mammalian RNA polymerase II.
  4. Redundant pathways for removal of defective RNA polymerase II complexes at a promoter-proximal pause checkpoint.
  5. Development of compact transcriptional effectors using high-throughput measurements in diverse contexts.
  6. Coordinated inheritance of extrachromosomal DNAs in cancer cells.
  7. Nucleolar Pol II interactome reveals TBPL1, PAF1, and Pol I at intergenic rDNA drive rRNA biogenesis.
  8. Recruitment of homodimeric proneural factors by conserved CAT-CAT E-boxes drives major epigenetic reconfiguration in cortical neurogenesis.
  9. Nucleosome flipping drives kinetic proofreading and processivity by SWR1.
  10. STAG2 loss in Ewing sarcoma alters enhancer-promoter contacts dependent and independent of EWS::FLI1.
  11. KLF13 promotes SLE pathogenesis by modifying chromatin accessibility of key proinflammatory cytokine genes.
  12. Molecular mechanism of parental H3/H4 recycling at a replication fork.
  13. Dynamic atlas of histone modifications and gene regulatory networks in endosperm of bread wheat.
  14. Type II topoisomerases shape multi-scale 3D chromatin folding in regions of positive supercoils.
  15. Recognition of BACH1 quaternary structure degrons by two F-box proteins under oxidative stress.
  16. FOXR2 targets LHX6+/DLX+ neural lineages to drive CNS neuroblastoma.
  17. NMR investigation of FOXO4-DNA interaction for discriminating target and non-target DNA sequences.
  18. Hypermethylation of CDKN2A CpG island drives resistance to PRC2 inhibitors in SWI/SNF loss-of-function tumors.
  19. EXPRESSO: a multi-omics database to explore multi-layered 3D genomic organization.
  20. Enhancing transcription-replication conflict targets ecDNA-positive cancers.
  21. A Foxf1-Wnt-Nr2f1 cascade promotes atrial cardiomyocyte differentiation in zebrafish.
  22. Epigenetic dynamics of partially methylated domains in human placenta and trophoblast stem cells.
  23. Transcriptomic analysis of the 12 major human breast cell types reveals mechanisms of cell and tissue function.
  24. LEDGF interacts with the NID domain of MeCP2 and modulates MeCP2 condensates.
  1. Nat Commun. 2024 Nov 03. 15(1): 9494
    Decoding the epigenetics and chromatin loop dynamics of androgen receptor-mediated transcription.
    Umut Berkay Altıntaş, Ji-Heui Seo, Claudia Giambartolomei, Dogancan Ozturan, Brad J Fortunato, Geoffrey M Nelson, Seth R Goldman, Karen Adelman, Faraz Hach, Matthew L Freedman, Nathan A Lack.
      Androgen receptor (AR)-mediated transcription plays a critical role in development and prostate cancer growth. AR drives gene expression by binding to thousands of cis-regulatory elements (CRE) that loop to hundreds of target promoters. With multiple CREs interacting with a single promoter, it remains unclear how individual AR bound CREs contribute to gene expression. To characterize the involvement of these CREs, we investigate the AR-driven epigenetic and chromosomal chromatin looping changes by generating a kinetic multi-omic dataset comprised of steady-state mRNA, chromatin accessibility, transcription factor binding, histone modifications, chromatin looping, and nascent RNA. Using an integrated regulatory network, we find that AR binding induces sequential changes in the epigenetic features at CREs, independent of gene expression. Further, we show that binding of AR does not result in a substantial rewiring of chromatin loops, but instead increases the contact frequency of pre-existing loops to target promoters. Our results show that gene expression strongly correlates to the changes in contact frequency. We then propose and experimentally validate an unbalanced multi-enhancer model where the impact on gene expression of AR-bound enhancers is heterogeneous, and is proportional to their contact frequency with target gene promoters. Overall, these findings provide insights into AR-mediated gene expression upon acute androgen simulation and develop a mechanistic framework to investigate nuclear receptor mediated perturbations.
    DOI:  https://doi.org/10.1038/s41467-024-53758-5
  2. Nat Commun. 2024 Nov 01. 15(1): 9432
    ChromaFold predicts the 3D contact map from single-cell chromatin accessibility.
    Vianne R Gao, Rui Yang, Arnav Das, Renhe Luo, Hanzhi Luo, Dylan R McNally, Ioannis Karagiannidis, Martin A Rivas, Zhong-Min Wang, Darko Barisic, Alireza Karbalayghareh, Wilfred Wong, Yingqian A Zhan, Christopher R Chin, William S Noble, Jeff A Bilmes, Effie Apostolou, Michael G Kharas, Wendy Béguelin, Aaron D Viny, Danwei Huangfu, Alexander Y Rudensky, Ari M Melnick, Christina S Leslie.
      Identifying cell-type-specific 3D chromatin interactions between regulatory elements can help decipher gene regulation and interpret disease-associated non-coding variants. However, achieving this resolution with current 3D genomics technologies is often infeasible given limited input cell numbers. We therefore present ChromaFold, a deep learning model that predicts 3D contact maps, including regulatory interactions, from single-cell ATAC sequencing (scATAC-seq) data alone. ChromaFold uses pseudobulk chromatin accessibility, co-accessibility across metacells, and a CTCF motif track as inputs and employs a lightweight architecture to train on standard GPUs. Trained on paired scATAC-seq and Hi-C data in human samples, ChromaFold accurately predicts the 3D contact map and peak-level interactions across diverse human and mouse test cell types. Compared to leading contact map prediction models that use ATAC-seq and CTCF ChIP-seq, ChromaFold achieves state-of-the-art performance using only scATAC-seq. Finally, fine-tuning ChromaFold on paired scATAC-seq and Hi-C in a complex tissue enables deconvolution of chromatin interactions across cell subpopulations.
    DOI:  https://doi.org/10.1038/s41467-024-53628-0
  3. Genes Dev. 2024 Nov 04.
    DNA-directed termination of mammalian RNA polymerase II.
    Lee Davidson, Jérôme O Rouvière, Rui Sousa-Luís, Takayuki Nojima, Nicholas J Proudfoot, Torben Heick Jensen, Steven West.
      The best-studied mechanism of eukaryotic RNA polymerase II (RNAPII) transcriptional termination involves polyadenylation site-directed cleavage of the nascent RNA. The RNAPII-associated cleavage product is then degraded by XRN2, dislodging RNAPII from the DNA template. In contrast, prokaryotic RNAP and eukaryotic RNAPIII often terminate directly at T-tracts in the coding DNA strand. Here, we demonstrate a similar and omnipresent capability for mammalian RNAPII. Importantly, this termination mechanism does not require upstream RNA cleavage. Accordingly, T-tract-dependent termination can take place when XRN2 cannot be engaged. We show that T-tracts can terminate snRNA transcription independently of RNA cleavage by the Integrator complex. Importantly, we found genome-wide termination at T-tracts in promoter-proximal regions but not within protein-coding gene bodies. XRN2-dependent termination dominates downstream from protein-coding genes, but the T-tract process is sometimes used. Overall, we demonstrate global DNA-directed attrition of RNAPII transcription, suggesting that RNAPs retain the potential to terminate over T-rich sequences throughout evolution.
    Keywords:  Integrator; RNA polymerase II; Xrn2; exosome; histone; snRNA; transcription termination
    DOI:  https://doi.org/10.1101/gad.351978.124
  4. Mol Cell. 2024 Oct 30. pii: S1097-2765(24)00835-9. [Epub ahead of print]
    Redundant pathways for removal of defective RNA polymerase II complexes at a promoter-proximal pause checkpoint.
    Daniel Blears, Jiangman Lou, Nova Fong, Richard Mitter, Ryan M Sheridan, Dandan He, A Barbara Dirac-Svejstrup, David Bentley, Jesper Q Svejstrup.
      The biological purpose of Integrator and RNA polymerase II (RNAPII) promoter-proximal pausing remains uncertain. Here, we show that loss of INTS6 in human cells results in increased interaction of RNAPII with proteins that can mediate its dissociation from the DNA template, including the CRL3ARMC5 E3 ligase, which ubiquitylates CTD serine5-phosphorylated RPB1 for degradation. ARMC5-dependent RNAPII ubiquitylation is activated by defects in factors acting at the promoter-proximal pause, including Integrator, DSIF, and capping enzyme. This ARMC5 checkpoint normally curtails a sizeable fraction of RNAPII transcription, and ARMC5 knockout cells produce more uncapped transcripts. When both the Integrator and CRL3ARMC5 turnover mechanisms are compromised, cell growth ceases and RNAPII with high pausing propensity disperses from the promoter-proximal pause site into the gene body. These data support a model in which CRL3ARMC5 functions alongside Integrator in a checkpoint mechanism that removes faulty RNAPII complexes at promoter-proximal pause sites to safeguard transcription integrity.
    Keywords:  ARMC5; CTD phosphorylation; CUL3; DRB; DSIF; INTS6; Integrator; NELF; RNA polymerase II; RNGTT; RPB1; TFIIH kinase; TFIIS; TT-seq; eNET-seq; mRNA capping; nascent transcription; pTEFb kinase; promoter-proximal pausing; protein phosphatase 2A; senataxin
    DOI:  https://doi.org/10.1016/j.molcel.2024.10.012
  5. Nat Biotechnol. 2024 Nov 01.
    Development of compact transcriptional effectors using high-throughput measurements in diverse contexts.
    Josh Tycko, Mike V Van, Aradhana, Nicole DelRosso, Hanrong Ye, David Yao, Raeline Valbuena, Alun Vaughan-Jackson, Xiaoshu Xu, Connor Ludwig, Kaitlyn Spees, Katherine Liu, Mingxin Gu, Venya Khare, Adi Xiyal Mukund, Peter H Suzuki, Sophia Arana, Catherine Zhang, Peter P Du, Thea S Ornstein, Gaelen T Hess, Roarke A Kamber, Lei S Qi, Ahmad S Khalil, Lacramioara Bintu, Michael C Bassik.
      Transcriptional effectors are protein domains known to activate or repress gene expression; however, a systematic understanding of which effector domains regulate transcription across genomic, cell type and DNA-binding domain (DBD) contexts is lacking. Here we develop dCas9-mediated high-throughput recruitment (HT-recruit), a pooled screening method for quantifying effector function at endogenous target genes and test effector function for a library containing 5,092 nuclear protein Pfam domains across varied contexts. We also map context dependencies of effectors drawn from unannotated protein regions using a larger library tiling chromatin regulators and transcription factors. We find that many effectors depend on target and DBD contexts, such as HLH domains that can act as either activators or repressors. To enable efficient perturbations, we select context-robust domains, including ZNF705 KRAB, that improve CRISPRi tools to silence promoters and enhancers. We engineer a compact human activator called NFZ, by combining NCOA3, FOXO3 and ZNF473 domains, which enables efficient CRISPRa with better viral delivery and inducible control of chimeric antigen receptor T cells.
    DOI:  https://doi.org/10.1038/s41587-024-02442-6
  6. Nature. 2024 Nov;635(8037): 201-209
    Coordinated inheritance of extrachromosomal DNAs in cancer cells.
    King L Hung, Matthew G Jones, Ivy Tsz-Lo Wong, Ellis J Curtis, Joshua T Lange, Britney Jiayu He, Jens Luebeck, Rachel Schmargon, Elisa Scanu, Lotte Brückner, Xiaowei Yan, Rui Li, Aditi Gnanasekar, Rocío Chamorro González, Julia A Belk, Zhonglin Liu, Bruno Melillo, Vineet Bafna, Jan R Dörr, Benjamin Werner, Weini Huang, Benjamin F Cravatt, Anton G Henssen, Paul S Mischel, Howard Y Chang.
      The chromosomal theory of inheritance dictates that genes on the same chromosome segregate together while genes on different chromosomes assort independently1. Extrachromosomal DNAs (ecDNAs) are common in cancer and drive oncogene amplification, dysregulated gene expression and intratumoural heterogeneity through random segregation during cell division2,3. Distinct ecDNA sequences, termed ecDNA species, can co-exist to facilitate intermolecular cooperation in cancer cells4. How multiple ecDNA species within a tumour cell are assorted and maintained across somatic cell generations is unclear. Here we show that cooperative ecDNA species are coordinately inherited through mitotic co-segregation. Imaging and single-cell analyses show that multiple ecDNAs encoding distinct oncogenes co-occur and are correlated in copy number in human cancer cells. ecDNA species are coordinately segregated asymmetrically during mitosis, resulting in daughter cells with simultaneous copy-number gains in multiple ecDNA species before any selection. Intermolecular proximity and active transcription at the start of mitosis facilitate the coordinated segregation of ecDNA species, and transcription inhibition reduces co-segregation. Computational modelling reveals the quantitative principles of ecDNA co-segregation and co-selection, predicting their observed distributions in cancer cells. Coordinated inheritance of ecDNAs enables co-amplification of specialized ecDNAs containing only enhancer elements and guides therapeutic strategies to jointly deplete cooperating ecDNA oncogenes. Coordinated inheritance of ecDNAs confers stability to oncogene cooperation and novel gene regulatory circuits, allowing winning combinations of epigenetic states to be transmitted across cell generations.
    DOI:  https://doi.org/10.1038/s41586-024-07861-8
  7. Nat Commun. 2024 Nov 06. 15(1): 9603
    Nucleolar Pol II interactome reveals TBPL1, PAF1, and Pol I at intergenic rDNA drive rRNA biogenesis.
    Negin Khosraviani, V Talya Yerlici, Jonathan St-Germain, Yi Yang Hou, Shi Bo Cao, Carla Ghali, Michael Bokros, Rehna Krishnan, Razqallah Hakem, Stephen Lee, Brian Raught, Karim Mekhail.
      Ribosomal DNA (rDNA) repeats harbor ribosomal RNA (rRNA) genes and intergenic spacers (IGS). RNA polymerase (Pol) I transcribes rRNA genes yielding rRNA components of ribosomes. IGS-associated Pol II prevents Pol I from excessively synthesizing IGS non-coding RNAs (ncRNAs) that can disrupt nucleoli and rRNA production. Here, compartment-enriched proximity-dependent biotin identification (compBioID) revealed the TATA-less-promoter-binding TBPL1 and transcription-regulatory PAF1 with nucleolar Pol II. TBPL1 localizes to TCT motifs, driving Pol II and Pol I and maintaining its baseline ncRNA levels. PAF1 promotes Pol II elongation, preventing unscheduled R-loops that hyper-restrain IGS Pol I-associated ncRNAs. PAF1 or TBPL1 deficiency disrupts nucleolar organization and rRNA biogenesis. In PAF1-deficient cells, repressing unscheduled IGS R-loops rescues nucleolar organization and rRNA production. Depleting IGS Pol I-dependent ncRNAs is sufficient to compromise nucleoli. We present the nucleolar interactome of Pol II and show that its regulation by TBPL1 and PAF1 ensures IGS Pol I ncRNAs maintaining nucleolar structure and function.
    DOI:  https://doi.org/10.1038/s41467-024-54002-w
  8. Nucleic Acids Res. 2024 Nov 04. pii: gkae950. [Epub ahead of print]
    Recruitment of homodimeric proneural factors by conserved CAT-CAT E-boxes drives major epigenetic reconfiguration in cortical neurogenesis.
    Xabier de Martin, Baldomero Oliva, Gabriel Santpere.
      Proneural factors of the basic helix-loop-helix family coordinate neurogenesis and neurodifferentiation. Among them, NEUROG2 and NEUROD2 subsequently act to specify neurons of the glutamatergic lineage. Disruption of these factors, their target genes and binding DNA motifs has been linked to various neuropsychiatric disorders. Proneural factors bind to specific DNA motifs called E-boxes (hexanucleotides of the form CANNTG, composed of two CAN half sites on opposed strands). While corticogenesis heavily relies on E-box activity, the collaboration of proneural factors on different E-box types and their chromatin remodeling mechanisms remain largely unknown. Here, we conducted a comprehensive analysis using chromatin immunoprecipitation followed by sequencing (ChIP-seq) data for NEUROG2 and NEUROD2, along with time-matched single-cell RNA-seq, ATAC-seq and DNA methylation data from the developing mouse cortex. Our findings show that these factors are highly enriched in transiently active genomic regions during intermediate stages of neuronal differentiation. Although they primarily bind CAG-containing E-boxes, their binding in dynamic regions is notably enriched in CAT-CAT E-boxes (i.e. CATATG, denoted as 5'3' half sites for dimers), which undergo significant DNA demethylation and exhibit the highest levels of evolutionary constraint. Aided by HT-SELEX data reanalysis, structural modeling and DNA footprinting, we propose that these proneural factors exert maximal chromatin remodeling influence during intermediate stages of neurogenesis by binding as homodimers to CAT-CAT motifs. This study provides an in-depth integrative analysis of the dynamic regulation of E-boxes during neuronal development, enhancing our understanding of the mechanisms underlying the binding specificity of critical proneural factors.
    DOI:  https://doi.org/10.1093/nar/gkae950
  9. Nature. 2024 Nov 06.
    Nucleosome flipping drives kinetic proofreading and processivity by SWR1.
    Paul Girvan, Adam S B Jalal, Elizabeth A McCormack, Michael T Skehan, Carol L Knight, Dale B Wigley, David S Rueda.
      The yeast SWR1 complex catalyses the exchange of histone H2A-H2B dimers in nucleosomes, with Htz1-H2B dimers1-3. Here we used single-molecule analysis to demonstrate two-step double exchange of the two H2A-H2B dimers in a canonical yeast nucleosome with Htz1-H2B dimers, and showed that double exchange can be processive without release of the nucleosome from the SWR1 complex. Further analysis showed that bound nucleosomes flip between two states, with each presenting a different face, and hence histone dimer, to SWR1. The bound dwell time is longer when an H2A-H2B dimer is presented for exchange than when presented with an Htz1-H2B dimer. A hexasome intermediate in the reaction is bound to the SWR1 complex in a single orientation with the 'empty' site presented for dimer insertion. Cryo-electron microscopy analysis revealed different populations of complexes showing nucleosomes caught 'flipping' between different conformations without release, each placing a different dimer into position for exchange, with the Swc2 subunit having a key role in this process. Together, the data reveal a processive mechanism for double dimer exchange that explains how SWR1 can 'proofread' the dimer identities within nucleosomes.
    DOI:  https://doi.org/10.1038/s41586-024-08152-y
  10. EMBO Rep. 2024 Nov 01.
    STAG2 loss in Ewing sarcoma alters enhancer-promoter contacts dependent and independent of EWS::FLI1.
    Daniel Giménez-Llorente, Ana Cuadrado, María José Andreu, Inmaculada Sanclemente-Alamán, Maria Solé-Ferran, Miriam Rodríguez-Corsino, Ana Losada.
      Cohesin complexes carrying STAG1 or STAG2 organize the genome into chromatin loops. STAG2 loss-of-function mutations promote metastasis in Ewing sarcoma, a pediatric cancer driven by the fusion transcription factor EWS::FLI1. We integrated transcriptomic data from patients and cellular models to identify a STAG2-dependent gene signature associated with worse prognosis. Subsequent genomic profiling and high-resolution chromatin interaction data from Capture Hi-C indicated that cohesin-STAG2 facilitates communication between EWS::FLI1-bound long GGAA repeats, presumably acting as neoenhancers, and their target promoters. Changes in CTCF-dependent chromatin contacts involving signature genes, unrelated to EWS::FLI1 binding, were also identified. STAG1 is unable to compensate for STAG2 loss and chromatin-bound cohesin is severely decreased, while levels of the processivity factor NIPBL remain unchanged, likely affecting DNA looping dynamics. These results illuminate how STAG2 loss modifies the chromatin interactome of Ewing sarcoma cells and provide a list of potential biomarkers and therapeutic targets.
    Keywords:  Cohesin; Genome Organization; Pediatric Cancer; Transcription
    DOI:  https://doi.org/10.1038/s44319-024-00303-6
  11. Commun Biol. 2024 Nov 06. 7(1): 1446
    KLF13 promotes SLE pathogenesis by modifying chromatin accessibility of key proinflammatory cytokine genes.
    Andrew Wang, Anna-Marie Fairhurst, Kui Liu, Benjamin Wakeland, Spencer Barnes, Venkat S Malladi, Kasthuribai Viswanathan, Carlos Arana, Igor Dozmorov, Amrita Singhar, Yong Du, Marjaan Imam, Angela Moses, Christian Chen, Ashwini Sunkavalli, Jose Casco, Dinesh Rakheja, Quan-Zhen Li, Chandra Mohan, Carol Clayberger, Edward K Wakeland, Shaheen Khan.
      Although significant progress has been achieved in elucidating the genetic architecture of systemic lupus erythematosus (SLE), identifying genes underlying the pathogenesis has been challenging. The NZM2410-derived lupus susceptibility Sle3 locus is associated with T cell hyperactivity and activated myeloid cells. However, candidate genes associated with these phenotypes have not been identified. Here, we narrow the Sle3 locus to a smaller genomic segment (Sle3k) and show that mice carrying Sle3k and Sle1 loci developed lupus nephritis. We identify Klf13 as the primary candidate gene that is associated with genome-wide transcription changes resulting in higher levels of proinflammatory cytokines, enhanced T cell activation, and hyperresponsiveness of myeloid cells. Correspondingly, Klf13 -/- mice display repression of genes involved in mediating immune activation, including key proinflammatory cytokines/chemokines in T cells and dysregulation in cytokine signaling pathways in myeloid cells in response to toll receptor ligands. Klf13 upregulation is associated with increased production of RANTES, a key chemokine in lupus nephritis, in activated T cells and the kidneys of lupus-prone mice. In sum, our findings reveal Klf13 as a key gene in the Sle3 interval in mediating lupus pathogenesis that may have implications in the rational design of new therapies for SLE.
    DOI:  https://doi.org/10.1038/s42003-024-07099-0
  12. Nat Commun. 2024 Nov 02. 15(1): 9485
    Molecular mechanism of parental H3/H4 recycling at a replication fork.
    Fritz Nagae, Yasuto Murayama, Tsuyoshi Terakawa.
      In chromatin replication, faithful recycling of histones from parental DNA to replicated strands is essential for maintaining epigenetic information across generations. A previous experiment has revealed that disrupting interactions between the N-terminal tail of Mcm2, a subunit in DNA replication machinery, and a histone H3/H4 tetramer perturb the recycling. However, the molecular pathways and the factors that regulate the ratio recycled to each strand and the destination location are yet to be revealed. Here, we performed molecular dynamics simulations of yeast DNA replication machinery, an H3/H4 tetramer, and replicated DNA strands. The simulations demonstrated that histones are recycled via Cdc45-mediated and unmediated pathways without histone chaperones, as our in vitro biochemical assays supported. Also, RPA binding regulated the ratio recycled to each strand, whereas DNA bending by Pol ε modulated the destination location. Together, the simulations provided testable hypotheses, which are vital for elucidating the molecular mechanisms of histone recycling.
    DOI:  https://doi.org/10.1038/s41467-024-53187-4
  13. Nat Commun. 2024 Nov 06. 15(1): 9572
    Dynamic atlas of histone modifications and gene regulatory networks in endosperm of bread wheat.
    Chao He, Siteng Bi, Yuqi Li, Chengxiang Song, Heping Zhang, Xintong Xu, Qiang Li, Sulaiman Saeed, Wei Chen, Chunjie Zhao, Caixia Lan, Handong Su, Hailiang Mao, Wenhao Yan.
      Dissecting the genetic basis of seed traits in wheat is impeded by limited genetic polymorphisms and significant variations caused by environmental conditions and seed position in a spikelet. Seed performance is largely determined by endosperm development controlled by spatiotemporal variation in gene activities, which is greatly affected by chromatin status. Here, we map genome-wide dynamic distributions of H3K27me3, H3K4me3 and H3K9ac modifications and profile gene transcription across wheat endosperm development. The combinatorial effects of active and repressive marks ensure spatiotemporal dynamic gene expression, especially for starch biosynthesis. By scanning the transcription factor binding motifs in the ATAC-seq peaks, hub regulators are identified from the regulatory network. In addition, we observe significant correlations between sequence polymorphisms of hub regulators and variations in seed traits in a germplasm population. Thus, the analysis of genomic regulatory activities together with genetic variation provides a robust approach to dissect seed traits in bread wheat.
    DOI:  https://doi.org/10.1038/s41467-024-53300-7
  14. Mol Cell. 2024 Oct 23. pii: S1097-2765(24)00830-X. [Epub ahead of print]
    Type II topoisomerases shape multi-scale 3D chromatin folding in regions of positive supercoils.
    Gabriel M C Longo, Sergi Sayols, Maria E Stefanova, Ting Xie, Waheba Elsayed, Anastasia Panagi, Amalia I Stavridou, Giuseppe Petrosino, Elizabeth Ing-Simmons, Uirá Souto Melo, Henrike J Gothe, Juan M Vaquerizas, Andriana G Kotini, Argyris Papantonis, Stefan Mundlos, Vassilis Roukos.
      Type II topoisomerases (TOP2s) resolve torsional stress accumulated during various cellular processes and are enriched at chromatin loop anchors and topologically associated domain (TAD) boundaries, where, when trapped, can lead to genomic instability promoting the formation of oncogenic fusions. Whether TOP2s relieve topological constraints at these positions and/or participate in 3D chromosome folding remains unclear. Here, we combine 3D genomics, imaging, and GapRUN, a method for the genome-wide profiling of positive supercoiling, to assess the role of TOP2s in shaping chromosome organization in human cells. Acute TOP2 depletion led to the emergence of new, large-scale contacts at the boundaries between active, positively supercoiled, and lamina-associated domains. TOP2-dependent changes at the higher-order chromatin folding were accompanied by remodeling of chromatin-nuclear lamina interactions and of gene expression, while at the chromatin loop level, TOP2 depletion predominantly remodeled transcriptionally anchored, positively supercoiled loops. We propose that TOP2s act as a fine regulator of chromosome folding at multiple scales.
    Keywords:  3D organization; DNA topology; GapR; TOP2 topoisomerases; chromosome organization; nuclear lamina; positive supercoiling; supercoiling; topoisomerases
    DOI:  https://doi.org/10.1016/j.molcel.2024.10.007
  15. Cell. 2024 Oct 30. pii: S0092-8674(24)01188-7. [Epub ahead of print]
    Recognition of BACH1 quaternary structure degrons by two F-box proteins under oxidative stress.
    Shiyun Cao, Sheena Faye Garcia, Huigang Shi, Ellie I James, Yuki Kito, Hui Shi, Haibin Mao, Sharon Kaisari, Gergely Rona, Sophia Deng, Hailey V Goldberg, Jackeline Ponce, Beatrix Ueberheide, Luca Lignitto, Miklos Guttman, Michele Pagano, Ning Zheng.
      Ubiquitin-dependent proteolysis regulates diverse cellular functions with high substrate specificity, which hinges on the ability of ubiquitin E3 ligases to decode the targets' degradation signals, i.e., degrons. Here, we show that BACH1, a transcription repressor of antioxidant response genes, features two distinct unconventional degrons encrypted in the quaternary structure of its homodimeric BTB domain. These two degrons are both functionalized by oxidative stress and are deciphered by two complementary E3s. FBXO22 recognizes a degron constructed by the BACH1 BTB domain dimer interface, which is unmasked from transcriptional co-repressors after oxidative stress releases BACH1 from chromatin. When this degron is impaired by oxidation, a second BACH1 degron manifested by its destabilized BTB dimer is probed by a pair of FBXL17 proteins that remodels the substrate into E3-bound monomers for ubiquitination. Our findings highlight the multidimensionality of protein degradation signals and the functional complementarity of different ubiquitin ligases targeting the same substrate.
    Keywords:  BACH1; BTB; FBXL17; FBXO22; SCF; cullin-RING ligases; degron; dimer; heme; nitric oxide; oxidative stress; protein degradation; quaternary structure; transcription factor; ubiquitin ligase
    DOI:  https://doi.org/10.1016/j.cell.2024.10.012
  16. Cancer Res. 2024 Nov 04.
    FOXR2 targets LHX6+/DLX+ neural lineages to drive CNS neuroblastoma.
    Selin Jessa, Antonella De Cola, Bhavyaa Chandarana, Michael McNicholas, Steven Hébert, Adam Ptack, Damien Faury, Jessica W Tsai, Andrey Korshunov, Timothy N Phoenix, Benjamin Ellezam, David T W Jones, Michael D Taylor, Pratiti Bandopadhayay, Manav Pathania, Nada Jabado, Claudia L Kleinman.
      Central nervous system neuroblastoma with FOXR2 activation (NB-FOXR2) is a high-grade tumor of the brain hemispheres and a newly identified molecular entity. Tumors express dual neuronal and glial markers, leading to frequent misdiagnoses, and limited information exists on the role of FOXR2 in their genesis. To identify their cellular origins, we profiled the transcriptomes of NB-FOXR2 tumors at the bulk and single-cell levels and integrated these profiles with large single-cell references of the normal brain. NB-FOXR2 tumors mapped to LHX6+/DLX+ lineages derived from the medial ganglionic eminence, a progenitor domain in the ventral telencephalon. In vivo prenatal Foxr2 targeting to the ganglionic eminences in mice induced postnatal cortical tumors recapitulating human NB-FOXR2 specific molecular signatures. Profiling of FOXR2 binding on chromatin in murine models revealed an association with ETS transcriptional networks, as well as direct binding of FOXR2 at key transcription factors that coordinate initiation of gliogenesis. These data indicate that NB-FOXR2 originate from LHX6+/DLX+ interneuron lineages, a lineage-of-origin distinct from that of other FOXR2-driven brain tumors, highlight the susceptibility of ventral telencephalon-derived interneurons to FOXR2-driven oncogenesis, and suggest that FOXR2-induced activation of glial programs may explain the mixed neuronal and oligodendroglial features in these tumors. More broadly, this work underscores systematic profiling of brain development as an efficient approach to orient oncogenic targeting for in vivo modeling, critical for the study of rare tumors and development of therapeutics.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-24-2248
  17. Commun Biol. 2024 Nov 01. 7(1): 1425
    NMR investigation of FOXO4-DNA interaction for discriminating target and non-target DNA sequences.
    Donghoon Kang, Min June Yang, Hae-Kap Cheong, Chin-Ju Park.
      Forkhead box O4 (FOXO4), a human transcription factor, recognizes target DNA through its forkhead domain (FHD) while maintaining comparable binding affinity to non-target DNA. The conserved region 3 (CR3), a transactivation domain, modulates DNA binding kinetics to FHD and contributes to target DNA selection, but the underlying mechanism of this selection remains elusive. Using paramagnetic relaxation enhancement analysis, we observed a minor state of CR3 close to FHD in the presence of non-target DNA, a state absent when FHD interacts with target DNA. This minor state suggests that CR3 effectively masks the non-target DNA-binding interface on FHD. The interaction weakens significantly under high salt concentration, implying that CR3 or high salt concentrations can modulate electrostatic interactions with non-target DNA. Our 15N relaxation measurements revealed FHD's flexibility with non-target DNA and increased rigidity with target DNA binding. Our findings offer insights into the role of FOXO4 as a transcription initiator.
    DOI:  https://doi.org/10.1038/s42003-024-07133-1
  18. Cell Death Dis. 2024 Nov 05. 15(11): 794
    Hypermethylation of CDKN2A CpG island drives resistance to PRC2 inhibitors in SWI/SNF loss-of-function tumors.
    Xinghao Wang, Yajun Wang, Min Xie, Shichao Ma, Yilin Zhang, Lele Wang, Yangfeng Ge, Guobin Li, Mengxi Zhao, Sheng Chen, Chenxi Yan, Hailong Zhang, Wei Sun.
      Polycomb repressive complex 2 (PRC2) catalyzes the writing of the tri-methylated histone H3 at Lys27 (H3K27me3) epigenetic marker and suppresses the expression of genes, including tumor suppressors. The function of the complex can be partially antagonized by the SWI/SNF chromatin-remodeling complex. Previous studies have suggested that PRC2 is important for the proliferation of tumors with SWI/SNF loss-of-function mutations. In the present study, we have developed an EED-directed allosteric inhibitor of PRC2 termed BR0063, which exhibits anti-proliferative properties in a subset of solid tumor cell lines harboring mutations of the SWI/SNF subunits, SMARCA4 or ARID1A. Tumor cells sensitive to BR0063 exhibited several distinct phenotypes, including cell senescence, which was mediated by the up-regulation of CDKN2A/p16. Further experiments revealed that the expression of p16 was suppressed in the BR0063-resistant cells via DNA hypermethylation in the CpG island (CGI) promoter region, rather than via PRC2 occupancy. The expression of TET1, which is required for DNA demethylation, was found to be inversely correlated with p16 CGI methylation, and this may serve as a biomarker for the prediction of resistance to PRC2 inhibitors in SWI/SNF LOF tumors.
    DOI:  https://doi.org/10.1038/s41419-024-07109-3
  19. Nucleic Acids Res. 2024 Nov 05. pii: gkae999. [Epub ahead of print]
    EXPRESSO: a multi-omics database to explore multi-layered 3D genomic organization.
    Liuyang Cai, Jun Qiao, Ruixin Zhou, Xinyi Wang, Yelan Li, Lei Jiang, Qiangwei Zhou, Guoliang Li, Tao Xu, Yuliang Feng.
      The three-dimensional (3D) organization of the human genome plays a crucial role in gene regulation. EXPloration of Regulatory Epigenome with Spatial and Sequence Observations (EXPRESSO) is a novel multi-omics database for exploration and visualization of multi-layered 3D genomic features across 46 different human tissues. Integrating 1360 3D genomic datasets (Hi-C, HiChIP, ChIA-PET) and 842 1D genomic and transcriptomic datasets (ChIP-seq, ATAC-seq, RNA-seq) from the same biosample, EXPRESSO provides a comprehensive resource for studying the interplay between 3D genome architecture and transcription regulation. This database offers diverse 3D genomic feature types (compartments, contact matrix, contact domains, stripes as diagonal lines extending from a genomic locus in contact matrix, chromatin loops, etc.) and user-friendly interface for both data exploration and download. Other key features include REpresentational State Transfer application programming interfaces for programmatic access, advanced visualization tools for 3D genomic features and web-based applications that correlate 3D genomic features with gene expression and epigenomic modifications. By providing extensive datasets and tools, EXPRESSO aims to deepen our understanding of 3D genomic architecture and its implications for human health and disease, serving as a vital resource for the research community. EXPRESSO is freely available at https://expresso.sustech.edu.cn.
    DOI:  https://doi.org/10.1093/nar/gkae999
  20. Nature. 2024 Nov;635(8037): 210-218
    Enhancing transcription-replication conflict targets ecDNA-positive cancers.
    Jun Tang, Natasha E Weiser, Guiping Wang, Sudhir Chowdhry, Ellis J Curtis, Yanding Zhao, Ivy Tsz-Lo Wong, Georgi K Marinov, Rui Li, Philip Hanoian, Edison Tse, Salvador Garcia Mojica, Ryan Hansen, Joshua Plum, Auzon Steffy, Snezana Milutinovic, S Todd Meyer, Jens Luebeck, Yanbo Wang, Shu Zhang, Nicolas Altemose, Christina Curtis, William J Greenleaf, Vineet Bafna, Stephen J Benkovic, Anthony B Pinkerton, Shailaja Kasibhatla, Christian A Hassig, Paul S Mischel, Howard Y Chang.
      Extrachromosomal DNA (ecDNA) presents a major challenge for cancer patients. ecDNA renders tumours treatment resistant by facilitating massive oncogene transcription and rapid genome evolution, contributing to poor patient survival1-7. At present, there are no ecDNA-specific treatments. Here we show that enhancing transcription-replication conflict enables targeted elimination of ecDNA-containing cancers. Stepwise analyses of ecDNA transcription reveal pervasive RNA transcription and associated single-stranded DNA, leading to excessive transcription-replication conflicts and replication stress compared with chromosomal loci. Nucleotide incorporation on ecDNA is markedly slower, and replication stress is significantly higher in ecDNA-containing tumours regardless of cancer type or oncogene cargo. pRPA2-S33, a mediator of DNA damage repair that binds single-stranded DNA, shows elevated localization on ecDNA in a transcription-dependent manner, along with increased DNA double strand breaks, and activation of the S-phase checkpoint kinase, CHK1. Genetic or pharmacological CHK1 inhibition causes extensive and preferential tumour cell death in ecDNA-containing tumours. We advance a highly selective, potent and bioavailable oral CHK1 inhibitor, BBI-2779, that preferentially kills ecDNA-containing tumour cells. In a gastric cancer model containing FGFR2 amplified on ecDNA, BBI-2779 suppresses tumour growth and prevents ecDNA-mediated acquired resistance to the pan-FGFR inhibitor infigratinib, resulting in potent and sustained tumour regression in mice. Transcription-replication conflict emerges as a target for ecDNA-directed therapy, exploiting a synthetic lethality of excess to treat cancer.
    DOI:  https://doi.org/10.1038/s41586-024-07802-5
  21. PLoS Genet. 2024 Nov 04. 20(11): e1011222
    A Foxf1-Wnt-Nr2f1 cascade promotes atrial cardiomyocyte differentiation in zebrafish.
    Ugo Coppola, Bitan Saha, Jennifer Kenney, Joshua S Waxman.
      Nr2f transcription factors (TFs) are conserved regulators of vertebrate atrial cardiomyocyte (AC) differentiation. However, little is known about the mechanisms directing Nr2f expression in ACs. Here, we identified a conserved enhancer 3' to the nr2f1a locus, which we call 3'reg1-nr2f1a (3'reg1), that can promote Nr2f1a expression in ACs. Sequence analysis of the enhancer identified putative Lef/Tcf and Foxf TF binding sites. Mutation of the Lef/Tcf sites within the 3'reg1 reporter, knockdown of Tcf7l1a, and manipulation of canonical Wnt signaling support that Tcf7l1a is derepressed via Wnt signaling to activate the transgenic enhancer and promote AC differentiation. Similarly, mutation of the Foxf binding sites in the 3'reg1 reporter, coupled with gain- and loss-of-function analysis supported that Foxf1 promotes expression of the enhancer and AC differentiation. Functionally, we find that Wnt signaling acts downstream of Foxf1 to promote expression of the 3'reg1 reporter within ACs and, importantly, both Foxf1 and Wnt signaling require Nr2f1a to promote a surplus of differentiated ACs. CRISPR-mediated deletion of the endogenous 3'reg1 abrogates the ability of Foxf1 and Wnt signaling to produce surplus ACs in zebrafish embryos. Together, our data support that downstream members of a conserved regulatory network involving Wnt signaling and Foxf1 function on a nr2f1a enhancer to promote AC differentiation in the zebrafish heart.
    DOI:  https://doi.org/10.1371/journal.pgen.1011222
  22. BMC Genomics. 2024 Nov 06. 25(1): 1050
    Epigenetic dynamics of partially methylated domains in human placenta and trophoblast stem cells.
    Hidehiro Toh, Hiroaki Okae, Kenjiro Shirane, Tetsuya Sato, Hirotaka Hamada, Chie Kikutake, Daisuke Saito, Takahiro Arima, Hiroyuki Sasaki, Mikita Suyama.
      BACKGROUND: The placenta is essential for nutrient exchange and hormone production between the mother and the developing fetus and serves as an invaluable model for epigenetic research. Most epigenetic studies of the human placenta have used whole placentas from term pregnancies and have identified the presence of partially methylated domains (PMDs). However, the origin of these domains, which are typically absent in most somatic cells, remains unclear in the placental context.RESULTS: Using whole-genome bisulfite sequencing and analysis of histone H3 modifications, we generated epigenetic profiles of human cytotrophoblasts during the first trimester and at term, as well as human trophoblast stem cells. Our study focused specifically on PMDs. We found that genomic regions likely to form PMDs are resistant to global DNA demethylation during trophectoderm reprogramming, and that PMDs arise through a slow methylation process within condensed chromatin near the nuclear lamina. In addition, we found significant differences in histone H3 modifications between PMDs in cytotrophoblasts and trophoblast stem cells.
    CONCLUSIONS: Our findings suggest that spatiotemporal genomic features shape megabase-scale DNA methylation patterns, including PMDs, in the human placenta and highlight distinct differences in PMDs between human cytotrophoblasts and trophoblast stem cells. These findings advance our understanding of placental biology and provide a basis for further research into human development and related diseases.
    Keywords:  Human cytotrophoblasts; Human trophoblast stem cells; Methylomes; Partially methylated domains
    DOI:  https://doi.org/10.1186/s12864-024-10986-9
  23. PLoS Biol. 2024 Nov;22(11): e3002820
    Transcriptomic analysis of the 12 major human breast cell types reveals mechanisms of cell and tissue function.
    Katelyn Del Toro, Rosalyn Sayaman, Kate Thi, Yamhilette Licon-Munoz, William Curtis Hines.
      A fundamental question in biology, central to our understanding of cancer and other pathologies, is determining how different cell types coordinate to form and maintain tissues. Recognizing the distinct features and capabilities of the cells that compose these tissues is critical. Unfortunately, the complexity of tissues often hinders our ability to distinguish between neighboring cell types and, in turn, scrutinize their transcriptomes and generate reliable and tractable cell models for studying their inherently different biologies. We have recently introduced a novel method that permits the identification and purification of the 12 cell types that compose the human breast-nearly all of which could be reliably propagated in the laboratory. Here, we explore the nature of these cell types. We sequence mRNAs from each purified population and investigate transcriptional patterns that reveal their distinguishing features. We describe the differentially expressed genes and enriched biological pathways that capture the essence of each cell type, and we highlight transcripts that display intriguing expression patterns. These data, analytic tools, and transcriptional analyses form a rich resource whose exploration provides remarkable insights into the inner workings of the cell types composing the breast, thus furthering our understanding of the rules governing normal cell and tissue function.
    DOI:  https://doi.org/10.1371/journal.pbio.3002820
  24. Structure. 2024 Oct 30. pii: S0969-2126(24)00452-0. [Epub ahead of print]
    LEDGF interacts with the NID domain of MeCP2 and modulates MeCP2 condensates.
    Saskia Lesire, Rodrigo Lata, Yannick Hoogvliets, Kune Herrebosch, Paulien Van De Velde, Anouk Speleers, Frauke Christ, Siska Van Belle, Zeger Debyser.
      Methyl-CpG-binding protein 2 (MeCP2) is a ubiquitously expressed nuclear protein involved in transcriptional regulation and chromatin remodeling. MeCP2 exists in two isoforms, MeCP2 E1 and E2, which share the same functional domains. Loss-of-function mutations in the MeCP2 gene are the main cause of Rett syndrome (RTT). Previous studies identified a complex formation between MeCP2 and lens epithelium derived growth factor (LEDGF), a transcriptional regulator that exists in two isoforms, LEDGF/p75 and LEDGF/p52. Here, we characterized the molecular and functional interaction between MeCP2 and LEDGF. The NCoR interaction domain (NID) domain in MeCP2 is essential for the direct binding to the PWWP-CR1 region of LEDGF. Introduction of R306C, an RTT mutation in the NID of MeCP2, reduced the interaction with LEDGF. Our data reveal mutual inhibition of MeCP2 and LEDGF multimerization due to overlapping binding sites. Aligning with this observation, LEDGF depletion resulted in larger MeCP2 and DNA foci in NIH3T3 cells, suggesting a role for the MeCP2-LEDGF complex in chromatin organization.
    Keywords:  LEDGF; MeCP2; Rett syndrome; chromatin
    DOI:  https://doi.org/10.1016/j.str.2024.10.016
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