bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2024–02–04
twenty-one papers selected by
Connor Rogerson, University of Cambridge
  1. HNF4A guides the MLL4 complex to establish and maintain H3K4me1 at gene regulatory elements.
  2. BORIS/CTCFL epigenetically reprograms clustered CTCF binding sites into alternative transcriptional start sites.
  3. Tet-mediated DNA methylation dynamics affect chromosome organization.
  4. Histone deacetylases maintain expression of the pluripotent gene network via recruitment of RNA polymerase II to coding and noncoding loci.
  5. Detection of new pioneer transcription factors as cell-type-specific nucleosome binders.
  6. Single-cell multiomics decodes regulatory programs for mouse secondary palate development.
  7. In vitro reconstitution of chromatin domains shows a role for nucleosome positioning in 3D genome organization.
  8. Specialized replication of heterochromatin domains ensures self-templated chromatin assembly and epigenetic inheritance.
  9. Genomic transcription factor binding site selection is edited by the chromatin remodeling factor CHD4.
  10. BCL7A and BCL7B potentiate SWI/SNF-complex-mediated chromatin accessibility to regulate gene expression and vegetative phase transition in plants.
  11. Using methylation data to improve transcription factor binding prediction.
  12. Asymmetric nucleosome PARylation at DNA breaks mediates directional nucleosome sliding by ALC1.
  13. Interaction network of human early embryonic transcription factors.
  14. An epigenetic barrier sets the timing of human neuronal maturation.
  15. Rat1 promotes premature transcription termination at R-loops.
  16. Nuclear to cytoplasmic transport is a druggable dependency in MYC-driven hepatocellular carcinoma.
  17. Gene regulatory patterning codes in early cell fate specification of the C. elegans embryo.
  18. H2A.Z histone variants facilitate HDACi-dependent removal of H3.3K27M mutant protein in pediatric high-grade glioma cells.
  19. Reconstitution of human PDAC using primary cells reveals oncogenic transcriptomic features at tumor onset.
  20. RNA polymerase II pausing is essential during spermatogenesis for appropriate gene expression and completion of meiosis.
  21. SHR and SCR coordinate root patterning and growth early in the cell cycle.
  1. Commun Biol. 2024 01 31. 7(1): 144
    HNF4A guides the MLL4 complex to establish and maintain H3K4me1 at gene regulatory elements.
    Avinash Thakur, Kwangjin Park, Rebecca Cullum, Bettina M Fuglerud, Mina Khoshnoodi, Sibyl Drissler, Tabea L Stephan, Jeremy Lotto, Donghwan Kim, Frank J Gonzalez, Pamela A Hoodless.
      Hepatocyte nuclear factor 4A (HNF4A/NR2a1), a transcriptional regulator of hepatocyte identity, controls genes that are crucial for liver functions, primarily through binding to enhancers. In mammalian cells, active and primed enhancers are marked by monomethylation of histone 3 (H3) at lysine 4 (K4) (H3K4me1) in a cell type-specific manner. How this modification is established and maintained at enhancers in connection with transcription factors (TFs) remains unknown. Using analysis of genome-wide histone modifications, TF binding, chromatin accessibility and gene expression, we show that HNF4A is essential for an active chromatin state. Using HNF4A loss and gain of function experiments in vivo and in cell lines in vitro, we show that HNF4A affects H3K4me1, H3K27ac and chromatin accessibility, highlighting its contribution to the establishment and maintenance of a transcriptionally permissive epigenetic state. Mechanistically, HNF4A interacts with the mixed-lineage leukaemia 4 (MLL4) complex facilitating recruitment to HNF4A-bound regions. Our findings indicate that HNF4A enriches H3K4me1, H3K27ac and establishes chromatin opening at transcriptional regulatory regions.
    DOI:  https://doi.org/10.1038/s42003-024-05835-0
  2. Genome Biol. 2024 Jan 31. 25(1): 40
    BORIS/CTCFL epigenetically reprograms clustered CTCF binding sites into alternative transcriptional start sites.
    Elena M Pugacheva, Dharmendra Nath Bhatt, Samuel Rivero-Hinojosa, Md Tajmul, Liron Fedida, Emma Price, Yon Ji, Dmitri Loukinov, Alexander V Strunnikov, Bing Ren, Victor V Lobanenkov.
       BACKGROUND: Pervasive usage of alternative promoters leads to the deregulation of gene expression in carcinogenesis and may drive the emergence of new genes in spermatogenesis. However, little is known regarding the mechanisms underpinning the activation of alternative promoters.
    RESULTS: Here we describe how alternative cancer-testis-specific transcription is activated. We show that intergenic and intronic CTCF binding sites, which are transcriptionally inert in normal somatic cells, could be epigenetically reprogrammed into active de novo promoters in germ and cancer cells. BORIS/CTCFL, the testis-specific paralog of the ubiquitously expressed CTCF, triggers the epigenetic reprogramming of CTCF sites into units of active transcription. BORIS binding initiates the recruitment of the chromatin remodeling factor, SRCAP, followed by the replacement of H2A histone with H2A.Z, resulting in a more relaxed chromatin state in the nucleosomes flanking the CTCF binding sites. The relaxation of chromatin around CTCF binding sites facilitates the recruitment of multiple additional transcription factors, thereby activating transcription from a given binding site. We demonstrate that the epigenetically reprogrammed CTCF binding sites can drive the expression of cancer-testis genes, long noncoding RNAs, retro-pseudogenes, and dormant transposable elements.
    CONCLUSIONS: Thus, BORIS functions as a transcription factor that epigenetically reprograms clustered CTCF binding sites into transcriptional start sites, promoting transcription from alternative promoters in both germ cells and cancer cells.
    Keywords:  Alternative transcription; CTCF; CTCFL/BORIS; Cancer; Germ cells
    DOI:  https://doi.org/10.1186/s13059-024-03175-0
  3. Nucleic Acids Res. 2024 Feb 01. pii: gkae054. [Epub ahead of print]
    Tet-mediated DNA methylation dynamics affect chromosome organization.
    Hao Tian, Pengfei Luan, Yaping Liu, Guoqiang Li.
      DNA Methylation is a significant epigenetic modification that can modulate chromosome states, but its role in orchestrating chromosome organization has not been well elucidated. Here we systematically assessed the effects of DNA Methylation on chromosome organization with a multi-omics strategy to capture DNA Methylation and high-order chromosome interaction simultaneously on mouse embryonic stem cells with DNA methylation dioxygenase Tet triple knock-out (Tet-TKO). Globally, upon Tet-TKO, we observed weakened compartmentalization, corresponding to decreased methylation differences between CpG island (CGI) rich and poor domains. Tet-TKO could also induce hypermethylation for the CTCF binding peaks in TAD boundaries and chromatin loop anchors. Accordingly, CTCF peak generally weakened upon Tet-TKO, which results in weakened TAD structure and depletion of long-range chromatin loops. Genes that lost enhancer-promoter looping upon Tet-TKO showed DNA hypermethylation in their gene bodies, which may compensate for the disruption of gene expression. We also observed distinct effects of Tet1 and Tet2 on chromatin organization and increased DNA methylation correlation on spatially interacted fragments upon Tet inactivation. Our work showed the broad effects of Tet inactivation and DNA methylation dynamics on chromosome organization.
    DOI:  https://doi.org/10.1093/nar/gkae054
  4. Genome Res. 2024 Jan 30.
    Histone deacetylases maintain expression of the pluripotent gene network via recruitment of RNA polymerase II to coding and noncoding loci.
    Richard D W Kelly, Kristy R Stengel, Aditya Chandru, Lyndsey C Johnson, Scott W Hiebert, Shaun M Cowley.
      Histone acetylation is a dynamic modification regulated by the opposing actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Deacetylation of histone tails results in chromatin tightening, and therefore, HDACs are generally regarded as transcriptional repressors. Counterintuitively, simultaneous deletion of Hdac1 and Hdac2 in embryonic stem cells (ESCs) reduces expression of the pluripotency-associated transcription factors Pou5f1, Sox2, and Nanog (PSN). By shaping global histone acetylation patterns, HDACs indirectly regulate the activity of acetyl-lysine readers, such as the transcriptional activator BRD4. Here, we use inhibitors of HDACs and BRD4 (LBH589 and JQ1, respectively) in combination with precision nuclear run-on and sequencing (PRO-seq) to examine their roles in defining the ESC transcriptome. Both LBH589 and JQ1 causes a marked reduction in the pluripotent gene network. However, although JQ1 treatment induces widespread transcriptional pausing, HDAC inhibition causes a reduction in both paused and elongating polymerase, suggesting an overall reduction in polymerase recruitment. Using enhancer RNA (eRNA) expression to measure enhancer activity, we find that LBH589-sensitive eRNAs are preferentially associated with superenhancers and PSN binding sites. These findings suggest that HDAC activity is required to maintain pluripotency by regulating the PSN enhancer network via the recruitment of RNA polymerase II.
    DOI:  https://doi.org/10.1101/gr.278050.123
  5. Elife. 2024 Jan 31. pii: RP88936. [Epub ahead of print]12
    Detection of new pioneer transcription factors as cell-type-specific nucleosome binders.
    Yunhui Peng, Wei Song, Vladimir B Teif, Ivan Ovcharenko, David Landsman, Anna R Panchenko.
      Wrapping of DNA into nucleosomes restricts accessibility to DNA and may affect the recognition of binding motifs by transcription factors. A certain class of transcription factors, the pioneer transcription factors, can specifically recognize their DNA binding sites on nucleosomes, initiate local chromatin opening, and facilitate the binding of co-factors in a cell-type-specific manner. For the majority of human pioneer transcription factors, the locations of their binding sites, mechanisms of binding, and regulation remain unknown. We have developed a computational method to predict the cell-type-specific ability of transcription factors to bind nucleosomes by integrating ChIP-seq, MNase-seq, and DNase-seq data with details of nucleosome structure. We have demonstrated the ability of our approach in discriminating pioneer from canonical transcription factors and predicted new potential pioneer transcription factors in H1, K562, HepG2, and HeLa-S3 cell lines. Last, we systematically analyzed the interaction modes between various pioneer transcription factors and detected several clusters of distinctive binding sites on nucleosomal DNA.
    Keywords:  chromatin; computational; computational biology; human; nucleosome; nucleosome binding; pioneer transcription factor; systems biology; transcription factor
    DOI:  https://doi.org/10.7554/eLife.88936
  6. Nat Commun. 2024 Jan 27. 15(1): 821
    Single-cell multiomics decodes regulatory programs for mouse secondary palate development.
    Fangfang Yan, Akiko Suzuki, Chihiro Iwaya, Guangsheng Pei, Xian Chen, Hiroki Yoshioka, Meifang Yu, Lukas M Simon, Junichi Iwata, Zhongming Zhao.
      Perturbations in gene regulation during palatogenesis can lead to cleft palate, which is among the most common congenital birth defects. Here, we perform single-cell multiome sequencing and profile chromatin accessibility and gene expression simultaneously within the same cells (n = 36,154) isolated from mouse secondary palate across embryonic days (E) 12.5, E13.5, E14.0, and E14.5. We construct five trajectories representing continuous differentiation of cranial neural crest-derived multipotent cells into distinct lineages. By linking open chromatin signals to gene expression changes, we characterize the underlying lineage-determining transcription factors. In silico perturbation analysis identifies transcription factors SHOX2 and MEOX2 as important regulators of the development of the anterior and posterior palate, respectively. In conclusion, our study charts epigenetic and transcriptional dynamics in palatogenesis, serving as a valuable resource for further cleft palate research.
    DOI:  https://doi.org/10.1038/s41467-024-45199-x
  7. Nat Genet. 2024 Jan 30.
    In vitro reconstitution of chromatin domains shows a role for nucleosome positioning in 3D genome organization.
    Elisa Oberbeckmann, Kimberly Quililan, Patrick Cramer, A Marieke Oudelaar.
      Eukaryotic genomes are organized into chromatin domains. The molecular mechanisms driving the formation of these domains are difficult to dissect in vivo and remain poorly understood. Here we reconstitute Saccharomyces cerevisiae chromatin in vitro and determine its 3D organization at subnucleosome resolution by micrococcal nuclease-based chromosome conformation capture and molecular dynamics simulations. We show that regularly spaced and phased nucleosome arrays form chromatin domains in vitro that resemble domains in vivo. This demonstrates that neither loop extrusion nor transcription is required for basic domain formation in yeast. In addition, we find that the boundaries of reconstituted domains correspond to nucleosome-free regions and that insulation strength scales with their width. Finally, we show that domain compaction depends on nucleosome linker length, with longer linkers forming more compact structures. Together, our results demonstrate that regular nucleosome positioning is important for the formation of chromatin domains and provide a proof-of-principle for bottom-up 3D genome studies.
    DOI:  https://doi.org/10.1038/s41588-023-01649-8
  8. Proc Natl Acad Sci U S A. 2024 Feb 06. 121(6): e2315596121
    Specialized replication of heterochromatin domains ensures self-templated chromatin assembly and epigenetic inheritance.
    Patroula Nathanailidou, Jothy Dhakshnamoorthy, Hua Xiao, Martin Zofall, Sahana Holla, Maura O'Neill, Thorkell Andresson, David Wheeler, Shiv I S Grewal.
      Heterochromatin, defined by histone H3 lysine 9 methylation (H3K9me), spreads across large domains and can be epigenetically inherited in a self-propagating manner. Heterochromatin propagation depends upon a read-write mechanism, where the Clr4/Suv39h methyltransferase binds to preexisting trimethylated H3K9 (H3K9me3) and further deposits H3K9me. How the parental methylated histone template is preserved during DNA replication is not well understood. Here, we demonstrate using Schizosaccharomyces pombe that heterochromatic regions are specialized replication domains demarcated by their surrounding boundary elements. DNA replication throughout these domains is distinguished by an abundance of replisome components and is coordinated by Swi6/HP1. Although mutations in the replicative helicase subunit Mcm2 that affect histone binding impede the maintenance of a heterochromatin domain at an artificially targeted ectopic site, they have only a modest impact on heterochromatin propagation via the read-write mechanism at an endogenous site. Instead, our findings suggest a crucial role for the replication factor Mcl1 in retaining parental histones and promoting heterochromatin propagation via a mechanism involving the histone chaperone FACT. Engagement of FACT with heterochromatin requires boundary elements, which position the heterochromatic domain at the nuclear peripheral subdomain enriched for heterochromatin factors. Our findings highlight the importance of replisome components and boundary elements in creating a specialized environment for the retention of parental methylated histones, which facilitates epigenetic inheritance of heterochromatin.
    Keywords:  epigenetic; gene silencing; heterochromatin; histone methylation
    DOI:  https://doi.org/10.1073/pnas.2315596121
  9. Nucleic Acids Res. 2024 Jan 28. pii: gkae025. [Epub ahead of print]
    Genomic transcription factor binding site selection is edited by the chromatin remodeling factor CHD4.
    Mika Saotome, Deepak B Poduval, Sara A Grimm, Aerica Nagornyuk, Sakuntha Gunarathna, Takashi Shimbo, Paul A Wade, Motoki Takaku.
      Biologically precise enhancer licensing by lineage-determining transcription factors enables activation of transcripts appropriate to biological demand and prevents deleterious gene activation. This essential process is challenged by the millions of matches to most transcription factor binding motifs present in many eukaryotic genomes, leading to questions about how transcription factors achieve the exquisite specificity required. The importance of chromatin remodeling factors to enhancer activation is highlighted by their frequent mutation in developmental disorders and in cancer. Here, we determine the roles of CHD4 in enhancer licensing and maintenance in breast cancer cells and during cellular reprogramming. In unchallenged basal breast cancer cells, CHD4 modulates chromatin accessibility. Its depletion leads to redistribution of transcription factors to previously unoccupied sites. During cellular reprogramming induced by the pioneer factor GATA3, CHD4 activity is necessary to prevent inappropriate chromatin opening. Mechanistically, CHD4 promotes nucleosome positioning over GATA3 binding motifs to compete with transcription factor-DNA interaction. We propose that CHD4 acts as a chromatin proof-reading enzyme that prevents unnecessary gene expression by editing chromatin binding activities of transcription factors.
    DOI:  https://doi.org/10.1093/nar/gkae025
  10. Nat Commun. 2024 Jan 31. 15(1): 935
    BCL7A and BCL7B potentiate SWI/SNF-complex-mediated chromatin accessibility to regulate gene expression and vegetative phase transition in plants.
    Yawen Lei, Yaoguang Yu, Wei Fu, Tao Zhu, Caihong Wu, Zhihao Zhang, Zewang Yu, Xin Song, Jianqu Xu, Zhenwei Liang, Peitao Lü, Chenlong Li.
      Switch defective/sucrose non-fermentable (SWI/SNF) chromatin remodeling complexes are multi-subunit machineries that establish and maintain chromatin accessibility and gene expression by regulating chromatin structure. However, how the remodeling activities of SWI/SNF complexes are regulated in eukaryotes remains elusive. B-cell lymphoma/leukemia protein 7 A/B/C (BCL7A/B/C) have been reported as subunits of SWI/SNF complexes for decades in animals and recently in plants; however, the role of BCL7 subunits in SWI/SNF function remains undefined. Here, we identify a unique role for plant BCL7A and BCL7B homologous subunits in potentiating the genome-wide chromatin remodeling activities of SWI/SNF complexes in plants. BCL7A/B require the catalytic ATPase BRAHMA (BRM) to assemble with the signature subunits of the BRM-Associated SWI/SNF complexes (BAS) and for genomic binding at a subset of target genes. Loss of BCL7A and BCL7B diminishes BAS-mediated genome-wide chromatin accessibility without changing the stability and genomic targeting of the BAS complex, highlighting the specialized role of BCL7A/B in regulating remodeling activity. We further show that BCL7A/B fine-tune the remodeling activity of BAS complexes to generate accessible chromatin at the juvenility resetting region (JRR) of the microRNAs MIR156A/C for plant juvenile identity maintenance. In summary, our work uncovers the function of previously elusive SWI/SNF subunits in multicellular eukaryotes and provides insights into the mechanisms whereby plants memorize the juvenile identity through SWI/SNF-mediated control of chromatin accessibility.
    DOI:  https://doi.org/10.1038/s41467-024-45250-x
  11. Epigenetics. 2024 Dec;19(1): 2309826
    Using methylation data to improve transcription factor binding prediction.
    Daniel Morgan, Dawn L DeMeo, Kimberly Glass.
      Modelling the regulatory mechanisms that determine cell fate, response to external perturbation, and disease state depends on measuring many factors, a task made more difficult by the plasticity of the epigenome. Scanning the genome for the sequence patterns defined by Position Weight Matrices (PWM) can be used to estimate transcription factor (TF) binding locations. However, this approach does not incorporate information regarding the epigenetic context necessary for TF binding. CpG methylation is an epigenetic mark influenced by environmental factors that is commonly assayed in human cohort studies. We developed a framework to score inferred TF binding locations using methylation data. We intersected motif locations identified using PWMs with methylation information captured in both whole-genome bisulfite sequencing and Illumina EPIC array data for six cell lines, scored motif locations based on these data, and compared with experimental data characterizing TF binding (ChIP-seq). We found that for most TFs, binding prediction improves using methylation-based scoring compared to standard PWM-scores. We also illustrate that our approach can be generalized to infer TF binding when methylation information is only proximally available, i.e. measured for nearby CpGs that do not directly overlap with a motif location. Overall, our approach provides a framework for inferring context-specific TF binding using methylation data. Importantly, the availability of DNA methylation data in existing patient populations provides an opportunity to use our approach to understand the impact of methylation on gene regulatory processes in the context of human disease.
    Keywords:  DNA methylation; Transcription factor binding prediction
    DOI:  https://doi.org/10.1080/15592294.2024.2309826
  12. Nat Commun. 2024 Feb 02. 15(1): 1000
    Asymmetric nucleosome PARylation at DNA breaks mediates directional nucleosome sliding by ALC1.
    Luka Bacic, Guillaume Gaullier, Jugal Mohapatra, Guanzhong Mao, Klaus Brackmann, Mikhail Panfilov, Glen Liszczak, Anton Sabantsev, Sebastian Deindl.
      The chromatin remodeler ALC1 is activated by DNA damage-induced poly(ADP-ribose) deposited by PARP1/PARP2 and their co-factor HPF1. ALC1 has emerged as a cancer drug target, but how it is recruited to ADP-ribosylated nucleosomes to affect their positioning near DNA breaks is unknown. Here we find that PARP1/HPF1 preferentially initiates ADP-ribosylation on the histone H2B tail closest to the DNA break. To dissect the consequences of such asymmetry, we generate nucleosomes with a defined ADP-ribosylated H2B tail on one side only. The cryo-electron microscopy structure of ALC1 bound to such an asymmetric nucleosome indicates preferential engagement on one side. Using single-molecule FRET, we demonstrate that this asymmetric recruitment gives rise to directed sliding away from the DNA linker closest to the ADP-ribosylation site. Our data suggest a mechanism by which ALC1 slides nucleosomes away from a DNA break to render it more accessible to repair factors.
    DOI:  https://doi.org/10.1038/s41467-024-45237-8
  13. EMBO Rep. 2024 Jan 31.
    Interaction network of human early embryonic transcription factors.
    Lisa Gawriyski, Zenglai Tan, Xiaonan Liu, Iftekhar Chowdhury, Dicle Malaymar Pinar, Qin Zhang, Jere Weltner, Eeva-Mari Jouhilahti, Gong-Hong Wei, Juha Kere, Markku Varjosalo.
      Embryonic genome activation (EGA) occurs during preimplantation development and is characterized by the initiation of de novo transcription from the embryonic genome. Despite its importance, the regulation of EGA and the transcription factors involved in this process are poorly understood. Paired-like homeobox (PRDL) family proteins are implicated as potential transcriptional regulators of EGA, yet the PRDL-mediated gene regulatory networks remain uncharacterized. To investigate the function of PRDL proteins, we are identifying the molecular interactions and the functions of a subset family of the Eutherian Totipotent Cell Homeobox (ETCHbox) proteins, seven PRDL family proteins and six other transcription factors (TFs), all suggested to participate in transcriptional regulation during preimplantation. Using mass spectrometry-based interactomics methods, AP-MS and proximity-dependent biotin labeling, and chromatin immunoprecipitation sequencing we derive the comprehensive regulatory networks of these preimplantation TFs. By these interactomics tools we identify more than a thousand high-confidence interactions for the 21 studied bait proteins with more than 300 interacting proteins. We also establish that TPRX2, currently assigned as pseudogene, is a transcriptional activator.
    Keywords:  BioID; ChIP-Seq; EGA; ETCHbox; PRDL
    DOI:  https://doi.org/10.1038/s44319-024-00074-0
  14. Nature. 2024 Jan 31.
    An epigenetic barrier sets the timing of human neuronal maturation.
    Gabriele Ciceri, Arianna Baggiolini, Hyein S Cho, Meghana Kshirsagar, Silvia Benito-Kwiecinski, Ryan M Walsh, Kelly A Aromolaran, Alberto J Gonzalez-Hernandez, Hermany Munguba, So Yeon Koo, Nan Xu, Kaylin J Sevilla, Peter A Goldstein, Joshua Levitz, Christina S Leslie, Richard P Koche, Lorenz Studer.
      The pace of human brain development is highly protracted compared with most other species1-7. The maturation of cortical neurons is particularly slow, taking months to years to develop adult functions3-5. Remarkably, such protracted timing is retained in cortical neurons derived from human pluripotent stem cells (hPSCs) during in vitro differentiation or upon transplantation into the mouse brain4,8,9. Those findings suggest the presence of a cell-intrinsic clock setting the pace of neuronal maturation, although the molecular nature of this clock remains unknown. Here we identify an epigenetic developmental programme that sets the timing of human neuronal maturation. First, we developed a hPSC-based approach to synchronize the birth of cortical neurons in vitro which enabled us to define an atlas of morphological, functional and molecular maturation. We observed a slow unfolding of maturation programmes, limited by the retention of specific epigenetic factors. Loss of function of several of those factors in cortical neurons enables precocious maturation. Transient inhibition of EZH2, EHMT1 and EHMT2 or DOT1L, at progenitor stage primes newly born neurons to rapidly acquire mature properties upon differentiation. Thus our findings reveal that the rate at which human neurons mature is set well before neurogenesis through the establishment of an epigenetic barrier in progenitor cells. Mechanistically, this barrier holds transcriptional maturation programmes in a poised state that is gradually released to ensure the prolonged timeline of human cortical neuron maturation.
    DOI:  https://doi.org/10.1038/s41586-023-06984-8
  15. Nucleic Acids Res. 2024 Jan 28. pii: gkae033. [Epub ahead of print]
    Rat1 promotes premature transcription termination at R-loops.
    José Antonio Mérida-Cerro, Pablo Maraver-Cárdenas, Ana G Rondón, Andrés Aguilera.
      Certain DNA sequences can adopt a non-B form in the genome that interfere with DNA-templated processes, including transcription. Among the sequences that are intrinsically difficult to transcribe are those that tend to form R-loops, three-stranded nucleic acid structures formed by a DNA-RNA hybrid and the displaced ssDNA. Here we compared the transcription of an endogenous gene with and without an R-loop-forming sequence inserted. We show that, in agreement with previous in vivo and in vitro analyses, transcription elongation is delayed by R-loops in yeast. Importantly, we demonstrate that the Rat1 transcription terminator factor facilitates transcription throughout such structures by inducing premature termination of arrested RNAPIIs. We propose that RNase H degrades the RNA moiety of the hybrid, providing an entry site for Rat1. Thus, we have uncovered an unanticipated function of Rat1 as a transcription restoring factor opening up the possibility that it may also promote transcription through other genomic DNA structures intrinsically difficult to transcribe. If R-loop-mediated transcriptional stress is not relieved by Rat1, it will cause genomic instability, probably through the increase of transcription-replication conflicts, a deleterious situation that could lead to cancer.
    DOI:  https://doi.org/10.1093/nar/gkae033
  16. Nat Commun. 2024 Feb 01. 15(1): 963
    Nuclear to cytoplasmic transport is a druggable dependency in MYC-driven hepatocellular carcinoma.
    Anja Deutzmann, Delaney K Sullivan, Renumathy Dhanasekaran, Wei Li, Xinyu Chen, Ling Tong, Wadie D Mahauad-Fernandez, John Bell, Adriane Mosley, Angela N Koehler, Yulin Li, Dean W Felsher.
      The MYC oncogene is often dysregulated in human cancer, including hepatocellular carcinoma (HCC). MYC is considered undruggable to date. Here, we comprehensively identify genes essential for survival of MYChigh but not MYClow cells by a CRISPR/Cas9 genome-wide screen in a MYC-conditional HCC model. Our screen uncovers novel MYC synthetic lethal (MYC-SL) interactions and identifies most MYC-SL genes described previously. In particular, the screen reveals nucleocytoplasmic transport to be a MYC-SL interaction. We show that the majority of MYC-SL nucleocytoplasmic transport genes are upregulated in MYChigh murine HCC and are associated with poor survival in HCC patients. Inhibiting Exportin-1 (XPO1) in vivo induces marked tumor regression in an autochthonous MYC-transgenic HCC model and inhibits tumor growth in HCC patient-derived xenografts. XPO1 expression is associated with poor prognosis only in HCC patients with high MYC activity. We infer that MYC may generally regulate and require altered expression of nucleocytoplasmic transport genes for tumorigenesis.
    DOI:  https://doi.org/10.1038/s41467-024-45128-y
  17. Elife. 2024 Jan 29. pii: RP87099. [Epub ahead of print]12
    Gene regulatory patterning codes in early cell fate specification of the C. elegans embryo.
    Alison G Cole, Tamar Hashimshony, Zhuo Du, Itai Yanai.
      Pattern formation originates during embryogenesis by a series of symmetry-breaking steps throughout an expanding cell lineage. In Drosophila, classic work has shown that segmentation in the embryo is established by morphogens within a syncytium, and the subsequent action of the gap, pair-rule, and segment polarity genes. This classic model however does not translate directly to species that lack a syncytium - such as Caenorhabditis elegans - where cell fate is specified by cell-autonomous cell lineage programs and their inter-signaling. Previous single-cell RNA-Seq studies in C. elegans have analyzed cells from a mixed suspension of cells from many embryos to study late differentiation stages, or individual early stage embryos to study early gene expression in the embryo. To study the intermediate stages of early and late gastrulation (28- to 102-cells stages) missed by these approaches, here we determine the transcriptomes of the 1- to 102-cell stage to identify 119 embryonic cell states during cell fate specification, including 'equivalence-group' cell identities. We find that gene expression programs are modular according to the sub-cell lineages, each establishing a set of stripes by combinations of transcription factor gene expression across the anterior-posterior axis. In particular, expression of the homeodomain genes establishes a comprehensive lineage-specific positioning system throughout the embryo beginning at the 28-cell stage. Moreover, we find that genes that segment the entire embryo in Drosophila have orthologs in C. elegans that exhibit sub-lineage-specific expression. These results suggest that the C. elegans embryo is patterned by a juxtaposition of distinct lineage-specific gene regulatory programs each with a unique encoding of cell location and fate. This use of homologous gene regulatory patterning codes suggests a deep homology of cell fate specification programs across diverse modes of development.
    Keywords:  C. elegans; developmental biology; embryogenesis; single-cell RNA-Seq
    DOI:  https://doi.org/10.7554/eLife.87099
  18. Cell Rep. 2024 Jan 31. pii: S2211-1247(24)00035-4. [Epub ahead of print]43(2): 113707
    H2A.Z histone variants facilitate HDACi-dependent removal of H3.3K27M mutant protein in pediatric high-grade glioma cells.
    Katarzyna B Leszczynska, Amanda Freitas-Huhtamäki, Chinchu Jayaprakash, Monika Dzwigonska, Francisca N L Vitorino, Cynthia Horth, Kamil Wojnicki, Bartlomiej Gielniewski, Paulina Szadkowska, Beata Kaza, Javad Nazarian, Maciej K Ciolkowski, Joanna Trubicka, Wieslawa Grajkowska, Benjamin A Garcia, Jacek Majewski, Bozena Kaminska, Jakub Mieczkowski.
      Diffuse intrinsic pontine gliomas (DIPGs) are deadly pediatric brain tumors, non-resectable due to brainstem localization and diffusive growth. Over 80% of DIPGs harbor a mutation in histone 3 (H3.3 or H3.1) resulting in a lysine-to-methionine substitution (H3K27M). Patients with DIPG have a dismal prognosis with no effective therapy. We show that histone deacetylase (HDAC) inhibitors lead to a significant reduction in the H3.3K27M protein (up to 80%) in multiple glioma cell lines. We discover that the SB939-mediated H3.3K27M loss is partially blocked by a lysosomal inhibitor, chloroquine. The H3.3K27M loss is facilitated by co-occurrence of H2A.Z, as evidenced by the knockdown of H2A.Z isoforms. Chromatin immunoprecipitation sequencing (ChIP-seq) analysis confirms the occupancy of H3.3K27M and H2A.Z at the same SB939-inducible genes. We discover a mechanism showing that HDAC inhibition in DIPG leads to pharmacological modulation of the oncogenic H3.3K27M protein levels. These findings show the possibility of directly targeting the H3.3K27M oncohistone.
    Keywords:  CP: Cancer; DIPG; H2A.Z; H3.3; H3.3K27M; HDAC inhibitors; SB939; histone variants; multiomics; pediatric high-grade gliomas
    DOI:  https://doi.org/10.1016/j.celrep.2024.113707
  19. Nat Commun. 2024 Jan 27. 15(1): 818
    Reconstitution of human PDAC using primary cells reveals oncogenic transcriptomic features at tumor onset.
    Yi Xu, Michael H Nipper, Angel A Dominguez, Zhenqing Ye, Naoki Akanuma, Kevin Lopez, Janice J Deng, Destiny Arenas, Ava Sanchez, Francis E Sharkey, Colin M Court, Aatur D Singhi, Huamin Wang, Martin E Fernandez-Zapico, Lu-Zhe Sun, Siyuan Zheng, Yidong Chen, Jun Liu, Pei Wang.
      Animal studies have demonstrated the ability of pancreatic acinar cells to transform into pancreatic ductal adenocarcinoma (PDAC). However, the tumorigenic potential of human pancreatic acinar cells remains under debate. To address this gap in knowledge, we expand sorted human acinar cells as 3D organoids and genetically modify them through introduction of common PDAC mutations. The acinar organoids undergo dramatic transcriptional alterations but maintain a recognizable DNA methylation signature. The transcriptomes of acinar organoids are similar to those of disease-specific cell populations. Oncogenic KRAS alone do not transform acinar organoids. However, acinar organoids can form PDAC in vivo after acquiring the four most common driver mutations of this disease. Similarly, sorted ductal cells carrying these genetic mutations can also form PDAC, thus experimentally proving that PDACs can originate from both human acinar and ductal cells. RNA-seq analysis reveal the transcriptional shift from normal acinar cells towards PDACs with enhanced proliferation, metabolic rewiring, down-regulation of MHC molecules, and alterations in the coagulation and complement cascade. By comparing PDAC-like cells with normal pancreas and PDAC samples, we identify a group of genes with elevated expression during early transformation which represent potential early diagnostic biomarkers.
    DOI:  https://doi.org/10.1038/s41467-024-45097-2
  20. Nat Commun. 2024 Jan 29. 15(1): 848
    RNA polymerase II pausing is essential during spermatogenesis for appropriate gene expression and completion of meiosis.
    Emily G Kaye, Kavyashree Basavaraju, Geoffrey M Nelson, Helena D Zomer, Debarun Roy, Irene Infancy Joseph, Reza Rajabi-Toustani, Huanyu Qiao, Karen Adelman, Prabhakara P Reddi.
      Male germ cell development requires precise regulation of gene activity in a cell-type and stage-specific manner, with perturbations in gene expression during spermatogenesis associated with infertility. Here, we use steady-state, nascent and single-cell RNA sequencing strategies to comprehensively characterize gene expression across male germ cell populations, to dissect the mechanisms of gene control and provide new insights towards therapy. We discover a requirement for pausing of RNA Polymerase II (Pol II) at the earliest stages of sperm differentiation to establish the landscape of gene activity across development. Accordingly, genetic knockout of the Pol II pause-inducing factor NELF in immature germ cells blocks differentiation to spermatids. Further, we uncover unanticipated roles for Pol II pausing in the regulation of meiosis during spermatogenesis, with the presence of paused Pol II associated with double-strand break (DSB) formation, and disruption of meiotic gene expression and DSB repair in germ cells lacking NELF.
    DOI:  https://doi.org/10.1038/s41467-024-45177-3
  21. Nature. 2024 Jan 31.
    SHR and SCR coordinate root patterning and growth early in the cell cycle.
    Cara M Winter, Pablo Szekely, Vladimir Popov, Heather Belcher, Raina Carter, Matthew Jones, Scott E Fraser, Thai V Truong, Philip N Benfey.
      Precise control of cell division is essential for proper patterning and growth during the development of multicellular organisms. Coordination of formative divisions that generate new tissue patterns with proliferative divisions that promote growth is poorly understood. SHORTROOT (SHR) and SCARECROW (SCR) are transcription factors that are required for formative divisions in the stem cell niche of Arabidopsis roots1,2. Here we show that levels of SHR and SCR early in the cell cycle determine the orientation of the division plane, resulting in either formative or proliferative cell division. We used 4D quantitative, long-term and frequent (every 15 min for up to 48 h) light sheet and confocal microscopy to probe the dynamics of SHR and SCR in tandem within single cells of living roots. Directly controlling their dynamics with an SHR induction system enabled us to challenge an existing bistable model3 of the SHR-SCR gene-regulatory network and to identify key features that are essential for rescue of formative divisions in shr mutants. SHR and SCR kinetics do not align with the expected behaviour of a bistable system, and only low transient levels, present early in the cell cycle, are required for formative divisions. These results reveal an uncharacterized mechanism by which developmental regulators directly coordinate patterning and growth.
    DOI:  https://doi.org/10.1038/s41586-023-06971-z
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