bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2022–08–21
twenty papers selected by
Connor Rogerson, University of Cambridge
  1. SPT6 functions in transcriptional pause/release via PAF1C recruitment.
  2. Chromatin-bound RB targets promoters, enhancers, and CTCF-bound loci and is redistributed by cell-cycle progression.
  3. Transcriptomics, regulatory syntax, and enhancer identification in mesoderm-induced ESCs at single-cell resolution.
  4. RNA Pol II pausing facilitates phased pluripotency transitions by buffering transcription.
  5. BindVAE: Dirichlet variational autoencoders for de novo motif discovery from accessible chromatin.
  6. The BASP1 transcriptional corepressor modifies chromatin through lipid-dependent and lipid-independent mechanisms.
  7. H3K9 tri-methylation at Nanog times differentiation commitment and enables the acquisition of primitive endoderm fate.
  8. Spatial profiling of chromatin accessibility in mouse and human tissues.
  9. Characterization and perturbation of CTCF-mediated chromatin interactions for enhancing myogenic transdifferentiation.
  10. TF-COMB - Discovering grammar of transcription factor binding sites.
  11. FACT modulates the conformations of histone H2A and H2B N-terminal tails within nucleosomes.
  12. Phase-separation antagonists potently inhibit transcription and broadly increase nucleosome density.
  13. Sall4 guides p53-mediated enhancer interference upon DNA damage in mouse embryonic stem cells.
  14. Inferring gene regulation from stochastic transcriptional variation across single cells at steady state.
  15. Transcriptional regulator Taf14 binds DNA and is required for the function of transcription factor TFIID in the absence of histone H2A.Z.
  16. EPIKOL, a chromatin-focused CRISPR/Cas9-based screening platform, to identify cancer-specific epigenetic vulnerabilities.
  17. Chromatin structure undergoes global and local reorganization during murine dendritic cell development and activation.
  18. SMARCE1 promotes neuroblastoma tumorigenesis through assisting MYCN-mediated transcriptional activation.
  19. Genome-wide functional perturbation of human microsatellite repeats using engineered zinc finger transcription factors.
  20. Live-seq enables temporal transcriptomic recording of single cells.
  1. Mol Cell. 2022 Aug 09. pii: S1097-2765(22)00655-4. [Epub ahead of print]
    SPT6 functions in transcriptional pause/release via PAF1C recruitment.
    Yuki Aoi, Avani P Shah, Sheetal Ganesan, Shimaa H A Soliman, Byoung-Kyu Cho, Young Ah Goo, Neil L Kelleher, Ali Shilatifard.
      It is unclear how various factors functioning in the transcriptional elongation by RNA polymerase II (RNA Pol II) cooperatively regulate pause/release and productive elongation in living cells. Using an acute protein-depletion approach, we report that SPT6 depletion results in the release of paused RNA Pol II into gene bodies through an impaired recruitment of PAF1C. Short genes demonstrate a release with increased mature transcripts, whereas long genes are released but fail to yield mature transcripts, due to a reduced processivity resulting from both SPT6 and PAF1C loss. Unexpectedly, SPT6 depletion causes an association of NELF with the elongating RNA Pol II on gene bodies, without any observed functional significance on transcriptional elongation pattern, arguing against a role for NELF in keeping RNA Pol II in the paused state. Furthermore, SPT6 depletion impairs heat-shock-induced pausing, pointing to a role for SPT6 in regulating RNA Pol II pause/release through PAF1C recruitment.
    Keywords:  NELF; PAF1; RNA polymerase II; SPT6; chromatin; elongation; gene expression; pause/release; promoter-proximal pausing; transcription
    DOI:  https://doi.org/10.1016/j.molcel.2022.06.037
  2. Mol Cell. 2022 Aug 12. pii: S1097-2765(22)00710-9. [Epub ahead of print]
    Chromatin-bound RB targets promoters, enhancers, and CTCF-bound loci and is redistributed by cell-cycle progression.
    Ioannis Sanidas, Hanjun Lee, Purva H Rumde, Gaylor Boulay, Robert Morris, Gabriel Golczer, Marcelo Stanzione, Soroush Hajizadeh, Jun Zhong, Meagan B Ryan, Ryan B Corcoran, Benjamin J Drapkin, Miguel N Rivera, Nicholas J Dyson, Michael S Lawrence.
      The interaction of RB with chromatin is key to understanding its molecular functions. Here, for first time, we identify the full spectrum of chromatin-bound RB. Rather than exclusively binding promoters, as is often described, RB targets three fundamentally different types of loci (promoters, enhancers, and insulators), which are largely distinguishable by the mutually exclusive presence of E2F1, c-Jun, and CTCF. While E2F/DP facilitates RB association with promoters, AP-1 recruits RB to enhancers. Although phosphorylation in CDK sites is often portrayed as releasing RB from chromatin, we show that the cell cycle redistributes RB so that it enriches at promoters in G1 and at non-promoter sites in cycling cells. RB-bound promoters include the classic E2F-targets and are similar between lineages, but RB-bound enhancers associate with different categories of genes and vary between cell types. Thus, RB has a well-preserved role controlling E2F in G1, and it targets cell-type-specific enhancers and CTCF sites when cells enter S-phase.
    Keywords:  AP-1; CTCF; E2F; PanChIP; RB phosphorylation; RB-bound enhancers; RB-bound promoters; cell-cycle regulation; regulation of RB activity; retinoblastoma protein
    DOI:  https://doi.org/10.1016/j.molcel.2022.07.014
  3. Cell Rep. 2022 Aug 16. pii: S2211-1247(22)01036-1. [Epub ahead of print]40(7): 111219
    Transcriptomics, regulatory syntax, and enhancer identification in mesoderm-induced ESCs at single-cell resolution.
    Mamduh Khateb, Jelena Perovanovic, Kyung Dae Ko, Kan Jiang, Xuesong Feng, Natalia Acevedo-Luna, Jérome Chal, Veronica Ciuffoli, Pavol Genzor, James Simone, Astrid D Haase, Olivier Pourquié, Stefania Dell'Orso, Vittorio Sartorelli.
      Embryonic stem cells (ESCs) can adopt lineage-specific gene-expression programs by stepwise exposure to defined factors, resulting in the generation of functional cell types. Bulk and single-cell-based assays were employed to catalog gene expression, histone modifications, chromatin conformation, and accessibility transitions in ESC populations and individual cells acquiring a presomitic mesoderm fate and undergoing further specification toward myogenic and neurogenic lineages. These assays identified cis-regulatory regions and transcription factors presiding over gene-expression programs occurring at defined ESC transitions and revealed the presence of heterogeneous cell populations within discrete ESC developmental stages. The datasets were employed to identify previously unappreciated genomic elements directing the initial activation of Pax7 and myogenic and neurogenic gene-expression programs. This study provides a resource for the discovery of genomic and transcriptional features of pluripotent, mesoderm-induced ESCs and ESC-derived cell lineages.
    Keywords:  CP: Developmental biology; Pax7; chromatin accessibility; embryonic stem cells; mesoderm; myogenesis; neurogenesis; single-cell omics
    DOI:  https://doi.org/10.1016/j.celrep.2022.111219
  4. Genes Dev. 2022 Aug 18.
    RNA Pol II pausing facilitates phased pluripotency transitions by buffering transcription.
    Abderhman Abuhashem, Alexandra G Chivu, Yixin Zhao, Edward J Rice, Adam Siepel, Charles G Danko, Anna-Katerina Hadjantonakis.
      Promoter-proximal RNA Pol II pausing is a critical step in transcriptional control. Pol II pausing has been predominantly studied in tissue culture systems. While Pol II pausing has been shown to be required for mammalian development, the phenotypic and mechanistic details of this requirement are unknown. Here, we found that loss of Pol II pausing stalls pluripotent state transitions within the epiblast of the early mouse embryo. Using Nelfb -/- mice and a NELFB degron mouse pluripotent stem cell model, we show that embryonic stem cells (ESCs) representing the naïve state of pluripotency successfully initiate a transition program but fail to balance levels of induced and repressed genes and enhancers in the absence of NELF. We found an increase in chromatin-associated NELF during transition from the naïve to later pluripotent states. Overall, our work defines the acute and long-term molecular consequences of NELF loss and reveals a role for Pol II pausing in the pluripotency continuum as a modulator of cell state transitions.
    Keywords:  NELF; dTAG; degron; embryonic stem cells; epiblast; mouse embryo; pausing; pluripotency; transcription
    DOI:  https://doi.org/10.1101/gad.349565.122
  5. Genome Biol. 2022 Aug 15. 23(1): 174
    BindVAE: Dirichlet variational autoencoders for de novo motif discovery from accessible chromatin.
    Meghana Kshirsagar, Han Yuan, Juan Lavista Ferres, Christina Leslie.
      We present a novel unsupervised deep learning approach called BindVAE, based on Dirichlet variational autoencoders, for jointly decoding multiple TF binding signals from open chromatin regions. BindVAE can disentangle an input DNA sequence into distinct latent factors that encode cell-type specific in vivo binding signals for individual TFs, composite patterns for TFs involved in cooperative binding, and genomic context surrounding the binding sites. On the task of retrieving the motifs of expressed TFs in a given cell type, BindVAE is competitive with existing motif discovery approaches.
    Keywords:  ATAC-seq; Dirichlet; Motifs; Transcription factor; VAE; Variational autoencoders; k-mers
    DOI:  https://doi.org/10.1186/s13059-022-02723-w
  6. iScience. 2022 Aug 19. 25(8): 104796
    The BASP1 transcriptional corepressor modifies chromatin through lipid-dependent and lipid-independent mechanisms.
    Alexander J Moorhouse, Amy E Loats, Kathryn F Medler, Stefan G E Roberts.
      The transcriptional corepressor BASP1 requires N-terminal myristoylation for its activity and functions through interactions with nuclear lipids. Here we determine the role of BASP1 lipidation in histone modification and the modulation of chromatin accessibility. We find that the removal of the active histone modifications H3K9ac and H3K4me3 by BASP1 requires the N-terminal myristoylation of BASP1. In contrast, the placement of the repressive histone modification, H3K27me3, by BASP1 does not require BASP1 lipidation. RNA-seq and ATAC-seq analysis finds that BASP1 regulates the activity of multiple transcription factors and induces extensive changes in chromatin accessibility. We find that ∼50% of BASP1 target genes show lipidation-dependent chromatin compaction and transcriptional repression. Our results suggest that BASP1 elicits both lipid-dependent and lipid-independent functions in histone modification and transcriptional repression. In accordance with this, we find that the tumor suppressor activity of BASP1 is also partially dependent on its myristoylation.
    Keywords:  Molecular Genetics; Molecular biology; Omics; Transcriptomics
    DOI:  https://doi.org/10.1016/j.isci.2022.104796
  7. Development. 2022 Aug 17. pii: dev.201074. [Epub ahead of print]
    H3K9 tri-methylation at Nanog times differentiation commitment and enables the acquisition of primitive endoderm fate.
    Agnès Dubois, Loris Vincenti, Almira Chervova, Maxim V C Greenberg, Sandrine Vandormael-Pournin, Déborah Bourc'his, Michel Cohen-Tannoudji, Pablo Navarro.
      Mouse Embryonic Stem (ES) cells have an inherent propensity to explore gene-regulatory states associated with either self-renewal or differentiation. This property depends on ERK, which downregulates pluripotency genes such as Nanog. Here, we aimed at identifying repressive histone modifications that would mark Nanog for inactivation in response to ERK activity. We found that the transcription factor ZFP57, which binds methylated DNA to nucleate heterochromatin, is recruited upstream of Nanog, within a region enriched for histone H3 lysine 9 tri-methylation (H3K9me3). While before differentiation H3K9me3 at Nanog depends on ERK, in somatic cells it becomes ERK-independent. Moreover, the loss of H3K9me3 at Nanog, induced by deleting the region or by knocking out DNA methyltransferases or Zfp57, is associated with reduced heterogeneity of NANOG, delayed commitment into differentiation and impaired ability to acquire a primitive endoderm fate. Hence, a network axis centred on DNA methylation, ZFP57 and H3K9me3, links Nanog regulation to ERK activity for the timely establishment of new cell identities. We suggest that establishment of irreversible H3K9me3 at specific master regulators allows the acquisition of particular cell fates during differentiation.
    Keywords:  ERK; H3K9me3; Heterogeneity; Nanog; Primitive endoderm; ZFP57
    DOI:  https://doi.org/10.1242/dev.201074
  8. Nature. 2022 Aug 17.
    Spatial profiling of chromatin accessibility in mouse and human tissues.
    Yanxiang Deng, Marek Bartosovic, Sai Ma, Di Zhang, Petra Kukanja, Yang Xiao, Graham Su, Yang Liu, Xiaoyu Qin, Gorazd B Rosoklija, Andrew J Dwork, J John Mann, Mina L Xu, Stephanie Halene, Joseph E Craft, Kam W Leong, Maura Boldrini, Gonçalo Castelo-Branco, Rong Fan.
      Cellular function in tissue is dependent on the local environment, requiring new methods for spatial mapping of biomolecules and cells in the tissue context1. The emergence of spatial transcriptomics has enabled genome-scale gene expression mapping2-5, but the ability to capture spatial epigenetic information of tissue at the cellular level and genome scale is lacking. Here we describe a method for spatially resolved chromatin accessibility profiling of tissue sections using next-generation sequencing (spatial-ATAC-seq) by combining in situ Tn5 transposition chemistry6 and microfluidic deterministic barcoding5. Profiling mouse embryos using spatial-ATAC-seq delineated tissue-region-specific epigenetic landscapes and identified gene regulators involved in the development of the central nervous system. Mapping the accessible genome in the mouse and human brain revealed the intricate arealization of brain regions. Applying spatial-ATAC-seq to tonsil tissue resolved the spatially distinct organization of immune cell types and states in lymphoid follicles and extrafollicular zones. This technology progresses spatial biology by enabling spatially resolved chromatin accessibility profiling to improve our understanding of cell identity, cell state and cell fate decision in relation to epigenetic underpinnings in development and disease.
    DOI:  https://doi.org/10.1038/s41586-022-05094-1
  9. Cell Rep. 2022 Aug 16. pii: S2211-1247(22)01023-3. [Epub ahead of print]40(7): 111206
    Characterization and perturbation of CTCF-mediated chromatin interactions for enhancing myogenic transdifferentiation.
    Ruimin Ren, Yu Fan, Zhelun Peng, Sheng Wang, Yunqi Jiang, Liangliang Fu, Jianhua Cao, Shuhong Zhao, Heng Wang.
      Expression of key transcription factors can induce transdifferentiation in somatic cells; however, this conversion is usually incomplete due to undefined intrinsic barriers. Here, we employ MyoD-induced transdifferentiation of fibroblasts as a model to illustrate the chromatin structures that impede the cell-fate transition. Focusing on the three-dimensional (3D) chromatin interactions, we show that MyoD directly establishes chromatin loops to activate myogenic transcriptional program. Similarly, dynamic changes of CTCF-mediated chromatin interactions are favorable for fibroblast-to-myoblast conversion. However, a substantial portion of CTCF-mediated chromatin interactions remain stable, and the associated genes are steady in expression and enriched for fibroblast function that may restrict cell-identity transformation. Temporal CTCF depletion can interrupt the resistant chromatin loops to enhance myogenic transdifferentiation in mice, pig, and chicken fibroblasts. Therefore, during induced transdifferentiation, the transcription factor can directly reorganize the 3D chromatin interactions, and perturbation of CTCF-mediated genome topology may resolve the limitations of cell fate transitions.
    Keywords:  CP: Molecular biology; CP: Stem cell research; CTCF; HiChIP; MyoD; chromatin interaction; fibroblast; myogenic; scRNA-seq; transdifferentiation
    DOI:  https://doi.org/10.1016/j.celrep.2022.111206
  10. Comput Struct Biotechnol J. 2022 ;20 4040-4051
    TF-COMB - Discovering grammar of transcription factor binding sites.
    Mette Bentsen, Vanessa Heger, Hendrik Schultheis, Carsten Kuenne, Mario Looso.
      Cooperativity between transcription factors is important to regulate target gene expression. In particular, the binding grammar of TFs in relation to each other, as well as in the context of other genomic elements, is crucial for TF functionality. However, tools to easily uncover co-occurrence between DNA-binding proteins, and investigate the regulatory modules of TFs, are limited. Here we present TF-COMB (Transcription Factor Co-Occurrence using Market Basket analysis) - a tool to investigate co-occurring TFs and binding grammar within regulatory regions. We found that TF-COMB can accurately identify known co-occurring TFs from ChIP-seq data, as well as uncover preferential localization to other genomic elements. With the use of ATAC-seq footprinting and TF motif locations, we found that TFs exhibit both preferred orientation and distance in relation to each other, and that these are biologically significant. Finally, we extended the analysis to not only investigate individual TF pairs, but also TF pairs in the context of networks, which enabled the investigation of TF complexes and TF hubs. In conclusion, TF-COMB is a flexible tool to investigate various aspects of TF binding grammar.
    Keywords:  Co-occurrence; Gene regulation; Grammar; Networks; Transcription factors
    DOI:  https://doi.org/10.1016/j.csbj.2022.07.025
  11. Commun Biol. 2022 Aug 13. 5(1): 814
    FACT modulates the conformations of histone H2A and H2B N-terminal tails within nucleosomes.
    Yasuo Tsunaka, Hideaki Ohtomo, Yoshifumi Nishimura.
      Gene expression is regulated by the modification and accessibility of histone tails within nucleosomes. The histone chaperone FACT (facilitate chromatin transcription), comprising SPT16 and SSRP1, interacts with nucleosomes through partial replacement of DNA with the phosphorylated acidic intrinsically disordered (pAID) segment of SPT16; pAID induces an accessible conformation of the proximal histone H3 N-terminal tail (N-tail) in the unwrapped nucleosome with FACT. Here, we use NMR to probe the histone H2A and H2B tails in the unwrapped nucleosome. Consequently, both the H2A and H2B N-tails on the pAID-proximal side bind to pAID with robust interactions, which are important for nucleosome assembly with FACT. Furthermore, the conformations of these N-tails on the distal DNA-contact site are altered from those in the canonical nucleosome. Our findings highlight that FACT both proximally and distally regulates the conformations of the H2A and H2B N-tails in the asymmetrically unwrapped nucleosome.
    DOI:  https://doi.org/10.1038/s42003-022-03785-z
  12. J Biol Chem. 2022 Aug 10. pii: S0021-9258(22)00808-0. [Epub ahead of print] 102365
    Phase-separation antagonists potently inhibit transcription and broadly increase nucleosome density.
    Rajyalakshmi Meduri, Linda S Rubio, Suman Mohajan, David S Gross.
      Biomolecular condensates are self-organized, membraneless bodies involved in many critical cellular activities including ribosome biogenesis, protein synthesis and gene transcription. Aliphatic alcohols are commonly used to study biomolecular condensates, but their effects on transcription are unclear. Here, we explore the impact of the aliphatic di-alcohol, 1,6-hexanediol, on Pol II transcription and nucleosome occupancy in budding yeast. As expected, 1,6-hexanediol, a reagent effective in disrupting biomolecular condensates, strongly suppressed the thermal stress-induced transcription of Heat Shock Factor 1 (Hsf1)-regulated genes that have previously been shown to physically interact and coalesce into intranuclear condensates. Surprisingly, the isomeric di-alcohol, 2,5-hexanediol, typically used as a negative control, abrogated Hsf1-target gene transcription under the same conditions. Each reagent also abolished the transcription of genes that do not detectably coalesce, including Msn2/Msn4-regulated heat-inducible genes and constitutively expressed housekeeping genes. Thus, at elevated temperature (39ºC), hexanediols potently inhibit the transcription of disparate genes and as demonstrated by ChIP do so by abolishing occupancy of RNA polymerase in chromatin. Concurrently, histone H3 density increased at least two-fold within all gene coding and regulatory regions examined, including quiescent euchromatic loci, silent heterochromatic loci and Pol III-transcribed loci. Our results offer a caveat for the use of hexanediols in studying the role of condensates in transcriptional control and provide evidence that exposure to these reagents elicits a widespread increase in nucleosome density and a concomitant loss of both Pol II and Pol III transcription.
    Keywords:  Heat Shock Factor 1 (Hsf1); Heat Shock Protein (HSP) gene coalescence; Msn2/Msn4; RNA Pol II; RNA Pol III; budding yeast; chromatin; hexanediol; nucleosome density; phase separation; transcriptional condensates
    DOI:  https://doi.org/10.1016/j.jbc.2022.102365
  13. Stem Cells. 2022 Aug 17. pii: sxac058. [Epub ahead of print]
    Sall4 guides p53-mediated enhancer interference upon DNA damage in mouse embryonic stem cells.
    Lei Wang, Xiaojun Tan, Lu Chen, Sisi Xu, Weiping Huang, Nan Chen, Yizhou Wu, Chunyan Wang, Daqiang Zhou, Mangmang Li.
      p53 plays pivotal roles in maintaining the genomic stability of mouse embryonic stem cells (mESCs) through transcriptionally activating and repressing target genes. However, how p53 recognizes its repressed targets remains largely unknown. Herein, we demonstrate that Sall4 negatively regulates DNA damage induced apoptosis (DIA) of mESCs through mediating p53 recruitment to enhancers of ESC-associated genes repressed by p53 from promoters of p53-activated genes. Upon DNA damage, Sall4 is transcriptionally repressed by p53, and plays an anti-apoptotic role without altering p53 activation. Moreover, Sall4 is identified as a novel p53-interacting partner. Consistently, Sall4 exerts its anti-apoptotic function in a p53-dependent manner. Intriguingly, Sall4 depletion not only promotes the transcriptional activation of several p53-regulated pro-apoptotic genes but also compromises p53-mediated repression of ESC master transcription factors in response to DNA damage. Mechanistically, Sall4 balances p53-binding affinity between p53-activated and -repressed genes through tethering p53 to ESC enhancers. In light of our study, Sall4 may contribute to tumorigenesis by antagonizing p53-mediated apoptosis.
    Keywords:  DNA damage; Sall4; enhancer interference; mouse embryonic stem cells; p53
    DOI:  https://doi.org/10.1093/stmcls/sxac058
  14. Proc Natl Acad Sci U S A. 2022 Aug 23. 119(34): e2207392119
    Inferring gene regulation from stochastic transcriptional variation across single cells at steady state.
    Anika Gupta, Jorge D Martin-Rufino, Thouis R Jones, Vidya Subramanian, Xiaojie Qiu, Emmanuelle I Grody, Alex Bloemendal, Chen Weng, Sheng-Yong Niu, Kyung Hoi Min, Arnav Mehta, Kaite Zhang, Layla Siraj, Aziz Al' Khafaji, Vijay G Sankaran, Soumya Raychaudhuri, Brian Cleary, Sharon Grossman, Eric S Lander.
      Regulatory relationships between transcription factors (TFs) and their target genes lie at the heart of cellular identity and function; however, uncovering these relationships is often labor-intensive and requires perturbations. Here, we propose a principled framework to systematically infer gene regulation for all TFs simultaneously in cells at steady state by leveraging the intrinsic variation in the transcriptional abundance across single cells. Through modeling and simulations, we characterize how transcriptional bursts of a TF gene are propagated to its target genes, including the expected ranges of time delay and magnitude of maximum covariation. We distinguish these temporal trends from the time-invariant covariation arising from cell states, and we delineate the experimental and technical requirements for leveraging these small but meaningful cofluctuations in the presence of measurement noise. While current technology does not yet allow adequate power for definitively detecting regulatory relationships for all TFs simultaneously in cells at steady state, we investigate a small-scale dataset to inform future experimental design. This study supports the potential value of mapping regulatory connections through stochastic variation, and it motivates further technological development to achieve its full potential.
    Keywords:  gene regulation; single-cell transcriptomics; transcriptional bursting
    DOI:  https://doi.org/10.1073/pnas.2207392119
  15. J Biol Chem. 2022 Aug 12. pii: S0021-9258(22)00812-2. [Epub ahead of print] 102369
    Transcriptional regulator Taf14 binds DNA and is required for the function of transcription factor TFIID in the absence of histone H2A.Z.
    Kadri Peil, Signe Värv, Ivar Ilves, Kersti Kristjuhan, Henel Jürgens, Arnold Kristjuhan.
      The transcriptional regulator Taf14 is a component of multiple protein complexes involved in transcription initiation and chromatin remodeling in yeast cells. Although Taf14 is not required for cell viability, it becomes essential in conditions where the formation of the transcription pre-initiation complex (PIC) is hampered. The specific role of Taf14 in mediating transcription initiation and PIC formation is unclear. Here, we explored its role in the general transcription factor TFIID by mapping Taf14 genetic and proteomic interactions and found that it was needed for the function of the complex if Htz1, the yeast homolog of histone H2A.Z, was absent from chromatin. Dissecting the functional domains of Taf14 revealed that the linker region between the YEATS and ET domains was required for cell viability in the absence of Htz1 protein. We further show that the linker region of Taf14 interacts with DNA. We propose that providing additional DNA binding capacity might be a general role of Taf14 in the recruitment of protein complexes to DNA and chromatin.
    Keywords:  H2A.Z; TFIID; Taf14; Taf2; Transcription; chromatin; pre-initiation complex (PIC)
    DOI:  https://doi.org/10.1016/j.jbc.2022.102369
  16. Cell Death Dis. 2022 Aug 16. 13(8): 710
    EPIKOL, a chromatin-focused CRISPR/Cas9-based screening platform, to identify cancer-specific epigenetic vulnerabilities.
    Ozlem Yedier-Bayram, Bengul Gokbayrak, Alisan Kayabolen, Ali Cenk Aksu, Ayse Derya Cavga, Ahmet Cingöz, Ezgi Yagmur Kala, Goktug Karabiyik, Rauf Günsay, Beril Esin, Tunc Morova, Fırat Uyulur, Hamzah Syed, Martin Philpott, Adam P Cribbs, Sonia H Y Kung, Nathan A Lack, Tamer T Onder, Tugba Bagci-Onder.
      Dysregulation of the epigenome due to alterations in chromatin modifier proteins commonly contribute to malignant transformation. To interrogate the roles of epigenetic modifiers in cancer cells, we generated an epigenome-wide CRISPR-Cas9 knockout library (EPIKOL) that targets a wide-range of epigenetic modifiers and their cofactors. We conducted eight screens in two different cancer types and showed that EPIKOL performs with high efficiency in terms of sgRNA distribution and depletion of essential genes. We discovered novel epigenetic modifiers that regulate triple-negative breast cancer (TNBC) and prostate cancer cell fitness. We confirmed the growth-regulatory functions of individual candidates, including SS18L2 and members of the NSL complex (KANSL2, KANSL3, KAT8) in TNBC cells. Overall, we show that EPIKOL, a focused sgRNA library targeting ~800 genes, can reveal epigenetic modifiers that are essential for cancer cell fitness under in vitro and in vivo conditions and enable the identification of novel anti-cancer targets. Due to its comprehensive epigenome-wide targets and relatively high number of sgRNAs per gene, EPIKOL will facilitate studies examining functional roles of epigenetic modifiers in a wide range of contexts, such as screens in primary cells, patient-derived xenografts as well as in vivo models.
    DOI:  https://doi.org/10.1038/s41419-022-05146-4
  17. Proc Natl Acad Sci U S A. 2022 Aug 23. 119(34): e2207009119
    Chromatin structure undergoes global and local reorganization during murine dendritic cell development and activation.
    Daisuke Kurotaki, Kenta Kikuchi, Kairong Cui, Wataru Kawase, Keita Saeki, Junpei Fukumoto, Akira Nishiyama, Kisaburo Nagamune, Keji Zhao, Keiko Ozato, Pedro P Rocha, Tomohiko Tamura.
      Classical dendritic cells (cDCs) are essential for immune responses and differentiate from hematopoietic stem cells via intermediate progenitors, such as monocyte-DC progenitors (MDPs) and common DC progenitors (CDPs). Upon infection, cDCs are activated and rapidly express host defense-related genes, such as those encoding cytokines and chemokines. Chromatin structures, including nuclear compartments and topologically associating domains (TADs), have been implicated in gene regulation. However, the extent and dynamics of their reorganization during cDC development and activation remain unknown. In this study, we comprehensively determined higher-order chromatin structures by Hi-C in DC progenitors and cDC subpopulations. During cDC differentiation, chromatin activation was initially induced at the MDP stage. Subsequently, a shift from inactive to active nuclear compartments occurred at the cDC gene loci in CDPs, which was followed by increased intra-TAD interactions and loop formation. Mechanistically, the transcription factor IRF8, indispensable for cDC differentiation, mediated chromatin activation and changes into the active compartments in DC progenitors, thereby possibly leading to cDC-specific gene induction. Using an infection model, we found that the chromatin structures of host defense-related gene loci were preestablished in unstimulated cDCs, indicating that the formation of higher-order chromatin structures prior to infection may contribute to the rapid responses to pathogens. Overall, these results suggest that chromatin structure reorganization is closely related to the establishment of cDC-specific gene expression and immune functions. This study advances the fundamental understanding of chromatin reorganization in cDC differentiation and activation.
    Keywords:  chromatin structure; dendritic cell; hematopoiesis; infection; transcription factor
    DOI:  https://doi.org/10.1073/pnas.2207009119
  18. Oncogene. 2022 Aug 17.
    SMARCE1 promotes neuroblastoma tumorigenesis through assisting MYCN-mediated transcriptional activation.
    Xiaosong Hu, Ruochen Liu, Jianbing Hou, Wen Peng, Sicheng Wan, Minghao Xu, Yongsen Li, Guanghui Zhang, Xuan Zhai, Ping Liang, Hongjuan Cui.
      SMARCE1 gene, encoding a core subunit of SWI/SNF chromatin remodeling complex, is situated on chromosome 17q21-ter region that is frequently gained in neuroblastoma. However, its role in the tumorigenesis remains unknown. Here, we showed that high expression of SMARCE1 was associated with poor prognosis of patients with neuroblastoma, especially those with MYCN amplification. Knockdown of SMARCE1 reduced proliferation, colony formation, and tumorigenicity of neuroblastoma cells. Mechanistically, SMARCE1 directly interacted with MYCN, which was necessary for MYCN-mediated transcriptional activation of downstream target genes including PLK1, ODC1, and E2F2. Overexpression of PLK1, ODC1 or E2F2 significantly reversed the inhibiting effect of SMARCE1 knockdown on the proliferation, colony formation, and tumorigenicity of MYCN-amplified neuroblastoma cells. Moreover, we revealed that MYCN directly regulated SMARCE1 transcription through binding to a non-canonical E-box of SMARCE1 promoter, thus enhancing SMARCE1-MYCN cooperativity. These findings establish SMARCE1 is a critical oncogenic factor in neuroblastoma and provide a new potential target for treatment of neuroblastoma with 17q21-ter gain and MYCN amplification.
    DOI:  https://doi.org/10.1038/s41388-022-02428-1
  19. Cell Genom. 2022 Apr 13. pii: 100119. [Epub ahead of print]2(4):
    Genome-wide functional perturbation of human microsatellite repeats using engineered zinc finger transcription factors.
    Y Esther Tak, Gaylor Boulay, Lukuo Lee, Sowmya Iyer, Nicholas T Perry, Hayley T Schultz, Sara P Garcia, Liliane Broye, Joy E Horng, Shruthi Rengarajan, Beverly Naigles, Angela Volorio, Jeffry D Sander, Jingyi Gong, Nicolὸ Riggi, J Keith Joung, Miguel N Rivera.
      Repeat elements can be dysregulated at a genome-wide scale in human diseases. For example, in Ewing sarcoma, hundreds of inert GGAA repeats can be converted into active enhancers when bound by EWS-FLI1. Here we show that fusions between EWS and GGAA-repeat-targeted engineered zinc finger arrays (ZFAs) can function at least as efficiently as EWS-FLI1 for converting hundreds of GGAA repeats into active enhancers in a Ewing sarcoma precursor cell model. Furthermore, a fusion of a KRAB domain to a ZFA can silence GGAA microsatellite enhancers genome wide in Ewing sarcoma cells, thereby reducing expression of EWS-FLI1-activated genes. Remarkably, this KRAB-ZFA fusion showed selective toxicity against Ewing sarcoma cells compared with non-Ewing cancer cells, consistent with its Ewing sarcoma-specific impact on the transcriptome. These findings demonstrate the value of ZFAs for functional annotation of repeats and illustrate how aberrant microsatellite activities might be regulated for potential therapeutic applications.
    DOI:  https://doi.org/10.1016/j.xgen.2022.100119
  20. Nature. 2022 Aug 17.
    Live-seq enables temporal transcriptomic recording of single cells.
    Wanze Chen, Orane Guillaume-Gentil, Pernille Yde Rainer, Christoph G Gäbelein, Wouter Saelens, Vincent Gardeux, Amanda Klaeger, Riccardo Dainese, Magda Zachara, Tomaso Zambelli, Julia A Vorholt, Bart Deplancke.
      Single-cell transcriptomics (scRNA-seq) has greatly advanced our ability to characterize cellular heterogeneity1. However, scRNA-seq requires lysing cells, which impedes further molecular or functional analyses on the same cells. Here, we established Live-seq, a single-cell transcriptome profiling approach that preserves cell viability during RNA extraction using fluidic force microscopy2,3, thus allowing to couple a cell's ground-state transcriptome to its downstream molecular or phenotypic behaviour. To benchmark Live-seq, we used cell growth, functional responses and whole-cell transcriptome read-outs to demonstrate that Live-seq can accurately stratify diverse cell types and states without inducing major cellular perturbations. As a proof of concept, we show that Live-seq can be used to directly map a cell's trajectory by sequentially profiling the transcriptomes of individual macrophages before and after lipopolysaccharide (LPS) stimulation, and of adipose stromal cells pre- and post-differentiation. In addition, we demonstrate that Live-seq can function as a transcriptomic recorder by preregistering the transcriptomes of individual macrophages that were subsequently monitored by time-lapse imaging after LPS exposure. This enabled the unsupervised, genome-wide ranking of genes on the basis of their ability to affect macrophage LPS response heterogeneity, revealing basal Nfkbia expression level and cell cycle state as important phenotypic determinants, which we experimentally validated. Thus, Live-seq can address a broad range of biological questions by transforming scRNA-seq from an end-point to a temporal analysis approach.
    DOI:  https://doi.org/10.1038/s41586-022-05046-9
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