bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2023–08–06
twenty-two papers selected by
Connor Rogerson, University of Cambridge
  1. FACT regulates pluripotency through proximal and distal regulation of gene expression in murine embryonic stem cells.
  2. The architecture of binding cooperativity between densely bound transcription factors.
  3. CEBPA phase separation links transcriptional activity and 3D chromatin hubs.
  4. Phase-separated nuclear bodies of nucleoporin fusions promote condensation of MLL1/CRM1 and rearrangement of 3D genome structure.
  5. The DNMT3A ADD domain is required for efficient de novo DNA methylation and maternal imprinting in mouse oocytes.
  6. Dictys: dynamic gene regulatory network dissects developmental continuum with single-cell multiomics.
  7. Dynamic nucleosome remodeling mediated by YY1 underlies early mouse development.
  8. Systematic benchmarking of single-cell ATAC-sequencing protocols.
  9. Histone H1 protects telomeric repeats from H3K27me3 invasion in Arabidopsis.
  10. Mammalian SWI/SNF chromatin remodeling complexes promote tyrosine kinase inhibitor resistance in EGFR-mutant lung cancer.
  11. The SWI/SNF complexes are required for retinal pigmented epithelium differentiation and the inhibition of cell proliferation and neural differentiation programs.
  12. The AT-hook is an evolutionarily conserved auto-regulatory domain of SWI/SNF required for cell lineage priming.
  13. Characterization of cell-fate decision landscapes by estimating transcription factor dynamics.
  14. The master growth regulator DELLA binding to histone H2A is essential for DELLA-mediated global transcription regulation.
  15. DSIF directly facilitates promoter proximal pausing of RNA Polymerase II.
  16. Transcription factors organize into functional groups on the linear genome and in 3D chromatin.
  17. Droplet-based bisulfite sequencing for high-throughput profiling of single-cell DNA methylomes.
  18. CRISPR/Cas9 screen for genome-wide interrogation of essential MYC-bound E-boxes in cancer cells.
  19. HEAP: a task adaptive-based explainable deep learning framework for enhancer activity prediction.
  20. Chromosome-level organization of the regulatory genome in the Drosophila nervous system.
  21. Tissue specificity of oncogenic BRAF targeted to lung and thyroid through a shared lineage factor.
  22. Transcription modulates chromatin dynamics and locus configuration sampling.
  1. BMC Biol. 2023 Aug 04. 21(1): 167
    FACT regulates pluripotency through proximal and distal regulation of gene expression in murine embryonic stem cells.
    David C Klein, Santana M Lardo, Kurtis N McCannell, Sarah J Hainer.
       BACKGROUND: The FACT complex is a conserved histone chaperone with critical roles in transcription and histone deposition. FACT is essential in pluripotent and cancer cells, but otherwise dispensable for most mammalian cell types. FACT deletion or inhibition can block induction of pluripotent stem cells, yet the mechanism through which FACT regulates cell fate decisions remains unclear.
    RESULTS: To explore the mechanism for FACT function, we generated AID-tagged murine embryonic cell lines for FACT subunit SPT16 and paired depletion with nascent transcription and chromatin accessibility analyses. We also analyzed SPT16 occupancy using CUT&RUN and found that SPT16 localizes to both promoter and enhancer elements, with a strong overlap in binding with OCT4, SOX2, and NANOG. Over a timecourse of SPT16 depletion, nucleosomes invade new loci, including promoters, regions bound by SPT16, OCT4, SOX2, and NANOG, and TSS-distal DNaseI hypersensitive sites. Simultaneously, transcription of Pou5f1 (encoding OCT4), Sox2, Nanog, and enhancer RNAs produced from these genes' associated enhancers are downregulated.
    CONCLUSIONS: We propose that FACT maintains cellular pluripotency through a precise nucleosome-based regulatory mechanism for appropriate expression of both coding and non-coding transcripts associated with pluripotency.
    Keywords:  Chromatin; Embryonic stem cells; FACT; Genomics; Histone chaperone; Histones; Nucleosome; Pluripotency; RNA; Transcription
    DOI:  https://doi.org/10.1186/s12915-023-01669-0
  2. Cell Syst. 2023 Jul 25. pii: S2405-4712(23)00185-0. [Epub ahead of print]
    The architecture of binding cooperativity between densely bound transcription factors.
    Offir Lupo, Divya Krishna Kumar, Rotem Livne, Michal Chappleboim, Idan Levy, Naama Barkai.
      The binding of transcription factors (TFs) along genomes is restricted to a subset of sites containing their preferred motifs. TF-binding specificity is often attributed to the co-binding of interacting TFs; however, apart from specific examples, this model remains untested. Here, we define dependencies among budding yeast TFs that localize to overlapping promoters by profiling the genome-wide consequences of co-depleting multiple TFs. We describe unidirectional interactions, revealing Msn2 as a central factor allowing TF binding at its target promoters. By contrast, no case of mutual cooperation was observed. Particularly, Msn2 retained binding at its preferred promoters upon co-depletion of fourteen similarly bound TFs. Overall, the consequences of TF co-depletions were moderate, limited to a subset of promoters, and failed to explain the role of regions outside the DNA-binding domain in directing TF-binding preferences. Our results call for re-evaluating the role of cooperative interactions in directing TF-binding preferences.
    Keywords:  binding specificity; intrinsically disordered regions; transcription factors
    DOI:  https://doi.org/10.1016/j.cels.2023.06.010
  3. Cell Rep. 2023 Jul 29. pii: S2211-1247(23)00908-7. [Epub ahead of print]42(8): 112897
    CEBPA phase separation links transcriptional activity and 3D chromatin hubs.
    Marie Christou-Kent, Sergi Cuartero, Carla Garcia-Cabau, Julia Ruehle, Julian Naderi, Julia Erber, Maria Victoria Neguembor, Marcos Plana-Carmona, Marc Alcoverro-Bertran, Luisa De Andres-Aguayo, Antonios Klonizakis, Eric Julià-Vilella, Cian Lynch, Manuel Serrano, Denes Hnisz, Xavier Salvatella, Thomas Graf, Grégoire Stik.
      Cell identity is orchestrated through an interplay between transcription factor (TF) action and genome architecture. The mechanisms used by TFs to shape three-dimensional (3D) genome organization remain incompletely understood. Here we present evidence that the lineage-instructive TF CEBPA drives extensive chromatin compartment switching and promotes the formation of long-range chromatin hubs during induced B cell-to-macrophage transdifferentiation. Mechanistically, we find that the intrinsically disordered region (IDR) of CEBPA undergoes in vitro phase separation (PS) dependent on aromatic residues. Both overexpressing B cells and native CEBPA-expressing cell types such as primary granulocyte-macrophage progenitors, liver cells, and trophectoderm cells reveal nuclear CEBPA foci and long-range 3D chromatin hubs at CEBPA-bound regions. In short, we show that CEBPA can undergo PS through its IDR, which may underlie in vivo foci formation and suggest a potential role of PS in regulating CEBPA function.
    Keywords:  3D genome organization; CEBPA; CP: Molecular biology; chromatin hubs; compartments; condensates; gene regulation; phase separation; transcription; transdifferentiation
    DOI:  https://doi.org/10.1016/j.celrep.2023.112897
  4. Cell Rep. 2023 Jul 28. pii: S2211-1247(23)00895-1. [Epub ahead of print]42(8): 112884
    Phase-separated nuclear bodies of nucleoporin fusions promote condensation of MLL1/CRM1 and rearrangement of 3D genome structure.
    Masahiro Oka, Mayumi Otani, Yoichi Miyamoto, Rieko Oshima, Jun Adachi, Takeshi Tomonaga, Munehiro Asally, Yuya Nagaoka, Kaori Tanaka, Atsushi Toyoda, Kazuki Ichikawa, Shinichi Morishita, Kyoichi Isono, Haruhiko Koseki, Ryuichiro Nakato, Yasuyuki Ohkawa, Yoshihiro Yoneda.
      NUP98 and NUP214 form chimeric fusion proteins that assemble into phase-separated nuclear bodies containing CRM1, a nuclear export receptor. However, these nuclear bodies' function in controlling gene expression remains elusive. Here, we demonstrate that the nuclear bodies of NUP98::HOXA9 and SET::NUP214 promote the condensation of mixed lineage leukemia 1 (MLL1), a histone methyltransferase essential for the maintenance of HOX gene expression. These nuclear bodies are robustly associated with MLL1/CRM1 and co-localized on chromatin. Furthermore, whole-genome chromatin-conformation capture analysis reveals that NUP98::HOXA9 induces a drastic alteration in high-order genome structure at target regions concomitant with the generation of chromatin loops and/or rearrangement of topologically associating domains in a phase-separation-dependent manner. Collectively, these results show that the phase-separated nuclear bodies of nucleoporin fusion proteins can enhance the activation of target genes by promoting the condensation of MLL1/CRM1 and rearrangement of the 3D genome structure.
    Keywords:  3D genome; CP: Molecular biology; CRM1; HOX cluster genes; MLL1; NUP214; NUP98; fusion gene; leukemia; molecular condensation; phase separation
    DOI:  https://doi.org/10.1016/j.celrep.2023.112884
  5. PLoS Genet. 2023 08;19(8): e1010855
    The DNMT3A ADD domain is required for efficient de novo DNA methylation and maternal imprinting in mouse oocytes.
    Ryuji Uehara, Wan Kin Au Yeung, Keisuke Toriyama, Hiroaki Ohishi, Naoki Kubo, Hidehiro Toh, Isao Suetake, Kenjiro Shirane, Hiroyuki Sasaki.
      Establishment of a proper DNA methylation landscape in mammalian oocytes is important for maternal imprinting and embryonic development. De novo DNA methylation in oocytes is mediated by the DNA methyltransferase DNMT3A, which has an ATRX-DNMT3-DNMT3L (ADD) domain that interacts with histone H3 tail unmethylated at lysine-4 (H3K4me0). The domain normally blocks the methyltransferase domain via intramolecular interaction and binding to histone H3K4me0 releases the autoinhibition. However, H3K4me0 is widespread in chromatin and the role of the ADD-histone interaction has not been studied in vivo. We herein show that amino-acid substitutions in the ADD domain of mouse DNMT3A cause dwarfism. Oocytes derived from homozygous females show mosaic loss of CG methylation and almost complete loss of non-CG methylation. Embryos derived from such oocytes die in mid-to-late gestation, with stochastic and often all-or-none-type CG-methylation loss at imprinting control regions and misexpression of the linked genes. The stochastic loss is a two-step process, with loss occurring in cleavage-stage embryos and regaining occurring after implantation. These results highlight an important role for the ADD domain in efficient, and likely processive, de novo CG methylation and pose a model for stochastic inheritance of epigenetic perturbations in germ cells to the next generation.
    DOI:  https://doi.org/10.1371/journal.pgen.1010855
  6. Nat Methods. 2023 Aug 03.
    Dictys: dynamic gene regulatory network dissects developmental continuum with single-cell multiomics.
    Lingfei Wang, Nikolaos Trasanidis, Ting Wu, Guanlan Dong, Michael Hu, Daniel E Bauer, Luca Pinello.
      Gene regulatory networks (GRNs) are key determinants of cell function and identity and are dynamically rewired during development and disease. Despite decades of advancement, challenges remain in GRN inference, including dynamic rewiring, causal inference, feedback loop modeling and context specificity. To address these challenges, we develop Dictys, a dynamic GRN inference and analysis method that leverages multiomic single-cell assays of chromatin accessibility and gene expression, context-specific transcription factor footprinting, stochastic process network and efficient probabilistic modeling of single-cell RNA-sequencing read counts. Dictys improves GRN reconstruction accuracy and reproducibility and enables the inference and comparative analysis of context-specific and dynamic GRNs across developmental contexts. Dictys' network analyses recover unique insights in human blood and mouse skin development with cell-type-specific and dynamic GRNs. Its dynamic network visualizations enable time-resolved discovery and investigation of developmental driver transcription factors and their regulated targets. Dictys is available as a free, open-source and user-friendly Python package.
    DOI:  https://doi.org/10.1038/s41592-023-01971-3
  7. Genes Dev. 2023 Aug 02.
    Dynamic nucleosome remodeling mediated by YY1 underlies early mouse development.
    Mizuki Sakamoto, Shusaku Abe, Yuka Miki, Yusuke Miyanari, Hiroyuki Sasaki, Takashi Ishiuchi.
      Nucleosome positioning can alter the accessibility of DNA-binding proteins to their cognate DNA elements, and thus its precise control is essential for cell identity and function. Mammalian preimplantation embryos undergo temporal changes in gene expression and cell potency, suggesting the involvement of dynamic epigenetic control during this developmental phase. However, the dynamics of nucleosome organization during early development are poorly understood. In this study, using a low-input MNase-seq method, we show that nucleosome positioning is globally obscure in zygotes but becomes well defined during subsequent development. Down-regulation of the chromatin assembly in embryonic stem cells can partially reverse nucleosome organization into a zygote-like pattern, suggesting a possible link between the chromatin assembly pathway and fuzzy nucleosomes in zygotes. We also reveal that YY1, a zinc finger-containing transcription factor expressed upon zygotic genome activation, regulates the de novo formation of well-positioned nucleosome arrays at the regulatory elements through identifying YY1-binding sites in eight-cell embryos. The YY1-binding regions acquire H3K27ac enrichment around the eight-cell and morula stages, and YY1 depletion impairs the morula-to-blastocyst transition. Thus, our study delineates the remodeling of nucleosome organization and its underlying mechanism during early mouse development.
    Keywords:  YY1; chromatin assembly; early mouse development; nucleosome organization
    DOI:  https://doi.org/10.1101/gad.350376.122
  8. Nat Biotechnol. 2023 Aug 03.
    Systematic benchmarking of single-cell ATAC-sequencing protocols.
    Florian V De Rop, Gert Hulselmans, Chris Flerin, Paula Soler-Vila, Albert Rafels, Valerie Christiaens, Carmen Bravo González-Blas, Domenica Marchese, Ginevra Caratù, Suresh Poovathingal, Orit Rozenblatt-Rosen, Michael Slyper, Wendy Luo, Christoph Muus, Fabiana Duarte, Rojesh Shrestha, S Tansu Bagdatli, M Ryan Corces, Lira Mamanova, Andrew Knights, Kerstin B Meyer, Ryan Mulqueen, Akram Taherinasab, Patrick Maschmeyer, Jörn Pezoldt, Camille Lucie Germaine Lambert, Marta Iglesias, Sebastián R Najle, Zain Y Dossani, Luciano G Martelotto, Zach Burkett, Ronald Lebofsky, José Ignacio Martin-Subero, Satish Pillai, Arnau Sebé-Pedrós, Bart Deplancke, Sarah A Teichmann, Leif S Ludwig, Theodore P Braun, Andrew C Adey, William J Greenleaf, Jason D Buenrostro, Aviv Regev, Stein Aerts, Holger Heyn.
      Single-cell assay for transposase-accessible chromatin by sequencing (scATAC-seq) has emerged as a powerful tool for dissecting regulatory landscapes and cellular heterogeneity. However, an exploration of systemic biases among scATAC-seq technologies has remained absent. In this study, we benchmark the performance of eight scATAC-seq methods across 47 experiments using human peripheral blood mononuclear cells (PBMCs) as a reference sample and develop PUMATAC, a universal preprocessing pipeline, to handle the various sequencing data formats. Our analyses reveal significant differences in sequencing library complexity and tagmentation specificity, which impact cell-type annotation, genotype demultiplexing, peak calling, differential region accessibility and transcription factor motif enrichment. Our findings underscore the importance of sample extraction, method selection, data processing and total cost of experiments, offering valuable guidance for future research. Finally, our data and analysis pipeline encompasses 169,000 PBMC scATAC-seq profiles and a best practices code repository for scATAC-seq data analysis, which are freely available to extend this benchmarking effort to future protocols.
    DOI:  https://doi.org/10.1038/s41587-023-01881-x
  9. Cell Rep. 2023 Jul 27. pii: S2211-1247(23)00905-1. [Epub ahead of print]42(8): 112894
    Histone H1 protects telomeric repeats from H3K27me3 invasion in Arabidopsis.
    Gianluca Teano, Lorenzo Concia, Léa Wolff, Léopold Carron, Ivona Biocanin, Kateřina Adamusová, Miloslava Fojtová, Michael Bourge, Amira Kramdi, Vincent Colot, Ueli Grossniklaus, Chris Bowler, Célia Baroux, Alessandra Carbone, Aline V Probst, Petra Procházková Schrumpfová, Jiří Fajkus, Simon Amiard, Stefan Grob, Clara Bourbousse, Fredy Barneche.
      While the pivotal role of linker histone H1 in shaping nucleosome organization is well established, its functional interplays with chromatin factors along the epigenome are just starting to emerge. Here we show that, in Arabidopsis, as in mammals, H1 occupies Polycomb Repressive Complex 2 (PRC2) target genes where it favors chromatin condensation and H3K27me3 deposition. We further show that, contrasting with its conserved function in PRC2 activation at genes, H1 selectively prevents H3K27me3 accumulation at telomeres and large pericentromeric interstitial telomeric repeat (ITR) domains by restricting DNA accessibility to Telomere Repeat Binding (TRB) proteins, a group of H1-related Myb factors mediating PRC2 cis recruitment. This study provides a mechanistic framework by which H1 avoids the formation of gigantic H3K27me3-rich domains at telomeric sequences and contributes to safeguard nucleus architecture.
    Keywords:  3D chromosome organization; Arabidopsis; CP: Molecular biology; CP: Plants; H3K27me3; Polycomb; TRB; Telomere Repeat Binding protein; chromatin accessibility; linker histone H1; telomere
    DOI:  https://doi.org/10.1016/j.celrep.2023.112894
  10. Cancer Cell. 2023 Aug 02. pii: S1535-6108(23)00245-3. [Epub ahead of print]
    Mammalian SWI/SNF chromatin remodeling complexes promote tyrosine kinase inhibitor resistance in EGFR-mutant lung cancer.
    Fernando J de Miguel, Claudia Gentile, William W Feng, Shannon J Silva, Akshay Sankar, Francisco Exposito, Wesley L Cai, Mary Ann Melnick, Camila Robles-Oteiza, Madeline M Hinkley, Jeanelle A Tsai, Antja-Voy Hartley, Jin Wei, Anna Wurtz, Fangyong Li, Maria I Toki, David L Rimm, Robert Homer, Craig B Wilen, Andrew Z Xiao, Jun Qi, Qin Yan, Don X Nguyen, Pasi A Jänne, Cigall Kadoch, Katerina A Politi.
      Acquired resistance to tyrosine kinase inhibitors (TKI), such as osimertinib used to treat EGFR-mutant lung adenocarcinomas, limits long-term efficacy and is frequently caused by non-genetic mechanisms. Here, we define the chromatin accessibility and gene regulatory signatures of osimertinib sensitive and resistant EGFR-mutant cell and patient-derived models and uncover a role for mammalian SWI/SNF chromatin remodeling complexes in TKI resistance. By profiling mSWI/SNF genome-wide localization, we identify both shared and cancer cell line-specific gene targets underlying the resistant state. Importantly, genetic and pharmacologic disruption of the SMARCA4/SMARCA2 mSWI/SNF ATPases re-sensitizes a subset of resistant models to osimertinib via inhibition of mSWI/SNF-mediated regulation of cellular programs governing cell proliferation, epithelial-to-mesenchymal transition, epithelial cell differentiation, and NRF2 signaling. These data highlight the role of mSWI/SNF complexes in supporting TKI resistance and suggest potential utility of mSWI/SNF inhibitors in TKI-resistant lung cancers.
    DOI:  https://doi.org/10.1016/j.ccell.2023.07.005
  11. Development. 2023 Jul 31. pii: dev.201488. [Epub ahead of print]
    The SWI/SNF complexes are required for retinal pigmented epithelium differentiation and the inhibition of cell proliferation and neural differentiation programs.
    Shai Ovadia, Cui Guizhong, Ran Elkon, Mazal Gulkar-Cohen, Nitay Zuk-Bar, Tran Touc, Naihe Jing, Ruth Ashery-Padan.
      During embryonic development, tissue-specific transcription factors and chromatin remodelers function together to ensure gradual, coordinated differentiation of multiple lineages. Here, we define this regulatory interplay in the developing retinal pigmented epithelium (RPE), a neuroectodermal lineage essential for the development, function, and maintenance of the adjacent retina. We present a high-resolution spatial transcriptomic atlas of the developing RPE and the adjacent ocular mesenchyme obtained by geographical position sequencing (Geo-seq) of a single developmental stage of the eye that encompasses young and more mature ocular progenitors. These transcriptomic data, available online, reveal the key transcription factors and their gene-regulatory networks during RPE and ocular mesenchyme differentiation. Moreover, conditional inactivation followed by Geo-seq revealed that this differentiation program is dependent on the activity of the SWI/SNF complexes, shown here to control the expression and activity of RPE transcription factors and, at the same time, inhibit the neural progenitor and cell proliferation genes. The findings reveal the roles of the SWI/SNF complexes in controlling the intersection between RPE and neural cell fates and the coupling of cell-cycle exit and differentiation.
    Keywords:  Eye development; Gene-regulatory networks; RPE; SWI/SNF
    DOI:  https://doi.org/10.1242/dev.201488
  12. Nat Commun. 2023 Aug 04. 14(1): 4682
    The AT-hook is an evolutionarily conserved auto-regulatory domain of SWI/SNF required for cell lineage priming.
    Dhurjhoti Saha, Solomon Hailu, Arjan Hada, Junwoo Lee, Jie Luo, Jeff A Ranish, Yuan-Chi Lin, Kyle Feola, Jim Persinger, Abhinav Jain, Bin Liu, Yue Lu, Payel Sen, Blaine Bartholomew.
      The SWI/SNF ATP-dependent chromatin remodeler is a master regulator of the epigenome, controlling pluripotency and differentiation. Towards the C-terminus of the catalytic subunit of SWI/SNF is a motif called the AT-hook that is evolutionary conserved. The AT-hook is present in many chromatin modifiers and generally thought to help anchor them to DNA. We observe however that the AT-hook regulates the intrinsic DNA-stimulated ATPase activity aside from promoting SWI/SNF recruitment to DNA or nucleosomes by increasing the reaction velocity a factor of 13 with no accompanying change in substrate affinity (KM). The changes in ATP hydrolysis causes an equivalent change in nucleosome movement, confirming they are tightly coupled. The catalytic subunit's AT-hook is required in vivo for SWI/SNF remodeling activity in yeast and mouse embryonic stem cells. The AT-hook in SWI/SNF is required for transcription regulation and activation of stage-specific enhancers critical in cell lineage priming. Similarly, growth assays suggest the AT-hook is required in yeast SWI/SNF for activation of genes involved in amino acid biosynthesis and metabolizing ethanol. Our findings highlight the importance of studying SWI/SNF attenuation versus eliminating the catalytic subunit or completely shutting down its enzymatic activity.
    DOI:  https://doi.org/10.1038/s41467-023-40386-8
  13. Cell Rep Methods. 2023 Jul 24. 3(7): 100512
    Characterization of cell-fate decision landscapes by estimating transcription factor dynamics.
    Sara Jiménez, Valérie Schreiber, Reuben Mercier, Gérard Gradwohl, Nacho Molina.
      Time-specific modulation of gene expression during differentiation by transcription factors promotes cell diversity. However, estimating their dynamic regulatory activity at the single-cell level and in a high-throughput manner remains challenging. We present FateCompass, an integrative approach that utilizes single-cell transcriptomics data to identify lineage-specific transcription factors throughout differentiation. By combining a probabilistic framework with RNA velocities or differentiation potential, we estimate transition probabilities, while a linear model of gene regulation is employed to compute transcription factor activities. Considering dynamic changes and correlations of expression and activities, FateCompass identifies lineage-specific regulators. Our validation using in silico data and application to pancreatic endocrine cell differentiation datasets highlight both known and potentially novel lineage-specific regulators. Notably, we uncovered undescribed transcription factors of an enterochromaffin-like population during in vitro differentiation toward ß-like cells. FateCompass provides a valuable framework for hypothesis generation, advancing our understanding of the gene regulatory networks driving cell-fate decisions.
    Keywords:  RNA velocity; cell-fate modeling; differentiation; endocrine cell; gene regulation
    DOI:  https://doi.org/10.1016/j.crmeth.2023.100512
  14. Nat Plants. 2023 Aug 03.
    The master growth regulator DELLA binding to histone H2A is essential for DELLA-mediated global transcription regulation.
    Xu Huang, Hao Tian, Jeongmoo Park, Dong-Ha Oh, Jianhong Hu, Rodolfo Zentella, Hong Qiao, Maheshi Dassanayake, Tai-Ping Sun.
      The DELLA genes, also known as 'Green Revolution' genes, encode conserved master growth regulators that control plant development in response to internal and environmental cues. Functioning as nuclear-localized transcription regulators, DELLAs modulate expression of target genes via direct protein-protein interaction of their carboxy-terminal GRAS domain with hundreds of transcription factors (TFs) and epigenetic regulators. However, the molecular mechanism of DELLA-mediated transcription reprogramming remains unclear. Here by characterizing new missense alleles of an Arabidopsis DELLA, repressor of ga1-3 (RGA), and co-immunoprecipitation assays, we show that RGA binds histone H2A via the PFYRE subdomain within its GRAS domain to form a TF-RGA-H2A complex at the target chromatin. Chromatin immunoprecipitation followed by sequencing analysis further shows that this activity is essential for RGA association with its target chromatin globally. Our results indicate that, although DELLAs are recruited to target promoters by binding to TFs via the LHR1 subdomain, DELLA-H2A interaction via the PFYRE subdomain is necessary to stabilize the TF-DELLA-H2A complex at the target chromatin. This study provides insights into the two distinct key modular functions in DELLA for its genome-wide transcription regulation in plants.
    DOI:  https://doi.org/10.1038/s41477-023-01477-y
  15. J Biol Chem. 2023 Jul 28. pii: S0021-9258(23)02134-8. [Epub ahead of print] 105106
    DSIF directly facilitates promoter proximal pausing of RNA Polymerase II.
    Roberta Dollinger, Eilene B Deng, Josie Schultz, Sharon Wu, Haley Reed Deorio, David S Gilmour.
      Promoter proximal pausing of RNA Polymerase II (Pol II) is a critical transcriptional regulatory mechanism in metazoans that requires the transcription factor DSIF and the inhibitory factor NELF. DSIF, composed of Spt4 and Spt5, establishes the pause by recruiting NELF to the elongation complex. However, the role of DSIF in pausing beyond NELF recruitment remains unclear. We used a highly purified in vitro system and Drosophila nuclear extract to investigate the role of DSIF in promoter proximal pausing. We identified two domains of Spt5, the KOW4 and NGN domains, that directly facilitate Pol II pausing. The KOW4 domain promotes pausing through its interaction with the nascent RNA while the NGN domain does so through a short helical motif that is in close proximity to the non-transcribed DNA template strand. Removal of this sequence in Drosophila has a male-specific dominant negative effect. The alpha helical motif is also needed to support fly viability. We also show that the interaction between the Spt5 KOW1 domain and the upstream DNA helix is required for DSIF association with the Pol II elongation complex. Disruption of the KOW1-DNA interaction is dominant lethal in vivo. Finally, we show that the KOW2-3 domain of Spt5 mediates the recruitment of NELF to the elongation complex. In summary, our results reveal additional roles for DSIF in transcription regulation and identify specific domains important for facilitating Pol II pausing.
    Keywords:  DNA-protein interaction; DSIF; NELF; NGN domain; RNA polymerase II; RNA-protein interaction; Spt5; mRNA; promoter proximal pausing; transcription
    DOI:  https://doi.org/10.1016/j.jbc.2023.105106
  16. Heliyon. 2023 Aug;9(8): e18211
    Transcription factors organize into functional groups on the linear genome and in 3D chromatin.
    Rakesh Netha Vadnala, Sridhar Hannenhalli, Leelavati Narlikar, Rahul Siddharthan.
      Transcription factors (TFs) and their binding sites have evolved to interact cooperatively or competitively with each other. Here we examine in detail, across multiple cell lines, such cooperation or competition among TFs both in sequential and spatial proximity (using chromatin conformation capture assays), considering in vivo binding data as well as TF binding motifs in DNA. We ascertain significantly co-occurring ("attractive") or avoiding ("repulsive") TF pairs using robust randomized models that retain the essential characteristics of the experimental data. Across human cell lines TFs organize into two groups, with intra-group attraction and inter-group repulsion. This is true for both sequential and spatial proximity, and for both in vivo binding and sequence motifs. Attractive TF pairs exhibit significantly more physical interactions suggesting an underlying mechanism. The two TF groups differ significantly in their genomic and network properties, as well in their function-while one group regulates housekeeping function, the other potentially regulates lineage-specific functions, that are disrupted in cancer. Weaker binding sites tend to occur in spatially interacting regions of the genome. Our results suggest that a complex pattern of spatial cooperativity of TFs and chromatin has evolved with the genome to support housekeeping and lineage-specific functions.
    Keywords:  Chromatin; Transcription factors
    DOI:  https://doi.org/10.1016/j.heliyon.2023.e18211
  17. Nat Commun. 2023 Aug 03. 14(1): 4672
    Droplet-based bisulfite sequencing for high-throughput profiling of single-cell DNA methylomes.
    Qiang Zhang, Sai Ma, Zhengzhi Liu, Bohan Zhu, Zirui Zhou, Gaoshan Li, J Javier Meana, Javier González-Maeso, Chang Lu.
      The genome-wide DNA methylation profile, or DNA methylome, is a critical component of the overall epigenomic landscape that modulates gene activities and cell fate. Single-cell DNA methylomic studies offer unprecedented resolution for detecting and profiling cell subsets based on methylomic features. However, existing single-cell methylomic technologies are based on use of tubes or well plates and these platforms are not easily scalable for handling a large number of single cells. Here we demonstrate a droplet-based microfluidic technology, Drop-BS, to construct single-cell bisulfite sequencing libraries for DNA methylome profiling. Drop-BS takes advantage of the ultrahigh throughput offered by droplet microfluidics to prepare bisulfite sequencing libraries of up to 10,000 single cells within 2 days. We apply the technology to profile mixed cell lines, mouse and human brain tissues to reveal cell type heterogeneity. Drop-BS offers a promising solution for single-cell methylomic studies requiring examination of a large cell population.
    DOI:  https://doi.org/10.1038/s41467-023-40411-w
  18. Mol Oncol. 2023 Jul 30.
    CRISPR/Cas9 screen for genome-wide interrogation of essential MYC-bound E-boxes in cancer cells.
    Marta Kazimierska, Marta Podralska, Magdalena Żurawek, Tomasz Woźniak, Marta Elżbieta Kasprzyk, Weronika Sura, Wojciech Łosiewski, Iwona Ziółkowska-Suchanek, Joost Kluiver, Anke van den Berg, Natalia Rozwadowska, Agnieszka Dzikiewicz-Krawczyk.
      The transcription factor MYC is a proto-oncogene with a well-documented essential role in the pathogenesis and maintenance of several types of cancer. MYC binds to specific E-box sequences in the genome to regulate gene expression in a cell-type- and developmental-stage-specific manner. To date, a combined analysis of essential MYC-bound E-boxes and their downstream target genes important for growth of different types of cancer is missing. In this study, we designed a CRISPR/Cas9 library to destroy E-box sequences in a genome-wide fashion. In parallel, we used the Brunello library to knock out protein-coding genes. We performed high-throughput screens with these libraries in four MYC-dependent cancer cell lines - K562, ST486, HepG2 and MCF7 - which revealed several essential E-boxes and genes. Among them we pinpointed crucial common and cell-type-specific MYC-regulated genes involved in pathways associated with cancer development. Extensive validation of our approach confirmed that E-box disruption affects MYC binding, target-gene expression and cell proliferation in vitro as well as tumor growth in vivo. Our unique, well-validated tool opens new possibilities to gain novel insights into MYC-dependent vulnerabilities in cancer cells.
    Keywords:  CRISPR/Cas9; MYC; MYC target genes; high-throughput screen; transcription factor
    DOI:  https://doi.org/10.1002/1878-0261.13493
  19. Brief Bioinform. 2023 Aug 03. pii: bbad286. [Epub ahead of print]
    HEAP: a task adaptive-based explainable deep learning framework for enhancer activity prediction.
    Yuhang Liu, Zixuan Wang, Hao Yuan, Guiquan Zhu, Yongqing Zhang.
      Enhancers are crucial cis-regulatory elements that control gene expression in a cell-type-specific manner. Despite extensive genetic and computational studies, accurately predicting enhancer activity in different cell types remains a challenge, and the grammar of enhancers is still poorly understood. Here, we present HEAP (high-resolution enhancer activity prediction), an explainable deep learning framework for predicting enhancers and exploring enhancer grammar. The framework includes three modules that use grammar-based reasoning for enhancer prediction. The algorithm can incorporate DNA sequences and epigenetic modifications to obtain better accuracy. We use a novel two-step multi-task learning method, task adaptive parameter sharing (TAPS), to efficiently predict enhancers in different cell types. We first train a shared model with all cell-type datasets. Then we adapt to specific tasks by adding several task-specific subset layers. Experiments demonstrate that HEAP outperforms published methods and showcases the effectiveness of the TAPS, especially for those with limited training samples. Notably, the explainable framework HEAP utilizes post-hoc interpretation to provide insights into the prediction mechanisms from three perspectives: data, model architecture and algorithm, leading to a better understanding of model decisions and enhancer grammar. To the best of our knowledge, HEAP will be a valuable tool for insight into the complex mechanisms of enhancer activity.
    Keywords:  bioinformatics; deep learning; enhancers; epigenetic modifications; multi-task learning
    DOI:  https://doi.org/10.1093/bib/bbad286
  20. Cell. 2023 Jul 25. pii: S0092-8674(23)00741-9. [Epub ahead of print]
    Chromosome-level organization of the regulatory genome in the Drosophila nervous system.
    Giriram Mohana, Julien Dorier, Xiao Li, Marion Mouginot, Rebecca C Smith, Héléna Malek, Marion Leleu, Daniel Rodriguez, Jenisha Khadka, Patrycja Rosa, Pascal Cousin, Christian Iseli, Simon Restrepo, Nicolas Guex, Brian D McCabe, Aleksander Jankowski, Michael S Levine, Maria Cristina Gambetta.
      Previous studies have identified topologically associating domains (TADs) as basic units of genome organization. We present evidence of a previously unreported level of genome folding, where distant TAD pairs, megabases apart, interact to form meta-domains. Within meta-domains, gene promoters and structural intergenic elements present in distant TADs are specifically paired. The associated genes encode neuronal determinants, including those engaged in axonal guidance and adhesion. These long-range associations occur in a large fraction of neurons but support transcription in only a subset of neurons. Meta-domains are formed by diverse transcription factors that are able to pair over long and flexible distances. We present evidence that two such factors, GAF and CTCF, play direct roles in this process. The relative simplicity of higher-order meta-domain interactions in Drosophila, compared with those previously described in mammals, allowed the demonstration that genomes can fold into highly specialized cell-type-specific scaffolds that enable megabase-scale regulatory associations.
    Keywords:  Drosophila; TAD; chromosomal loop; gene regulation; genome architecture; genome organization; nervous system; neuron; transcription
    DOI:  https://doi.org/10.1016/j.cell.2023.07.008
  21. iScience. 2023 Jul 21. 26(7): 107071
    Tissue specificity of oncogenic BRAF targeted to lung and thyroid through a shared lineage factor.
    Elin Schoultz, Shawn Liang, Therese Carlsson, Stefan Filges, Anders Ståhlberg, Henrik Fagman, Clotilde Wiel, Volkan Sayin, Mikael Nilsson.
      Cells of origin in cancer determine tumor phenotypes, but whether lineage-defining transcription factors might influence tissue specificity of tumorigenesis among organs with similar developmental traits are unknown. We demonstrate here that tumor development and progression markedly differ in lung and thyroid targeted by Braf mutation in Nkx2.1CreERT2 mice heterozygous for Nkx2-1. In absence of tamoxifen, non-induced Nkx2.1CreERT2;BrafCA/+ mutants developed multiple full-blown lung adenocarcinomas with a latency of 1-3 months whereas thyroid tumors were rare and constrained, although minute BrafCA activation documented by variant allele sequencing was similar in both tissues. Induced oncogene activation accelerated neoplastic growth only in the lungs. By contrast, NKX2-1+ progenitor cells were equally responsive to constitutive expression of mutant Braf during lung and thyroid development. Both lung and thyroid cells transiently downregulated NKX2-1 in early tumor stages. These results indicate that BRAFV600E-induced tumorigenesis obey organ-specific traits that might be differentially modified by a shared lineage factor.
    Keywords:  Cancer; Cell biology; Genetics
    DOI:  https://doi.org/10.1016/j.isci.2023.107071
  22. Nat Struct Mol Biol. 2023 Aug 03.
    Transcription modulates chromatin dynamics and locus configuration sampling.
    Giada Forte, Adam Buckle, Shelagh Boyle, Davide Marenduzzo, Nick Gilbert, Chris A Brackley.
      In living cells, the 3D structure of gene loci is dynamic, but this is not revealed by 3C and FISH experiments in fixed samples, leaving a notable gap in our understanding. To overcome these limitations, we applied the highly predictive heteromorphic polymer (HiP-HoP) model to determine chromatin fiber mobility at the Pax6 locus in three mouse cell lines with different transcription states. While transcriptional activity minimally affects movement of 40-kbp regions, we observed that motion of smaller 1-kbp regions depends strongly on local disruption to chromatin fiber structure marked by H3K27 acetylation. This also substantially influenced locus configuration dynamics by modulating protein-mediated promoter-enhancer loops. Importantly, these simulations indicate that chromatin dynamics are sufficiently fast to sample all possible locus conformations within minutes, generating wide dynamic variability within single cells. This combination of simulation and experimental validation provides insight into how transcriptional activity influences chromatin structure and gene dynamics.
    DOI:  https://doi.org/10.1038/s41594-023-01059-8
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