bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2021‒01‒10
twenty-four papers selected by
Connor Rogerson
University of Cambridge, MRC Cancer Unit
  1. Promoter-proximal CTCF binding promotes distal enhancer-dependent gene activation.
  2. Nonlinear relationship between chromatin accessibility and estradiol-regulated gene expression.
  3. Zinc Finger Protein SALL4 Functions through an AT-Rich Motif to Regulate Gene Expression.
  4. An interpretable bimodal neural network characterizes the sequence and preexisting chromatin predictors of induced transcription factor binding.
  5. KDM1A maintains genome-wide homeostasis of transcriptional enhancers.
  6. Chromatin regulatory dynamics of early human small intestinal development using a directed differentiation model.
  7. A genome-wide CRISPR-based screen identifies KAT7 as a driver of cellular senescence.
  8. Order and stochasticity in the folding of individual Drosophila genomes.
  9. Motif-driven interactions between RNA and PRC2 are rheostats that regulate transcription elongation.
  10. The Hox Transcription Factor Ubx Ensures Somatic Myogenesis by Suppressing the Mesodermal Master Regulator Twist.
  11. A transcriptional regulatory loop of master regulator transcription factors, PPARG, and fatty acid synthesis promotes esophageal adenocarcinoma.
  12. ZNF143 mediates CTCF-bound promoter-enhancer loops required for murine hematopoietic stem and progenitor cell function.
  13. Paternal age affects offspring via an epigenetic mechanism involving REST/NRSF.
  14. Spatiotemporal analysis of human intestinal development at single-cell resolution.
  15. Comprehensive integrative profiling of upper tract urothelial carcinomas.
  16. Global computational alignment of tumor and cell line transcriptional profiles.
  17. Intestinal differentiation involves cleavage of histone H3 N-terminal tails by multiple proteases.
  18. Single-cell analysis reveals transcriptomic remodellings in distinct cell types that contribute to human prostate cancer progression.
  19. Hyperosmotic stress alters the RNA polymerase II interactome and induces readthrough transcription despite widespread transcriptional repression.
  20. HiDeF: identifying persistent structures in multiscale 'omics data.
  21. Chromatin accessibility maps provide evidence of multilineage gene priming in hematopoietic stem cells.
  22. SALL4 controls cell fate in response to DNA base composition.
  23. High-resolution single-cell 3D-models of chromatin ensembles during Drosophila embryogenesis.
  24. An integrated multi-omics approach to identify regulatory mechanisms in cancer metastatic processes.
  1. Nat Struct Mol Biol. 2021 Jan 04.
    Promoter-proximal CTCF binding promotes distal enhancer-dependent gene activation.
    Naoki Kubo, Haruhiko Ishii, Xiong Xiong, Simona Bianco, Franz Meitinger, Rong Hu, James D Hocker, Mattia Conte, David Gorkin, Miao Yu, Bin Li, Jesse R Dixon, Ming Hu, Mario Nicodemi, Huimin Zhao, Bing Ren.
      The CCCTC-binding factor (CTCF) works together with the cohesin complex to drive the formation of chromatin loops and topologically associating domains, but its role in gene regulation has not been fully defined. Here, we investigated the effects of acute CTCF loss on chromatin architecture and transcriptional programs in mouse embryonic stem cells undergoing differentiation to neural precursor cells. We identified CTCF-dependent enhancer-promoter contacts genome-wide and found that they disproportionately affect genes that are bound by CTCF at the promoter and are dependent on long-distance enhancers. Disruption of promoter-proximal CTCF binding reduced both long-range enhancer-promoter contacts and transcription, which were restored by artificial tethering of CTCF to the promoter. Promoter-proximal CTCF binding is correlated with the transcription of over 2,000 genes across a diverse set of adult tissues. Taken together, the results of our study show that CTCF binding to promoters may promote long-distance enhancer-dependent transcription at specific genes in diverse cell types.
    DOI:  https://doi.org/10.1038/s41594-020-00539-5
  2. Oncogene. 2021 Jan 08.
    Nonlinear relationship between chromatin accessibility and estradiol-regulated gene expression.
    Duojiao Chen, Taylor M Parker, Poornima Bhat-Nakshatri, Xiaona Chu, Yunlong Liu, Yue Wang, Harikrishna Nakshatri.
      Chromatin accessibility is central to basal and inducible gene expression. Through ATAC-seq experiments in estrogen receptor-positive (ER+) breast cancer cell line MCF-7 and integration with multi-omics data, we found estradiol (E2) induced chromatin accessibility changes in a small number of breast cancer-relevant E2-regulated genes. As expected, open chromatin regions associated with E2-inducible gene expression showed enrichment of estrogen response element (ERE) and those associated with E2-repressible gene expression were enriched for ERE, PBX1, and PBX3. While a significant number of open chromatin regions showed pioneer factor FOXA1 occupancy in the absence of E2, E2-treatment further enhanced FOXA1 occupancy suggesting that ER-E2 enhances chromatin occupancy of FOXA1 to a subset of E2-regulated genes. Surprisingly, promoters of 80% and enhancers of 60% of E2-inducible genes displayed closed chromatin configuration both in the absence and presence of E2. Integration of ATAC-seq data with ERα ChIP-seq data revealed that ~40% ERα binding sites in the genome are found in chromatin regions that are not accessible as per ATAC-seq. Such ERα binding regions were enriched for binding sites of multiple nuclear receptors including ER, ESRRB, ERRγ, COUP-TFII (NR2F2), RARα, EAR2 as well as traditional pioneer factors FOXA1 and GATA3. Similar data were also obtained when ERα ChIP-seq data were integrated with MNase-seq and DNase-seq data sets. In summation, our results reveal complex mechanisms of ER-E2 interaction with nucleosomes. Notably, "closed chromatin" configuration as defined by ATAC-seq or by other techniques is not necessarily associated with lack of gene expression and technical limitations may preclude ATAC-seq to demonstrate accessibility of chromatin regions that are bound by ERα.
    DOI:  https://doi.org/10.1038/s41388-020-01607-2
  3. Cell Rep. 2021 Jan 05. pii: S2211-1247(20)31563-1. [Epub ahead of print]34(1): 108574
    Zinc Finger Protein SALL4 Functions through an AT-Rich Motif to Regulate Gene Expression.
    Nikki R Kong, Mahmoud A Bassal, Hong Kee Tan, Jesse V Kurland, Kol Jia Yong, John J Young, Yang Yang, Fudong Li, Jonathan D Lee, Yue Liu, Chan-Shuo Wu, Alicia Stein, Hongbo R Luo, Leslie E Silberstein, Martha L Bulyk, Daniel G Tenen, Li Chai.
      The zinc finger transcription factor SALL4 is highly expressed in embryonic stem cells, downregulated in most adult tissues, but reactivated in many aggressive cancers. This unique expression pattern makes SALL4 an attractive therapeutic target. However, whether SALL4 binds DNA directly to regulate gene expression is unclear, and many of its targets in cancer cells remain elusive. Here, through an unbiased screen of protein binding microarray (PBM) and cleavage under targets and release using nuclease (CUT&RUN) experiments, we identify and validate the DNA binding domain of SALL4 and its consensus binding sequence. Combined with RNA sequencing (RNA-seq) analyses after SALL4 knockdown, we discover hundreds of new SALL4 target genes that it directly regulates in aggressive liver cancer cells, including genes encoding a family of histone 3 lysine 9-specific demethylases (KDMs). Taken together, these results elucidate the mechanism of SALL4 DNA binding and reveal pathways and molecules to target in SALL4-dependent tumors.
    Keywords:  CUT&RUN; KDM; SALL4; heterochromatin; liver cancer; protein binding microarray; transcription
    DOI:  https://doi.org/10.1016/j.celrep.2020.108574
  4. Genome Biol. 2021 Jan 07. 22(1): 20
    An interpretable bimodal neural network characterizes the sequence and preexisting chromatin predictors of induced transcription factor binding.
    Divyanshi Srivastava, Begüm Aydin, Esteban O Mazzoni, Shaun Mahony.
      BACKGROUND: Transcription factor (TF) binding specificity is determined via a complex interplay between the transcription factor's DNA binding preference and cell type-specific chromatin environments. The chromatin features that correlate with transcription factor binding in a given cell type have been well characterized. For instance, the binding sites for a majority of transcription factors display concurrent chromatin accessibility. However, concurrent chromatin features reflect the binding activities of the transcription factor itself and thus provide limited insight into how genome-wide TF-DNA binding patterns became established in the first place. To understand the determinants of transcription factor binding specificity, we therefore need to examine how newly activated transcription factors interact with sequence and preexisting chromatin landscapes.RESULTS: Here, we investigate the sequence and preexisting chromatin predictors of TF-DNA binding by examining the genome-wide occupancy of transcription factors that have been induced in well-characterized chromatin environments. We develop Bichrom, a bimodal neural network that jointly models sequence and preexisting chromatin data to interpret the genome-wide binding patterns of induced transcription factors. We find that the preexisting chromatin landscape is a differential global predictor of TF-DNA binding; incorporating preexisting chromatin features improves our ability to explain the binding specificity of some transcription factors substantially, but not others. Furthermore, by analyzing site-level predictors, we show that transcription factor binding in previously inaccessible chromatin tends to correspond to the presence of more favorable cognate DNA sequences.
    CONCLUSIONS: Bichrom thus provides a framework for modeling, interpreting, and visualizing the joint sequence and chromatin landscapes that determine TF-DNA binding dynamics.
    DOI:  https://doi.org/10.1186/s13059-020-02218-6
  5. Genome Res. 2021 Jan 07.
    KDM1A maintains genome-wide homeostasis of transcriptional enhancers.
    Saurabh Agarwal, Katherine M Bonefas, Patricia M Garay, Emily Brookes, Yumie Murata-Nakamura, Robert S Porter, Todd S Macfarlan, Bing Ren, Shigeki Iwase.
      Transcriptional enhancers enable exquisite spatiotemporal control of gene expression in metazoans. Enrichment of monomethylation of histone H3 lysine 4 (H3K4me1) is a major chromatin signature of transcriptional enhancers. Lysine (K)-specific demethylase 1A (KDM1A, also known as LSD1), an H3K4me2/me1 demethylase, inactivates stem-cell enhancers during the differentiation of mouse embryonic stem cells (mESCs). However, its role in undifferentiated mESCs remains obscure. Here, we show that KDM1A actively maintains the optimal enhancer status in both undifferentiated and lineage-committed cells. KDM1A occupies a majority of enhancers in undifferentiated mESCs. KDM1A levels at enhancers exhibit clear positive correlations with its substrate H3K4me2, H3K27ac, and transcription at enhancers. In Kdm1a-deficient mESCs, a large fraction of these enhancers gains additional H3K4 methylation, which is accompanied by increases in H3K27 acetylation and increased expression of both enhancer RNAs (eRNAs) and target genes. In postmitotic neurons, loss of KDM1A leads to premature activation of neuronal activity-dependent enhancers and genes. Taken together, these results suggest that KDM1A is a versatile regulator of enhancers and acts as a rheostat to maintain optimal enhancer activity by counterbalancing H3K4 methylation at enhancers.
    DOI:  https://doi.org/10.1101/gr.234559.118
  6. Nucleic Acids Res. 2021 Jan 06. pii: gkaa1204. [Epub ahead of print]
    Chromatin regulatory dynamics of early human small intestinal development using a directed differentiation model.
    Yu-Han Hung, Sha Huang, Michael K Dame, Qianhui Yu, Qing C Yu, Yi A Zeng, J Gray Camp, Jason R Spence, Praveen Sethupathy.
      The establishment of the small intestinal (SI) lineage during human embryogenesis ensures functional integrity of the intestine after birth. The chromatin dynamics that drive SI lineage formation and regional patterning in humans are essentially unknown. To fill this knowledge void, we apply a cutting-edge genomic technology to a state-of-the-art human model of early SI development. Specifically, we leverage chromatin run-on sequencing (ChRO-seq) to define the landscape of active promoters, enhancers and gene bodies across distinct stages of directed differentiation of human pluripotent stem cells into SI spheroids with regional specification. Through comprehensive ChRO-seq analysis we identify candidate stage-specific chromatin activity states, novel markers and enhancer hotspots during the directed differentiation. Moreover, we propose a detailed transcriptional network associated with SI lineage formation or regional patterning. Our ChRO-seq analyses uncover a previously undescribed pattern of enhancer activity and transcription at HOX gene loci underlying SI regional patterning. We also validated this unique HOX dynamics by the analysis of single cell RNA-seq data from human fetal SI. Overall, the results lead to a new proposed working model for the regulatory underpinnings of human SI development, thereby adding a novel dimension to the literature that has relied almost exclusively on non-human models.
    DOI:  https://doi.org/10.1093/nar/gkaa1204
  7. Sci Transl Med. 2021 Jan 06. pii: eabd2655. [Epub ahead of print]13(575):
    A genome-wide CRISPR-based screen identifies KAT7 as a driver of cellular senescence.
    Wei Wang, Yuxuan Zheng, Shuhui Sun, Wei Li, Moshi Song, Qianzhao Ji, Zeming Wu, Zunpeng Liu, Yanling Fan, Feifei Liu, Jingyi Li, Concepcion Rodriguez Esteban, Si Wang, Qi Zhou, Juan Carlos Izpisua Belmonte, Weiqi Zhang, Jing Qu, Fuchou Tang, Guang-Hui Liu.
      Understanding the genetic and epigenetic bases of cellular senescence is instrumental in developing interventions to slow aging. We performed genome-wide CRISPR-Cas9-based screens using two types of human mesenchymal precursor cells (hMPCs) exhibiting accelerated senescence. The hMPCs were derived from human embryonic stem cells carrying the pathogenic mutations that cause the accelerated aging diseases Werner syndrome and Hutchinson-Gilford progeria syndrome. Genes whose deficiency alleviated cellular senescence were identified, including KAT7, a histone acetyltransferase, which ranked as a top hit in both progeroid hMPC models. Inactivation of KAT7 decreased histone H3 lysine 14 acetylation, repressed p15INK4b transcription, and alleviated hMPC senescence. Moreover, lentiviral vectors encoding Cas9/sg-Kat7, given intravenously, alleviated hepatocyte senescence and liver aging and extended life span in physiologically aged mice as well as progeroid Zmpste24-/- mice that exhibit a premature aging phenotype. CRISPR-Cas9-based genetic screening is a robust method for systematically uncovering senescence genes such as KAT7, which may represent a therapeutic target for developing aging interventions.
    DOI:  https://doi.org/10.1126/scitranslmed.abd2655
  8. Nat Commun. 2021 01 04. 12(1): 41
    Order and stochasticity in the folding of individual Drosophila genomes.
    Sergey V Ulianov, Vlada V Zakharova, Aleksandra A Galitsyna, Pavel I Kos, Kirill E Polovnikov, Ilya M Flyamer, Elena A Mikhaleva, Ekaterina E Khrameeva, Diego Germini, Mariya D Logacheva, Alexey A Gavrilov, Alexander S Gorsky, Sergey K Nechaev, Mikhail S Gelfand, Yegor S Vassetzky, Alexander V Chertovich, Yuri Y Shevelyov, Sergey V Razin.
      Mammalian and Drosophila genomes are partitioned into topologically associating domains (TADs). Although this partitioning has been reported to be functionally relevant, it is unclear whether TADs represent true physical units located at the same genomic positions in each cell nucleus or emerge as an average of numerous alternative chromatin folding patterns in a cell population. Here, we use a single-nucleus Hi-C technique to construct high-resolution Hi-C maps in individual Drosophila genomes. These maps demonstrate chromatin compartmentalization at the megabase scale and partitioning of the genome into non-hierarchical TADs at the scale of 100 kb, which closely resembles the TAD profile in the bulk in situ Hi-C data. Over 40% of TAD boundaries are conserved between individual nuclei and possess a high level of active epigenetic marks. Polymer simulations demonstrate that chromatin folding is best described by the random walk model within TADs and is most suitably approximated by a crumpled globule build of Gaussian blobs at longer distances. We observe prominent cell-to-cell variability in the long-range contacts between either active genome loci or between Polycomb-bound regions, suggesting an important contribution of stochastic processes to the formation of the Drosophila 3D genome.
    DOI:  https://doi.org/10.1038/s41467-020-20292-z
  9. Nat Struct Mol Biol. 2021 Jan 04.
    Motif-driven interactions between RNA and PRC2 are rheostats that regulate transcription elongation.
    Michael Rosenberg, Roy Blum, Barry Kesner, Eric Aeby, Jean-Michel Garant, Attila Szanto, Jeannie T Lee.
      Although polycomb repressive complex 2 (PRC2) is now recognized as an RNA-binding complex, the full range of binding motifs and why PRC2-RNA complexes often associate with active genes have not been elucidated. Here, we identify high-affinity RNA motifs whose mutations weaken PRC2 binding and attenuate its repressive function in mouse embryonic stem cells. Interactions occur at promoter-proximal regions and frequently coincide with pausing of RNA polymerase II (POL-II). Surprisingly, while PRC2-associated nascent transcripts are highly expressed, ablating PRC2 further upregulates expression via loss of pausing and enhanced transcription elongation. Thus, PRC2-nascent RNA complexes operate as rheostats to fine-tune transcription by regulating transitions between pausing and elongation, explaining why PRC2-RNA complexes frequently occur within active genes. Nascent RNA also targets PRC2 in cis and downregulates neighboring genes. We propose a unifying model in which RNA specifically recruits PRC2 to repress genes through POL-II pausing and, more classically, trimethylation of histone H3 at Lys27.
    DOI:  https://doi.org/10.1038/s41594-020-00535-9
  10. Cell Rep. 2021 Jan 05. pii: S2211-1247(20)31566-7. [Epub ahead of print]34(1): 108577
    The Hox Transcription Factor Ubx Ensures Somatic Myogenesis by Suppressing the Mesodermal Master Regulator Twist.
    Katrin Domsch, Julia Schröder, Matthias Janeschik, Christoph Schaub, Ingrid Lohmann.
      Early lineage-specific master regulators are essential for the specification of cell types. However, once cells are committed to a specific fate, it is critical to restrict the activity of such factors to enable differentiation. To date, it remains unclear how these factors are silenced. Using the Drosophila mesoderm as a model and a comparative genomic approach, we identify the Hox transcription factor Ultrabithorax (Ubx) to be critical for the repression of the master regulator Twist. Mesoderm-specific Ubx loss-of-function experiments using CRISPR-Cas9 and overexpression studies demonstrate that Ubx majorly impacts twist transcription. A mechanistic analysis reveals that Ubx requires the NK-homeodomain protein Tinman to bind to the twist promoter. Furthermore, we find these factor interactions to be critical for silencing by recruiting the Polycomb DNA binding protein Pleiohomeotic. Altogether, our data reveal that Ubx is a critical player in mediating the silencing of Twist, which is crucial for coordinated muscle differentiation.
    Keywords:  Hox genes; Mef2; Tinman; Ultrabithorax (Ubx); mesoderm; repression; twist
    DOI:  https://doi.org/10.1016/j.celrep.2020.108577
  11. Cancer Res. 2021 Jan 05. pii: canres.0652.2020. [Epub ahead of print]
    A transcriptional regulatory loop of master regulator transcription factors, PPARG, and fatty acid synthesis promotes esophageal adenocarcinoma.
    Sai Ma, Bo Zhou, Qian Yang, Yunzhi Pan, Wei Yang, Stephen J Freedland, Ling-Wen Ding, Michael R Freeman, Joshua J Breunig, Neil A Bhowmick, Jian Pan, H Phillip Koeffler, De-Chen Lin.
      Although obesity is one of the strongest risk factors for esophageal adenocarcinoma (EAC), the molecular mechanisms underlying this association remain unclear. We recently identified 4 EAC-specific master regulator transcription factors (MRTF) ELF3, KLF5, GATA6, and EHF. In the present study, Gene Set Enrichment Analysis (GSEA) of both EAC patient samples and cell line models unbiasedly underscore fatty acid synthesis as the central pathway downstream of three MRTF (ELF3, KLF5, GATA6). Further characterizations unexpectedly identified a transcriptional feedback loop between MRTF and fatty acid synthesis, which mutually activated each other through the nuclear receptor PPARG. MRTF cooperatively promoted PPARG transcription by directly regulating its promoter and a distal EAC-specific enhancer, leading to PPARG overexpression in EAC. PPARG was also elevated in Barrett's esophagus, a recognized precursor to EAC, implying that PPARG might play a role in the intestinal metaplasia of esophageal squamous epithelium. Upregulation of PPARG increased de novo synthesis of fatty acids, phospholipids, and sphingolipids as revealed by mass spectrometry-based lipidomics. Moreover, ChIP-Seq, 4C-Seq, and a high-fat diet murine model together characterized a novel, noncanonical, and cancer-specific function of PPARG in EAC. PPARG directly regulated the ELF3 super-enhancer, subsequently activating the transcription of other MRTF through an interconnected regulatory circuitry. Together, elucidation of this novel transcriptional feedback loop of MRTF/PPARG/fatty acid synthesis advances our understanding of the mechanistic foundation for epigenomic dysregulation and metabolic alterations in EAC. More importantly, this work identifies a potential avenue for prevention and early intervention of EAC by blocking this feedback loop.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-20-0652
  12. Nat Commun. 2021 01 04. 12(1): 43
    ZNF143 mediates CTCF-bound promoter-enhancer loops required for murine hematopoietic stem and progenitor cell function.
    Qiling Zhou, Miao Yu, Roberto Tirado-Magallanes, Bin Li, Lingshi Kong, Mingrui Guo, Zi Hui Tan, Sanghoon Lee, Li Chai, Akihiko Numata, Touati Benoukraf, Melissa Jane Fullwood, Motomi Osato, Bing Ren, Daniel G Tenen.
      CCCTC binding factor (CTCF) is an important factor in the maintenance of chromatin-chromatin interactions, yet the mechanism regulating its binding to chromatin is unknown. We demonstrate that zinc finger protein 143 (ZNF143) is a key regulator for CTCF-bound promoter-enhancer loops. In the murine genome, a large percentage of CTCF and ZNF143 DNA binding motifs are distributed 37 bp apart in the convergent orientation. Furthermore, deletion of ZNF143 leads to loss of CTCF binding on promoter and enhancer regions associated with gene expression changes. CTCF-bound promoter-enhancer loops are also disrupted after excision of ZNF143. ZNF143-CTCF-bound promoter-enhancer loops regulate gene expression patterns essential for maintenance of murine hematopoietic stem and progenitor cell integrity. Our data suggest a common feature of gene regulation is that ZNF143 is a critical factor for CTCF-bound promoter-enhancer loops.
    DOI:  https://doi.org/10.1038/s41467-020-20282-1
  13. EMBO Rep. 2021 Jan 05. e51524
    Paternal age affects offspring via an epigenetic mechanism involving REST/NRSF.
    Kaichi Yoshizaki, Ryuichi Kimura, Hisato Kobayashi, Shinya Oki, Takako Kikkawa, Lingling Mai, Kohei Koike, Kentaro Mochizuki, Hitoshi Inada, Yasuhisa Matsui, Tomohiro Kono, Noriko Osumi.
      Advanced paternal age can have deleterious effects on various traits in the next generation. Here, we establish a paternal-aging model in mice to understand the molecular mechanisms of transgenerational epigenetics. Whole-genome target DNA methylome analyses of sperm from aged mice reveal more hypo-methylated genomic regions enriched in REST/NRSF binding motifs. Gene set enrichment analyses also reveal the upregulation of REST/NRSF target genes in the forebrain of embryos from aged fathers. Offspring derived from young mice administrated with a DNA de-methylation drug phenocopy the abnormal vocal communication of pups derived from aged fathers. In conclusion, hypo-methylation of sperm DNA can be a key molecular feature modulating neurodevelopmental programs in offspring by causing fluctuations in the expression of REST/NRSF target genes.
    Keywords:  DNA hypo-methylation; REST/NRSF; paternal aging; transgenerational epigenetic inheritance; ultrasonic vocalization
    DOI:  https://doi.org/10.15252/embr.202051524
  14. Cell. 2021 Jan 04. pii: S0092-8674(20)31686-X. [Epub ahead of print]
    Spatiotemporal analysis of human intestinal development at single-cell resolution.
    David Fawkner-Corbett, Agne Antanaviciute, Kaushal Parikh, Marta Jagielowicz, Ana Sousa Gerós, Tarun Gupta, Neil Ashley, Doran Khamis, Darren Fowler, Edward Morrissey, Chris Cunningham, Paul R V Johnson, Hashem Koohy, Alison Simmons.
      Development of the human intestine is not well understood. Here, we link single-cell RNA sequencing and spatial transcriptomics to characterize intestinal morphogenesis through time. We identify 101 cell states including epithelial and mesenchymal progenitor populations and programs linked to key morphogenetic milestones. We describe principles of crypt-villus axis formation; neural, vascular, mesenchymal morphogenesis, and immune population of the developing gut. We identify the differentiation hierarchies of developing fibroblast and myofibroblast subtypes and describe diverse functions for these including as vascular niche cells. We pinpoint the origins of Peyer's patches and gut-associated lymphoid tissue (GALT) and describe location-specific immune programs. We use our resource to present an unbiased analysis of morphogen gradients that direct sequential waves of cellular differentiation and define cells and locations linked to rare developmental intestinal disorders. We compile a publicly available online resource, spatio-temporal analysis resource of fetal intestinal development (STAR-FINDer), to facilitate further work.
    Keywords:  congenital disease; gene expression; human development; human developmental cell atlas; intestinal crypt; intestinal development; mesenchymal cells; single-cell RNA-sequencing; spatial transcriptomics; stem cells
    DOI:  https://doi.org/10.1016/j.cell.2020.12.016
  15. Genome Biol. 2021 Jan 04. 22(1): 7
    Comprehensive integrative profiling of upper tract urothelial carcinomas.
    Xiaoping Su, Xiaofan Lu, Sehrish Khan Bazai, Eva Compérat, Roger Mouawad, Hui Yao, Morgan Rouprêt, Jean-Philippe Spano, David Khayat, Irwin Davidson, Nizar N Tannir, Fangrong Yan, Gabriel G Malouf.
      BACKGROUND: Crosstalk between genetic, epigenetic, and immune alterations in upper tract urothelial carcinomas and their role in shaping muscle invasiveness and patient outcome are poorly understood.RESULTS: We perform an integrative genome- and methylome-wide profiling of diverse non-muscle-invasive and muscle-invasive upper tract urothelial carcinomas. In addition to mutations of FGFR3 and KDM6A, we identify ZFP36L1 as a novel, significantly mutated tumor suppressor gene. Overall, mutations of ZFP36 family genes (ZFP36, ZFP36L1, and ZFP36L2) are identified in 26.7% of cases, which display a high mutational load. Unsupervised DNA methylation subtype classification identifies two epi-clusters associated with distinct muscle-invasive status and patient outcome, namely, EpiC-low and EpiC-high. While the former is hypomethylated, immune-depleted, and enriched for FGFR3-mutated, the latter is hypermethylated, immune-infiltrated, and tightly associated with somatic mutations of SWI/SNF genes.
    CONCLUSIONS: Our study delineates for the first time the key role for convergence between genetic and epigenetic alterations in shaping clinicopathological and immune upper tract urothelial carcinoma features.
    Keywords:  DNA methylation; Epigenetics; Immunity; SWI/SNF gene mutations; Sequencing; Upper tract urothelial carcinomas; ZFP36L1
    DOI:  https://doi.org/10.1186/s13059-020-02230-w
  16. Nat Commun. 2021 01 04. 12(1): 22
    Global computational alignment of tumor and cell line transcriptional profiles.
    Allison Warren, Yejia Chen, Andrew Jones, Tsukasa Shibue, William C Hahn, Jesse S Boehm, Francisca Vazquez, Aviad Tsherniak, James M McFarland.
      Cell lines are key tools for preclinical cancer research, but it remains unclear how well they represent patient tumor samples. Direct comparisons of tumor and cell line transcriptional profiles are complicated by several factors, including the variable presence of normal cells in tumor samples. We thus develop an unsupervised alignment method (Celligner) and apply it to integrate several large-scale cell line and tumor RNA-Seq datasets. Although our method aligns the majority of cell lines with tumor samples of the same cancer type, it also reveals large differences in tumor similarity across cell lines. Using this approach, we identify several hundred cell lines from diverse lineages that present a more mesenchymal and undifferentiated transcriptional state and that exhibit distinct chemical and genetic dependencies. Celligner could be used to guide the selection of cell lines that more closely resemble patient tumors and improve the clinical translation of insights gained from cell lines.
    DOI:  https://doi.org/10.1038/s41467-020-20294-x
  17. Nucleic Acids Res. 2021 Jan 04. pii: gkaa1228. [Epub ahead of print]
    Intestinal differentiation involves cleavage of histone H3 N-terminal tails by multiple proteases.
    Karin Johanna Ferrari, Simona Amato, Roberta Noberini, Cecilia Toscani, Daniel Fernández-Pérez, Alessandra Rossi, Pasquale Conforti, Marika Zanotti, Tiziana Bonaldi, Simone Tamburri, Diego Pasini.
      The proteolytic cleavage of histone tails, also termed histone clipping, has been described as a mechanism for permanent removal of post-translational modifications (PTMs) from histone proteins. Such activity has been ascribed to ensure regulatory function in key cellular processes such as differentiation, senescence and transcriptional control, for which different histone-specific proteases have been described. However, all these studies were exclusively performed using cell lines cultured in vitro and no clear evidence that histone clipping is regulated in vivo has been reported. Here we show that histone H3 N-terminal tails undergo extensive cleavage in the differentiated cells of the villi in mouse intestinal epithelium. Combining biochemical methods, 3D organoid cultures and in vivo approaches, we demonstrate that intestinal H3 clipping is the result of multiple proteolytic activities. We identified Trypsins and Cathepsin L as specific H3 tail proteases active in small intestinal differentiated cells and showed that their proteolytic activity is differentially affected by the PTM pattern of histone H3 tails. Together, our findings provide in vivo evidence of H3 tail proteolysis in mammalian tissues, directly linking H3 clipping to cell differentiation.
    DOI:  https://doi.org/10.1093/nar/gkaa1228
  18. Nat Cell Biol. 2021 Jan;23(1): 87-98
    Single-cell analysis reveals transcriptomic remodellings in distinct cell types that contribute to human prostate cancer progression.
    Sujun Chen, Guanghui Zhu, Yue Yang, Fubo Wang, Yu-Tian Xiao, Na Zhang, Xiaojie Bian, Yasheng Zhu, Yongwei Yu, Fei Liu, Keqin Dong, Javier Mariscal, Yin Liu, Fraser Soares, Helen Loo Yau, Bo Zhang, Weidong Chen, Chao Wang, Dai Chen, Qinghua Guo, Zhengfang Yi, Mingyao Liu, Michael Fraser, Daniel D De Carvalho, Paul C Boutros, Dolores Di Vizio, Zhou Jiang, Theodorus van der Kwast, Alejandro Berlin, Song Wu, Jianhua Wang, Housheng Hansen He, Shancheng Ren.
      Prostate cancer shows remarkable clinical heterogeneity, which manifests in spatial and clonal genomic diversity. By contrast, the transcriptomic heterogeneity of prostate tumours is poorly understood. Here we have profiled the transcriptomes of 36,424 single cells from 13 prostate tumours and identified the epithelial cells underlying disease aggressiveness. The tumour microenvironment (TME) showed activation of multiple progression-associated transcriptomic programs. Notably, we observed promiscuous KLK3 expression and validated the ability of cancer cells in altering T-cell transcriptomes. Profiling of a primary tumour and two matched lymph nodes provided evidence that KLK3 ectopic expression is associated with micrometastases. Close cell-cell communication exists among cells. We identified an endothelial subset harbouring active communication (activated endothelial cells, aECs) with tumour cells. Together with sequencing of an additional 11 samples, we showed that aECs are enriched in castration-resistant prostate cancer and promote cancer cell invasion. Finally, we created a user-friendly web interface for users to explore the sequenced data.
    DOI:  https://doi.org/10.1038/s41556-020-00613-6
  19. Mol Cell. 2020 Dec 24. pii: S1097-2765(20)30894-7. [Epub ahead of print]
    Hyperosmotic stress alters the RNA polymerase II interactome and induces readthrough transcription despite widespread transcriptional repression.
    Nicolle A Rosa-Mercado, Joshua T Zimmer, Maria Apostolidi, Jesse Rinehart, Matthew D Simon, Joan A Steitz.
      Stress-induced readthrough transcription results in the synthesis of downstream-of-gene (DoG)-containing transcripts. The mechanisms underlying DoG formation during cellular stress remain unknown. Nascent transcription profiles during DoG induction in human cell lines using TT-TimeLapse sequencing revealed widespread transcriptional repression upon hyperosmotic stress. Yet, DoGs are produced regardless of the transcriptional level of their upstream genes. ChIP sequencing confirmed that stress-induced redistribution of RNA polymerase (Pol) II correlates with the transcriptional output of genes. Stress-induced alterations in the Pol II interactome are observed by mass spectrometry. While certain cleavage and polyadenylation factors remain Pol II associated, Integrator complex subunits dissociate from Pol II under stress leading to a genome-wide loss of Integrator on DNA. Depleting the catalytic subunit of Integrator using siRNAs induces hundreds of readthrough transcripts, whose parental genes partially overlap those of stress-induced DoGs. Our results provide insights into the mechanisms underlying DoG production and how Integrator activity influences DoG transcription.
    Keywords:  Integrator complex; RNA polymerase II; Transient Transcriptome TimeLapse sequencing; downstream-of-gene transcripts; hyperosmotic stress; stress response
    DOI:  https://doi.org/10.1016/j.molcel.2020.12.002
  20. Genome Biol. 2021 Jan 07. 22(1): 21
    HiDeF: identifying persistent structures in multiscale 'omics data.
    Fan Zheng, She Zhang, Christopher Churas, Dexter Pratt, Ivet Bahar, Trey Ideker.
      In any 'omics study, the scale of analysis can dramatically affect the outcome. For instance, when clustering single-cell transcriptomes, is the analysis tuned to discover broad or specific cell types? Likewise, protein communities revealed from protein networks can vary widely in sizes depending on the method. Here, we use the concept of persistent homology, drawn from mathematical topology, to identify robust structures in data at all scales simultaneously. Application to mouse single-cell transcriptomes significantly expands the catalog of identified cell types, while analysis of SARS-COV-2 protein interactions suggests hijacking of WNT. The method, HiDeF, is available via Python and Cytoscape.
    Keywords:  Community detection; Multiscale; Persistent homology; Protein-protein interaction network; Resolution; Single-cell clustering; Systems biology
    DOI:  https://doi.org/10.1186/s13059-020-02228-4
  21. Epigenetics Chromatin. 2021 Jan 06. 14(1): 2
    Chromatin accessibility maps provide evidence of multilineage gene priming in hematopoietic stem cells.
    Eric W Martin, Jana Krietsch, Roman E Reggiardo, Rebekah Sousae, Daniel H Kim, E Camilla Forsberg.
      Hematopoietic stem cells (HSCs) have the capacity to differentiate into vastly different types of mature blood cells. The epigenetic mechanisms regulating the multilineage ability, or multipotency, of HSCs are not well understood. To test the hypothesis that cis-regulatory elements that control fate decisions for all lineages are primed in HSCs, we used ATAC-seq to compare chromatin accessibility of HSCs with five unipotent cell types. We observed the highest similarity in accessibility profiles between megakaryocyte progenitors and HSCs, whereas B cells had the greatest number of regions with de novo gain in accessibility during differentiation. Despite these differences, we identified cis-regulatory elements from all lineages that displayed epigenetic priming in HSCs. These findings provide new insights into the regulation of stem cell multipotency, as well as a resource to identify functional drivers of lineage fate.
    DOI:  https://doi.org/10.1186/s13072-020-00377-1
  22. Mol Cell. 2020 Dec 16. pii: S1097-2765(20)30888-1. [Epub ahead of print]
    SALL4 controls cell fate in response to DNA base composition.
    Raphaël Pantier, Kashyap Chhatbar, Timo Quante, Konstantina Skourti-Stathaki, Justyna Cholewa-Waclaw, Grace Alston, Beatrice Alexander-Howden, Heng Yang Lee, Atlanta G Cook, Cornelia G Spruijt, Michiel Vermeulen, Jim Selfridge, Adrian Bird.
      Mammalian genomes contain long domains with distinct average compositions of A/T versus G/C base pairs. In a screen for proteins that might interpret base composition by binding to AT-rich motifs, we identified the stem cell factor SALL4, which contains multiple zinc fingers. Mutation of the domain responsible for AT binding drastically reduced SALL4 genome occupancy and prematurely upregulated genes in proportion to their AT content. Inactivation of this single AT-binding zinc-finger cluster mimicked defects seen in Sall4 null cells, including precocious differentiation of embryonic stem cells (ESCs) and embryonic lethality in mice. In contrast, deletion of two other zinc-finger clusters was phenotypically neutral. Our data indicate that loss of pluripotency is triggered by downregulation of SALL4, leading to de-repression of a set of AT-rich genes that promotes neuronal differentiation. We conclude that base composition is not merely a passive byproduct of genome evolution and constitutes a signal that aids control of cell fate.
    Keywords:  DNA base composition; SALL4; differentiation; embryonic stem cells; gene regulation; pluripotency
    DOI:  https://doi.org/10.1016/j.molcel.2020.11.046
  23. Nat Commun. 2021 Jan 08. 12(1): 205
    High-resolution single-cell 3D-models of chromatin ensembles during Drosophila embryogenesis.
    Qiu Sun, Alan Perez-Rathke, Daniel M Czajkowsky, Zhifeng Shao, Jie Liang.
      Single-cell chromatin studies provide insights into how chromatin structure relates to functions of individual cells. However, balancing high-resolution and genome wide-coverage remains challenging. We describe a computational method for the reconstruction of large 3D-ensembles of single-cell (sc) chromatin conformations from population Hi-C that we apply to study embryogenesis in Drosophila. With minimal assumptions of physical properties and without adjustable parameters, our method generates large ensembles of chromatin conformations via deep-sampling. Our method identifies specific interactions, which constitute 5-6% of Hi-C frequencies, but surprisingly are sufficient to drive chromatin folding, giving rise to the observed Hi-C patterns. Modeled sc-chromatins quantify chromatin heterogeneity, revealing significant changes during embryogenesis. Furthermore, >50% of modeled sc-chromatin maintain topologically associating domains (TADs) in early embryos, when no population TADs are perceptible. Domain boundaries become fixated during development, with strong preference at binding-sites of insulator-complexes upon the midblastula transition. Overall, high-resolution 3D-ensembles of sc-chromatin conformations enable further in-depth interpretation of population Hi-C, improving understanding of the structure-function relationship of genome organization.
    DOI:  https://doi.org/10.1038/s41467-020-20490-9
  24. Genome Biol. 2021 Jan 07. 22(1): 19
    An integrated multi-omics approach to identify regulatory mechanisms in cancer metastatic processes.
    Saba Ghaffari, Casey Hanson, Remington E Schmidt, Kelly J Bouchonville, Steven M Offer, Saurabh Sinha.
      BACKGROUND: Metastatic progress is the primary cause of death in most cancers, yet the regulatory dynamics driving the cellular changes necessary for metastasis remain poorly understood. Multi-omics approaches hold great promise for addressing this challenge; however, current analysis tools have limited capabilities to systematically integrate transcriptomic, epigenomic, and cistromic information to accurately define the regulatory networks critical for metastasis.RESULTS: To address this limitation, we use a purposefully generated cellular model of colon cancer invasiveness to generate multi-omics data, including expression, accessibility, and selected histone modification profiles, for increasing levels of invasiveness. We then adopt a rigorous probabilistic framework for joint inference from the resulting heterogeneous data, along with transcription factor binding profiles. Our approach uses probabilistic graphical models to leverage the functional information provided by specific epigenomic changes, models the influence of multiple transcription factors simultaneously, and automatically learns the activating or repressive roles of cis-regulatory events. Global analysis of these relationships reveals key transcription factors driving invasiveness, as well as their likely target genes. Disrupting the expression of one of the highly ranked transcription factors JunD, an AP-1 complex protein, confirms functional relevance to colon cancer cell migration and invasion. Transcriptomic profiling confirms key regulatory targets of JunD, and a gene signature derived from the model demonstrates strong prognostic potential in TCGA colorectal cancer data.
    CONCLUSIONS: Our work sheds new light into the complex molecular processes driving colon cancer metastasis and presents a statistically sound integrative approach to analyze multi-omics profiles of a dynamic biological process.
    Keywords:  Colon cancer; Metastasis; Multi-omics; Probabilistic model; Transcriptional regulation
    DOI:  https://doi.org/10.1186/s13059-020-02213-x
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