bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2024–01–14
twenty-two papers selected by
Connor Rogerson, University of Cambridge
  1. ZNF143 deletion alters enhancer/promoter looping and CTCF/cohesin geometry.
  2. Pioneer and PRDM transcription factors coordinate bivalent epigenetic states to safeguard cell fate.
  3. Mitotic bookmarking redundancy by nuclear receptors in pluripotent cells.
  4. Transcription factor co-expression mediates lineage priming for embryonic and extra-embryonic differentiation.
  5. Direct observation of a condensate effect on super-enhancer controlled gene bursting.
  6. Pluripotency state transition of embryonic stem cells requires the turnover of histone chaperone FACT on chromatin.
  7. Modeling methyl-sensitive transcription factor motifs with an expanded epigenetic alphabet.
  8. NELF focuses sites of initiation and maintains promoter architecture.
  9. ChIPr: accurate prediction of cohesin-mediated 3D genome organization from 2D chromatin features.
  10. Multi-omics integration identifies cell-state-specific repression by PBRM1-PIAS1 cooperation.
  11. Notch signaling and Bsh homeodomain activity are integrated to diversify Drosophila lamina neuron types.
  12. Genome-wide mapping and cryo-EM structural analyses of the overlapping tri-nucleosome composed of hexasome-hexasome-octasome moieties.
  13. Thiolutin has complex effects in vivo but is a direct inhibitor of RNA polymerase II in vitro.
  14. The chromatin landscape of healthy and injured cell types in the human kidney.
  15. Disruption of H3K36 methylation provokes cellular plasticity to drive aberrant glandular formation and squamous carcinogenesis.
  16. Sequencing of N6-methyl-deoxyadenosine at single-base resolution across the mammalian genome.
  17. Minimal requirements for the epigenetic inheritance of engineered silent chromatin domains.
  18. Less-is-more: selecting transcription factor binding regions informative for motif inference.
  19. A full-body transcription factor expression atlas with completely resolved cell identities in C. elegans.
  20. Multiplexed CRISPR gene editing in primary human islet cells with Cas9 ribonucleoprotein.
  21. Aberrant DNA repair reveals a vulnerability in histone H3.3-mutant brain tumors.
  22. Integrative functional genomic analyses identify genetic variants influencing skin pigmentation in Africans.
  1. Cell Rep. 2024 Jan 10. pii: S2211-1247(23)01674-1. [Epub ahead of print]43(1): 113663
    ZNF143 deletion alters enhancer/promoter looping and CTCF/cohesin geometry.
    Mo Zhang, Haiyan Huang, Jingwei Li, Qiang Wu.
      The transcription factor ZNF143 contains a central domain of seven zinc fingers in a tandem array and is involved in 3D genome construction. However, the mechanism by which ZNF143 functions in chromatin looping remains unclear. Here, we show that ZNF143 directionally recognizes a diverse range of genomic sites directly within enhancers and promoters and is required for chromatin looping between these sites. In addition, ZNF143 is located between CTCF and cohesin at numerous CTCF sites, and ZNF143 removal narrows the space between CTCF and cohesin. Moreover, genetic deletion of ZNF143, in conjunction with acute CTCF degradation, reveals that ZNF143 and CTCF collaborate to regulate higher-order topological chromatin organization. Finally, CTCF depletion enlarges direct ZNF143 chromatin looping. Thus, ZNF143 is recruited by CTCF to the CTCF sites to regulate CTCF/cohesin configuration and TAD (topologically associating domain) formation, whereas directional recognition of genomic DNA motifs directly by ZNF143 itself regulates promoter activity via chromatin looping.
    Keywords:  CP: Molecular biology; CTCF; TAD; ZNF143; chromatin loop; cohesin; compartment; enhancer; promoter; topological insulator; transcription
    DOI:  https://doi.org/10.1016/j.celrep.2023.113663
  2. Mol Cell. 2024 Jan 04. pii: S1097-2765(23)01026-2. [Epub ahead of print]
    Pioneer and PRDM transcription factors coordinate bivalent epigenetic states to safeguard cell fate.
    Satoshi Matsui, Marissa Granitto, Morgan Buckley, Katie Ludwig, Sandra Koigi, Joseph Shiley, William J Zacharias, Christopher N Mayhew, Hee-Woong Lim, Makiko Iwafuchi.
      Pioneer transcription factors (TFs) regulate cell fate by establishing transcriptionally primed and active states. However, cell fate control requires the coordination of both lineage-specific gene activation and repression of alternative-lineage programs, a process that is poorly understood. Here, we demonstrate that the pioneer TF FOXA coordinates with PRDM1 TF to recruit nucleosome remodeling and deacetylation (NuRD) complexes and Polycomb repressive complexes (PRCs), which establish highly occupied, accessible nucleosome conformation with bivalent epigenetic states, thereby preventing precocious and alternative-lineage gene expression during human endoderm differentiation. Similarly, the pioneer TF OCT4 coordinates with PRDM14 to form bivalent enhancers and repress cell differentiation programs in human pluripotent stem cells, suggesting that this may be a common and critical function of pioneer TFs. We propose that pioneer and PRDM TFs coordinate to safeguard cell fate through epigenetic repression mechanisms.
    Keywords:  FOXA; NuRD; OCT4; PRC; PRDM1; PRDM14; Polycomb repressive complex; bivalent epigenetic state; cell fate control; complex; nucleosome remodeling and deacetylation; pioneer transcription factor
    DOI:  https://doi.org/10.1016/j.molcel.2023.12.007
  3. Nat Struct Mol Biol. 2024 Jan 09.
    Mitotic bookmarking redundancy by nuclear receptors in pluripotent cells.
    Almira Chervova, Amandine Molliex, H Irem Baymaz, Rémi-Xavier Coux, Thaleia Papadopoulou, Florian Mueller, Eslande Hercul, David Fournier, Agnès Dubois, Nicolas Gaiani, Petra Beli, Nicola Festuccia, Pablo Navarro.
      Mitotic bookmarking transcription factors (TFs) are thought to mediate rapid and accurate reactivation after mitotic gene silencing. However, the loss of individual bookmarking TFs often leads to the deregulation of only a small proportion of their mitotic targets, raising doubts on the biological significance and importance of their bookmarking function. Here we used targeted proteomics of the mitotic bookmarking TF ESRRB, an orphan nuclear receptor, to discover a large redundancy in mitotic binding among members of the protein super-family of nuclear receptors. Focusing on the nuclear receptor NR5A2, which together with ESRRB is essential in maintaining pluripotency in mouse embryonic stem cells, we demonstrate conjoint bookmarking activity of both factors on promoters and enhancers of a large fraction of active genes, particularly those most efficiently reactivated in G1. Upon fast and simultaneous degradation of both factors during mitotic exit, hundreds of mitotic targets of ESRRB/NR5A2, including key players of the pluripotency network, display attenuated transcriptional reactivation. We propose that redundancy in mitotic bookmarking TFs, especially nuclear receptors, confers robustness to the reestablishment of gene regulatory networks after mitosis.
    DOI:  https://doi.org/10.1038/s41594-023-01195-1
  4. Stem Cell Reports. 2024 Jan 06. pii: S2213-6711(23)00495-2. [Epub ahead of print]
    Transcription factor co-expression mediates lineage priming for embryonic and extra-embryonic differentiation.
    Alba Redó-Riveiro, Jasmina Al-Mousawi, Madeleine Linneberg-Agerholm, Martin Proks, Marta Perera, Nazmus Salehin, Joshua M Brickman.
      In early mammalian development, cleavage stage blastomeres and inner cell mass (ICM) cells co-express embryonic and extra-embryonic transcriptional determinants. Using a protein-based double reporter we identify an embryonic stem cell (ESC) population that co-expresses the extra-embryonic factor GATA6 alongside the embryonic factor SOX2. Based on single cell transcriptomics, we find this population resembles the unsegregated ICM, exhibiting enhanced differentiation potential for endoderm while maintaining epiblast competence. To relate transcription factor binding in these cells to future fate, we describe a complete enhancer set in both ESCs and naive extra-embryonic endoderm stem cells and assess SOX2 and GATA6 binding at these elements in the ICM-like ESC sub-population. Both factors support cooperative recognition in these lineages, with GATA6 bound alongside SOX2 on a fraction of pluripotency enhancers and SOX2 alongside GATA6 more extensively on endoderm enhancers, suggesting that cooperative binding between these antagonistic factors both supports self-renewal and prepares progenitor cells for later differentiation.
    Keywords:  Blastocyst; Cooperativity; Embryonic Stem Cells; Endoderm; Enhancer; Epiblast; Lineage Priming; Pluripotency; Transcription; nEnd
    DOI:  https://doi.org/10.1016/j.stemcr.2023.12.002
  5. Cell. 2023 Dec 29. pii: S0092-8674(23)01337-5. [Epub ahead of print]
    Direct observation of a condensate effect on super-enhancer controlled gene bursting.
    Manyu Du, Simon Hendrik Stitzinger, Jan-Hendrik Spille, Won-Ki Cho, Choongman Lee, Mohammed Hijaz, Andrea Quintana, Ibrahim I Cissé.
      Enhancers are distal DNA elements believed to loop and contact promoters to control gene expression. Recently, we found diffraction-sized transcriptional condensates at genes controlled by clusters of enhancers (super-enhancers). However, a direct function of endogenous condensates in controlling gene expression remains elusive. Here, we develop live-cell super-resolution and multi-color 3D-imaging approaches to investigate putative roles of endogenous condensates in the regulation of super-enhancer controlled gene Sox2. In contrast to enhancer distance, we find instead that the condensate's positional dynamics are a better predictor of gene expression. A basal gene bursting occurs when the condensate is far (>1 μm), but burst size and frequency are enhanced when the condensate moves in proximity (<1 μm). Perturbations of cohesin and local DNA elements do not prevent basal bursting but affect the condensate and its burst enhancement. We propose a three-way kissing model whereby the condensate interacts transiently with gene locus and regulatory DNA elements to control gene bursting.
    Keywords:  RNA polymerase II; Sox2; biomolecular condensate; cluster; cohesin; function; nuclear organization; phase separation; super-enhancer; transcription
    DOI:  https://doi.org/10.1016/j.cell.2023.12.005
  6. iScience. 2024 Jan 19. 27(1): 108537
    Pluripotency state transition of embryonic stem cells requires the turnover of histone chaperone FACT on chromatin.
    Hang Zhao, Di Li, Xue Xiao, Cuifang Liu, Guifang Chen, Xiaoyu Su, Zhenxin Yan, Shijia Gu, Yizhou Wang, Guohong Li, Jianxun Feng, Wei Li, Ping Chen, Jiayi Yang, Qing Li.
      The differentiation of embryonic stem cells (ESCs) begins with the transition from the naive to the primed state. The formative state was recently established as a critical intermediate between the two states. Here, we demonstrate the role of the histone chaperone FACT in regulating the naive-to-formative transition. We found that the Q265K mutation in the FACT subunit SSRP1 increased the binding of FACT to histone H3-H4, impaired nucleosome disassembly in vitro, and reduced the turnover of FACT on chromatin in vivo. Strikingly, mouse ESCs harboring this mutation showed elevated naive-to-formative transition. Mechanistically, the SSRP1-Q265K mutation enriched FACT at the enhancers of formative-specific genes to increase targeted gene expression. Together, these findings suggest that the turnover of FACT on chromatin is crucial for regulating the enhancers of formative-specific genes, thereby mediating the naive-to-formative transition. This study highlights the significance of FACT in fine-tuning cell fate transition during early development.
    Keywords:  Epigenetics; Molecular biology; Stem cells research
    DOI:  https://doi.org/10.1016/j.isci.2023.108537
  7. Genome Biol. 2024 Jan 08. 25(1): 11
    Modeling methyl-sensitive transcription factor motifs with an expanded epigenetic alphabet.
    Coby Viner, Charles A Ishak, James Johnson, Nicolas J Walker, Hui Shi, Marcela K Sjöberg-Herrera, Shu Yi Shen, Santana M Lardo, David J Adams, Anne C Ferguson-Smith, Daniel D De Carvalho, Sarah J Hainer, Timothy L Bailey, Michael M Hoffman.
       BACKGROUND: Transcription factors bind DNA in specific sequence contexts. In addition to distinguishing one nucleobase from another, some transcription factors can distinguish between unmodified and modified bases. Current models of transcription factor binding tend not to take DNA modifications into account, while the recent few that do often have limitations. This makes a comprehensive and accurate profiling of transcription factor affinities difficult.
    RESULTS: Here, we develop methods to identify transcription factor binding sites in modified DNA. Our models expand the standard A/C/G/T DNA alphabet to include cytosine modifications. We develop Cytomod to create modified genomic sequences and we also enhance the MEME Suite, adding the capacity to handle custom alphabets. We adapt the well-established position weight matrix (PWM) model of transcription factor binding affinity to this expanded DNA alphabet. Using these methods, we identify modification-sensitive transcription factor binding motifs. We confirm established binding preferences, such as the preference of ZFP57 and C/EBPβ for methylated motifs and the preference of c-Myc for unmethylated E-box motifs.
    CONCLUSIONS: Using known binding preferences to tune model parameters, we discover novel modified motifs for a wide array of transcription factors. Finally, we validate our binding preference predictions for OCT4 using cleavage under targets and release using nuclease (CUT&RUN) experiments across conventional, methylation-, and hydroxymethylation-enriched sequences. Our approach readily extends to other DNA modifications. As more genome-wide single-base resolution modification data becomes available, we expect that our method will yield insights into altered transcription factor binding affinities across many different modifications.
    DOI:  https://doi.org/10.1186/s13059-023-03070-0
  8. Nucleic Acids Res. 2024 Jan 09. pii: gkad1253. [Epub ahead of print]
    NELF focuses sites of initiation and maintains promoter architecture.
    Juan F Santana, Benjamin M Spector, Gustavo A Suarez, Donal S Luse, David H Price.
      Many factors control the elongation phase of transcription by RNA polymerase II (Pol II), a process that plays an essential role in regulating gene expression. We utilized cells expressing degradation tagged subunits of NELFB, PAF1 and RTF1 to probe the effects of depletion of the factors on nascent transcripts using PRO-Seq and on chromatin architecture using DFF-ChIP. Although NELF is involved in promoter proximal pausing, depletion of NELFB had only a minimal effect on the level of paused transcripts and almost no effect on control of productive elongation. Instead, NELF depletion increased the utilization of downstream transcription start sites and caused a dramatic, genome-wide loss of H3K4me3 marked nucleosomes. Depletion of PAF1 and RTF1 both had major effects on productive transcript elongation in gene bodies and also caused initiation site changes like those seen with NELFB depletion. Our study confirmed that the first nucleosome encountered during initiation and early elongation is highly positioned with respect to the major TSS. In contrast, the positions of H3K4me3 marked nucleosomes in promoter regions are heterogeneous and are influenced by transcription. We propose a model defining NELF function and a general role of the H3K4me3 modification in blocking transcription initiation.
    DOI:  https://doi.org/10.1093/nar/gkad1253
  9. Genome Biol. 2024 Jan 12. 25(1): 15
    ChIPr: accurate prediction of cohesin-mediated 3D genome organization from 2D chromatin features.
    Ahmed Abbas, Khyati Chandratre, Yunpeng Gao, Jiapei Yuan, Michael Q Zhang, Ram S Mani.
      The three-dimensional genome organization influences diverse nuclear processes. Here we present Chromatin Interaction Predictor (ChIPr), a suite of regression models based on deep neural networks, random forest, and gradient boosting to predict cohesin-mediated chromatin interaction strength between any two loci in the genome. The predictions of ChIPr correlate well with ChIA-PET data in four cell lines. The standard ChIPr model requires three experimental inputs: ChIP-Seq signals for RAD21, H3K27ac, and H3K27me3 but works well with just RAD21 signal. Integrative analysis reveals novel insights into the role of CTCF motif, its orientation, and CTCF binding on cohesin-mediated chromatin interactions.
    DOI:  https://doi.org/10.1186/s13059-023-03158-7
  10. Cell Genom. 2023 Dec 22. pii: S2666-979X(23)00315-4. [Epub ahead of print] 100471
    Multi-omics integration identifies cell-state-specific repression by PBRM1-PIAS1 cooperation.
    Patric J Ho, Junghun Kweon, Laura A Blumensaadt, Amy E Neely, Elizabeth Kalika, Daniel B Leon, Sanghyon Oh, Cooper W P Stringer, Sarah M Lloyd, Ziyou Ren, Xiaomin Bao.
      PBRM1 is frequently mutated in cancers of epithelial origin. How PBRM1 regulates normal epithelial homeostasis, prior to cancer initiation, remains unclear. Here, we show that PBRM1's gene regulatory roles differ drastically between cell states, leveraging human skin epithelium (epidermis) as a research platform. In progenitors, PBRM1 predominantly functions to repress terminal differentiation to sustain progenitors' regenerative potential; in the differentiation state, however, PBRM1 switches toward an activator. Between these two cell states, PBRM1 retains its genomic binding but associates with differential interacting proteins. Our targeted screen identified the E3 SUMO ligase PIAS1 as a key interactor. PIAS1 co-localizes with PBRM1 on chromatin to directly repress differentiation genes in progenitors, and PIAS1's chromatin binding drastically diminishes in differentiation. Furthermore, SUMOylation contributes to PBRM1's repressive function in progenitor maintenance. Thus, our findings highlight PBRM1's cell-state-specific regulatory roles influenced by its protein interactome despite its stable chromatin binding.
    Keywords:  BAF; PBRM1; PIAS1; SUMO; SWI/SNF; differentiation; epidermis; epithelial; keratinocytes; progenitor; repression; skin
    DOI:  https://doi.org/10.1016/j.xgen.2023.100471
  11. Elife. 2024 Jan 09. pii: RP90136. [Epub ahead of print]12
    Notch signaling and Bsh homeodomain activity are integrated to diversify Drosophila lamina neuron types.
    Chundi Xu, Tyler B Ramos, Owen J Marshall, Chris Q Doe.
      Notch signaling is an evolutionarily conserved pathway for specifying binary neuronal fates, yet how it specifies different fates in different contexts remains elusive. In our accompanying paper, using the Drosophila lamina neuron types (L1-L5) as a model, we show that the primary homeodomain transcription factor (HDTF) Bsh activates secondary HDTFs Ap (L4) and Pdm3 (L5) and specifies L4/L5 neuronal fates. Here we test the hypothesis that Notch signaling enables Bsh to differentially specify L4 and L5 fates. We show asymmetric Notch signaling between newborn L4 and L5 neurons, but they are not siblings; rather, Notch signaling in L4 is due to Delta expression in adjacent L1 neurons. While Notch signaling and Bsh expression are mutually independent, Notch is necessary and sufficient for Bsh to specify L4 fate over L5. The NotchON L4, compared to NotchOFF L5, has a distinct open chromatin landscape which allows Bsh to bind distinct genomic loci, leading to L4-specific identity gene transcription. We propose a novel model in which Notch signaling is integrated with the primary HDTF activity to diversify neuron types by directly or indirectly generating a distinct open chromatin landscape that constrains the pool of genes that a primary HDTF can activate.
    Keywords:  Bsh; D. melanogaster; chromatin landscape; developmental biology; homeodomain; lamina; neuronal fate; notch
    DOI:  https://doi.org/10.7554/eLife.90136
  12. Commun Biol. 2024 Jan 08. 7(1): 61
    Genome-wide mapping and cryo-EM structural analyses of the overlapping tri-nucleosome composed of hexasome-hexasome-octasome moieties.
    Masahiro Nishimura, Takeru Fujii, Hiroki Tanaka, Kazumitsu Maehara, Ken Morishima, Masahiro Shimizu, Yuki Kobayashi, Kayo Nozawa, Yoshimasa Takizawa, Masaaki Sugiyama, Yasuyuki Ohkawa, Hitoshi Kurumizaka.
      The nucleosome is a fundamental unit of chromatin in which about 150 base pairs of DNA are wrapped around a histone octamer. The overlapping di-nucleosome has been proposed as a product of chromatin remodeling around the transcription start site, and previously found as a chromatin unit, in which about 250 base pairs of DNA continuously bind to the histone core composed of a hexamer and an octamer. In the present study, our genome-wide analysis of human cells suggests another higher nucleosome stacking structure, the overlapping tri-nucleosome, which wraps about 300-350 base-pairs of DNA in the region downstream of certain transcription start sites of actively transcribed genes. We determine the cryo-electron microscopy (cryo-EM) structure of the overlapping tri-nucleosome, in which three subnucleosome moieties, hexasome, hexasome, and octasome, are associated by short connecting DNA segments. Small angle X-ray scattering and coarse-grained molecular dynamics simulation analyses reveal that the cryo-EM structure of the overlapping tri-nucleosome may reflect its structure in solution. Our findings suggest that nucleosome stacking structures composed of hexasome and octasome moieties may be formed by nucleosome remodeling factors around transcription start sites for gene regulation.
    DOI:  https://doi.org/10.1038/s42003-023-05694-1
  13. Nucleic Acids Res. 2024 Jan 12. pii: gkad1258. [Epub ahead of print]
    Thiolutin has complex effects in vivo but is a direct inhibitor of RNA polymerase II in vitro.
    Chenxi Qiu, Payal Arora, Indranil Malik, Amber J Laperuta, Emily M Pavlovic, Scott Ugochukwu, Mandar Naik, Craig D Kaplan.
      Thiolutin is a natural product transcription inhibitor with an unresolved mode of action. Thiolutin and the related dithiolopyrrolone holomycin chelate Zn2+ and previous studies have concluded that RNA Polymerase II (Pol II) inhibition in vivo is indirect. Here, we present chemicogenetic and biochemical approaches to investigate thiolutin's mode of action in Saccharomyces cerevisiae. We identify mutants that alter sensitivity to thiolutin. We provide genetic evidence that thiolutin causes oxidation of thioredoxins in vivo and that thiolutin both induces oxidative stress and interacts functionally with multiple metals including Mn2+ and Cu2+, and not just Zn2+. Finally, we show direct inhibition of RNA polymerase II (Pol II) transcription initiation by thiolutin in vitro in support of classical studies that thiolutin can directly inhibit transcription in vitro. Inhibition requires both Mn2+ and appropriate reduction of thiolutin as excess DTT abrogates its effects. Pause prone, defective elongation can be observed in vitro if inhibition is bypassed. Thiolutin effects on Pol II occupancy in vivo are widespread but major effects are consistent with prior observations for Tor pathway inhibition and stress induction, suggesting that thiolutin use in vivo should be restricted to studies on its modes of action and not as an experimental tool.
    DOI:  https://doi.org/10.1093/nar/gkad1258
  14. Nat Commun. 2024 Jan 10. 15(1): 433
    The chromatin landscape of healthy and injured cell types in the human kidney.
    Kidney Precision Medicine Project (KPMP)
      There is a need to define regions of gene activation or repression that control human kidney cells in states of health, injury, and repair to understand the molecular pathogenesis of kidney disease and design therapeutic strategies. Comprehensive integration of gene expression with epigenetic features that define regulatory elements remains a significant challenge. We measure dual single nucleus RNA expression and chromatin accessibility, DNA methylation, and H3K27ac, H3K4me1, H3K4me3, and H3K27me3 histone modifications to decipher the chromatin landscape and gene regulation of the kidney in reference and adaptive injury states. We establish a spatially-anchored epigenomic atlas to define the kidney's active, silent, and regulatory accessible chromatin regions across the genome. Using this atlas, we note distinct control of adaptive injury in different epithelial cell types. A proximal tubule cell transcription factor network of ELF3, KLF6, and KLF10 regulates the transition between health and injury, while in thick ascending limb cells this transition is regulated by NR2F1. Further, combined perturbation of ELF3, KLF6, and KLF10 distinguishes two adaptive proximal tubular cell subtypes, one of which manifested a repair trajectory after knockout. This atlas will serve as a foundation to facilitate targeted cell-specific therapeutics by reprogramming gene regulatory networks.
    DOI:  https://doi.org/10.1038/s41467-023-44467-6
  15. Dev Cell. 2023 Dec 27. pii: S1534-5807(23)00657-3. [Epub ahead of print]
    Disruption of H3K36 methylation provokes cellular plasticity to drive aberrant glandular formation and squamous carcinogenesis.
    Eun Kyung Ko, Amy Anderson, Carina D'souza, Jonathan Zou, Sijia Huang, Sohyun Cho, Faizan Alawi, Stephen Prouty, Vivian Lee, Sora Yoon, Keegan Krick, Yoko Horiuchi, Kai Ge, John T Seykora, Brian C Capell.
      Chromatin organization is essential for maintaining cell-fate trajectories and developmental programs. Here, we find that disruption of H3K36 methylation dramatically impairs normal epithelial differentiation and development, which promotes increased cellular plasticity and enrichment of alternative cell fates. Specifically, we observe a striking increase in the aberrant generation of excessive epithelial glandular tissues, including hypertrophic salivary, sebaceous, and meibomian glands, as well as enhanced squamous tumorigenesis. These phenotypic and gene expression manifestations are associated with loss of H3K36me2 and rewiring of repressive H3K27me3, changes we also observe in human patients with glandular hyperplasia. Collectively, these results have identified a critical role for H3K36 methylation in both in vivo epithelial cell-fate decisions and the prevention of squamous carcinogenesis and suggest that H3K36 methylation modulation may offer new avenues for the treatment of numerous common disorders driven by altered glandular function, which collectively affect large segments of the human population.
    Keywords:  chromatin; development; epigenetics; epithelial plasticity; gene regulation; meibomian gland; salivary gland; sebaceous gland; squamous cell carcinoma
    DOI:  https://doi.org/10.1016/j.devcel.2023.12.007
  16. Mol Cell. 2024 Jan 06. pii: S1097-2765(23)01040-7. [Epub ahead of print]
    Sequencing of N6-methyl-deoxyadenosine at single-base resolution across the mammalian genome.
    Xinran Feng, Xiaolong Cui, Li-Sheng Zhang, Chang Ye, Pingluan Wang, Yuhao Zhong, Tong Wu, Zhong Zheng, Chuan He.
      Although DNA N6-methyl-deoxyadenosine (6mA) is abundant in bacteria and protists, its presence and function in mammalian genomes have been less clear. We present Direct-Read 6mA sequencing (DR-6mA-seq), an antibody-independent method, to measure 6mA at base resolution. DR-6mA-seq employs a unique mutation-based strategy to reveal 6mA sites as misincorporation signatures without any chemical or enzymatic modulation of 6mA. We validated DR-6mA-seq through the successful mapping of the well-characterized G(6mA)TC motif in the E. coli DNA. As expected, when applying DR-6mA-seq to mammalian systems, we found that genomic DNA (gDNA) 6mA abundance is generally low in most mammalian tissues and cells; however, we did observe distinct gDNA 6mA sites in mouse testis and glioblastoma cells. DR-6mA-seq provides an enabling tool to detect 6mA at single-base resolution for a comprehensive understanding of DNA 6mA in eukaryotes.
    Keywords:  DNA modification; N(6)-methyl-deoxyadenosine; epigenetics; glioblastoma; single-base resolution sequencing
    DOI:  https://doi.org/10.1016/j.molcel.2023.12.021
  17. Proc Natl Acad Sci U S A. 2024 Jan 16. 121(3): e2318455121
    Minimal requirements for the epigenetic inheritance of engineered silent chromatin domains.
    Andy H Yuan, Danesh Moazed.
      Mechanisms enabling genetically identical cells to differentially regulate gene expression are complex and central to organismal development and evolution. While gene silencing pathways involving DNA sequence-specific recruitment of histone-modifying enzymes are prevalent in nature, examples of sequence-independent heritable gene silencing are scarce. Studies of the fission yeast Schizosaccharomyces pombe indicate that sequence-independent propagation of heterochromatin can occur but requires numerous multisubunit protein complexes and their diverse activities. Such complexity has so far precluded a coherent articulation of the minimal requirements for heritable gene silencing by conventional in vitro reconstitution approaches. Here, we take an unconventional approach to defining these requirements by engineering sequence-independent silent chromatin inheritance in budding yeast Saccharomyces cerevisiae cells. The mechanism conferring memory upon these cells is remarkably simple and requires only two proteins, one that recognizes histone H3 lysine 9 methylation (H3K9me) and catalyzes the deacetylation of histone H4 lysine 16 (H4K16), and another that recognizes deacetylated H4K16 and catalyzes H3K9me. Together, these bilingual "read-write" proteins form an interdependent positive feedback loop that is sufficient for the transmission of DNA sequence-independent silent information over multiple generations.
    Keywords:  HP1; SIR complex; epigenetic inheritance; heterochromatin; histone deacetylation
    DOI:  https://doi.org/10.1073/pnas.2318455121
  18. Nucleic Acids Res. 2024 Jan 12. pii: gkad1240. [Epub ahead of print]
    Less-is-more: selecting transcription factor binding regions informative for motif inference.
    Jinrui Xu, Jiahao Gao, Pengyu Ni, Mark Gerstein.
      Numerous statistical methods have emerged for inferring DNA motifs for transcription factors (TFs) from genomic regions. However, the process of selecting informative regions for motif inference remains understudied. Current approaches select regions with strong ChIP-seq signal for a given TF, assuming that such strong signal primarily results from specific interactions between the TF and its motif. Additionally, these selection approaches do not account for non-target motifs, i.e. motifs of other TFs; they presume the occurrence of these non-target motifs infrequent compared to that of the target motif, and thus assume these have minimal interference with the identification of the target. Leveraging extensive ChIP-seq datasets, we introduced the concept of TF signal 'crowdedness', referred to as C-score, for each genomic region. The C-score helps in highlighting TF signals arising from non-specific interactions. Moreover, by considering the C-score (and adjusting for the length of genomic regions), we can effectively mitigate interference of non-target motifs. Using these tools, we find that in many instances, strong ChIP-seq signal stems mainly from non-specific interactions, and the occurrence of non-target motifs significantly impacts the accurate inference of the target motif. Prioritizing genomic regions with reduced crowdedness and short length markedly improves motif inference. This 'less-is-more' effect suggests that ChIP-seq region selection warrants more attention.
    DOI:  https://doi.org/10.1093/nar/gkad1240
  19. Nat Commun. 2024 Jan 09. 15(1): 358
    A full-body transcription factor expression atlas with completely resolved cell identities in C. elegans.
    Yongbin Li, Siyu Chen, Weihong Liu, Di Zhao, Yimeng Gao, Shipeng Hu, Hanyu Liu, Yuanyuan Li, Lei Qu, Xiao Liu.
      Invariant cell lineage in C. elegans enables spatiotemporal resolution of transcriptional regulatory mechanisms controlling the fate of each cell. Here, we develop RAPCAT (Robust-point-matching- And Piecewise-affine-based Cell Annotation Tool) to automate cell identity assignment in three-dimensional image stacks of L1 larvae and profile reporter expression of 620 transcription factors in every cell. Transcription factor profile-based clustering analysis defines 80 cell types distinct from conventional phenotypic cell types and identifies three general phenotypic modalities related to these classifications. First, transcription factors are broadly downregulated in quiescent stage Hermaphrodite Specific Neurons, suggesting stage- and cell type-specific variation in transcriptome size. Second, transcription factor expression is more closely associated with morphology than other phenotypic modalities in different pre- and post-differentiation developmental stages. Finally, embryonic cell lineages can be associated with specific transcription factor expression patterns and functions that persist throughout postembryonic life. This study presents a comprehensive transcription factor atlas for investigation of intra-cell type heterogeneity.
    DOI:  https://doi.org/10.1038/s41467-023-42677-6
  20. iScience. 2024 Jan 19. 27(1): 108693
    Multiplexed CRISPR gene editing in primary human islet cells with Cas9 ribonucleoprotein.
    Romina J Bevacqua, Weichen Zhao, Emilio Merheb, Seung Hyun Kim, Alexander Marson, Anna L Gloyn, Seung K Kim.
      Successful genome editing in primary human islets could reveal features of the genetic regulatory landscape underlying β cell function and diabetes risk. Here, we describe a CRISPR-based strategy to interrogate functions of predicted regulatory DNA elements using electroporation of a complex of Cas9 ribonucleoprotein (Cas9 RNP) and guide RNAs into primary human islet cells. We successfully targeted coding regions including the PDX1 exon 1, and non-coding DNA linked to diabetes susceptibility. CRISPR-Cas9 RNP approaches revealed genetic targets of regulation by DNA elements containing candidate diabetes risk SNPs, including an in vivo enhancer of the MPHOSPH9 gene. CRISPR-Cas9 RNP multiplexed targeting of two cis-regulatory elements linked to diabetes risk in PCSK1, which encodes an endoprotease crucial for Insulin processing, also demonstrated efficient simultaneous editing of PCSK1 regulatory elements, resulting in impaired β cell PCSK1 regulation and Insulin secretion. Multiplex CRISPR-Cas9 RNP provides powerful approaches to investigate and elucidate human islet cell gene regulation in health and diabetes.
    Keywords:  Techniques in genetics; biology experimental methods; cell biology; human genetics
    DOI:  https://doi.org/10.1016/j.isci.2023.108693
  21. Nucleic Acids Res. 2024 Jan 12. pii: gkad1257. [Epub ahead of print]
    Aberrant DNA repair reveals a vulnerability in histone H3.3-mutant brain tumors.
    Giulia Giacomini, Sandra Piquet, Odile Chevallier, Juliette Dabin, Siau-Kun Bai, Byungjin Kim, Robert Siddaway, Brian Raught, Etienne Coyaud, Chun-Min Shan, Robert J D Reid, Takenori Toda, Rodney Rothstein, Viviana Barra, Therese Wilhelm, Sabah Hamadat, Chloé Bertin, Alexander Crane, Frank Dubois, Ignasi Forne, Axel Imhof, Pratiti Bandopadhayay, Rameen Beroukhim, Valeria Naim, Songtao Jia, Cynthia Hawkins, Beatrice Rondinelli, Sophie E Polo.
      Pediatric high-grade gliomas (pHGG) are devastating and incurable brain tumors with recurrent mutations in histone H3.3. These mutations promote oncogenesis by dysregulating gene expression through alterations of histone modifications. We identify aberrant DNA repair as an independent mechanism, which fosters genome instability in H3.3 mutant pHGG, and opens new therapeutic options. The two most frequent H3.3 mutations in pHGG, K27M and G34R, drive aberrant repair of replication-associated damage by non-homologous end joining (NHEJ). Aberrant NHEJ is mediated by the DNA repair enzyme polynucleotide kinase 3'-phosphatase (PNKP), which shows increased association with mutant H3.3 at damaged replication forks. PNKP sustains the proliferation of cells bearing H3.3 mutations, thus conferring a molecular vulnerability, specific to mutant cells, with potential for therapeutic targeting.
    DOI:  https://doi.org/10.1093/nar/gkad1257
  22. Nat Genet. 2024 Jan 10.
    Integrative functional genomic analyses identify genetic variants influencing skin pigmentation in Africans.
    Yuanqing Feng, Ning Xie, Fumitaka Inoue, Shaohua Fan, Joshua Saskin, Chao Zhang, Fang Zhang, Matthew E B Hansen, Thomas Nyambo, Sununguko Wata Mpoloka, Gaonyadiwe George Mokone, Charles Fokunang, Gurja Belay, Alfred K Njamnshi, Michael S Marks, Elena Oancea, Nadav Ahituv, Sarah A Tishkoff.
      Skin color is highly variable in Africans, yet little is known about the underlying molecular mechanism. Here we applied massively parallel reporter assays to screen 1,157 candidate variants influencing skin pigmentation in Africans and identified 165 single-nucleotide polymorphisms showing differential regulatory activities between alleles. We combine Hi-C, genome editing and melanin assays to identify regulatory elements for MFSD12, HMG20B, OCA2, MITF, LEF1, TRPS1, BLOC1S6 and CYB561A3 that impact melanin levels in vitro and modulate human skin color. We found that independent mutations in an OCA2 enhancer contribute to the evolution of human skin color diversity and detect signals of local adaptation at enhancers of MITF, LEF1 and TRPS1, which may contribute to the light skin color of Khoesan-speaking populations from Southern Africa. Additionally, we identified CYB561A3 as a novel pigmentation regulator that impacts genes involved in oxidative phosphorylation and melanogenesis. These results provide insights into the mechanisms underlying human skin color diversity and adaptive evolution.
    DOI:  https://doi.org/10.1038/s41588-023-01626-1
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