bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2024–12–01
23 papers selected by
Connor Rogerson, University of Cambridge
  1. The enhancer module of Integrator controls cell identity and early neural fate commitment.
  2. The PNUTS phosphatase complex controls transcription pause release.
  3. Chromatin endogenous cleavage provides a global view of yeast RNA polymerase II transcription kinetics.
  4. Multiple allelic configurations govern long-range Shh enhancer-promoter communication in the embryonic forebrain.
  5. The yeast genome is globally accessible in living cells.
  6. High-throughput capture of transcription factor-driven epigenome dynamics using PHILO ChIP-seq.
  7. CXXC5 stabilizes DNA methylation patterns in mouse embryonic stem cells.
  8. Integrative analysis identifies the atypical repressor E2F8 as a targetable transcriptional activator driving lethal prostate cancer.
  9. ARID1A governs the silencing of sex-linked transcription during male meiosis in the mouse.
  10. Zfp260 choreographs the early stage osteo-lineage commitment of skeletal stem cells.
  11. EZH2 directly methylates PARP1 and regulates its activity in cancer.
  12. Chromatin Helicase CHD6 Establishes Pro-inflammatory Enhancers and is a Synthetic Lethal Target in FH-Deficient Renal Cell Carcinoma.
  13. Identification of co-localised transcription factors based on paired motifs analysis.
  14. ChIP-DIP maps binding of hundreds of proteins to DNA simultaneously and identifies diverse gene regulatory elements.
  15. Heterochromatin-dependent transcription links the PRC2 complex to small RNA-mediated DNA elimination.
  16. PGC-1α drives small cell neuroendocrine cancer progression toward an ASCL1-expressing subtype with increased mitochondrial capacity.
  17. The phosphatase PP1 sustains global transcription by promoting RNA polymerase II pause release.
  18. Engineered transcription-associated Cas9 targeting in eukaryotic cells.
  19. UHRF1 ubiquitin ligase activity supports the maintenance of low-density CpG methylation.
  20. Frizzled5 controls murine intestinal epithelial cell plasticity through organization of chromatin accessibility.
  21. Recovery of biological signals lost in single-cell batch integration with CellANOVA.
  22. A massively parallel reporter assay library to screen short synthetic promoters in mammalian cells.
  23. Transcription factors ASCL1 and OLIG2 drive glioblastoma initiation and co-regulate tumor cell types and migration.
  1. Nat Cell Biol. 2024 Nov 26.
    The enhancer module of Integrator controls cell identity and early neural fate commitment.
    Yingjie Zhang, Connor M Hill, Kelsey A Leach, Luca Grillini, Sandra Deliard, Sarah R Offley, Martina Gatto, Francis Picone, Avery Zucco, Alessandro Gardini.
      Lineage-specific transcription factors operate as master orchestrators of developmental processes by activating select cis-regulatory enhancers and proximal promoters. Direct DNA binding of transcription factors ultimately drives context-specific recruitment of the basal transcriptional machinery that comprises RNA polymerase II (RNAPII) and a host of polymerase-associated multiprotein complexes, including the metazoan-specific Integrator complex. Integrator is primarily known to modulate RNAPII processivity and to surveil RNA integrity across coding genes. Here we describe an enhancer module of Integrator that directs cell fate specification by promoting epigenetic changes and transcription factor binding at neural enhancers. Depletion of Integrator's INTS10 subunit upends neural traits and derails cells towards mesenchymal identity. Commissioning of neural enhancers relies on Integrator's enhancer module, which stabilizes SOX2 binding at chromatin upon exit from pluripotency. We propose that Integrator is a functional bridge between enhancers and promoters and a main driver of early development, providing new insight into a growing family of neurodevelopmental syndromes.
    DOI:  https://doi.org/10.1038/s41556-024-01556-y
  2. Mol Cell. 2024 Nov 21. pii: S1097-2765(24)00886-4. [Epub ahead of print]
    The PNUTS phosphatase complex controls transcription pause release.
    Jessica R Kelley, Emilia Dimitrova, Maciej Maciuszek, Hieu T Nguyen, Aleksander T Szczurek, Amy L Hughes, Neil P Blackledge, Arminja N Kettenbach, Robert J Klose.
      Gene expression is regulated by controlling distinct steps of the transcriptional cycle, including initiation, pausing, elongation, and termination. Kinases phosphorylate RNA polymerase II (RNA Pol II) and associated factors to control transitions between these steps and to act as central gene regulatory nodes. Similarly, phosphatases that dephosphorylate these components are emerging as important regulators of transcription, although their roles remain less well understood. Here, we discover that the mouse PNUTS-PP1 phosphatase complex plays an essential role in controlling transcription pause release in addition to its previously described function in transcription termination. Transcription pause release by the PNUTS complex is essential for almost all RNA Pol II-dependent gene transcription, relies on its PP1 phosphatase subunit, and controls the phosphorylation of factors required for pause release and elongation. Together, these observations reveal an essential new role for a phosphatase complex in transcription pause release and show that the PNUTS complex is essential for RNA Pol II-dependent transcription.
    Keywords:  PNUTS; PP1 phosphatase; RNA Pol II; pause release; termination; transcription
    DOI:  https://doi.org/10.1016/j.molcel.2024.10.045
  3. Elife. 2024 Nov 28. pii: RP100764. [Epub ahead of print]13
    Chromatin endogenous cleavage provides a global view of yeast RNA polymerase II transcription kinetics.
    Jake VanBelzen, Bennet Sakelaris, Donna G Brickner, Nikita Marcou, Hermann Riecke, Niall M Mangan, Jason H Brickner.
      Chromatin immunoprecipitation (ChIP-seq) is the most common approach to observe global binding of proteins to DNA in vivo. The occupancy of transcription factors (TFs) from ChIP-seq agrees well with an alternative method, chromatin endogenous cleavage (ChEC-seq2). However, ChIP-seq and ChEC-seq2 reveal strikingly different patterns of enrichment of yeast RNA polymerase II (RNAPII). We hypothesized that this reflects distinct populations of RNAPII, some of which are captured by ChIP-seq and some of which are captured by ChEC-seq2. RNAPII association with enhancers and promoters - predicted from biochemical studies - is detected well by ChEC-seq2 but not by ChIP-seq. Enhancer/promoter-bound RNAPII correlates with transcription levels and matches predicted occupancy based on published rates of enhancer recruitment, preinitiation assembly, initiation, elongation, and termination. The occupancy from ChEC-seq2 allowed us to develop a stochastic model for global kinetics of RNAPII transcription which captured both the ChEC-seq2 data and changes upon chemical-genetic perturbations to transcription. Finally, RNAPII ChEC-seq2 and kinetic modeling suggests that a mutation in the Gcn4 transcription factor that blocks interaction with the NPC destabilizes promoter-associated RNAPII without altering its recruitment to the enhancer.
    Keywords:  ChEC; ChIP; S. cerevisiae; chromosomes; gene expression; genomics; kinetics; regulation; transcription
    DOI:  https://doi.org/10.7554/eLife.100764
  4. Mol Cell. 2024 Nov 14. pii: S1097-2765(24)00882-7. [Epub ahead of print]
    Multiple allelic configurations govern long-range Shh enhancer-promoter communication in the embryonic forebrain.
    Jailynn Harke, Jeewon R Lee, Son C Nguyen, Arian Arab, Staci M Rakowiecki, Siewert Hugelier, Christina Paliou, Antonella Rauseo, Rebecca Yunker, Kellen Xu, Yao Yao, Melike Lakadamyali, Guillaume Andrey, Douglas J Epstein, Eric F Joyce.
      Developmental gene regulation requires input from enhancers spread over large genomic distances. Our understanding of long-range enhancer-promoter (E-P) communication, characterized as loops, remains incomplete without addressing the role of intervening chromatin. Here, we examine the topology of the entire Sonic hedgehog (Shh) regulatory domain in individual alleles from the mouse embryonic forebrain. Through sequential Oligopaint labeling and super-resolution microscopy, we find that the Shh locus maintains a compact structure that adopts several diverse configurations independent of Shh expression. The most frequent configuration contained distal E-P contacts at the expense of those more proximal to Shh, consistent with an interconnected loop. Genetic perturbations demonstrate that this long-range E-P communication operates by Shh-expression-independent and dependent mechanisms, involving CTCF binding sites and active enhancers, respectively. We propose a model whereby gene regulatory elements secure long-range E-P interactions amid an inherent architectural framework to coordinate spatiotemporal patterns of gene expression.
    Keywords:  3D genome organization; CTCF; E-P; Shh; allelic configurations; developmental gene regulation; enhancer activity; enhancer-promoter communication; sequential DNA-FISH; super-resolution microscopy
    DOI:  https://doi.org/10.1016/j.molcel.2024.10.042
  5. Nat Struct Mol Biol. 2024 Nov 25.
    The yeast genome is globally accessible in living cells.
    Hemant K Prajapati, Peter R Eriksson, Paul A Elizalde, Christopher T Coey, Zhuwei Xu, David J Clark.
      Eukaryotic genomes are packaged into chromatin, which is composed of condensed filaments of regularly spaced nucleosomes, resembling beads on a string. The nucleosome contains ~147 bp of DNA wrapped almost twice around a central core histone octamer. The packaging of DNA into chromatin represents a challenge to transcription factors and other proteins requiring access to their binding sites. Consequently, control of DNA accessibility is thought to play a key role in gene regulation. Here we measure DNA accessibility genome wide in living budding yeast cells by inducible expression of DNA methyltransferases. We find that the genome is globally accessible in living cells, unlike in isolated nuclei, where DNA accessibility is severely restricted. Gene bodies are methylated at only slightly slower rates than promoters, indicating that yeast chromatin is highly dynamic in vivo. In contrast, silenced loci and centromeres are strongly protected. Global shifts in nucleosome positions occur in cells as they are depleted of ATP-dependent chromatin remodelers, suggesting that nucleosome dynamics result from competition among these enzymes. We conclude that chromatin is in a state of continuous flux in living cells, but static in nuclei, suggesting that DNA packaging in yeast is not generally repressive.
    DOI:  https://doi.org/10.1038/s41594-024-01318-2
  6. Nucleic Acids Res. 2024 Nov 26. pii: gkae1123. [Epub ahead of print]
    High-throughput capture of transcription factor-driven epigenome dynamics using PHILO ChIP-seq.
    Aanchal Choudhary, Moonia Ammari, Hyuk Sung Yoon, Mark Zander.
      Assessing the dynamics of chromatin features and transcription factor (TF) binding at scale remains a significant challenge in plants. Here, we present PHILO (Plant HIgh-throughput LOw input) ChIP-seq, a high-throughput ChIP-seq platform that enables the cost-effective and extensive capture of TF binding and genome-wide distributions of histone modifications. The PHILO ChIP-seq pipeline is adaptable to many plant species, requires very little starting material (1mg), and provides the option to use MNase (micrococcal nuclease) for chromatin fragmentation. By employing H3K9ac PHILO ChIP-seq on eight Arabidopsis thaliana jasmonic acid (JA) pathway mutants, with the simultaneous processing of over 100 samples, we not only recapitulated but also expanded the current understanding of the intricate interplay between the master TFs MYC2/3/4 and various chromatin regulators. Additionally, our analyses brought to light previously unknown histone acetylation patterns within the regulatory regions of MYC2 target genes in Arabidopsis, which is also conserved in tomato (Solanum lycopersicum). In summary, our PHILO ChIP-seq platform demonstrates its high effectiveness in investigating TF binding and chromatin dynamics on a large scale in plants, paving the way for the cost-efficient realization of complex experimental setups.
    DOI:  https://doi.org/10.1093/nar/gkae1123
  7. Epigenomics. 2024 Nov 25. 1-13
    CXXC5 stabilizes DNA methylation patterns in mouse embryonic stem cells.
    Seung-Gi Jin, Jennifer Johnson, Zhijun Huang, Wei Cui, Thomas Dunwell, Gerd P Pfeifer.
       AIMS: Mammalian genomes encode 12 proteins that contain a CXXC zinc finger domain. Most members of this family are large multi-domain proteins that function in the control of DNA methylation and histone methylation patterns. CXXC5 is a smaller member of the family, along with its closest homologue CXXC4. These two proteins lack known catalytic domains. Here, we have characterized CXXC5 in mouse embryonic stem (ES) cells.
    MATERIALS & METHODS: We used gene knockouts, RNA sequencing, and DNA methylation analysis by whole-genome bisulfite sequencing.
    RESULTS & CONCLUSIONS: We show that CXXC5 is a nuclear protein that interacts with 5-methylcytosine oxidases (TET proteins). Removal of CXXC5 from ES cells leads to very few changes in gene expression. CXXC5 extensively colocalizes with TET1 and TET2 at CpG islands. CXXC5 inactivation leads to a substantial reduction of DNA methylation levels that affects all genomic compartments including genic and intergenic regions and CpG island shores. We propose a model in which CXXC5 serves as an anchor for TET proteins at CpG islands. In the absence of CXXC5, the 5-methylcytosine oxidases become dislodged from CpG islands and are enabled to induce genome-scale DNA demethylation. Thus, CXXC5 serves as a stabilizer of DNA methylation patterns.
    Keywords:  5-methylcytosine; CXXC4; CXXC5; DNA methylation; TET1
    DOI:  https://doi.org/10.1080/17501911.2024.2426450
  8. Oncogene. 2024 Nov 29.
    Integrative analysis identifies the atypical repressor E2F8 as a targetable transcriptional activator driving lethal prostate cancer.
    Furong Huang, Kexin Li, Zhong Chen, Zhifen Cui, William Hankey, Kun Fang, Jingyue Yan, Hongyan Wang, Victor X Jin, Yizhou Dong, Qianben Wang.
      Acquired resistance to androgen receptor (AR)-targeted therapies underscores the need to identify alternative therapeutic targets for treating lethal prostate cancer. In this study, we evaluated the prognostic significance of 1635 human transcription factors (TFs) by analyzing castration-resistant prostate cancer (CRPC) datasets from the West and East Stand Up to Cancer (SU2C) cohorts. Through this screening approach, we identified E2F8, a putative transcriptional repressor, as a TF consistently associated with poorer patient outcomes in both cohorts. Notably, E2F8 is highly expressed and active in AR-negative CRPC compared to AR-positive CRPC. Integrative profiling of E2F8 cistromes and transcriptomes in AR-negative CRPC cells revealed that E2F8 directly and non-canonically activates target oncogenes involved in cancer-associated pathways. To target E2F8 in CRPC, we employed the CRISPR/CasRx system to knockdown E2F8 mRNA, resulting in effective and specific downregulation of E2F8 and its target oncogenes, as well as significant growth inhibition in AR-negative CRPC in both cultured cells and xenograft models. Our findings identify and characterize E2F8 as a targetable transcriptional activator driving CRPC, particularly the growth of AR-negative CRPC.
    DOI:  https://doi.org/10.1038/s41388-024-03239-2
  9. Elife. 2024 Nov 26. pii: RP88024. [Epub ahead of print]12
    ARID1A governs the silencing of sex-linked transcription during male meiosis in the mouse.
    Debashish U Menon, Prabuddha Chakraborty, Noel Murcia, Terry Magnuson.
      We present evidence implicating the BAF (BRG1/BRM Associated Factor) chromatin remodeler in meiotic sex chromosome inactivation (MSCI). By immunofluorescence (IF), the putative BAF DNA binding subunit, ARID1A (AT-rich Interaction Domain 1 a), appeared enriched on the male sex chromosomes during diplonema of meiosis I. Germ cells showing a Cre-induced loss of ARID1A arrested in pachynema and failed to repress sex-linked genes, indicating a defective MSCI. Mutant sex chromosomes displayed an abnormal presence of elongating RNA polymerase II coupled with an overall increase in chromatin accessibility detectable by ATAC-seq. We identified a role for ARID1A in promoting the preferential enrichment of the histone variant, H3.3, on the sex chromosomes, a known hallmark of MSCI. Without ARID1A, the sex chromosomes appeared depleted of H3.3 at levels resembling autosomes. Higher resolution analyses by CUT&RUN revealed shifts in sex-linked H3.3 associations from discrete intergenic sites and broader gene-body domains to promoters in response to the loss of ARID1A. Several sex-linked sites displayed ectopic H3.3 occupancy that did not co-localize with DMC1 (DNA meiotic recombinase 1). This observation suggests a requirement for ARID1A in DMC1 localization to the asynapsed sex chromatids. We conclude that ARID1A-directed H3.3 localization influences meiotic sex chromosome gene regulation and DNA repair.
    Keywords:  ARID1A; Mouse; chromatin remodelers; developmental biology; gene regulation; genetics; genomics; meiotic sex chromosome; mouse; sex-linked DNA repair; spermatogenesis
    DOI:  https://doi.org/10.7554/eLife.88024
  10. Nat Commun. 2024 Nov 24. 15(1): 10186
    Zfp260 choreographs the early stage osteo-lineage commitment of skeletal stem cells.
    Yuteng Weng, Yanhuizhi Feng, Zeyuan Li, Shuyu Xu, Di Wu, Jie Huang, Haicheng Wang, Zuolin Wang.
      The initial fine-tuning processes are crucial for successful bone regeneration, as they guide skeletal stem cells through progenitor differentiation toward osteo- or chondrogenic fate. While fate determination processes are well-documented, the mechanisms preceding progenitor commitment remain poorly understood. Here, we identified a transcription factor, Zfp260, as pivotal for stem cell maturation into progenitors and directing osteogenic differentiation. Zfp260 is markedly up-regulated as cells transition from stem to progenitor stages; its dysfunction causes lineage arrest at the progenitor stage, impairing bone repair. Zfp260 is required for maintaining chromatin accessibility and regulates Runx2 expression by forming super-enhancer complexes. Furthermore, the PKCα kinase phosphorylates Zfp260 at residues Y173, S182, and S197, which are essential for its functional activity. Mutations at these residues significantly impair its functionality. These findings position Zfp260 as a vital factor bridging stem cell activation with progenitor cell fate determination, unveiling a element fundamental to successful bone regeneration.
    DOI:  https://doi.org/10.1038/s41467-024-54640-0
  11. Sci Adv. 2024 Nov 29. 10(48): eadl2804
    EZH2 directly methylates PARP1 and regulates its activity in cancer.
    Qingshu Meng, Jiangchuan Shen, Yanan Ren, Qi Liu, Rui Wang, Qiaqia Li, Weihua Jiang, Quan Wang, Yixiang Zhang, Jonathan C Trinidad, Xiaotong Lu, Tingyou Wang, Yanqiang Li, Chaehyun Yum, Yang Yi, Yongyong Yang, Dongyu Zhao, Clair Harris, Sundeep Kalantry, Kaifu Chen, Rendong Yang, Hengyao Niu, Qi Cao.
      DNA repair dysregulation is a key driver of cancer development. Understanding the molecular mechanisms underlying DNA repair dysregulation in cancer cells is crucial for cancer development and therapies. Here, we report that enhancer of zeste homolog 2 (EZH2) directly methylates poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1), an essential enzyme involved in DNA repair, and regulates its activity. Functionally, EZH2-catalyzed methylation represses PARP1 catalytic activity, down-regulates the recruitment of x-ray repair cross-complementing group-1 to DNA lesions and its associated DNA damage repair; on the other hand, it protects the cells from nicotinamide adenine dinucleotide overconsumption upon DNA damage formation. Meanwhile, EZH2-mediated methylation regulates PARP1 transcriptional and oncogenic activity, at least in part, through impairing PARP1-E2F1 interaction and E2F1 transcription factor activity. EZH2 and PARP1 inhibitors synergistically suppress prostate cancer growth. Collectively, our findings uncover an insight of EZH2 functions in fine-tuning PARP1 activity during DNA damage repair and cancer progression, which provides a rationale for combinational targeting EZH2 and PARP1 in cancer.
    DOI:  https://doi.org/10.1126/sciadv.adl2804
  12. Cancer Res. 2024 Nov 26.
    Chromatin Helicase CHD6 Establishes Pro-inflammatory Enhancers and is a Synthetic Lethal Target in FH-Deficient Renal Cell Carcinoma.
    Juan Jin, Jun Luo, Xiaodong Jin, Kiat Shenq Lim, Yang He, Jiawei Ding, Yan Shen, Yuchen Hou, Hanqing Liu, Xiaoyu Zhu, Jing Zhao, Wenjie Zhou, Hai Huang, Yi Gao, Jun Xiao, Hongchao He, Qunyi Li, Lianxin Liu, Li Chen, Qiang He, Chuanjie Zhang.
      Fumarate hydratase (FH) deficiency causes hereditary leiomyomatosis and renal cell carcinoma (RCC). FH-deficient tumors lack effective therapeutic options. Here, we utilized an epigenetic-focused single-guide RNA library to elucidate potential drug targets in FH-deficient tumors. The screen identified chromodomain helicase DNA binding protein 6 (CHD6) as an essential regulator of the growth of FH-mutated RCC. Mechanically, FH loss induced fumarate-mediated succinylation and inactivation of KEAP1, blocking subsequent ubiquitin-proteasome degradation of CHD6. Stabilized CHD6 formed a complex with p65 to establish pro-inflammatory enhancers and thereby regulate NF-κB-mediated transcription. Moreover, CHD6 recruited mSWI/SNF ATPases to maintain chromatin accessibility at CHD6-bound enhancers. The PROTAC degrader of SMARCA2/4 AU-15330 effectively abolished structures of cis-regulatory elements bound by CHD6 and suppressed the growth of FH-mutated, but not FH-intact, RCC in vivo. Collectively, these data indicate that CHD6 is a molecular bridge between FH deficiency and pro-inflammatory enhancers assembly that endows FH-deficient tumors with epigenetic vulnerabilities.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-24-0787
  13. IET Syst Biol. 2024 Nov 26.
    Identification of co-localised transcription factors based on paired motifs analysis.
    Li Liu, Lu Han, Kaiyuan Han, Zheng Zhang, Haojiang Zhang, Lirong Zhang.
      The interaction of transcription factors (TFs) with DNA precisely regulates gene transcription. In mammalian cells, thousands of TFs often interact with DNA cis-regulatory elements in a combinatorial manner rather than act alone. The identification of cooperativity between TFs can help to explore the mechanism of transcriptional regulation. However, little is known about the cooperative patterns of TFs in the genome. To identify which TFs prefer co-localisation, the authors conducted a paired motif analysis in the accessible regions of the human genome based on the Poisson background model. Especially, the authors distinguish the cooperative binding TFs and the competitive binding TFs according to the distance between TF motifs. In the K562 cell line, the authors find that TFs from a same family are always competing the same binding sites, such as FOS_JUN family, whereas KLF family TFs show significant cooperative binding in the adjacency region. Furthermore, the comparative analysis across 16 human cell lines indicates that most TF combination patterns are conserved, but there are still some cell-line-specific patterns. Finally, in human prostate cancer cells (PC-3) and human prostate normal cells (RWPE-2), the authors investigate the specific TF combination patterns in the disease cell and normal cell. The results show that the cooperative binding TF pairs shared by PC-3 and RWPE-2 account for over 90%. Simultaneously, the authors also identify 26 specific TF combination pairs in PC-3 cancer cells.
    Keywords:  DNA; bioinformatics; data mining; genomics; statistical analysis
    DOI:  https://doi.org/10.1049/syb2.12104
  14. Nat Genet. 2024 Nov 25.
    ChIP-DIP maps binding of hundreds of proteins to DNA simultaneously and identifies diverse gene regulatory elements.
    Andrew A Perez, Isabel N Goronzy, Mario R Blanco, Benjamin T Yeh, Jimmy K Guo, Carolina S Lopes, Olivia Ettlin, Alex Burr, Mitchell Guttman.
      Gene expression is controlled by dynamic localization of thousands of regulatory proteins to precise genomic regions. Understanding this cell type-specific process has been a longstanding goal yet remains challenging because DNA-protein mapping methods generally study one protein at a time. Here, to address this, we developed chromatin immunoprecipitation done in parallel (ChIP-DIP) to generate genome-wide maps of hundreds of diverse regulatory proteins in a single experiment. ChIP-DIP produces highly accurate maps within large pools (>160 proteins) for all classes of DNA-associated proteins, including modified histones, chromatin regulators and transcription factors and across multiple conditions simultaneously. First, we used ChIP-DIP to measure temporal chromatin dynamics in primary dendritic cells following LPS stimulation. Next, we explored quantitative combinations of histone modifications that define distinct classes of regulatory elements and characterized their functional activity in human and mouse cell lines. Overall, ChIP-DIP generates context-specific protein localization maps at consortium scale within any molecular biology laboratory and experimental system.
    DOI:  https://doi.org/10.1038/s41588-024-02000-5
  15. EMBO Rep. 2024 Nov 29.
    Heterochromatin-dependent transcription links the PRC2 complex to small RNA-mediated DNA elimination.
    Therese Solberg, Chundi Wang, Ryuma Matsubara, Zhiwei Wen, Mariusz Nowacki.
      Facultative heterochromatin is marked by the repressive histone modification H3K27me3 in eukaryotes. Deposited by the PRC2 complex, H3K27me3 is essential for regulating gene expression during development, and chromatin bearing this mark is generally considered transcriptionally inert. The PRC2 complex has also been linked to programmed DNA elimination during development in ciliates such as Paramecium. Due to a lack of mechanistic insight, a direct involvement has been questioned as most eliminated DNA segments in Paramecium are shorter than the size of a nucleosome. Here, we identify two sets of histone methylation readers essential for PRC2-mediated DNA elimination in Paramecium: Firefly1/2 and Mayfly1-4. The chromodomain proteins Firefly1/2 act in tight association with TFIIS4, a transcription elongation factor required for noncoding RNA transcription. These noncoding transcripts act as scaffolds for sequence-specific targeting by PIWI-bound sRNAs, resulting in local nucleosome depletion and DNA elimination. Our findings elucidate the molecular mechanism underlying the role of PRC2 in PIWI-mediated DNA elimination and suggest that its role in IES elimination may be to activate rather than repress transcription.
    Keywords:  H3K27me3; H3K9me3; Heterochromatin; PRC2; Small RNA
    DOI:  https://doi.org/10.1038/s44319-024-00332-1
  16. Proc Natl Acad Sci U S A. 2024 Dec 03. 121(49): e2416882121
    PGC-1α drives small cell neuroendocrine cancer progression toward an ASCL1-expressing subtype with increased mitochondrial capacity.
    Grigor Varuzhanyan, Chia-Chun Chen, Jack Freeland, Tian He, Wendy Tran, Kai Song, Liang Wang, Donghui Cheng, Shili Xu, Gabriella A Dibernardo, Favour N Esedebe, Vipul Bhatia, Mingqi Han, Evan R Abt, Jung Wook Park, Sanaz Memarzadeh, David B Shackelford, John K Lee, Thomas G Graeber, Orian S Shirihai, Owen N Witte.
      Adenocarcinomas from multiple tissues can converge to treatment-resistant small cell neuroendocrine (SCN) cancers composed of ASCL1, POU2F3, NEUROD1, and YAP1 subtypes. We investigated how mitochondrial metabolism influences SCN cancer (SCNC) progression. Extensive bioinformatics analyses encompassing thousands of patient tumors and human cancer cell lines uncovered enhanced expression of proliferator-activatedreceptor gamma coactivator 1-alpha (PGC-1α), a potent regulator of mitochondrial oxidative phosphorylation (OXPHOS), across several SCNCs. PGC-1α correlated tightly with increased expression of the lineage marker Achaete-scute homolog 1, (ASCL1) through a positive feedback mechanism. Analyses using a human prostate tissue-based SCN transformation system showed that the ASCL1 subtype has heightened PGC-1α expression and OXPHOS activity. PGC-1α inhibition diminished OXPHOS, reduced SCNC cell proliferation, and blocked SCN prostate tumor formation. Conversely, PGC-1α overexpression enhanced OXPHOS, validated by small-animal Positron Emission Tomography mitochondrial imaging, tripled the SCN prostate tumor formation rate, and promoted commitment to the ASCL1 lineage. These results establish PGC-1α as a driver of SCNC progression and subtype determination, highlighting metabolic vulnerabilities in SCNCs across different tissues.
    Keywords:  ASCL1; PGC-1a; lung cancer; oxidative phosphorylation; prostate cancer
    DOI:  https://doi.org/10.1073/pnas.2416882121
  17. Mol Cell. 2024 Nov 20. pii: S1097-2765(24)00885-2. [Epub ahead of print]
    The phosphatase PP1 sustains global transcription by promoting RNA polymerase II pause release.
    Zhenning Wang, Aixia Song, Bolin Tao, Maojian Miao, Yi-Qing Luo, Jingwen Wang, Zhinang Yin, Ruijing Xiao, Xinwen Zhou, Xue-Ying Shang, Shibin Hu, Kaiwei Liang, Charles G Danko, Fei Xavier Chen.
      RNA polymerase II progression from initiation to elongation is driven in part by a cascade of protein kinases acting on the core transcription machinery. Conversely, the corresponding phosphatases, notably PP2A and PP1-the most abundant serine-threonine phosphatases in cells-are thought to mainly impede polymerase progression, respectively restraining pause release at promoters and elongation at terminators. Here, we reveal an unexpected role of PP1, within the phosphatase 1 nuclear targeting subunit (PNUTS)-PP1 complex, in sustaining global transcriptional activation in human cells. Acute disruption of PNUTS-PP1 leads to severe defects in the release of paused polymerase and subsequent downregulation for the majority of transcribed genes. PNUTS-PP1 promotes pause release by dephosphorylating multiple substrates, including the 7SK small nuclear ribonucleoprotein particle (snRNP) subunit MEPCE, a known pausing regulator. PNUTS-PP1 exhibits antagonistic functions compared with Integrator-PP2A (INTAC) phosphatase, which generally inhibits pause release. Our research thus highlights opposing roles of PP1 and PP2A in modulating genome-wide transcriptional pausing and gene expression.
    Keywords:  7SK snRNP; INTAC; PNUTS; PNUTS-PP1; PP1; PP2A; RNA polymerase II; pausing; phosphatase; transcription
    DOI:  https://doi.org/10.1016/j.molcel.2024.10.046
  18. Nat Commun. 2024 Nov 27. 15(1): 10287
    Engineered transcription-associated Cas9 targeting in eukaryotic cells.
    Gregory W Goldberg, Manjunatha Kogenaru, Sarah Keegan, Max A B Haase, Larisa Kagermazova, Mauricio A Arias, Kenenna Onyebeke, Samantha Adams, Daniel K Beyer, David Fenyö, Marcus B Noyes, Jef D Boeke.
      DNA targeting Class 2 CRISPR-Cas effector nucleases, including the well-studied Cas9 proteins, evolved protospacer-adjacent motif (PAM) and guide RNA interactions that sequentially license their binding and cleavage activities at protospacer target sites. Both interactions are nucleic acid sequence specific but function constitutively; thus, they provide intrinsic spatial control over DNA targeting activities but naturally lack temporal control. Here we show that engineered Cas9 fusion proteins which bind to nascent RNAs near a protospacer can facilitate spatiotemporal coupling between transcription and DNA targeting at that protospacer: Transcription-associated Cas9 Targeting (TraCT). Engineered TraCT is enabled in eukaryotic yeast or human cells when suboptimal PAM interactions limit basal activity and when one or more nascent RNA substrates are still tethered to the actively transcribed target DNA in cis. Using yeast, we further show that this phenomenon can be applied for selective editing at one of two identical targets in distinct gene loci, or, in diploid allelic loci that are differentially transcribed. Our work demonstrates that temporal control over Cas9's targeting activity at specific DNA sites may be engineered without modifying Cas9's core domains and guide RNA components or their expression levels. More broadly, it establishes co-transcriptional RNA binding as a cis-acting mechanism that can conditionally stimulate CRISPR-Cas DNA targeting in eukaryotic cells.
    DOI:  https://doi.org/10.1038/s41467-024-54629-9
  19. Nucleic Acids Res. 2024 Nov 28. pii: gkae1105. [Epub ahead of print]
    UHRF1 ubiquitin ligase activity supports the maintenance of low-density CpG methylation.
    Rochelle L Tiedemann, Joel Hrit, Qian Du, Ashley K Wiseman, Hope E Eden, Bradley M Dickson, Xiangqian Kong, Alison A Chomiak, Robert M Vaughan, Bailey M Tibben, Jakob M Hebert, Yael David, Wanding Zhou, Stephen B Baylin, Peter A Jones, Susan J Clark, Scott B Rothbart.
      The RING E3 ubiquitin ligase UHRF1 is an established cofactor for DNA methylation inheritance. The model posits that nucleosomal engagement through histone and DNA interactions directs UHRF1 ubiquitin ligase activity toward lysines on histone H3 tails, creating binding sites for DNMT1 through ubiquitin interacting motifs (UIM1 and UIM2). However, the extent to which DNMT1 relies on ubiquitin signaling through UHRF1 in support of DNA methylation maintenance remains unclear. Here, with integrative epigenomic and biochemical analyses, we reveal that DNA methylation maintenance at low-density cytosine-guanine dinucleotides (CpGs) is particularly vulnerable to disruption of UHRF1 ubiquitin ligase activity and DNMT1 ubiquitin reading activity through UIM1. Hypomethylation of low-density CpGs in this manner induces formation of partially methylated domains (PMDs), a methylation signature observed across human cancers. In contrast, UIM2 disruption completely abolishes the DNA methylation maintenance function of DNMT1 in a CpG density-independent manner. In the context of DNA methylation recovery following acute DNMT1 depletion, we further reveal a 'bookmarking' function for UHRF1 ubiquitin ligase activity in support of DNA re-methylation. Collectively, these studies show that DNMT1-dependent DNA methylation inheritance is a ubiquitin-regulated process that is partially reliant on UHRF1 and suggest a disrupted UHRF1-DNMT1 ubiquitin signaling axis contributes to PMD formation in cancers.
    DOI:  https://doi.org/10.1093/nar/gkae1105
  20. Dev Cell. 2024 Nov 15. pii: S1534-5807(24)00661-0. [Epub ahead of print]
    Frizzled5 controls murine intestinal epithelial cell plasticity through organization of chromatin accessibility.
    Lu Deng, Xi C He, Shiyuan Chen, Ning Zhang, Fengyan Deng, Allison Scott, Yanfeng He, Dai Tsuchiya, Sarah E Smith, Michael Epp, Seth Malloy, Fang Liu, Mark Hembree, Qinghui Mu, Jeffrey S Haug, Ermanno Malagola, Huzaifa Hassan, Kaitlyn Petentler, Rhonda Egidy, Lucinda Maddera, Jonathon Russell, Yan Wang, Hua Li, Chongbei Zhao, Anoja Perera, Timothy C Wang, Calvin J Kuo, Linheng Li.
      The homeostasis of the intestinal epithelium relies on intricate yet insufficiently understood mechanisms of intestinal epithelial plasticity. Here, we elucidate the pivotal role of Frizzled5 (Fzd5), a Wnt pathway receptor, as a determinant of murine intestinal epithelial cell fate. Deletion of Fzd5 in Lgr5+ intestinal stem cells (ISCs) impairs their self-renewal, whereas its deletion in Krt19+ cells disrupts lineage generation, without affecting crypt integrity in either case. However, a broader deletion of Fzd5 across the epithelium leads to substantial crypt deterioration. Integrated analysis of single-cell RNA sequencing (scRNA-seq) and single-cell ATAC-seq (scATAC-seq) identifies that Fzd5 governs chromatin accessibility, orchestrating the regulation of stem- and lineage-related gene expression mainly in ISCs and progenitor cells. In summary, our findings provide insights into the regulatory role of Fzd5 in governing intestinal epithelial plasticity.
    Keywords:  Fzd5; Wnt pathway; chromatin accessibility; intestinal stem cell; plasticity; single-cell transcriptome and epigenome
    DOI:  https://doi.org/10.1016/j.devcel.2024.10.021
  21. Nat Biotechnol. 2024 Nov 26.
    Recovery of biological signals lost in single-cell batch integration with CellANOVA.
    Zhaojun Zhang, Divij Mathew, Tristan L Lim, Kaishu Mason, Clara Morral Martinez, Sijia Huang, E John Wherry, Katalin Susztak, Andy J Minn, Zongming Ma, Nancy R Zhang.
      Data integration to align cells across batches has become a cornerstone of single-cell data analysis, critically affecting downstream results. Currently, there are no guidelines for when the biological differences between samples are separable from batch effects. Here we show that current paradigms for single-cell data integration remove biologically meaningful variation and introduce distortion. We present a statistical model and computationally scalable algorithm, CellANOVA (cell state space analysis of variance), that harnesses experimental design to explicitly recover biological signals that are erased during single-cell data integration. CellANOVA uses a 'pool-of-controls' design concept, applicable across diverse settings, to separate unwanted variation from biological variation of interest and allow the recovery of subtle biological signals. We apply CellANOVA to diverse contexts and validate the recovered biological signals by orthogonal assays. In particular, we show that CellANOVA is effective in the challenging case of single-cell and single-nucleus data integration, where it recovers subtle biological signals that can be validated and replicated by external data.
    DOI:  https://doi.org/10.1038/s41587-024-02463-1
  22. Nat Commun. 2024 Nov 28. 15(1): 10353
    A massively parallel reporter assay library to screen short synthetic promoters in mammalian cells.
    Adam M Zahm, William S Owens, Samuel R Himes, Braden S Fallon, Kathleen E Rondem, Alexa N Gormick, Joshua S Bloom, Sriram Kosuri, Henry Chan, Justin G English.
      Cellular responses to stimuli underpin discoveries in drug development, synthetic biology, and general life sciences. We introduce a library comprising 6144 synthetic promoters, each shorter than 250 bp, designed as transcriptional readouts of cellular stimulus responses in massively parallel reporter assay format. This library facilitates precise detection and amplification of transcriptional activity from our promoters, enabling the systematic development of tunable reporters with dynamic ranges of 50-100 fold. Our library proved functional in numerous cell lines and responsive to a variety of stimuli, including metabolites, mitogens, toxins, and pharmaceutical agents, generating robust and scalable reporters effective in screening assays, biomarkers, and synthetic circuits attuned to endogenous cellular activities. Particularly valuable in therapeutic development, our library excels in capturing candidate reporters to signals mediated by drug targets, a feature we illustrate across nine diverse G-protein coupled receptors (GPCRs), critical targets in drug development. We detail how this tool isolates and defines discrete signaling pathways associated with specific GPCRs, elucidating their transcriptional signatures. With its ease of implementation, broad utility, publicly available data, and comprehensive documentation, our library will be beneficial in synthetic biology, cellular engineering, ligand exploration, and drug development.
    DOI:  https://doi.org/10.1038/s41467-024-54502-9
  23. Nat Commun. 2024 Nov 28. 15(1): 10363
    Transcription factors ASCL1 and OLIG2 drive glioblastoma initiation and co-regulate tumor cell types and migration.
    Bianca L Myers, Kathryn J Brayer, Luis E Paez-Beltran, Estrella Villicana, Matthew S Keith, Hideaki Suzuki, Jessie Newville, Rebekka H Anderson, Yunee Lo, Conner M Mertz, Rahul K Kollipara, Mark D Borromeo, Q Richard Lu, Robert M Bachoo, Jane E Johnson, Tou Yia Vue.
      Glioblastomas (GBMs) are highly aggressive, infiltrative, and heterogeneous brain tumors driven by complex genetic alterations. The basic-helix-loop-helix (bHLH) transcription factors ASCL1 and OLIG2 are dynamically co-expressed in GBMs; however, their combinatorial roles in regulating the plasticity and heterogeneity of GBM cells are unclear. Here, we show that induction of somatic mutations in subventricular zone (SVZ) progenitor cells leads to the dysregulation of ASCL1 and OLIG2, which then function redundantly and are required for brain tumor formation in a mouse model of GBM. Subsequently, the binding of ASCL1 and OLIG2 to each other's loci and to downstream target genes then determines the cell types and degree of migration of tumor cells. Single-cell RNA sequencing (scRNA-seq) reveals that a high level of ASCL1 is key in specifying highly migratory neural stem cell (NSC)/astrocyte-like tumor cell types, which are marked by upregulation of ribosomal protein, oxidative phosphorylation, cancer metastasis, and therapeutic resistance genes.
    DOI:  https://doi.org/10.1038/s41467-024-54750-9
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