bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2023–02–12
24 papers selected by
Connor Rogerson, University of Cambridge
  1. Single-molecule footprinting identifies context-dependent regulation of enhancers by DNA methylation.
  2. H2B ubiquitination recruits FACT to maintain a stable altered nucleosome state for transcriptional activation.
  3. A cis-regulatory lexicon of DNA motif combinations mediating cell-type-specific gene regulation.
  4. Transcription factor SOX15 regulates stem cell pluripotency and promotes neural fate during differentiation by activating the neurogenic gene Hes5.
  5. DNA methylation entropy is associated with DNA sequence features and developmental epigenetic divergence.
  6. Systematic identification of factors involved in the silencing of germline genes in mouse embryonic stem cells.
  7. Role of the histone tails in histone octamer transfer.
  8. A CpG island-encoded mechanism protects genes from premature transcription termination.
  9. Using single cell atlas data to reconstruct regulatory networks.
  10. Tumor-suppressive disruption of cancer subtype-associated super enhancer circuits by small molecule treatment.
  11. Recycling of modified H2A-H2B provides short-term memory of chromatin states.
  12. Widespread contribution of transposable elements to the rewiring of mammalian 3D genomes.
  13. Application of high-throughput single-nucleus DNA sequencing in pancreatic cancer.
  14. Histone variant H2A.Z modulates nucleosome dynamics to promote DNA accessibility.
  15. MORF and MOZ acetyltransferases target unmethylated CpG islands through the winged helix domain.
  16. SLAMseq resolves the kinetics of maternal and zygotic gene expression during early zebrafish embryogenesis.
  17. Aberrant phase separation and nucleolar dysfunction in rare genetic diseases.
  18. Single-cell multi-omic analysis profiles defective genome activation and epigenetic reprogramming associated with human pre-implantation embryo arrest.
  19. The Integrator complex regulates microRNA abundance through RISC loading.
  20. Dynamic activity in cis-regulatory elements of leukocytes identifies transcription factor activation and stratifies COVID-19 severity in ICU patients.
  21. Epigenetic and chromosomal features drive transposon insertion in Drosophila melanogaster.
  22. HiConfidence: a novel approach uncovering the biological signal in Hi-C data affected by technical biases.
  23. Dissecting cell identity via network inference and in silico gene perturbation.
  24. Transcription factors underlying photoreceptor diversity.
  1. Mol Cell. 2023 Feb 01. pii: S1097-2765(23)00040-0. [Epub ahead of print]
    Single-molecule footprinting identifies context-dependent regulation of enhancers by DNA methylation.
    Elisa Kreibich, Rozemarijn Kleinendorst, Guido Barzaghi, Sarah Kaspar, Arnaud R Krebs.
      Enhancers are cis-regulatory elements that control the establishment of cell identities during development. In mammals, enhancer activation is tightly coupled with DNA demethylation. However, whether this epigenetic remodeling is necessary for enhancer activation is unknown. Here, we adapted single-molecule footprinting to measure chromatin accessibility and transcription factor binding as a function of the presence of methylation on the same DNA molecules. We leveraged natural epigenetic heterogeneity at active enhancers to test the impact of DNA methylation on their chromatin accessibility in multiple cell lineages. Although reduction of DNA methylation appears dispensable for the activity of most enhancers, we identify a class of cell-type-specific enhancers where DNA methylation antagonizes the binding of transcription factors. Genetic perturbations reveal that chromatin accessibility and transcription factor binding require active demethylation at these loci. Thus, in addition to safeguarding the genome from spurious activation, DNA methylation directly controls transcription factor occupancy at active enhancers.
    Keywords:  DNA methylation; enhancers; epigenetics; gene regulation; genomics; single-molecule footprinting; transcription factors
    DOI:  https://doi.org/10.1016/j.molcel.2023.01.017
  2. Nat Commun. 2023 Feb 10. 14(1): 741
    H2B ubiquitination recruits FACT to maintain a stable altered nucleosome state for transcriptional activation.
    Anfeng Luo, Jingwei Kong, Jun Chen, Xue Xiao, Jie Lan, Xiaorong Li, Cuifang Liu, Peng-Ye Wang, Guohong Li, Wei Li, Ping Chen.
      Histone H2B mono-ubiquitination at lysine 120 (ubH2B) has been found to regulate transcriptional elongation by collaborating with the histone chaperone FACT (Facilitates Chromatin Transcription) and plays essential roles in chromatin-based transcriptional processes. However, the mechanism of how ubH2B directly collaborates with FACT at the nucleosome level still remains elusive. In this study, we demonstrate that ubH2B impairs the mechanical stability of the nucleosome and helps to recruit FACT by enhancing the binding of FACT on the nucleosome. FACT prefers to bind and deposit H2A-ubH2B dimers to form an intact nucleosome. Strikingly, the preferable binding of FACT on ubH2B-nucleosome greatly enhances nucleosome stability and maintains its integrity. The stable altered nucleosome state obtained by ubH2B and FACT provides a key platform for gene transcription, as revealed by genome-wide and time-course ChIP-qPCR analyses. Our findings provide mechanistic insights of how ubH2B directly collaborates with FACT to regulate nucleosome dynamics for gene transcription.
    DOI:  https://doi.org/10.1038/s41467-023-36467-3
  3. Cell Genom. 2022 Nov 09. pii: 100191. [Epub ahead of print]2(11):
    A cis-regulatory lexicon of DNA motif combinations mediating cell-type-specific gene regulation.
    Laura K H Donohue, Margaret G Guo, Yang Zhao, Namyoung Jung, Rose T Bussat, Daniel S Kim, Poornima H Neela, Laura N Kellman, Omar S Garcia, Robin M Meyers, Russ B Altman, Paul A Khavari.
      Gene expression is controlled by transcription factors (TFs) that bind cognate DNA motif sequences in cis-regulatory elements (CREs). The combinations of DNA motifs acting within homeostasis and disease, however, are unclear. Gene expression, chromatin accessibility, TF footprinting, and H3K27ac-dependent DNA looping data were generated and a random-forest-based model was applied to identify 7,531 cell-type-specific cis-regulatory modules (CRMs) across 15 diploid human cell types. A co-enrichment framework within CRMs nominated 838 cell-type-specific, recurrent heterotypic DNA motif combinations (DMCs), which were functionally validated using massively parallel reporter assays. Cancer cells engaged DMCs linked to neoplasia-enabling processes operative in normal cells while also activating new DMCs only seen in the neoplastic state. This integrative approach identifies cell-type-specific cis-regulatory combinatorial DNA motifs in diverse normal and diseased human cells and represents a general framework for deciphering cis-regulatory sequence logic in gene regulation.
    DOI:  https://doi.org/10.1016/j.xgen.2022.100191
  4. J Biol Chem. 2023 Feb 08. pii: S0021-9258(23)00128-X. [Epub ahead of print] 102996
    Transcription factor SOX15 regulates stem cell pluripotency and promotes neural fate during differentiation by activating the neurogenic gene Hes5.
    Eun-Bee Choi, Munender Vodnala, Prince Saini, Sharath Anugula, Madeleine Zerbato, Jaclyn J Ho, Jianing Wang, Shannan J Ho Sui, Joon Yoon, Marielle Roels, Carla Inouye, Yick W Fong.
      SOX2 and SOX15 are Sox family transcription factors enriched in embryonic stem cells (ESCs). The role of SOX2 in activating gene expression programs essential for stem cell self-renewal and acquisition of pluripotency during somatic cell reprogramming is well-documented. However, the contribution of SOX15 to these processes is unclear and often presumed redundant with SOX2 largely because overexpression of SOX15 can partially restore self-renewal in SOX2-deficient ESCs. Here, we show that SOX15 contributes to stem cell maintenance by cooperating with ESC-enriched transcriptional coactivators to ensure optimal expression of pluripotency-associated genes. We demonstrate that SOX15 depletion compromises reprogramming of fibroblasts to pluripotency which cannot be compensated by SOX2. Ectopic expression of SOX15 promotes the reversion of a post-implantation, epiblast stem cell state back to a pre-implantation, ESC-like identity even though SOX2 is expressed in both cell states. We also uncover a role of SOX15 in lineage specification, by showing that loss of SOX15 leads to defects in commitment of ESCs to neural fates. SOX15 promotes neural differentiation by binding to and activating a previously uncharacterized distal enhancer of a key neurogenic regulator, Hes5. Together, these findings identify a multifaceted role of SOX15 in induction and maintenance of pluripotency and neural differentiation.
    DOI:  https://doi.org/10.1016/j.jbc.2023.102996
  5. Nucleic Acids Res. 2023 Feb 10. pii: gkad050. [Epub ahead of print]
    DNA methylation entropy is associated with DNA sequence features and developmental epigenetic divergence.
    Yuqi Fang, Zhicheng Ji, Weiqiang Zhou, Jordi Abante, Michael A Koldobskiy, Hongkai Ji, Andrew P Feinberg.
      Epigenetic information defines tissue identity and is largely inherited in development through DNA methylation. While studied mostly for mean differences, methylation also encodes stochastic change, defined as entropy in information theory. Analyzing allele-specific methylation in 49 human tissue sample datasets, we find that methylation entropy is associated with specific DNA binding motifs, regulatory DNA, and CpG density. Then applying information theory to 42 mouse embryo methylation datasets, we find that the contribution of methylation entropy to time- and tissue-specific patterns of development is comparable to the contribution of methylation mean, and methylation entropy is associated with sequence and chromatin features conserved with human. Moreover, methylation entropy is directly related to gene expression variability in development, suggesting a role for epigenetic entropy in developmental plasticity.
    DOI:  https://doi.org/10.1093/nar/gkad050
  6. Nucleic Acids Res. 2023 Feb 11. pii: gkad071. [Epub ahead of print]
    Systematic identification of factors involved in the silencing of germline genes in mouse embryonic stem cells.
    Hala Al Adhami, Judith Vallet, Celia Schaal, Paul Schumacher, Anaïs Flore Bardet, Michael Dumas, Johana Chicher, Philippe Hammann, Sylvain Daujat, Michael Weber.
      In mammals, many germline genes are epigenetically repressed to prevent their illegitimate expression in somatic cells. To advance our understanding of the mechanisms restricting the expression of germline genes, we analyzed their chromatin signature and performed a CRISPR-Cas9 knock-out screen for genes involved in germline gene repression using a Dazl-GFP reporter system in mouse embryonic stem cells (mESCs). We show that the repression of germline genes mainly depends on the polycomb complex PRC1.6 and DNA methylation, which function additively in mESCs. Furthermore, we validated novel genes involved in the repression of germline genes and characterized three of them: Usp7, Shfm1 (also known as Sem1) and Erh. Inactivation of Usp7, Shfm1 or Erh led to the upregulation of germline genes, as well as retrotransposons for Shfm1, in mESCs. Mechanistically, USP7 interacts with PRC1.6 components, promotes PRC1.6 stability and presence at germline genes, and facilitates DNA methylation deposition at germline gene promoters for long term repression. Our study provides a global view of the mechanisms and novel factors required for silencing germline genes in embryonic stem cells.
    DOI:  https://doi.org/10.1093/nar/gkad071
  7. Nucleic Acids Res. 2023 Feb 11. pii: gkad079. [Epub ahead of print]
    Role of the histone tails in histone octamer transfer.
    Yahli Lorch, Roger D Kornberg, Barbara Maier-Davis.
      The exceptionally high positive charge of the histones, concentrated in the N- and C-terminal tails, is believed to contribute to the stability of the nucleosome by neutralizing the negative charge of the nucleosomal DNA. We find, on the contrary, that the high positive charge contributes to instability, performing an essential function in chromatin remodeling. We show that the tails are required for removal of the histone octamer by the RSC chromatin remodeling complex, and this function is not due to direct RSC-tail interaction. We also show that the tails are required for histone octamer transfer from nucleosomes to DNA, and this activity of the tails is a consequence of their positive charge. Thus, the histone tails, intrinsically disordered protein regions, perform a critical role in chromatin structure and transcription, unrelated to their well-known role in regulation through posttranscriptional modification.
    DOI:  https://doi.org/10.1093/nar/gkad079
  8. Nat Commun. 2023 Feb 09. 14(1): 726
    A CpG island-encoded mechanism protects genes from premature transcription termination.
    Amy L Hughes, Aleksander T Szczurek, Jessica R Kelley, Anna Lastuvkova, Anne H Turberfield, Emilia Dimitrova, Neil P Blackledge, Robert J Klose.
      Transcription must be tightly controlled to regulate gene expression and development. However, our understanding of the molecular mechanisms that influence transcription and how these are coordinated in cells to ensure normal gene expression remains rudimentary. Here, by dissecting the function of the SET1 chromatin-modifying complexes that bind to CpG island-associated gene promoters, we discover that they play a specific and essential role in enabling the expression of low to moderately transcribed genes. Counterintuitively, this effect can occur independently of SET1 complex histone-modifying activity and instead relies on an interaction with the RNA Polymerase II-binding protein WDR82. Unexpectedly, we discover that SET1 complexes enable gene expression by antagonising premature transcription termination by the ZC3H4/WDR82 complex at CpG island-associated genes. In contrast, at extragenic sites of transcription, which typically lack CpG islands and SET1 complex occupancy, we show that the activity of ZC3H4/WDR82 is unopposed. Therefore, we reveal a gene regulatory mechanism whereby CpG islands are bound by a protein complex that specifically protects genic transcripts from premature termination, effectively distinguishing genic from extragenic transcription and enabling normal gene expression.
    DOI:  https://doi.org/10.1038/s41467-023-36236-2
  9. Nucleic Acids Res. 2023 Feb 10. pii: gkad053. [Epub ahead of print]
    Using single cell atlas data to reconstruct regulatory networks.
    Qi Song, Matthew Ruffalo, Ziv Bar-Joseph.
      Inference of global gene regulatory networks from omics data is a long-term goal of systems biology. Most methods developed for inferring transcription factor (TF)-gene interactions either relied on a small dataset or used snapshot data which is not suitable for inferring a process that is inherently temporal. Here, we developed a new computational method that combines neural networks and multi-task learning to predict RNA velocity rather than gene expression values. This allows our method to overcome many of the problems faced by prior methods leading to more accurate and more comprehensive set of identified regulatory interactions. Application of our method to atlas scale single cell data from 6 HuBMAP tissues led to several validated and novel predictions and greatly improved on prior methods proposed for this task.
    DOI:  https://doi.org/10.1093/nar/gkad053
  10. NAR Cancer. 2023 Mar;5(1): zcad007
    Tumor-suppressive disruption of cancer subtype-associated super enhancer circuits by small molecule treatment.
    Anke Koeniger, Pierfrancesco Polo, Anna Brichkina, Florian Finkernagel, Alexander Visekruna, Andrea Nist, Thorsten Stiewe, Michael Daude, Wibke E Diederich, Thomas M Gress, Till Adhikary, Matthias Lauth.
      Transcriptional cancer subtypes which correlate with traits such as tumor growth, drug sensitivity or the chances of relapse and metastasis, have been described for several malignancies. The core regulatory circuits (CRCs) defining these subtypes are established by chromatin super enhancers (SEs) driving key transcription factors (TFs) specific for the particular cell state. In neuroblastoma (NB), one of the most frequent solid pediatric cancer entities, two major SE-directed molecular subtypes have been described: A more lineage-committed adrenergic (ADRN) and a mesenchymal (MES) subtype. Here, we found that a small isoxazole molecule (ISX), a frequently used pro-neural drug, reprogrammed SE activity and switched NB cells from an ADRN subtype towards a growth-retarded MES-like state. The MES-like state shared strong transcriptional overlap with ganglioneuroma (GN), a benign and highly differentiated tumor of the neural crest. Mechanistically, ISX suppressed chromatin binding of N-MYC, a CRC-amplifying transcription factor, resulting in loss of key ADRN subtype-enriched components such as N-MYC itself, PHOX2B and ALK, while concomitently, MES subtype markers were induced. Globally, ISX treatment installed a chromatin accessibility landscape typically associated with low risk NB. In summary, we provide evidence that CRCs and cancer subtype reprogramming might be amenable to future therapeutic targeting.
    DOI:  https://doi.org/10.1093/narcan/zcad007
  11. Cell. 2023 Feb 02. pii: S0092-8674(23)00007-7. [Epub ahead of print]
    Recycling of modified H2A-H2B provides short-term memory of chromatin states.
    Valentin Flury, Nazaret Reverón-Gómez, Nicolas Alcaraz, Kathleen R Stewart-Morgan, Alice Wenger, Robert J Klose, Anja Groth.
      Chromatin landscapes are disrupted during DNA replication and must be restored faithfully to maintain genome regulation and cell identity. The histone H3-H4 modification landscape is restored by parental histone recycling and modification of new histones. How DNA replication impacts on histone H2A-H2B is currently unknown. Here, we measure H2A-H2B modifications and H2A.Z during DNA replication and across the cell cycle using quantitative genomics. We show that H2AK119ub1, H2BK120ub1, and H2A.Z are recycled accurately during DNA replication. Modified H2A-H2B are segregated symmetrically to daughter strands via POLA1 on the lagging strand, but independent of H3-H4 recycling. Post-replication, H2A-H2B modification and variant landscapes are quickly restored, and H2AK119ub1 guides accurate restoration of H3K27me3. This work reveals epigenetic transmission of parental H2A-H2B during DNA replication and identifies cross talk between H3-H4 and H2A-H2B modifications in epigenome propagation. We propose that rapid short-term memory of recycled H2A-H2B modifications facilitates restoration of stable H3-H4 chromatin states.
    Keywords:  DNA replication; H2A; H2A.Z; H2B; chromatin; histone PTM cross talk; histone recycling; polycomb; post-translational modifications; ubiquitination
    DOI:  https://doi.org/10.1016/j.cell.2023.01.007
  12. Nat Commun. 2023 Feb 06. 14(1): 634
    Widespread contribution of transposable elements to the rewiring of mammalian 3D genomes.
    Mayank N K Choudhary, Kara Quaid, Xiaoyun Xing, Heather Schmidt, Ting Wang.
      Transposable elements (TEs) are major contributors of genetic material in mammalian genomes. These often include binding sites for architectural proteins, including the multifarious master protein, CTCF, which shapes the 3D genome by creating loops, domains, compartment borders, and RNA-DNA interactions. These play a role in the compact packaging of DNA and have the potential to facilitate regulatory function. In this study, we explore the widespread contribution of TEs to mammalian 3D genomes by quantifying the extent to which they give rise to loops and domain border differences across various cell types and species using several 3D genome mapping technologies. We show that specific families and subfamilies of TEs have contributed to lineage-specific 3D chromatin structures across mammalian species. In many cases, these loops may facilitate sustained interaction between distant cis-regulatory elements and target genes, and domains may segregate chromatin state to impact gene expression in a lineage-specific manner. An experimental validation of our analytical findings using CRISPR-Cas9 to delete a candidate TE resulted in disruption of species-specific 3D chromatin structure. Taken together, we comprehensively quantify and selectively validate our finding that TEs contribute to shaping 3D genome organization and may, in some cases, impact gene regulation during the course of mammalian evolution.
    DOI:  https://doi.org/10.1038/s41467-023-36364-9
  13. Nat Commun. 2023 Feb 10. 14(1): 749
    Application of high-throughput single-nucleus DNA sequencing in pancreatic cancer.
    Haochen Zhang, Elias-Ramzey Karnoub, Shigeaki Umeda, Ronan Chaligné, Ignas Masilionis, Caitlin A McIntyre, Palash Sashittal, Akimasa Hayashi, Amanda Zucker, Katelyn Mullen, Jungeui Hong, Alvin Makohon-Moore, Christine A Iacobuzio-Donahue.
      Despite insights gained by bulk DNA sequencing of cancer it remains challenging to resolve the admixture of normal and tumor cells, and/or of distinct tumor subclones; high-throughput single-cell DNA sequencing circumvents these and brings cancer genomic studies to higher resolution. However, its application has been limited to liquid tumors or a small batch of solid tumors, mainly because of the lack of a scalable workflow to process solid tumor samples. Here we optimize a highly automated nuclei extraction workflow that achieves fast and reliable targeted single-nucleus DNA library preparation of 38 samples from 16 pancreatic ductal adenocarcinoma patients, with an average library yield per sample of 2867 single nuclei. We demonstrate that this workflow not only performs well using low cellularity or low tumor purity samples but reveals genomic evolution patterns of pancreatic ductal adenocarcinoma as well.
    DOI:  https://doi.org/10.1038/s41467-023-36344-z
  14. Nat Commun. 2023 Feb 11. 14(1): 769
    Histone variant H2A.Z modulates nucleosome dynamics to promote DNA accessibility.
    Shuxiang Li, Tiejun Wei, Anna R Panchenko.
      Nucleosomes, containing histone variants H2A.Z, are important for gene transcription initiation and termination, chromosome segregation and DNA double-strand break repair, among other functions. However, the underlying mechanisms of how H2A.Z influences nucleosome stability, dynamics and DNA accessibility are not well understood, as experimental and computational evidence remains inconclusive. Our modeling efforts of human nucleosome stability and dynamics, along with comparisons with experimental data show that the incorporation of H2A.Z results in a substantial decrease of the energy barrier for DNA unwrapping. This leads to the spontaneous DNA unwrapping of about forty base pairs from both ends, nucleosome gapping and increased histone plasticity, which otherwise is not observed for canonical nucleosomes. We demonstrate that both N- and C-terminal tails of H2A.Z play major roles in these events, whereas the H3.3 variant exerts a negligible impact in modulating the DNA end unwrapping. In summary, our results indicate that H2A.Z deposition makes nucleosomes more mobile and DNA more accessible to transcriptional machinery and other chromatin components.
    DOI:  https://doi.org/10.1038/s41467-023-36465-5
  15. Nat Commun. 2023 Feb 08. 14(1): 697
    MORF and MOZ acetyltransferases target unmethylated CpG islands through the winged helix domain.
    Dustin C Becht, Brianna J Klein, Akinori Kanai, Suk Min Jang, Khan L Cox, Bing-Rui Zhou, Sabrina K Phanor, Yi Zhang, Ruo-Wen Chen, Christopher C Ebmeier, Catherine Lachance, Maxime Galloy, Amelie Fradet-Turcotte, Martha L Bulyk, Yawen Bai, Michael G Poirier, Jacques Côté, Akihiko Yokoyama, Tatiana G Kutateladze.
      Human acetyltransferases MOZ and MORF are implicated in chromosomal translocations associated with aggressive leukemias. Oncogenic translocations involve the far amino terminus of MOZ/MORF, the function of which remains unclear. Here, we identified and characterized two structured winged helix (WH) domains, WH1 and WH2, in MORF and MOZ. WHs bind DNA in a cooperative manner, with WH1 specifically recognizing unmethylated CpG sequences. Structural and genomic analyses show that the DNA binding function of WHs targets MORF/MOZ to gene promoters, stimulating transcription and H3K23 acetylation, and WH1 recruits oncogenic fusions to HOXA genes that trigger leukemogenesis. Cryo-EM, NMR, mass spectrometry and mutagenesis studies provide mechanistic insight into the DNA-binding mechanism, which includes the association of WH1 with the CpG-containing linker DNA and binding of WH2 to the dyad of the nucleosome. The discovery of WHs in MORF and MOZ and their DNA binding functions could open an avenue in developing therapeutics to treat diseases associated with aberrant MOZ/MORF acetyltransferase activities.
    DOI:  https://doi.org/10.1038/s41467-023-36368-5
  16. Cell Rep. 2023 Feb 08. pii: S2211-1247(23)00081-5. [Epub ahead of print]42(2): 112070
    SLAMseq resolves the kinetics of maternal and zygotic gene expression during early zebrafish embryogenesis.
    Pooja Bhat, Luis E Cabrera-Quio, Veronika A Herzog, Nina Fasching, Andrea Pauli, Stefan L Ameres.
      The maternal-to-zygotic transition (MZT) is a key developmental process in metazoan embryos that involves the activation of zygotic transcription (ZGA) and degradation of maternal transcripts. We employed metabolic mRNA sequencing (SLAMseq) to deconvolute the compound embryonic transcriptome in zebrafish. While mitochondrial zygotic transcripts prevail prior to MZT, we uncover the spurious transcription of hundreds of short and intron-poor genes as early as the 2-cell stage. Upon ZGA, most zygotic transcripts originate from thousands of maternal-zygotic (MZ) genes that are transcribed at rates comparable to those of hundreds of purely zygotic genes and replenish maternal mRNAs at distinct timescales. Rapid replacement of MZ transcripts involves transcript decay features unrelated to major maternal degradation pathways and promotes de novo synthesis of the core gene expression machinery by increasing poly(A)-tail length and translation efficiency. SLAMseq hence provides insights into the timescales, molecular features, and regulation of MZT during zebrafish embryogenesis.
    Keywords:  CP: Developmental biology; embryogenesis; maternal-to-zygotic transition; metabolic RNA sequencing; zebrafish; zygotic genome activation
    DOI:  https://doi.org/10.1016/j.celrep.2023.112070
  17. Nature. 2023 Feb 08.
    Aberrant phase separation and nucleolar dysfunction in rare genetic diseases.
    Martin A Mensah, Henri Niskanen, Alexandre P Magalhaes, Shaon Basu, Martin Kircher, Henrike L Sczakiel, Alisa M V Reiter, Jonas Elsner, Peter Meinecke, Saskia Biskup, Brian H Y Chung, Gregor Dombrowsky, Christel Eckmann-Scholz, Marc Phillip Hitz, Alexander Hoischen, Paul-Martin Holterhus, Wiebke Hülsemann, Kimia Kahrizi, Vera M Kalscheuer, Anita Kan, Mandy Krumbiegel, Ingo Kurth, Jonas Leubner, Ann Carolin Longardt, Jörg D Moritz, Hossein Najmabadi, Karolina Skipalova, Lot Snijders Blok, Andreas Tzschach, Eberhard Wiedersberg, Martin Zenker, Carla Garcia-Cabau, René Buschow, Xavier Salvatella, Matthew L Kraushar, Stefan Mundlos, Almuth Caliebe, Malte Spielmann, Denise Horn, Denes Hnisz.
      Thousands of genetic variants in protein-coding genes have been linked to disease. However, the functional impact of most variants is unknown as they occur within intrinsically disordered protein regions that have poorly defined functions1-3. Intrinsically disordered regions can mediate phase separation and the formation of biomolecular condensates, such as the nucleolus4,5. This suggests that mutations in disordered proteins may alter condensate properties and function6-8. Here we show that a subset of disease-associated variants in disordered regions alter phase separation, cause mispartitioning into the nucleolus and disrupt nucleolar function. We discover de novo frameshift variants in HMGB1 that cause brachyphalangy, polydactyly and tibial aplasia syndrome, a rare complex malformation syndrome. The frameshifts replace the intrinsically disordered acidic tail of HMGB1 with an arginine-rich basic tail. The mutant tail alters HMGB1 phase separation, enhances its partitioning into the nucleolus and causes nucleolar dysfunction. We built a catalogue of more than 200,000 variants in disordered carboxy-terminal tails and identified more than 600 frameshifts that create arginine-rich basic tails in transcription factors and other proteins. For 12 out of the 13 disease-associated variants tested, the mutation enhanced partitioning into the nucleolus, and several variants altered rRNA biogenesis. These data identify the cause of a rare complex syndrome and suggest that a large number of genetic variants may dysregulate nucleoli and other biomolecular condensates in humans.
    DOI:  https://doi.org/10.1038/s41586-022-05682-1
  18. Cell Rep. 2023 Feb 09. pii: S2211-1247(23)00111-0. [Epub ahead of print]42(2): 112100
    Single-cell multi-omic analysis profiles defective genome activation and epigenetic reprogramming associated with human pre-implantation embryo arrest.
    Jose Ramon Hernandez Mora, Claudia Buhigas, Stephen Clark, Raquel Del Gallego Bonilla, Dagne Daskeviciute, Ana Monteagudo-Sánchez, Maria Eugenia Poo-Llanillo, Jose Vicente Medrano, Carlos Simón, Marcos Meseguer, Gavin Kelsey, David Monk.
      During pre-implantation stages of mammalian development, maternally stored material promotes both the erasure of the sperm and oocyte epigenetic profiles and is responsible for concomitant genome activation. Here, we have utilized single-cell methylome and transcriptome sequencing (scM&T-seq) to quantify both mRNA expression and DNA methylation in oocytes and a developmental series of human embryos at single-cell resolution. We fully characterize embryonic genome activation and maternal transcript degradation and map key epigenetic reprogramming events in developmentally high-quality embryos. By comparing these signatures with early embryos that have undergone spontaneous cleavage-stage arrest, as determined by time-lapse imaging, we identify embryos that fail to appropriately activate their genomes or undergo epigenetic reprogramming. Our results indicate that a failure to successfully accomplish these essential milestones impedes the developmental potential of pre-implantation embryos and is likely to have important implications, similar to aneuploidy, for the success of assisted reproductive cycles.
    Keywords:  CP: Stem cell research; DNA methylation; embryo arrest; embryonic genome activation; single cell; trophectoderm differentiation
    DOI:  https://doi.org/10.1016/j.celrep.2023.112100
  19. Sci Adv. 2023 Feb 10. 9(6): eadf0597
    The Integrator complex regulates microRNA abundance through RISC loading.
    Nina Kirstein, Sadat Dokaneheifard, Pradeep Reddy Cingaram, Monica Guiselle Valencia, Felipe Beckedorff, Helena Gomes Dos Santos, Ezra Blumenthal, Mina Masoumeh Tayari, Gabriel Stephen Gaidosh, Ramin Shiekhattar.
      MicroRNA (miRNA) homeostasis is crucial for the posttranscriptional regulation of their target genes during development and in disease states. miRNAs are derived from primary transcripts and are processed from a hairpin precursor intermediary to a mature 22-nucleotide duplex RNA. Loading of the duplex into the Argonaute (AGO) protein family is pivotal to miRNA abundance and its posttranscriptional function. The Integrator complex plays a key role in protein coding and noncoding RNA maturation, RNA polymerase II pause-release, and premature transcriptional termination. Here, we report that loss of Integrator results in global destabilization of mature miRNAs. Enhanced ultraviolet cross-linking and immunoprecipitation of Integrator uncovered an association with duplex miRNAs before their loading onto AGOs. Tracing miRNA fate from biogenesis to stabilization by incorporating 4-thiouridine in nascent transcripts pinpointed a critical role for Integrator in miRNA assembly into AGOs.
    DOI:  https://doi.org/10.1126/sciadv.adf0597
  20. Cell Rep Med. 2023 Jan 25. pii: S2666-3791(23)00027-7. [Epub ahead of print] 100935
    Dynamic activity in cis-regulatory elements of leukocytes identifies transcription factor activation and stratifies COVID-19 severity in ICU patients.
    Michael Tun Yin Lam, Sascha H Duttke, Mazen F Odish, Hiep D Le, Emily A Hansen, Celina T Nguyen, Samantha Trescott, Roy Kim, Shaunak Deota, Max W Chang, Arjun Patel, Mark Hepokoski, Mona Alotaibi, Mark Rolfsen, Katherine Perofsky, Anna S Warden, Jennifer Foley, Sydney I Ramirez, Jennifer M Dan, Robert K Abbott, Shane Crotty, Laura E Crotty Alexander, Atul Malhotra, Satchidananda Panda, Christopher W Benner, Nicole G Coufal.
      Transcription factor programs mediating the immune response to coronavirus disease 2019 (COVID-19) are not fully understood. Capturing active transcription initiation from cis-regulatory elements such as enhancers and promoters by capped small RNA sequencing (csRNA-seq), in contrast to capturing steady-state transcripts by conventional RNA-seq, allows unbiased identification of the underlying transcription factor activity and regulatory pathways. Here, we profile transcription initiation in critically ill COVID-19 patients, identifying transcription factor motifs that correlate with clinical lung injury and disease severity. Unbiased clustering reveals distinct subsets of cis-regulatory elements that delineate the cell type, pathway-specific, and combinatorial transcription factor activity. We find evidence of critical roles of regulatory networks, showing that STAT/BCL6 and E2F/MYB regulatory programs from myeloid cell populations are activated in patients with poor disease outcomes and associated with COVID-19 susceptibility genetic variants. More broadly, we demonstrate how capturing acute, disease-mediated changes in transcription initiation can provide insight into the underlying molecular mechanisms and stratify patient disease severity.
    Keywords:  COVID-19; active cistrome; acute respiratory distress syndrome; biomarkers; critical care; disease stratification; endotyping; enhancer RNA; transcription factor activity; transcriptional regulation
    DOI:  https://doi.org/10.1016/j.xcrm.2023.100935
  21. Nucleic Acids Res. 2023 Feb 10. pii: gkad054. [Epub ahead of print]
    Epigenetic and chromosomal features drive transposon insertion in Drosophila melanogaster.
    Jichuan Cao, Tianxiong Yu, Bo Xu, Zhongren Hu, Xiao-Ou Zhang, William E Theurkauf, Zhiping Weng.
      Transposons are mobile genetic elements prevalent in the genomes of most species. The distribution of transposons within a genome reflects the actions of two opposing processes: initial insertion site selection, and selective pressure from the host. By analyzing whole-genome sequencing data from transposon-activated Drosophila melanogaster, we identified 43 316 de novo and 237 germline insertions from four long-terminal-repeat (LTR) transposons, one LINE transposon (I-element), and one DNA transposon (P-element). We found that all transposon types favored insertion into promoters de novo, but otherwise displayed distinct insertion patterns. De novo and germline P-element insertions preferred replication origins, often landing in a narrow region around transcription start sites and in regions of high chromatin accessibility. De novo LTR transposon insertions preferred regions with high H3K36me3, promoters and exons of active genes; within genes, LTR insertion frequency correlated with gene expression. De novo I-element insertion density increased with distance from the centromere. Germline I-element and LTR transposon insertions were depleted in promoters and exons, suggesting strong selective pressure to remove transposons from functional elements. Transposon movement is associated with genome evolution and disease; therefore, our results can improve our understanding of genome and disease biology.
    DOI:  https://doi.org/10.1093/nar/gkad054
  22. Brief Bioinform. 2023 Feb 09. pii: bbad044. [Epub ahead of print]
    HiConfidence: a novel approach uncovering the biological signal in Hi-C data affected by technical biases.
    Victoria A Kobets, Sergey V Ulianov, Aleksandra A Galitsyna, Semen A Doronin, Elena A Mikhaleva, Mikhail S Gelfand, Yuri Y Shevelyov, Sergey V Razin, Ekaterina E Khrameeva.
      The chromatin interaction assays, particularly Hi-C, enable detailed studies of genome architecture in multiple organisms and model systems, resulting in a deeper understanding of gene expression regulation mechanisms mediated by epigenetics. However, the analysis and interpretation of Hi-C data remain challenging due to technical biases, limiting direct comparisons of datasets obtained in different experiments and laboratories. As a result, removing biases from Hi-C-generated chromatin contact matrices is a critical data analysis step. Our novel approach, HiConfidence, eliminates biases from the Hi-C data by weighing chromatin contacts according to their consistency between replicates so that low-quality replicates do not substantially influence the result. The algorithm is effective for the analysis of global changes in chromatin structures such as compartments and topologically associating domains. We apply the HiConfidence approach to several Hi-C datasets with significant technical biases, that could not be analyzed effectively using existing methods, and obtain meaningful biological conclusions. In particular, HiConfidence aids in the study of how changes in histone acetylation pattern affect chromatin organization in Drosophila melanogaster S2 cells. The method is freely available at GitHub: https://github.com/victorykobets/HiConfidence.
    Keywords:  Hi-C; chromatin; normalization; technical biases
    DOI:  https://doi.org/10.1093/bib/bbad044
  23. Nature. 2023 Feb 08.
    Dissecting cell identity via network inference and in silico gene perturbation.
    Kenji Kamimoto, Blerta Stringa, Christy M Hoffmann, Kunal Jindal, Lilianna Solnica-Krezel, Samantha A Morris.
      Cell identity is governed by the complex regulation of gene expression, represented as gene-regulatory networks1. Here we use gene-regulatory networks inferred from single-cell multi-omics data to perform in silico transcription factor perturbations, simulating the consequent changes in cell identity using only unperturbed wild-type data. We apply this machine-learning-based approach, CellOracle, to well-established paradigms-mouse and human haematopoiesis, and zebrafish embryogenesis-and we correctly model reported changes in phenotype that occur as a result of transcription factor perturbation. Through systematic in silico transcription factor perturbation in the developing zebrafish, we simulate and experimentally validate a previously unreported phenotype that results from the loss of noto, an established notochord regulator. Furthermore, we identify an axial mesoderm regulator, lhx1a. Together, these results show that CellOracle can be used to analyse the regulation of cell identity by transcription factors, and can provide mechanistic insights into development and differentiation.
    DOI:  https://doi.org/10.1038/s41586-022-05688-9
  24. Elife. 2023 Feb 06. pii: e81579. [Epub ahead of print]12
    Transcription factors underlying photoreceptor diversity.
    Juan M Angueyra, Vincent P Kunze, Laura K Patak, Hailey Kim, Katie Kindt, Wei Li.
      During development, retinal progenitors navigate a complex landscape of fate decisions to generate the major cell classes necessary for proper vision. Transcriptional regulation is critical to generate diversity within these major cell classes. Here, we aim to provide the resources and techniques required to identify transcription factors necessary to generate and maintain diversity in photoreceptor subtypes, which are critical for vision. First, we generate a key resource: a high-quality and deep transcriptomic profile of each photoreceptor subtype in adult zebrafish. We make this resource openly accessible, easy to explore, and have integrated it with other currently available photoreceptor transcriptomic datasets. Second, using our transcriptomic profiles, we derive an in-depth map of expression of transcription factors in photoreceptors. Third, we use efficient CRISPR-Cas9 based mutagenesis to screen for null phenotypes in F0 larvae (F0 screening) as a fast, efficient, and versatile technique to assess the involvement of candidate transcription factors in the generation of photoreceptor subtypes. We first show that known phenotypes can be easily replicated using this method: loss of S cones in foxq2 mutants and loss of rods in nr2e3 mutants. We then identify novel functions for the transcription factor Tbx2, demonstrating that it plays distinct roles in controlling the generation of all photoreceptor subtypes within the retina. Our study provides a roadmap to discover additional factors involved in this process. Additionally, we explore four transcription factors of unknown function (Skor1a, Sall1a, Lrrfip1a, and Xbp1), and find no evidence for their involvement in the generation of photoreceptor subtypes. This dataset and screening method will be a valuable way to explore the genes involved in many other essential aspects of photoreceptor biology.
    Keywords:  cone subtypes; developmental biology; neuroscience; photoreceptor fate; retina; transcription factor; transcriptomics; zebrafish
    DOI:  https://doi.org/10.7554/eLife.81579
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