bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2024–12–08
twenty-six papers selected by
Connor Rogerson, University of Cambridge
  1. Long-range regulation of transcription scales with genomic distance in a gene-specific manner.
  2. Enhancer cooperativity can compensate for loss of activity over large genomic distances.
  3. Arabidopsis histone acetyltransferase complex coordinates cytoplasmic histone acetylation and nuclear chromatin accessibility.
  4. Transcription-coupled changes in genomic region proximities during transcriptional bursting.
  5. Single chromatin fiber profiling and nucleosome position mapping in the human brain.
  6. DNA hypomethylation promotes UHRF1-and SUV39H1/H2-dependent crosstalk between H3K18ub and H3K9me3 to reinforce heterochromatin states.
  7. Osmotic disruption of chromatin induces Topoisomerase 2 activity at sites of transcriptional stress.
  8. Chromatin loops gather targets of upstream regulators together for efficient gene transcription regulation during vernalization in wheat.
  9. An integrative TAD catalog in lymphoblastoid cell lines discloses the functional impact of deletions and insertions in human genomes.
  10. Structure and methyl-lysine binding selectivity of the HUSH complex subunit MPP8.
  11. EpiCHAOS: a metric to quantify epigenomic heterogeneity in single-cell data.
  12. SAM-DNMT3A, a strategy for induction of genome-wide DNA methylation, identifies DNA methylation as a vulnerability in ER-positive breast cancers.
  13. Single-nucleus CUT&RUN elucidates the function of intrinsic and genomics-driven epigenetic heterogeneity in head and neck cancer progression.
  14. A Pax3 lineage gives rise to transient haematopoietic progenitors.
  15. Arrayed CRISPR libraries for the genome-wide activation, deletion and silencing of human protein-coding genes.
  16. Characterization of DNA methylation reader proteins of Arabidopsis thaliana.
  17. A unified-field theory of genome organization and gene regulation.
  18. SiRCle (Signature Regulatory Clustering) model integration reveals mechanisms of phenotype regulation in renal cancer.
  19. IFRD1 is required for maintenance of bladder epithelial homeostasis.
  20. Histone methyltransferases MLL2 and SETD1A/B play distinct roles in H3K4me3 deposition during the transition from totipotency to pluripotency.
  21. Tracking the gene expression programs and clonal relationships that underlie mast, myeloid, and T lineage specification from stem cells.
  22. Castor is a temporal transcription factor that specifies early born central complex neuron identity.
  23. DPF2 reads histone lactylation to drive transcription and tumorigenesis.
  24. RANK drives structured intestinal epithelial expansion during pregnancy.
  25. Tracking transcription-translation coupling in real time.
  26. Rarγ-Foxa1 signaling promotes luminal identity in prostate progenitors and is disrupted in prostate cancer.
  1. Mol Cell. 2024 Nov 19. pii: S1097-2765(24)00861-X. [Epub ahead of print]
    Long-range regulation of transcription scales with genomic distance in a gene-specific manner.
    Christina L Jensen, Liang-Fu Chen, Tomek Swigut, Olivia J Crocker, David Yao, Mike C Bassik, James E Ferrell, Alistair N Boettiger, Joanna Wysocka.
      Although critical for tuning the timing and level of transcription, enhancer communication with distal promoters is not well understood. Here, we bypass the need for sequence-specific transcription factors (TFs) and recruit activators directly using a chimeric array of gRNA oligos to target dCas9 fused to the activator VP64-p65-Rta (CARGO-VPR). We show that this approach achieves effective activator recruitment to arbitrary genomic sites, even those inaccessible when targeted with a single guide. We utilize CARGO-VPR across the Prdm8-Fgf5 locus in mouse embryonic stem cells (mESCs), where neither gene is expressed. Although activator recruitment to any tested region results in the transcriptional induction of at least one gene, the expression level strongly depends on the genomic distance between the promoter and activator recruitment site. However, the expression-distance relationship for each gene scales distinctly in a manner not attributable to differences in 3D contact frequency, promoter DNA sequence, or the presence of repressive chromatin marks at the locus.
    Keywords:  CRISPR activation; dCas9; enhancer; gene regulation; genomic distance; promoter
    DOI:  https://doi.org/10.1016/j.molcel.2024.10.021
  2. Mol Cell. 2024 Nov 23. pii: S1097-2765(24)00913-4. [Epub ahead of print]
    Enhancer cooperativity can compensate for loss of activity over large genomic distances.
    Henry F Thomas, Songjie Feng, Felix Haslhofer, Marie Huber, María García Gallardo, Vincent Loubiere, Daria Vanina, Mattia Pitasi, Alexander Stark, Christa Buecker.
      Enhancers are short DNA sequences that activate their target promoter from a distance; however, increasing the genomic distance between the enhancer and the promoter decreases expression levels. Many genes are controlled by combinations of multiple enhancers, yet the interaction and cooperation of individual enhancer elements are not well understood. Here, we developed a synthetic platform in mouse embryonic stem cells that allows building complex regulatory landscapes from the bottom up. We tested the system by integrating individual enhancers at different distances and confirmed that the strength of an enhancer contributes to how strongly it is affected by increased genomic distance. Furthermore, synergy between two enhancer elements depends on the distance at which the two elements are integrated: introducing a weak enhancer between a strong enhancer and the promoter strongly increases reporter gene expression, allowing enhancers to activate from increased genomic distances.
    Keywords:  enhancer; facilitator; synergy of enhancer elements; transcriptional regulation
    DOI:  https://doi.org/10.1016/j.molcel.2024.11.008
  3. Sci Adv. 2024 Dec 06. 10(49): eadp1840
    Arabidopsis histone acetyltransferase complex coordinates cytoplasmic histone acetylation and nuclear chromatin accessibility.
    Chan-Juan Wu, Xin Xu, Dan-Yang Yuan, Zhen-Zhen Liu, Lian-Mei Tan, Yin-Na Su, Lin Li, She Chen, Xin-Jian He.
      Conserved type B histone acetyltransferases are recognized for their role in acetylating newly synthesized histones in the cytoplasm of eukaryotes. However, their involvement in regulating chromatin within the nucleus remains unclear. Our study shows that the Arabidopsis thaliana type B histone acetyltransferase HAG2 interacts with the histone chaperones MSI2, MSI3, and NASP, as well as the histones H3 and H4, forming a complex in both the cytoplasm and the nucleus. Within this complex, HAG2 and MSI2/3 constitute a histone acetylation module essential for acetylating histone H4 in the cytoplasm. Furthermore, this module works together with NASP to regulate histone acetylation, chromatin accessibility, and gene transcription in the nucleus. This complex enhances chromatin accessibility near transcription start sites while reducing accessibility near transcription termination sites. Our findings reveal a distinct role for the Arabidopsis type B histone acetyltransferase in the nucleus, shedding light on the coordination between cytoplasmic histone acetylation and nuclear chromatin regulation in plants.
    DOI:  https://doi.org/10.1126/sciadv.adp1840
  4. Sci Adv. 2024 Dec 06. 10(49): eadn0020
    Transcription-coupled changes in genomic region proximities during transcriptional bursting.
    Hiroaki Ohishi, Soya Shinkai, Hitoshi Owada, Takeru Fujii, Kazufumi Hosoda, Shuichi Onami, Takashi Yamamoto, Yasuyuki Ohkawa, Hiroshi Ochiai.
      The orchestration of our genes heavily relies on coordinated communication between enhancers and promoters, yet the mechanisms behind this dynamic interplay during active transcription remain unclear. Here, we investigated enhancer-promoter (E-P) interactions in relation to transcriptional bursting in mouse embryonic stem cells using sequential DNA/RNA/immunofluorescence-fluorescence in situ hybridization analyses. Our data reveal that the active state of specific genes is characterized by specific proximities between different genomic regions and the accumulation of transcriptional regulatory factors. Mathematical simulations suggest that an increase in local viscosity could potentially contribute to stabilizing the duration of these E-P proximities. Our study provides insights into the association among E-P proximity, protein accumulation, and transcriptional dynamics, paving the way for a more nuanced understanding of gene-specific regulatory mechanisms.
    DOI:  https://doi.org/10.1126/sciadv.adn0020
  5. Cell Rep Methods. 2024 Nov 25. pii: S2667-2375(24)00301-1. [Epub ahead of print] 100911
    Single chromatin fiber profiling and nucleosome position mapping in the human brain.
    Cyril J Peter, Aman Agarwal, Risa Watanabe, Bibi S Kassim, Xuedi Wang, Tova Y Lambert, Behnam Javidfar, Viviana Evans, Travis Dawson, Maya Fridrikh, Kiran Girdhar, Panos Roussos, Sathiji K Nageshwaran, Nadejda M Tsankova, Robert P Sebra, Mitchell R Vollger, Andrew B Stergachis, Dan Hasson, Schahram Akbarian.
      We apply a single-molecule chromatin fiber sequencing (Fiber-seq) protocol designed for amplification-free cell-type-specific mapping of the regulatory architecture at nucleosome resolution along extended ∼10-kb chromatin fibers to neuronal and non-neuronal nuclei sorted from human brain tissue. Specifically, application of this method enables the resolution of cell-selective promoter and enhancer architectures on single fibers, including transcription factor footprinting and position mapping, with sequence-specific fixation of nucleosome arrays flanking transcription start sites and regulatory motifs. We uncover haplotype-specific chromatin patterns, multiple regulatory elements cis-aligned on individual fibers, and accessible chromatin at 20,000 unique sites encompassing retrotransposons and other repeat sequences hitherto "unmappable" by short-read epigenomic sequencing. Overall, we show that Fiber-seq is applicable to human brain tissue, offering sharp demarcation of nucleosome-depleted regions at sites of open chromatin in conjunction with multi-kilobase nucleosomal positioning at single-fiber resolution on a genome-wide scale.
    Keywords:  CP: Biotechnology; CP: Neuroscience; Hia5 methyltransferase; adenine methylation; cytosine methylation; long-read sequencing; nucleosomal array; nucleosomal offset; postmortem brain; transcription factor footprint
    DOI:  https://doi.org/10.1016/j.crmeth.2024.100911
  6. Mol Cell. 2024 Nov 25. pii: S1097-2765(24)00914-6. [Epub ahead of print]
    DNA hypomethylation promotes UHRF1-and SUV39H1/H2-dependent crosstalk between H3K18ub and H3K9me3 to reinforce heterochromatin states.
    Yanqing Liu, Joel A Hrit, Alison A Chomiak, Stephanie Stransky, Jordan R Hoffman, Rochelle L Tiedemann, Ashley K Wiseman, Leena S Kariapper, Bradley M Dickson, Evan J Worden, Christopher J Fry, Simone Sidoli, Scott B Rothbart.
      Mono-ubiquitination of lysine 18 on histone H3 (H3K18ub), catalyzed by UHRF1, is a DNMT1 docking site that facilitates replication-coupled DNA methylation maintenance. Its functions beyond this are unknown. Here, we genomically map simultaneous increases in UHRF1-dependent H3K18ub and SUV39H1/H2-dependent H3K9me3 following DNMT1 inhibition. Mechanistically, transient accumulation of hemi-methylated DNA at CpG islands facilitates UHRF1 recruitment and E3 ligase activity toward H3K18. Notably, H3K18ub enhances SUV39H1/H2 methyltransferase activity and, in colon cancer cells, nucleates new H3K9me3 domains at CpG island promoters of DNA methylation-silenced tumor suppressor genes (TSGs). Disrupting UHRF1 enzyme activity prevents H3K9me3 accumulation while promoting PRC2-dependent H3K27me3 as a tertiary layer of gene repression in these regions. By contrast, disrupting H3K18ub-dependent SUV39H1/H2 activity enhances the transcriptional activating and antiproliferative effects of DNMT1 inhibition. Collectively, these findings reveal roles for UHRF1 and H3K18ub in regulating a hierarchy of repressive histone methylation signaling and rationalize a combination strategy for epigenetic cancer therapy.
    Keywords:  DNA methylation; SUV39H1/H2; UHRF1; epigenetic therapy; histone methylation; histone ubiquitination
    DOI:  https://doi.org/10.1016/j.molcel.2024.11.009
  7. Nat Commun. 2024 Dec 05. 15(1): 10606
    Osmotic disruption of chromatin induces Topoisomerase 2 activity at sites of transcriptional stress.
    William H Gittens, Rachal M Allison, Ellie M Wright, George G B Brown, Matthew J Neale.
      Transcription generates superhelical stress in DNA that poses problems for genome stability, but determining when and where such stress arises within chromosomes is challenging. Here, using G1-arrested S. cerevisiae cells, and employing rapid fixation and ultra-sensitive enrichment, we utilise the physiological activity of endogenous topoisomerase 2 (Top2) as a probe of transcription-induced superhelicity. We demonstrate that Top2 activity is surprisingly uncorrelated with transcriptional activity, suggesting that superhelical stress is obscured from Top2 within chromatin in vivo. We test this idea using osmotic perturbation-a treatment that transiently destabilises chromatin in vivo-revealing that Top2 activity redistributes within sub-minute timescales into broad zones patterned by long genes, convergent gene arrays, and transposon elements-and also by acute transcriptional induction. We propose that latent superhelical stress is normally absorbed by the intrinsic topological buffering capacity of chromatin, helping to avoid spurious topoisomerase activity arising within the essential coding regions of the genome.
    DOI:  https://doi.org/10.1038/s41467-024-54567-6
  8. Genome Biol. 2024 Dec 03. 25(1): 306
    Chromatin loops gather targets of upstream regulators together for efficient gene transcription regulation during vernalization in wheat.
    Yanyan Liu, Xintong Xu, Chao He, Liujie Jin, Ziru Zhou, Jie Gao, Minrong Guo, Xin Wang, Chuanye Chen, Mohammed H Ayaad, Xingwang Li, Wenhao Yan.
       BACKGROUND: Plants respond to environmental stimuli by altering gene transcription that is highly related with chromatin status, including histone modification, chromatin accessibility, and three-dimensional chromatin interaction. Vernalization is essential for the transition to reproductive growth for winter wheat. How wheat reshapes its chromatin features, especially chromatin interaction during vernalization, remains unknown.
    RESULTS: Combinatory analysis of gene transcription and histone modifications in winter wheat under different vernalization conditions identifies 17,669 differential expressed genes and thousands of differentially enriched peaks of H3K4me3, H3K27me3, and H3K9ac. We find dynamic gene expression across the vernalization process is highly associated with H3K4me3. More importantly, the dynamic H3K4me3- and H3K9ac-associated chromatin-chromatin interactions demonstrate that vernalization leads to increased chromatin interactions and gene activation. Remarkably, spatially distant targets of master regulators like VRN1 and VRT2 are gathered together by chromatin loops to achieve efficient transcription regulation, which is designated as a "shepherd" model. Furthermore, by integrating gene regulatory network for vernalization and natural variation of flowering time, TaZNF10 is identified as a negative regulator for vernalization-related flowering time in wheat.
    CONCLUSIONS: We reveal dynamic gene transcription network during vernalization and find that the spatially distant genes can be recruited together via chromatin loops associated with active histone mark thus to be more efficiently found and bound by upstream regulator. It provides new insights into understanding vernalization and response to environmental stimuli in wheat and other plants.
    Keywords:  Chromatin interaction; Epigenome; Gene transcription regulation; Vernalization; Wheat
    DOI:  https://doi.org/10.1186/s13059-024-03437-x
  9. Genome Res. 2024 Dec 05.
    An integrative TAD catalog in lymphoblastoid cell lines discloses the functional impact of deletions and insertions in human genomes.
    Human Genome Structural Variation Consortium (HGSVC)
      The human genome is packaged within a three-dimensional (3D) nucleus and organized into structural units known as compartments, topologically associating domains (TADs), and loops. TAD boundaries, separating adjacent TADs, have been found to be well conserved across mammalian species and more evolutionarily constrained than TADs themselves. Recent studies show that structural variants (SVs) can modify 3D genomes through the disruption of TADs, which play an essential role in insulating genes from outside regulatory elements' aberrant regulation. However, how SV affects the 3D genome structure and their association among different aspects of gene regulation and candidate cis-regulatory elements (cCREs) have rarely been studied systematically. Here, we assess the impact of SVs intersecting with TAD boundaries by developing an integrative Hi-C analysis pipeline, which enables the generation of an in-depth catalog of TADs and TAD boundaries in human lymphoblastoid cell lines (LCLs) to fill the gap of limited resources. Our catalog contains 18,865 TADs, including 4596 sub-TADs, with 185 SVs (TAD-SVs) that alter chromatin architecture. By leveraging the ENCODE registry of cCREs in humans, we determine that 34 of 185 TAD-SVs intersect with cCREs and observe significant enrichment of TAD-SVs within cCREs. This study provides a database of TADs and TAD-SVs in the human genome that will facilitate future investigations of the impact of SVs on chromatin structure and gene regulation in health and disease.
    DOI:  https://doi.org/10.1101/gr.279419.124
  10. J Mol Biol. 2024 Dec 03. pii: S0022-2836(24)00520-5. [Epub ahead of print] 168890
    Structure and methyl-lysine binding selectivity of the HUSH complex subunit MPP8.
    Nikos Nikolopoulos, Shun-Ichiro Oda, Daniil M Prigozhin, Yorgo Modis.
      The Human Silencing Hub (HUSH) guards the genome from the pathogenic effects of retroelement expression. Composed of MPP8, TASOR, and Periphilin-1, HUSH recognizes actively transcribed retrotransposed sequences by the presence of long (>1.5-kb) nascent transcripts without introns. HUSH recruits effectors that alter chromatin structure, degrade transcripts, and deposit transcriptionally repressive epigenetic marks. Here, we report the crystal structure of the C-terminal domain (CTD) of MPP8 necessary for HUSH activity. The MPP8 CTD consists of five ankyrin repeats followed by a domain with structural homology to the PINIT domains of Siz/PIAS-family SUMO E3 ligases. AlphaFold3 modeling of the MPP8-TASOR complex predicts that a SPOC domain and a domain with a novel fold in TASOR form extended interaction interfaces with the MPP8 CTD. Point mutations at these interfaces resulted in loss of HUSH-dependent transcriptional repression in a cell-based reporter assay, validating the AlphaFold3 model. The MPP8 chromodomain, known to bind the repressive mark H3K9me3, bound with similar or higher affinity to sequences in the H3K9 methyltransferase subunits SETDB1, ATF7IP, G9a, and GLP. Hence, MPP8 promotes heterochromatinization by recruiting H3K9 methyltransferases. Our work identifies novel structural elements in MPP8 required for HUSH complex assembly and silencing, thereby fulfilling vital functions in controlling retrotransposons.
    Keywords:  H3K9 methyltransferase; LINE-1 retrotransposon; Transcriptional repression; epigenetic silencing; histone H3 lysine 9 methylation (H3K9me3)
    DOI:  https://doi.org/10.1016/j.jmb.2024.168890
  11. Genome Biol. 2024 Dec 03. 25(1): 305
    EpiCHAOS: a metric to quantify epigenomic heterogeneity in single-cell data.
    Katherine Kelly, Michael Scherer, Martina Maria Braun, Pavlo Lutsik, Christoph Plass.
      Epigenetic heterogeneity is a fundamental property of biological systems and is recognized as a potential driver of tumor plasticity and therapy resistance. Single-cell epigenomics technologies have been widely employed to study epigenetic variation between-but not within-cellular clusters. We introduce epiCHAOS: a quantitative metric of cell-to-cell heterogeneity, applicable to any single-cell epigenomics data type. After validation in synthetic datasets, we apply epiCHAOS to investigate global and region-specific patterns of epigenetic heterogeneity across diverse biological systems. EpiCHAOS provides an excellent approximation of stemness and plasticity in development and malignancy, making it a valuable addition to single-cell cancer epigenomics analyses.
    Keywords:  Cancer; Chromatin; DNA methylation; Epigenetics; Heterogeneity; Plasticity; Single-cell epigenomics
    DOI:  https://doi.org/10.1186/s13059-024-03446-w
  12. Nat Commun. 2024 Dec 01. 15(1): 10449
    SAM-DNMT3A, a strategy for induction of genome-wide DNA methylation, identifies DNA methylation as a vulnerability in ER-positive breast cancers.
    Mahnaz Hosseinpour, Xinqi Xi, Ling Liu, Luis Malaver-Ortega, Laura Perlaza-Jimenez, Jihoon E Joo, Harrison M York, Jonathan Beesley, C Elizabeth Caldon, Pierre-Antoine Dugué, James G Dowty, Senthil Arumugam, Melissa C Southey, Joseph Rosenbluh.
      DNA methylation is an epigenetic mark that plays a critical role in regulating gene expression. DNA methyltransferase (DNMT) inhibitors, inhibit global DNA methylation and have been a key tool in studies of DNA methylation. A major bottleneck is the lack of tools to induce global DNA methylation. Here, we engineered a CRISPR based approach, that we initially designed, to enable site-specific DNA methylation. Using the synergistic activation mediator (SAM) system, we unexpectedly find that regardless of the targeted sequence any sgRNA induces global genome-wide DNA methylation. We term this method SAM-DNMT3A and show that induction of global DNA methylation is a unique vulnerability in ER-positive breast cancer suggesting a therapeutic approach. Our findings highlight the need of caution when using CRISPR based approaches for inducing DNA methylation and demonstrate a method for global induction of DNA methylation.
    DOI:  https://doi.org/10.1038/s41467-024-54824-8
  13. Genome Res. 2024 Dec 02. pii: gr.279105.124. [Epub ahead of print]
    Single-nucleus CUT&RUN elucidates the function of intrinsic and genomics-driven epigenetic heterogeneity in head and neck cancer progression.
    Howard Womersley, Daniel Muliaditan, Ramanuj DasGupta, Lih Feng Cheow.
      Interrogating regulatory epigenetic alterations during tumor progression at the resolution of single cells has remained an understudied area of research. Here we developed a highly sensitive single-nucleus CUT&RUN (snCUT&RUN) assay to profile histone modifications in isogenic primary, metastatic, and cisplatin-resistant head and neck squamous cell carcinoma (HNSCC) patient-derived tumor cell lines. We find that the epigenome can be involved in diverse modes to contribute towards HNSCC progression. First, we demonstrate that gene expression changes during HNSCC progression can be comodulated by alterations in both copy number and chromatin activity, driving epigenetic rewiring of cell states. Furthermore, intratumour epigenetic heterogeneity (ITeH) may predispose subclonal populations within the primary tumour to adapt to selective pressures and foster the acquisition of malignant characteristics. In conclusion, snCUT&RUN serves as a valuable addition to the existing toolkit of single-cell epigenomic assays and can be used to dissect the functionality of the epigenome during cancer progression.
    DOI:  https://doi.org/10.1101/gr.279105.124
  14. Development. 2024 Dec 01. pii: dev202924. [Epub ahead of print]151(23):
    A Pax3 lineage gives rise to transient haematopoietic progenitors.
    Giovanni Canu, Rosamaria Correra, Guillermo Diez-Pinel, Raphaël F P Castellan, Laura Denti, Alessandro Fantin, Christiana Ruhrberg.
      During embryonic development, muscle tissues, skin, and a subset of vascular endothelial cells arise from Pax3-expressing embryonic progenitors defined as paraxial mesoderm. By contrast, haemogenic potential is well established for extra-embryonic mesoderm and intra-embryonic lateral plate mesoderm, which do not express Pax3. To date, it is not known whether the haematopoietic system also contains Pax3 lineage cells. Here, we show that the mouse foetal liver and foetal circulation contain a transient population of Pax3 lineage cells with hallmarks of haematopoietic progenitors and the potential to generate both myeloid and erythroid cells. We propose that Pax3 lineage haematopoietic cells should be investigated to better understand normal haematopoietic development from different mesodermal derivatives. Further, genetic alterations of Pax3 lineage haematopoietic cells should be investigated for their potential to cause haematopoietic malignancies.
    Keywords:  Foetal liver; Hematopoietic development; Macrophage origin; Mouse; Paraxial mesoderm; Pax3; Vascular endothelial cell
    DOI:  https://doi.org/10.1242/dev.202924
  15. Nat Biomed Eng. 2024 Dec 04.
    Arrayed CRISPR libraries for the genome-wide activation, deletion and silencing of human protein-coding genes.
    Jiang-An Yin, Lukas Frick, Manuel C Scheidmann, Tingting Liu, Chiara Trevisan, Ashutosh Dhingra, Anna Spinelli, Yancheng Wu, Longping Yao, Dalila Laura Vena, Britta Knapp, Jingjing Guo, Elena De Cecco, Kathi Ging, Andrea Armani, Edward J Oakeley, Florian Nigsch, Joel Jenzer, Jasmin Haegele, Michal Pikusa, Joachim Täger, Salvador Rodriguez-Nieto, Vangelis Bouris, Rafaela Ribeiro, Federico Baroni, Manmeet Sakshi Bedi, Scott Berry, Marco Losa, Simone Hornemann, Martin Kampmann, Lucas Pelkmans, Dominic Hoepfner, Peter Heutink, Adriano Aguzzi.
      Arrayed CRISPR libraries extend the scope of gene-perturbation screens to non-selectable cell phenotypes. However, library generation requires assembling thousands of vectors expressing single-guide RNAs (sgRNAs). Here, by leveraging massively parallel plasmid-cloning methodology, we show that arrayed libraries can be constructed for the genome-wide ablation (19,936 plasmids) of human protein-coding genes and for their activation and epigenetic silencing (22,442 plasmids), with each plasmid encoding an array of four non-overlapping sgRNAs designed to tolerate most human DNA polymorphisms. The quadruple-sgRNA libraries yielded high perturbation efficacies in deletion (75-99%) and silencing (76-92%) experiments and substantial fold changes in activation experiments. Moreover, an arrayed activation screen of 1,634 human transcription factors uncovered 11 novel regulators of the cellular prion protein PrPC, screening with a pooled version of the ablation library led to the identification of 5 novel modifiers of autophagy that otherwise went undetected, and 'post-pooling' individually produced lentiviruses eliminated template-switching artefacts and enhanced the performance of pooled screens for epigenetic silencing. Quadruple-sgRNA arrayed libraries are a powerful and versatile resource for targeted genome-wide perturbations.
    DOI:  https://doi.org/10.1038/s41551-024-01278-4
  16. Genome Res. 2024 Dec 04.
    Characterization of DNA methylation reader proteins of Arabidopsis thaliana.
    Jonathan Cahn, James P B Lloyd, Ino D Karemaker, Pascal W T C Jansen, Jahnvi Pflueger, Owen Duncan, Jakob Petereit, Ozren Bogdanovic, A Harvey Millar, Michiel Vermeulen, Ryan Lister.
      In plants, cytosine DNA methylation (mC) is largely associated with transcriptional repression of transposable elements, but it can also be found in the body of expressed genes, referred to as gene body methylation (gbM). gbM is correlated with ubiquitously expressed genes; however, its function, or absence thereof, is highly debated. The different outputs that mC can have raise questions as to how it is interpreted-or read-differently in these sequence and genomic contexts. To screen for potential mC-binding proteins, we performed an unbiased DNA affinity pull-down assay combined with quantitative mass spectrometry using methylated DNA probes for each DNA sequence context. All mC readers known to date preferentially bind to the methylated probes, along with a range of new mC-binding protein candidates. Functional characterization of these mC readers, focused on the MBD and SUVH families, was undertaken by ChIP-seq mapping of genome-wide binding sites, their protein interactors, and the impact of high-order mutations on transcriptomic and epigenomic profiles. Together, these results highlight specific context preferences for these proteins, and in particular the ability of MBD2 to bind predominantly to gbM. This comprehensive analysis of Arabidopsis mC readers emphasizes the complexity and interconnectivity between DNA methylation and chromatin remodeling processes in plants.
    DOI:  https://doi.org/10.1101/gr.279379.124
  17. iScience. 2024 Dec 20. 27(12): 111218
    A unified-field theory of genome organization and gene regulation.
    Giuseppe Negro, Massimiliano Semeraro, Peter R Cook, Davide Marenduzzo.
      Our aim is to predict how often genic and non-genic promoters fire within a cell. We first review a parsimonious pan-genomic model for genome organization and gene regulation, where transcription rate is determined by proximity in 3D space of promoters to clusters containing appropriate factors and RNA polymerases. This model reconciles conflicting results indicating that regulatory mammalian networks are both simple (as over-expressing just 4 transcription factors switches cell state) and complex (as genome-wide association studies show phenotypes like cell type are determined by thousands of loci rarely encoding such factors). We then present 3D polymer simulations, and a proximity formula based on our biological model that enables prediction of transcriptional activities of all promoters in three human cell types. This simple fitting-free formula contains just one variable (distance on the genetic map to the nearest active promoter), and we suggest it can in principle be applied to any organism.
    Keywords:  Biological sciences; Data processing in systems biology; Natural sciences; Systems biology
    DOI:  https://doi.org/10.1016/j.isci.2024.111218
  18. Genome Med. 2024 Dec 04. 16(1): 144
    SiRCle (Signature Regulatory Clustering) model integration reveals mechanisms of phenotype regulation in renal cancer.
    Ariane Mora, Christina Schmidt, Brad Balderson, Christian Frezza, Mikael Bodén.
       BACKGROUND: Clear cell renal cell carcinoma (ccRCC) tumours develop and progress via complex remodelling of the kidney epigenome, transcriptome, proteome and metabolome. Given the subsequent tumour and inter-patient heterogeneity, drug-based treatments report limited success, calling for multi-omics studies to extract regulatory relationships, and ultimately, to develop targeted therapies. Yet, methods for multi-omics integration to reveal mechanisms of phenotype regulation are lacking.
    METHODS: Here, we present SiRCle (Signature Regulatory Clustering), a method to integrate DNA methylation, RNA-seq and proteomics data at the gene level by following central dogma of biology, i.e. genetic information proceeds from DNA, to RNA, to protein. To identify regulatory clusters across the different omics layers, we group genes based on the layer where the gene's dysregulation first occurred. We combine the SiRCle clusters with a variational autoencoder (VAE) to reveal key features from omics' data for each SiRCle cluster and compare patient subpopulations in a ccRCC and a PanCan cohort.
    RESULTS: Applying SiRCle to a ccRCC cohort, we showed that glycolysis is upregulated by DNA hypomethylation, whilst mitochondrial enzymes and respiratory chain complexes are translationally suppressed. Additionally, we identify metabolic enzymes associated with survival along with the possible molecular driver behind the gene's perturbations. By using the VAE to integrate omics' data followed by statistical comparisons between tumour stages on the integrated space, we found a stage-dependent downregulation of proximal renal tubule genes, hinting at a loss of cellular identity in cancer cells. We also identified the regulatory layers responsible for their suppression. Lastly, we applied SiRCle to a PanCan cohort and found common signatures across ccRCC and PanCan in addition to the regulatory layer that defines tissue identity.
    CONCLUSIONS: Our results highlight SiRCle's ability to reveal mechanisms of phenotype regulation in cancer, both specifically in ccRCC and broadly in a PanCan context. SiRCle ranks genes according to biological features. https://github.com/ArianeMora/SiRCle_multiomics_integration .
    Keywords:  Clear cell renal cell carcinoma; Integration; Machine learning; Metabolism; Multi-omics; PanCan; Regulation; Variational autoencoder
    DOI:  https://doi.org/10.1186/s13073-024-01415-3
  19. iScience. 2024 Dec 20. 27(12): 111282
    IFRD1 is required for maintenance of bladder epithelial homeostasis.
    Bisiayo E Fashemi, Amala K Rougeau, Arnold M Salazar, Steven J Bark, Rayvanth Chappidi, Jeffrey W Brown, Charles J Cho, Jason C Mills, Indira U Mysorekar.
      The maintenance of homeostasis and rapid regeneration of the urothelium following stress are critical for bladder function. Here, we identify a key role for IFRD1 in maintaining urothelial homeostasis in a mouse model. We demonstrate that the murine bladder expresses IFRD1 at homeostasis, particularly in the urothelium, and its loss alters the global transcriptome with significant accumulation of endolysosomes and dysregulated uroplakin expression pattern. We show that IFRD1 interacts with mRNA-translation-regulating factors in human urothelial cells. Loss of Ifrd1 leads to disrupted proteostasis, enhanced endoplasmic reticulum (ER stress) with activation of the PERK arm of the unfolded protein response pathway, and increased oxidative stress. Ifrd1-deficient bladders exhibit urothelial cell apoptosis/exfoliation, enhanced basal cell proliferation, reduced differentiation into superficial cells, increased urothelial permeability, and aberrant voiding behavior. These findings highlight a crucial role for IFRD1 in urothelial homeostasis, suggesting its potential as a therapeutic target for bladder dysfunction.
    Keywords:  Cell biology; Physiology; Transcriptomics
    DOI:  https://doi.org/10.1016/j.isci.2024.111282
  20. EMBO J. 2024 Dec 05.
    Histone methyltransferases MLL2 and SETD1A/B play distinct roles in H3K4me3 deposition during the transition from totipotency to pluripotency.
    Jingjing Zhang, Qiaoran Sun, Liang Liu, Shichun Yang, Xia Zhang, Yi-Liang Miao, Xin Liu.
      In early mammalian embryogenesis, a shift from non-canonical histone H3 lysine 4 trimethylation (H3K4me3) linked to transcriptional repression to canonical H3K4me3 indicating active promoters occurs during zygotic genome activation (ZGA). However, the mechanisms and roles of these H3K4me3 states in embryogenesis remain poorly understood. Our research reveals that the histone methyltransferase MLL2 is responsible for installing H3K4me3 (both non-canonical and canonical) in totipotent embryos, while a transition to SETD1A/B-deposited H3K4me3 occurs in pluripotent embryos. Interestingly, MLL2-mediated H3K4me3 operates independently of transcription, fostering a relaxed chromatin state conducive to totipotency rather than directly influencing transcription. Conversely, SETD1A/B-mediated H3K4me3, which depends on transcription, is crucial for facilitating expression of genes essential for pluripotency and pre-implantation development. Our findings highlight the role of the H3K4me3 transition, mediated by an MLL2-to-SETD1A/B relay mechanism, in the regulation of transition from totipotency to pluripotency during early embryogenesis.
    Keywords:  First Lineage Segregation; H3K4me3; MLL2; SETD1A/B; Zygotic Genome Activation
    DOI:  https://doi.org/10.1038/s44318-024-00329-5
  21. Cell Syst. 2024 Nov 26. pii: S2405-4712(24)00310-7. [Epub ahead of print]
    Tracking the gene expression programs and clonal relationships that underlie mast, myeloid, and T lineage specification from stem cells.
    Yale S Michaels, Matthew C Major, Becca Bonham-Carter, Jingqi Zhang, Tiam Heydari, John M Edgar, Mona M Siu, Laura Greenstreet, Roser Vilarrasa-Blasi, Seungjoon Kim, Elizabeth L Castle, Aden Forrow, M Iliana Ibanez-Rios, Carla Zimmerman, Yvonne Chung, Tara Stach, Nico Werschler, David J H F Knapp, Roser Vento-Tormo, Geoffrey Schiebinger, Peter W Zandstra.
      T cells develop from hematopoietic progenitors in the thymus and protect against pathogens and cancer. However, the emergence of human T cell-competent blood progenitors and their subsequent specification to the T lineage have been challenging to capture in real time. Here, we leveraged a pluripotent stem cell differentiation system to understand the transcriptional dynamics and cell fate restriction events that underlie this critical developmental process. Time-resolved single-cell RNA sequencing revealed that downregulation of the multipotent hematopoietic program, upregulation of >90 lineage-associated transcription factors, and cell-cycle exit all occur within a highly coordinated developmental window. Gene-regulatory network inference uncovered a role for YBX1 in T lineage specification. We mapped the differentiation cell fate hierarchy using transcribed lineage barcoding and discovered that mast and myeloid potential bifurcate from each other early in hematopoiesis, upstream of T lineage restriction. Our systems-level analyses provide a quantitative, time-resolved model of human T cell fate specification. A record of this paper's transparent peer review process is included in the supplemental information.
    Keywords:  T cell; induced pluripotent stem cell; lineage tracing; mast cell; molecular barcoding; myeloid; single-cell RNA sequencing; systems biology
    DOI:  https://doi.org/10.1016/j.cels.2024.11.001
  22. Development. 2024 Dec 02. pii: dev.204318. [Epub ahead of print]
    Castor is a temporal transcription factor that specifies early born central complex neuron identity.
    Noah R Dillon, Chris Q Doe.
      The generation of neuronal diversity is important for brain function, but how diversity is generated is incompletely understood. We used the development of the Drosophila central complex (CX) to address this question. The CX develops from eight bilateral Type 2 neuroblasts (T2NBs), which generate hundreds of different neuronal types. T2NBs express broad opposing temporal gradients of RNA-binding proteins. It remains unknown whether these protein gradients are sufficient to directly generate all known neuronal diversity, or whether there are temporal transcription factors (TTFs) with narrow expression windows that each specify a small subset of CX neuron identities. Multiple candidate TTFs have been identified, but their function remains uncharacterized. Here, we show that: (i) the adult E-PG neurons are born from early larval T2NBs; (ii) the candidate TTF Castor is expressed transiently in early larval T2NBs when E-PG and P-EN neurons are born; and (iii) that Castor is required to specify early born E-PG and P-EN neuron identities. We conclude that Castor is a TTF in larval T2NB lineages that specifies multiple, early born CX neuron identities.
    Keywords:   Drosophila ; Castor; Central complex; Neuroblast; Temporal specification
    DOI:  https://doi.org/10.1242/dev.204318
  23. Proc Natl Acad Sci U S A. 2024 Dec 10. 121(50): e2421496121
    DPF2 reads histone lactylation to drive transcription and tumorigenesis.
    Guijin Zhai, Ziping Niu, Zixin Jiang, Fei Zhao, Siyu Wang, Chen Chen, Wei Zheng, Aiyuan Wang, Yong Zang, Yanpu Han, Kai Zhang.
      Lysine lactylation (Kla) is a new type of histone mark implicated in the regulation of various functional processes such as transcription. However, how this histone mark acts in cancers remains unexplored due in part to a lack of knowledge about its reader proteins. Here, we observe that cervical cancer (CC) cells undergo metabolic reprogram by which lactate accumulation and thereby boosts histone lactylation, particularly H3K14la. Utilizing a multivalent photoaffinity probe in combination with quantitative proteomics approach, we identify DPF2 as a candidate target of H3K14la. Biochemical studies as well as CUT&Tag analysis reveal that DPF2 is capable of binding to H3K14la and colocalizes with it on promoters of oncogenic genes. Notably, disrupting the DPF2-H3K14la interaction through structure-guided mutation blunts those cancer-related gene expression along with cell survival. Together, our findings reveal DPF2 as a bona fide H3K14la effector that couples histone lactylation to gene transcription and cell survival, offering insight into how histone Kla engages in transcription and tumorigenesis.
    Keywords:  chemical proteomics; epigenetics; histone modifications; histone reader; lysine lactylation
    DOI:  https://doi.org/10.1073/pnas.2421496121
  24. Nature. 2024 Dec 04.
    RANK drives structured intestinal epithelial expansion during pregnancy.
    Masahiro Onji, Verena Sigl, Thomas Lendl, Maria Novatchkova, Asier Ullate-Agote, Amanda Andersson-Rolf, Ivona Kozieradzki, Rubina Koglgruber, Tsung-Pin Pai, Dominic Lichtscheidl, Komal Nayak, Matthias Zilbauer, Natalia A Carranza García, Laura Katharina Sievers, Maren Falk-Paulsen, Shane J F Cronin, Astrid Hagelkruys, Shinichiro Sawa, Lisa C Osborne, Philip Rosenstiel, Manolis Pasparakis, Jürgen Ruland, Hiroshi Takayanagi, Hans Clevers, Bon-Kyoung Koo, Josef M Penninger.
      During reproduction, multiple species such as insects and all mammals undergo extensive physiological and morphological adaptions to ensure health and survival of the mother and optimal development of the offspring. Here we report that the intestinal epithelium undergoes expansion during pregnancy and lactation in mammals. This enlargement of the intestinal surface area results in a novel geometry of expanded villi. Receptor activator of nuclear factor-κΒ (RANK, encoded by TNFRSF11A) and its ligand RANKL were identified as a molecular pathway involved in this villous expansion of the small intestine in vivo in mice and in intestinal mouse and human organoids. Mechanistically, RANK-RANKL protects gut epithelial cells from cell death and controls the intestinal stem cell niche through BMP receptor signalling, resulting in the elongation of villi and a prominent increase in the intestinal surface. As a transgenerational consequence, babies born to female mice that lack Rank in the intestinal epithelium show reduced weight and develop glucose intolerance after metabolic stress. Whereas gut epithelial remodelling in pregnancy/lactation is reversible, constitutive expression of an active form of RANK is sufficient to drive intestinal expansion followed by loss of villi and stem cells, and prevents the formation of Apcmin-driven small intestinal stem cell tumours. These data identify RANK-RANKL as a pathway that drives intestinal epithelial expansion in pregnancy/lactation, one of the most elusive and fundamental tissue remodelling events in mammalian life history and evolution.
    DOI:  https://doi.org/10.1038/s41586-024-08284-1
  25. Nature. 2024 Dec 04.
    Tracking transcription-translation coupling in real time.
    Nusrat Shahin Qureshi, Olivier Duss.
      A central question in biology is how macromolecular machines function cooperatively. In bacteria, transcription and translation occur in the same cellular compartment, and can be physically and functionally coupled1-4. Although high-resolution structures of the ribosome-RNA polymerase (RNAP) complex have provided initial mechanistic insights into the coupling process5-10, we lack knowledge of how these structural snapshots are placed along a dynamic reaction trajectory. Here we reconstitute a complete and active transcription-translation system and develop multi-colour single-molecule fluorescence microscopy experiments to directly and simultaneously track transcription elongation, translation elongation and the physical and functional coupling between the ribosome and the RNAP in real time. Our data show that physical coupling between ribosome and RNAP can occur over hundreds of nucleotides of intervening mRNA by mRNA looping, a process facilitated by NusG. We detect active transcription elongation during mRNA looping and show that NusA-paused RNAPs can be activated by the ribosome by long-range physical coupling. Conversely, the ribosome slows down while colliding with the RNAP. We hereby provide an alternative explanation for how the ribosome can efficiently rescue RNAP from frequent pausing without requiring collisions by a closely trailing ribosome. Overall, our dynamic data mechanistically highlight an example of how two central macromolecular machineries, the ribosome and RNAP, can physically and functionally cooperate to optimize gene expression.
    DOI:  https://doi.org/10.1038/s41586-024-08308-w
  26. EMBO Rep. 2024 Dec 04.
    Rarγ-Foxa1 signaling promotes luminal identity in prostate progenitors and is disrupted in prostate cancer.
    Dario De Felice, Alessandro Alaimo, Davide Bressan, Sacha Genovesi, Elisa Marmocchi, Nicole Annesi, Giulia Beccaceci, Davide Dalfovo, Federico Cutrupi, Stefano Medaglia, Veronica Foletto, Marco Lorenzoni, Francesco Gandolfi, Srinivasaraghavan Kannan, Chandra S Verma, Alessandro Vasciaveo, Michael M Shen, Alessandro Romanel, Fulvio Chiacchiera, Francesco Cambuli, Andrea Lunardi.
      Retinoic acid (RA) signaling is a master regulator of vertebrate development with crucial roles in body axis orientation and tissue differentiation, including in the reproductive system. However, a mechanistic understanding of how RA signaling governs cell lineage identity is often missing. Here, leveraging prostate organoid technology, we show that RA signaling orchestrates the commitment of adult mouse prostate progenitors to glandular identity, epithelial barrier integrity, and specification of prostatic lumen. RA-dependent RARγ activation promotes the expression of Foxa1, which synergizes with the androgen pathway for luminal expansion, cytoarchitecture and function. FOXA1 mutations are common in prostate and breast cancers, though their pathogenic mechanism is incompletely understood. Combining functional genetics with structural modeling of FOXA1 folding and chromatin binding analyses, we discover that FOXA1F254E255 is a loss-of-function mutation compromising its transcriptional function and luminal fate commitment of prostate progenitors. Overall, we define RA as an instructive signal for glandular identity in adult prostate progenitors. Importantly, we identify cancer-associated FOXA1 indels affecting residue F254 as loss-of-function mutations promoting dedifferentiation of adult prostate progenitors.
    Keywords:  FOXA1; Organoids; Prostate; Retinoic Acid
    DOI:  https://doi.org/10.1038/s44319-024-00335-y
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