bims-gerecp Biomed News
on Gene regulatory networks of epithelial cell plasticity
Issue of 2024‒07‒21
fifteen papers selected by
Xiao Qin, University of Oxford
  1. Symbolic recording of signalling and cis-regulatory element activity to DNA.
  2. HyperCas12a enables highly-multiplexed epigenome editing screens.
  3. Single-cell EpiChem jointly measures drug-chromatin binding and multimodal epigenome.
  4. Progress in multifactorial single-cell chromatin profiling methods.
  5. In vivo single-cell CRISPR uncovers distinct TNF programmes in tumour evolution.
  6. Is There a Mathematician on Board?
  7. Enhancer-promoter hubs organize transcriptional networks promoting oncogenesis and drug resistance.
  8. Clonal inactivation of TERT impairs stem cell competition.
  9. Leveraging neighborhood representations of single-cell data to achieve sensitive DE testing with miloDE.
  10. JAK/STAT signaling promotes the emergence of unique cell states in ulcerative colitis.
  11. Transcriptome-wide characterization of genetic perturbations.
  12. Transcriptomic subtyping of gastrointestinal malignancies.
  13. Position-dependent function of human sequence-specific transcription factors.
  14. Non-coding variants impact cis-regulatory coordination in a cell type-specific manner.
  15. Precision oncology: current and future platforms for treatment selection.
  1. Nature. 2024 Jul 17.
    Symbolic recording of signalling and cis-regulatory element activity to DNA.
    Wei Chen, Junhong Choi, Xiaoyi Li, Jenny F Nathans, Beth Martin, Wei Yang, Nobuhiko Hamazaki, Chengxiang Qiu, Jean-Benoît Lalanne, Samuel Regalado, Haedong Kim, Vikram Agarwal, Eva Nichols, Anh Leith, Choli Lee, Jay Shendure.
      Measurements of gene expression or signal transduction activity are conventionally performed using methods that require either the destruction or live imaging of a biological sample within the timeframe of interest. Here we demonstrate an alternative paradigm in which such biological activities are stably recorded to the genome. Enhancer-driven genomic recording of transcriptional activity in multiplex (ENGRAM) is based on the signal-dependent production of prime editing guide RNAs that mediate the insertion of signal-specific barcodes (symbols) into a genomically encoded recording unit. We show how this strategy can be used for multiplex recording of the cell-type-specific activities of dozens to hundreds of cis-regulatory elements with high fidelity, sensitivity and reproducibility. Leveraging signal transduction pathway-responsive cis-regulatory elements, we also demonstrate time- and concentration-dependent genomic recording of WNT, NF-κB and Tet-On activities. By coupling ENGRAM to sequential genome editing via DNA Typewriter1, we stably record information about the temporal dynamics of two orthogonal signalling pathways to genomic DNA. Finally we apply ENGRAM to integratively record the transient activity of nearly 100 transcription factor consensus motifs across daily windows spanning the differentiation of mouse embryonic stem cells into gastruloids, an in vitro model of early mammalian development. Although these are proof-of-concept experiments and much work remains to fully realize the possibilities, the symbolic recording of biological signals or states within cells, to the genome and over time, has broad potential to complement contemporary paradigms for how we make measurements in biological systems.
    DOI:  https://doi.org/10.1038/s41586-024-07706-4
  2. bioRxiv. 2024 Jul 09. pii: 2024.07.08.602263. [Epub ahead of print]
    HyperCas12a enables highly-multiplexed epigenome editing screens.
    Schuyler M Melore, Marisa C Hamilton, Timothy E Reddy.
      Interactions between multiple genes or cis-regulatory elements (CREs) underlie a wide range of biological processes in both health and disease. High-throughput screens using dCas9 fused to epigenome editing domains have allowed researchers to assess the impact of activation or repression of both coding and non-coding genomic regions on a phenotype of interest, but assessment of genetic interactions between those elements has been limited to pairs. Here, we combine a hyper-efficient version of Lachnospiraceae bacterium dCas12a (dHyperLbCas12a) with RNA Polymerase II expression of long CRISPR RNA (crRNA) arrays to enable efficient highly-multiplexed epigenome editing. We demonstrate that this system is compatible with several activation and repression domains, including the P300 histone acetyltransferase domain and SIN3A interacting domain (SID). We also show that the dCas12a platform can perform simultaneous activation and repression using a single crRNA array via co-expression of multiple dCas12a orthologues. Lastly, demonstrate that the dCas12a system is highly effective for high-throughput screens. We use dHyperLbCas12a-KRAB and a ∼19,000-member barcoded library of crRNA arrays containing six crRNAs each to dissect the independent and combinatorial contributions of CREs to the dose-dependent control of gene expression at a glucocorticoid-responsive locus. The tools and methods introduced here create new possibilities for highly multiplexed control of gene expression in a wide variety of biological systems.
    DOI:  https://doi.org/10.1101/2024.07.08.602263
  3. Nat Methods. 2024 Jul 18.
    Single-cell EpiChem jointly measures drug-chromatin binding and multimodal epigenome.
    Chao Dong, Xiaoxuan Meng, Tong Zhang, Zhifang Guo, Yaxi Liu, Peihuang Wu, Shiwei Chen, Fanqi Zhou, Yanni Ma, Haiqing Xiong, Shaokun Shu, Aibin He.
      Studies of molecular and cellular functions of small-molecule inhibitors in cancer treatment, eliciting effects by targeting genome and epigenome associated proteins, requires measurement of drug-target engagement in single-cell resolution. Here we present EpiChem for in situ single-cell joint mapping of small molecules and multimodal epigenomic landscape. We demonstrate single-cell co-assays of three small molecules together with histone modifications, chromatin accessibility or target proteins in human colorectal cancer (CRC) organoids. Integrated multimodal analysis reveals diverse drug interactions in the context of chromatin states within heterogeneous CRC organoids. We further reveal drug genomic binding dynamics and adaptive epigenome across cell types after small-molecule drug treatment in CRC organoids. This method provides a unique tool to exploit the mechanisms of cell type-specific drug actions.
    DOI:  https://doi.org/10.1038/s41592-024-02360-0
  4. Biochem Soc Trans. 2024 Jul 18. pii: BST20231471. [Epub ahead of print]
    Progress in multifactorial single-cell chromatin profiling methods.
    Tim Stuart.
      Chromatin states play a key role in shaping overall cellular states and fates. Building a complete picture of the functional state of chromatin in cells requires the co-detection of several distinct biochemical aspects. These span DNA methylation, chromatin accessibility, chromosomal conformation, histone posttranslational modifications, and more. While this certainly presents a challenging task, over the past few years many new and creative methods have been developed that now enable co-assay of these different aspects of chromatin at single cell resolution. This field is entering an exciting phase, where a confluence of technological improvements, decreased sequencing costs, and computational innovation are presenting new opportunities to dissect the diversity of chromatin states present in tissues, and how these states may influence gene regulation. In this review, I discuss the spectrum of current experimental approaches for multifactorial chromatin profiling, highlight some of the experimental and analytical challenges, as well as some areas for further innovation.
    Keywords:  chromatin; epigenetics; single-cell
    DOI:  https://doi.org/10.1042/BST20231471
  5. Nature. 2024 Jul 17.
    In vivo single-cell CRISPR uncovers distinct TNF programmes in tumour evolution.
    Peter F Renz, Umesh Ghoshdastider, Simona Baghai Sain, Fabiola Valdivia-Francia, Ameya Khandekar, Mark Ormiston, Martino Bernasconi, Clara Duré, Jonas A Kretz, Minkyoung Lee, Katie Hyams, Merima Forny, Marcel Pohly, Xenia Ficht, Stephanie J Ellis, Andreas E Moor, Ataman Sendoel.
      The tumour evolution model posits that malignant transformation is preceded by randomly distributed driver mutations in cancer genes, which cause clonal expansions in phenotypically normal tissues. Although clonal expansions can remodel entire tissues1-3, the mechanisms that result in only a small number of clones transforming into malignant tumours remain unknown. Here we develop an in vivo single-cell CRISPR strategy to systematically investigate tissue-wide clonal dynamics of the 150 most frequently mutated squamous cell carcinoma genes. We couple ultrasound-guided in utero lentiviral microinjections, single-cell RNA sequencing and guide capture to longitudinally monitor clonal expansions and document their underlying gene programmes at single-cell transcriptomic resolution. We uncover a tumour necrosis factor (TNF) signalling module, which is dependent on TNF receptor 1 and involving macrophages, that acts as a generalizable driver of clonal expansions in epithelial tissues. Conversely, during tumorigenesis, the TNF signalling module is downregulated. Instead, we identify a subpopulation of invasive cancer cells that switch to an autocrine TNF gene programme associated with epithelial-mesenchymal transition. Finally, we provide in vivo evidence that the autocrine TNF gene programme is sufficient to mediate invasive properties and show that the TNF signature correlates with shorter overall survival of patients with squamous cell carcinoma. Collectively, our study demonstrates the power of applying in vivo single-cell CRISPR screening to mammalian tissues, unveils distinct TNF programmes in tumour evolution and highlights the importance of understanding the relationship between clonal expansions in epithelia and tumorigenesis.
    DOI:  https://doi.org/10.1038/s41586-024-07663-y
  6. Cancer Res. 2024 Jul 15. 84(14): 2229-2230
    Is There a Mathematician on Board?
    Elana J Fertig.
      As convergence science is emerging as a theme in cancer research, scientists from diverse backgrounds, including mathematics, are increasingly entering our research community. The captain of a Southwest Airlines flight recently paged in jest for a mathematician to help support fuel calculations that would enable a flight to arrive safely at the American Association for Cancer Research (AACR) Annual Meeting, epitomizing the need for well-trained mathematicians to address pressing problems. Here, we summarize the roles mathematicians can play in cancer research and the support needed to facilitate their entry into the cancer research field. The inclusion of scientific diversity across quantitative and engineering disciplines is critical for advancing the understanding and treatment of cancer.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-24-1396
  7. bioRxiv. 2024 Jul 02. pii: 2024.07.02.601745. [Epub ahead of print]
    Enhancer-promoter hubs organize transcriptional networks promoting oncogenesis and drug resistance.
    Brent S Perlman, Noah Burget, Yeqiao Zhou, Gregory W Schwartz, Jelena Petrovic, Zora Modrusan, Robert B Faryabi.
      Recent advances in high-resolution mapping of spatial interactions among regulatory elements support the existence of complex topological assemblies of enhancers and promoters known as enhancer-promoter hubs or cliques. Yet, organization principles of these multi-interacting enhancer-promoter hubs and their potential role in regulating gene expression in cancer remains unclear. Here, we systematically identified enhancer-promoter hubs in breast cancer, lymphoma, and leukemia. We found that highly interacting enhancer-promoter hubs form at key oncogenes and lineage-associated transcription factors potentially promoting oncogenesis of these diverse cancer types. Genomic and optical mapping of interactions among enhancer and promoter elements further showed that topological alterations in hubs coincide with transcriptional changes underlying acquired resistance to targeted therapy in T cell leukemia and B cell lymphoma. Together, our findings suggest that enhancer-promoter hubs are dynamic and heterogeneous topological assemblies with the potential to control gene expression circuits promoting oncogenesis and drug resistance.
    DOI:  https://doi.org/10.1101/2024.07.02.601745
  8. Nature. 2024 Jul 17.
    Clonal inactivation of TERT impairs stem cell competition.
    Kazuteru Hasegawa, Yang Zhao, Alina Garbuzov, M Ryan Corces, Patrick Neuhöfer, Victoria M Gillespie, Peggie Cheung, Julia A Belk, Yung-Hsin Huang, Yuning Wei, Lu Chen, Howard Y Chang, Steven E Artandi.
      Telomerase is intimately associated with stem cells and cancer, because it catalytically elongates telomeres-nucleoprotein caps that protect chromosome ends1. Overexpression of telomerase reverse transcriptase (TERT) enhances the proliferation of cells in a telomere-independent manner2-8, but so far, loss-of-function studies have provided no evidence that TERT has a direct role in stem cell function. In many tissues, homeostasis is shaped by stem cell competition, a process in which stem cells compete on the basis of inherent fitness. Here we show that conditional deletion of Tert in the spermatogonial stem cell (SSC)-containing population in mice markedly impairs competitive clone formation. Using lineage tracing from the Tert locus, we find that TERT-expressing SSCs yield long-lived clones, but that clonal inactivation of TERT promotes stem cell differentiation and a genome-wide reduction in open chromatin. This role for TERT in competitive clone formation occurs independently of both its reverse transcriptase activity and the canonical telomerase complex. Inactivation of TERT causes reduced activity of the MYC oncogene, and transgenic expression of MYC in the TERT-deleted pool of SSCs efficiently rescues clone formation. Together, these data reveal a catalytic-activity-independent requirement for TERT in enhancing stem cell competition, uncover a genetic connection between TERT and MYC and suggest that a selective advantage for stem cells with high levels of TERT contributes to telomere elongation in the male germline during homeostasis and ageing.
    DOI:  https://doi.org/10.1038/s41586-024-07700-w
  9. Genome Biol. 2024 Jul 18. 25(1): 189
    Leveraging neighborhood representations of single-cell data to achieve sensitive DE testing with miloDE.
    Alsu Missarova, Emma Dann, Leah Rosen, Rahul Satija, John Marioni.
      Single-cell RNA-sequencing enables testing for differential expression (DE) between conditions at a cell type level. While powerful, one of the limitations of such approaches is that the sensitivity of DE testing is dictated by the sensitivity of clustering, which is often suboptimal. To overcome this, we present miloDE-a cluster-free framework for DE testing (available as an open-source R package). We illustrate the performance of miloDE on both simulated and real data. Using miloDE, we identify a transient hemogenic endothelia-like state in mouse embryos lacking Tal1 and detect distinct programs during macrophage activation in idiopathic pulmonary fibrosis.
    DOI:  https://doi.org/10.1186/s13059-024-03334-3
  10. Stem Cell Reports. 2024 Jul 08. pii: S2213-6711(24)00186-3. [Epub ahead of print]
    JAK/STAT signaling promotes the emergence of unique cell states in ulcerative colitis.
    Grzegorz Maciag, Stine Lind Hansen, Kata Krizic, Lauge Kellermann, Maureen Joy Inventor Zøylner, Svetlana Ulyanchenko, Martti Maimets, Astrid Møller Baattrup, Lene Buhl Riis, Konstantin Khodosevich, Toshiro Sato, Raul Bardini Bressan, Ole Haagen Nielsen, Kim B Jensen.
      The intestinal epithelium ensures uptake of vital nutrients and acts as a barrier between luminal contents and the underlying immune system. In inflammatory bowel diseases, such as ulcerative colitis (UC), this barrier is compromised, and patients experience debilitating symptoms. Here, we perform single-cell RNA profiling of epithelial cells and outline patterns of cell fate decisions in healthy individuals and UC patients. We demonstrate that patterns of hierarchical behavior are altered in UC patients and identify unique cellular states associated with Janus kinase/signal transducer and activator of transcription (JAK/STAT) activation in ulcerated and non-ulcerated areas of the colonic epithelium. These transcriptional changes could be recapitulated in human colonic organoids, wherein cytokine-mediated activation of JAK/STAT led to the emergence of cell populations with augmented regenerative properties. Altogether, our findings indicate that intricate relationships between epithelial and cytokine signaling regulate cell fate during epithelial tissue regeneration in humans and have important implications for the understanding of UC biology.
    DOI:  https://doi.org/10.1016/j.stemcr.2024.06.006
  11. bioRxiv. 2024 Jul 03. pii: 2024.07.03.601903. [Epub ahead of print]
    Transcriptome-wide characterization of genetic perturbations.
    Ajay Nadig, Joseph M Replogle, Angela N Pogson, Steven A McCarroll, Jonathan S Weissman, Elise B Robinson, Luke J O'Connor.
      Single cell CRISPR screens such as Perturb-seq enable transcriptomic profiling of genetic perturbations at scale. However, the data produced by these screens are often noisy due to cost and technical constraints, limiting power to detect true effects with conventional differential expression analyses. Here, we introduce TRanscriptome-wide Analysis of Differential Expression (TRADE), a statistical framework which estimates the transcriptome-wide distribution of true differential expression effects from noisy gene-level measurements. Within TRADE, we derive multiple novel, interpretable statistical metrics, including the "transcriptome-wide impact", an estimator of the overall transcriptional effect of a perturbation which is stable across sampling depths. We analyze new and published large-scale Perturb-seq datasets to show that many true transcriptional effects are not statistically significant, but detectable in aggregate with TRADE. In a genome-scale Perturb-seq screen, we find that a typical gene perturbation affects an estimated 45 genes, whereas a typical essential gene perturbation affects over 500 genes. An advantage of our approach is its ability to compare the transcriptomic effects of genetic perturbations across contexts and dosages despite differences in power. We use this ability to identify perturbations with cell-type dependent effects and to find examples of perturbations where transcriptional responses are not only larger in magnitude, but also qualitatively different, as a function of dosage. Lastly, we expand our analysis to case/control comparison of gene expression for neuropsychiatric conditions, finding that transcriptomic effect correlations are greater than genetic correlations for these diagnoses. TRADE lays an analytic foundation for the systematic comparison of genetic perturbation atlases, as well as differential expression experiments more broadly.
    DOI:  https://doi.org/10.1101/2024.07.03.601903
  12. Trends Cancer. 2024 Jul 16. pii: S2405-8033(24)00120-1. [Epub ahead of print]
    Transcriptomic subtyping of gastrointestinal malignancies.
    Tim R de Back, Sander R van Hooff, Dirkje W Sommeijer, Louis Vermeulen.
      Gastrointestinal (GI) cancers are highly heterogeneous at multiple levels. Tumor heterogeneity can be captured by molecular profiling, such as genetic, epigenetic, proteomic, and transcriptomic classification. Transcriptomic subtyping has the advantage of combining genetic and epigenetic information, cancer cell-intrinsic properties, and the tumor microenvironment (TME). Unsupervised transcriptomic subtyping systems of different GI malignancies have gained interest because they reveal shared biological features across cancers and bear prognostic and predictive value. Importantly, transcriptomic subtypes accurately reflect complex phenotypic states varying not only per tumor region, but also throughout disease progression, with consequences for clinical management. Here, we discuss methodologies of transcriptomic subtyping, proposed taxonomies for GI malignancies, and the challenges posed to clinical implementation, highlighting opportunities for future transcriptomic profiling efforts to optimize clinical impact.
    Keywords:  colorectal cancer; gastric cancer; gastrointestinal malignancies; pancreatic ductal adenocarcinoma; personalized medicine; transcriptomic subtyping
    DOI:  https://doi.org/10.1016/j.trecan.2024.06.007
  13. Nature. 2024 Jul 17.
    Position-dependent function of human sequence-specific transcription factors.
    Sascha H Duttke, Carlos Guzman, Max Chang, Nathaniel P Delos Santos, Bayley R McDonald, Jialei Xie, Aaron F Carlin, Sven Heinz, Christopher Benner.
      Patterns of transcriptional activity are encoded in our genome through regulatory elements such as promoters or enhancers that, paradoxically, contain similar assortments of sequence-specific transcription factor (TF) binding sites1-3. Knowledge of how these sequence motifs encode multiple, often overlapping, gene expression programs is central to understanding gene regulation and how mutations in non-coding DNA manifest in disease4,5. Here, by studying gene regulation from the perspective of individual transcription start sites (TSSs), using natural genetic variation, perturbation of endogenous TF protein levels and massively parallel analysis of natural and synthetic regulatory elements, we show that the effect of TF binding on transcription initiation is position dependent. Analysing TF-binding-site occurrences relative to the TSS, we identified several motifs with highly preferential positioning. We show that these patterns are a combination of a TF's distinct functional profiles-many TFs, including canonical activators such as NRF1, NFY and Sp1, activate or repress transcription initiation depending on their precise position relative to the TSS. As such, TFs and their spacing collectively guide the site and frequency of transcription initiation. More broadly, these findings reveal how similar assortments of TF binding sites can generate distinct gene regulatory outcomes depending on their spatial configuration and how DNA sequence polymorphisms may contribute to transcription variation and disease and underscore a critical role for TSS data in decoding the regulatory information of our genome.
    DOI:  https://doi.org/10.1038/s41586-024-07662-z
  14. Genome Biol. 2024 Jul 18. 25(1): 190
    Non-coding variants impact cis-regulatory coordination in a cell type-specific manner.
    Olga Pushkarev, Guido van Mierlo, Judith Franziska Kribelbauer, Wouter Saelens, Vincent Gardeux, Bart Deplancke.
      BACKGROUND: Interactions among cis-regulatory elements (CREs) play a crucial role in gene regulation. Various approaches have been developed to map these interactions genome-wide, including those relying on interindividual epigenomic variation to identify groups of covariable regulatory elements, referred to as chromatin modules (CMs). While CM mapping allows to investigate the relationship between chromatin modularity and gene expression, the computational principles used for CM identification vary in their application and outcomes.RESULTS: We comprehensively evaluate and streamline existing CM mapping tools and present guidelines for optimal utilization of epigenome data from a diverse population of individuals to assess regulatory coordination across the human genome. We showcase the effectiveness of our recommended practices by analyzing distinct cell types and demonstrate cell type specificity of CRE interactions in CMs and their relevance for gene expression. Integration of genotype information revealed that many non-coding disease-associated variants affect the activity of CMs in a cell type-specific manner by affecting the binding of cell type-specific transcription factors. We provide example cases that illustrate in detail how CMs can be used to deconstruct GWAS loci, assess variable expression of cell surface receptors in immune cells, and reveal how genetic variation can impact the expression of prognostic markers in chronic lymphocytic leukemia.
    CONCLUSIONS: Our study presents an optimal strategy for CM mapping and reveals how CMs capture the coordination of CREs and its impact on gene expression. Non-coding genetic variants can disrupt this coordination, and we highlight how this may lead to disease predisposition in a cell type-specific manner.
    Keywords:   Cis-regulatory interactions; Epigenomics; Gene regulation; Genome-wide association studies; Quantitative trait loci
    DOI:  https://doi.org/10.1186/s13059-024-03333-4
  15. Trends Cancer. 2024 Jul 18. pii: S2405-8033(24)00135-3. [Epub ahead of print]
    Precision oncology: current and future platforms for treatment selection.
    Xinran Tang, Michael F Berger, David B Solit.
      Genomic profiling of hundreds of cancer-associated genes is now a component of routine cancer care. DNA sequencing can identify mutations, mutational signatures, and structural alterations predictive of therapy response and assess for heritable cancer risk, but it has been less useful for identifying predictive biomarkers of sensitivity to cytotoxic chemotherapies, antibody drug conjugates, and immunotherapies. The clinical adoption of molecular profiling platforms such as RNA sequencing better suited to identifying those patients most likely to respond to immunotherapies and drug combinations will be critical to expanding the benefits of precision oncology. This review discusses the potential advantages of innovative molecular and functional profiling platforms designed to replace or complement targeted DNA sequencing and the major hurdles to their clinical adoption.
    Keywords:  DNA sequencing; RNA sequencing; cell free DNA; functional precision oncology; targeted cancer therapy
    DOI:  https://doi.org/10.1016/j.trecan.2024.06.009
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