bims-nutges Biomed News
on Nucleolar transcription and genomic stability
Issue of 2021–08–01
thirteen papers selected by
Kanwal Tariq, Stockholm University
  1. Mechanisms of BRCA1-BARD1 nucleosome recognition and ubiquitylation.
  2. BARD1 reads H2A lysine 15 ubiquitination to direct homologous recombination.
  3. Epigenetic rewriting at centromeric DNA repeats leads to increased chromatin accessibility and chromosomal instability.
  4. Chromatin remodeler ARID1A binds IRF3 to selectively induce antiviral interferon production in macrophages.
  5. KDM3A regulates alternative splicing of cell-cycle genes following DNA damage.
  6. TDRD3 promotes DHX9 chromatin recruitment and R-loop resolution.
  7. Loss of mitochondrial transcription factor A in neural stem cells leads to immature brain development and triggers the activation of the integral stress response in vivo.
  8. A Brownian ratchet model for DNA loop extrusion by the cohesin complex.
  9. SMARCA4 deficiency-associated heterochromatin induces intrinsic DNA replication stress and susceptibility to ATR inhibition in lung adenocarcinoma.
  10. FACS-based isolation of fixed mouse neuronal nuclei for ATAC-seq and Hi-C.
  11. Multiplexed Imaging of Posttranslational Modifications of Endogenous Proteins in Live Cells.
  12. APC/CFZR-1 regulates centrosomal ZYG-1 to limit centrosome number.
  13. Defining R-loop classes and their contributions to genome instability.
  1. Nature. 2021 Jul 28.
    Mechanisms of BRCA1-BARD1 nucleosome recognition and ubiquitylation.
    Qi Hu, Maria Victoria Botuyan, Debiao Zhao, Gaofeng Cui, Elie Mer, Georges Mer.
      The BRCA1-BARD1 tumour suppressor is an E3 ubiquitin ligase necessary for the repair of DNA double-strand breaks by homologous recombination1-10. The BRCA1-BARD1 complex localizes to damaged chromatin after DNA replication and catalyses the ubiquitylation of histone H2A and other cellular targets11-14. The molecular bases for the recruitment to double-strand breaks and target recognition of BRCA1-BARD1 remain unknown. Here we use cryo-electron microscopy to show that the ankyrin repeat and tandem BRCT domains in BARD1 adopt a compact fold and bind to nucleosomal histones, DNA and monoubiquitin attached to H2A amino-terminal K13 or K15, two signals known to be specific for double-strand breaks15,16. We further show that RING domains17 in BRCA1-BARD1 orient an E2 ubiquitin-conjugating enzyme atop the nucleosome in a dynamic conformation, primed for ubiquitin transfer to the flexible carboxy-terminal tails of H2A and variant H2AX. Our work reveals a regulatory crosstalk in which recognition of monoubiquitin by BRCA1-BARD1 at the N terminus of H2A blocks the formation of polyubiquitin chains and cooperatively promotes ubiquitylation at the C terminus of H2A. These findings elucidate the mechanisms of BRCA1-BARD1 chromatin recruitment and ubiquitylation specificity, highlight key functions of BARD1 in both processes and explain how BRCA1-BARD1 promotes homologous recombination by opposing the DNA repair protein 53BP1 in post-replicative chromatin18-22. These data provide a structural framework to evaluate BARD1 variants and help to identify mutations that drive the development of cancer.
    DOI:  https://doi.org/10.1038/s41586-021-03716-8
  2. Nature. 2021 Jul 28.
    BARD1 reads H2A lysine 15 ubiquitination to direct homologous recombination.
    Jordan R Becker, Gillian Clifford, Clara Bonnet, Anja Groth, Marcus D Wilson, J Ross Chapman.
      Protein ubiquitination at sites of DNA double-strand breaks (DSBs) by RNF168 recruits BRCA1 and 53BP11,2, which are mediators of the homologous recombination and non-homologous end joining DSB repair pathways, respectively3. Non-homologous end joining relies on 53BP1 binding directly to ubiquitinated lysine 15 on H2A-type histones (H2AK15ub)4,5 (which is an RNF168-dependent modification6), but how RNF168 promotes BRCA1 recruitment and function remains unclear. Here we identify a tandem BRCT-domain-associated ubiquitin-dependent recruitment motif (BUDR) in BRCA1-associated RING domain protein 1 (BARD1) (the obligate partner protein of BRCA1) that, by engaging H2AK15ub, recruits BRCA1 to DSBs. Disruption of the BUDR of BARD1 compromises homologous recombination and renders cells hypersensitive to PARP inhibition and cisplatin. We further show that BARD1 binds nucleosomes through multivalent interactions: coordinated binding of H2AK15ub and unmethylated H4 lysine 20 by its adjacent BUDR and ankyrin repeat domains, respectively, provides high-affinity recognition of DNA lesions in replicated chromatin and promotes the homologous recombination activities of the BRCA1-BARD1 complex. Finally, our genetic epistasis experiments confirm that the need for BARD1 chromatin-binding activities can be entirely relieved upon deletion of RNF168 or 53BP1. Thus, our results demonstrate that by sensing DNA-damage-dependent and post-replication histone post-translation modification states, BRCA1-BARD1 complexes coordinate the antagonization of the 53BP1 pathway with promotion of homologous recombination, establishing a simple paradigm for the governance of the choice of DSB repair pathway.
    DOI:  https://doi.org/10.1038/s41586-021-03776-w
  3. Epigenetics Chromatin. 2021 Jul 28. 14(1): 35
    Epigenetic rewriting at centromeric DNA repeats leads to increased chromatin accessibility and chromosomal instability.
    Sheldon Decombe, François Loll, Laura Caccianini, Kévin Affannoukoué, Ignacio Izeddin, Julien Mozziconacci, Christophe Escudé, Judith Lopes.
       BACKGROUND: Centromeric regions of human chromosomes contain large numbers of tandemly repeated α-satellite sequences. These sequences are covered with constitutive heterochromatin which is enriched in trimethylation of histone H3 on lysine 9 (H3K9me3). Although well studied using artificial chromosomes and global perturbations, the contribution of this epigenetic mark to chromatin structure and genome stability remains poorly known in a more natural context.
    RESULTS: Using transcriptional activator-like effectors (TALEs) fused to a histone lysine demethylase (KDM4B), we were able to reduce the level of H3K9me3 on the α-satellites repeats of human chromosome 7. We show that the removal of H3K9me3 affects chromatin structure by increasing the accessibility of DNA repeats to the TALE protein. Tethering TALE-demethylase to centromeric repeats impairs the recruitment of HP1α and proteins of Chromosomal Passenger Complex (CPC) on this specific centromere without affecting CENP-A loading. Finally, the epigenetic re-writing by the TALE-KDM4B affects specifically the stability of chromosome 7 upon mitosis, highlighting the importance of H3K9me3 in centromere integrity and chromosome stability, mediated by the recruitment of HP1α and the CPC.
    CONCLUSION: Our cellular model allows to demonstrate the direct role of pericentromeric H3K9me3 epigenetic mark on centromere integrity and function in a natural context and opens interesting possibilities for further studies regarding the role of the H3K9me3 mark.
    Keywords:  Centromere; Chromosomal instability; Epigenetics; Heterochromatin; Histone methylation
    DOI:  https://doi.org/10.1186/s13072-021-00410-x
  4. Cell Death Dis. 2021 Jul 27. 12(8): 743
    Chromatin remodeler ARID1A binds IRF3 to selectively induce antiviral interferon production in macrophages.
    Ye Hu, Xin Wang, Jiaying Song, Jiacheng Wu, Jia Xu, Yangyang Chai, Yuanyuan Ding, Bingjing Wang, Chunmei Wang, Yong Zhao, Zhongyang Shen, Xiaoqing Xu, Xuetao Cao.
      Transcription factor IRF3 is critical for the induction of antiviral type I interferon (IFN-I). The epigenetic regulation of IFN-I production in antiviral innate immunity needs to be further identified. Here, we reported that epigenetic remodeler ARID1A, a critical component of the mSWI/SNF complex, could bind IRF3 and then was recruited to the Ifn-I promoter by IRF3, thus selectively promoting IFN-I but not TNF-α, IL-6 production in macrophages upon viral infection. Myeloid cell-specific deficiency of Arid1a rendered mice more susceptible to viral infection, accompanied with less IFN-I production. Mechanistically, ARID1A facilitates chromatin accessibility of IRF3 at the Ifn-I promoters by interacting with histone methyltransferase NSD2, which methylates H3K4 and H3K36 of the promoter regions. Our findings demonstrated the new roles of ARID1A and NSD2 in innate immunity, providing insight into the crosstalks of chromatin remodeling, histone modification, and transcription factors in the epigenetic regulation of antiviral innate immunity.
    DOI:  https://doi.org/10.1038/s41419-021-04032-9
  5. RNA. 2021 Jul 28. pii: rna.078796.121. [Epub ahead of print]
    KDM3A regulates alternative splicing of cell-cycle genes following DNA damage.
    Mai Baker, Mayra Petasny, Nadeen Taqatqa, Mercedes Bentata, Gillian Kay, Eden Engal, Yuval Nevo, Ahmad Siam, Sara Preiser, Maayan Salton.
      Changes in the cellular environment result in chromatin structure alteration, which in turn regulates gene expression. To learn about the effect of the cellular environment on the transcriptome, we studied the H3K9 de-methylase KDM3A. Using RNA-seq, we found that KDM3A regulates the transcription and alternative splicing of genes associated with cell cycle and DNA damage. We showed that KDM3A undergoes phosphorylation by PKA at serine 265 following DNA damage, and that the phosphorylation is important for a proper cell cycle regulation. We demonstrated that SAT1 alternative splicing, regulated by KDM3A, plays a role in cell cycle regulation. Furthermore we found that KDM3A's demethylase activity is not needed for SAT1 alternative splicing regulation. In addition, we identified KDM3A's protein partner ARID1A, the SWI/SNF subunit, and SRSF3 as regulators of SAT1 alternative splicing and showed that KDM3A is essential for SRSF3 binding to SAT1 pre-mRNA. These results suggest that KDM3A serves as a sensor of the environment and an adaptor for splicing factor binding. Our work reveals chromatin sensing of the environment in the regulation of alternative splicing.
    Keywords:  alternative splicing; chromatin; pre-mRNA Splicing
    DOI:  https://doi.org/10.1261/rna.078796.121
  6. Nucleic Acids Res. 2021 Jul 30. pii: gkab642. [Epub ahead of print]
    TDRD3 promotes DHX9 chromatin recruitment and R-loop resolution.
    Wei Yuan, Qais Al-Hadid, Zhihao Wang, Lei Shen, Hyejin Cho, Xiwei Wu, Yanzhong Yang.
      R-loops, which consist of a DNA/RNA hybrid and a displaced single-stranded DNA (ssDNA), are increasingly recognized as critical regulators of chromatin biology. R-loops are particularly enriched at gene promoters, where they play important roles in regulating gene expression. However, the molecular mechanisms that control promoter-associated R-loops remain unclear. The epigenetic 'reader' Tudor domain-containing protein 3 (TDRD3), which recognizes methylarginine marks on histones and on the C-terminal domain of RNA polymerase II, was previously shown to recruit DNA topoisomerase 3B (TOP3B) to relax negatively supercoiled DNA and prevent R-loop formation. Here, we further characterize the function of TDRD3 in R-loop metabolism and introduce the DExH-box helicase 9 (DHX9) as a novel interaction partner of the TDRD3/TOP3B complex. TDRD3 directly interacts with DHX9 via its Tudor domain. This interaction is important for recruiting DHX9 to target gene promoters, where it resolves R-loops in a helicase activity-dependent manner to facilitate gene expression. Additionally, TDRD3 also stimulates the helicase activity of DHX9. This stimulation relies on the OB-fold of TDRD3, which likely binds the ssDNA in the R-loop structure. Thus, DHX9 functions together with TOP3B to suppress promoter-associated R-loops. Collectively, these findings reveal new functions of TDRD3 and provide important mechanistic insights into the regulation of R-loop metabolism.
    DOI:  https://doi.org/10.1093/nar/gkab642
  7. PLoS One. 2021 ;16(7): e0255355
    Loss of mitochondrial transcription factor A in neural stem cells leads to immature brain development and triggers the activation of the integral stress response in vivo.
    Rintaro Kuroda, Kaoru Tominaga, Katsumi Kasashima, Kenji Kuroiwa, Eiji Sakashita, Hiroko Hayakawa, Tom Kouki, Nobuhiko Ohno, Kensuke Kawai, Hitoshi Endo.
      Mitochondrial dysfunction is significantly associated with neurological deficits and age-related neurological diseases. While mitochondria are dynamically regulated and properly maintained during neurogenesis, the manner in which mitochondrial activities are controlled and contribute to these processes is not fully understood. Mitochondrial transcription factor A (TFAM) contributes to mitochondrial function by maintaining mitochondrial DNA (mtDNA). To clarify how mitochondrial dysfunction affects neurogenesis, we induced mitochondrial dysfunction specifically in murine neural stem cells (NSCs) by inactivating Tfam. Tfam inactivation in NSCs resulted in mitochondrial dysfunction by reducing respiratory chain activities and causing a severe deficit in neural differentiation and maturation both in vivo and in vitro. Brain tissue from Tfam-deficient mice exhibited neuronal cell death primarily at layer V and microglia were activated prior to cell death. Cultured Tfam-deficient NSCs showed a reduction in reactive oxygen species produced by the mitochondria. Tfam inactivation during neurogenesis resulted in the accumulation of ATF4 and activation of target gene expression. Therefore, we propose that the integrated stress response (ISR) induced by mitochondrial dysfunction in neurogenesis is activated to protect the progression of neurodegenerative diseases.
    DOI:  https://doi.org/10.1371/journal.pone.0255355
  8. Elife. 2021 Jul 26. pii: e67530. [Epub ahead of print]10
    A Brownian ratchet model for DNA loop extrusion by the cohesin complex.
    Torahiko L Higashi, Georgii Pobegalov, Minzhe Tang, Maxim I Molodtsov, Frank Uhlmann.
      The cohesin complex topologically encircles DNA to promote sister chromatid cohesion. Alternatively, cohesin extrudes DNA loops, thought to reflect chromatin domain formation. Here, we propose a structure-based model explaining both activities. ATP and DNA binding promote cohesin conformational changes that guide DNA through a kleisin N-gate into a DNA gripping state. Two HEAT-repeat DNA binding modules, associated with cohesin's heads and hinge, are now juxtaposed. Gripping state disassembly, following ATP hydrolysis, triggers unidirectional hinge module movement, which completes topological DNA entry by directing DNA through the ATPase head gate. If head gate passage fails, hinge module motion creates a Brownian ratchet that, instead, drives loop extrusion. Molecular-mechanical simulations of gripping state formation and resolution cycles recapitulate experimentally observed DNA loop extrusion characteristics. Our model extends to asymmetric and symmetric loop extrusion, as well as z-loop formation. Loop extrusion by biased Brownian motion has important implications for chromosomal cohesin function.
    Keywords:  Cohesin; DNA loop extrusion; S. pombe; SMC complexes; biophysical simulation; chromosomes; computational biology; gene expression; sister chromatid cohesion; structural biology; systems biology
    DOI:  https://doi.org/10.7554/eLife.67530
  9. NAR Cancer. 2020 Jun;2(2): zcaa005
    SMARCA4 deficiency-associated heterochromatin induces intrinsic DNA replication stress and susceptibility to ATR inhibition in lung adenocarcinoma.
    Kiminori Kurashima, Hideto Kashiwagi, Iwao Shimomura, Ayako Suzuki, Fumitaka Takeshita, Marianne Mazevet, Masahiko Harata, Takayuki Yamashita, Yusuke Yamamoto, Takashi Kohno, Bunsyo Shiotani.
      The SWI/SNF chromatin remodeling complex regulates transcription through the control of chromatin structure and is increasingly thought to play an important role in human cancer. Lung adenocarcinoma (LADC) patients frequently harbor mutations in SMARCA4, a core component of this multisubunit complex. Most of these mutations are loss-of-function mutations, which disrupt critical functions in the regulation of chromatin architecture and can cause DNA replication stress. This study reports that LADC cells deficient in SMARCA4 showed increased DNA replication stress and greater sensitivity to the ATR inhibitor (ATRi) in vitro and in vivo. Mechanistically, loss of SMARCA4 increased heterochromatin formation, resulting in stalled forks, a typical DNA replication stress. In the absence of SMARCA4, severe ATRi-induced single-stranded DNA, which caused replication catastrophe, was generated on nascent DNA near the reversed forks around heterochromatin in an Mre11-dependent manner. Thus, loss of SMARCA4 confers susceptibility to ATRi, both by increasing heterochromatin-associated replication stress and by allowing Mre11 to destabilize reversed forks. These two mechanisms synergistically increase susceptibility of SMARCA4-deficient LADC cells to ATRi. These results provide a preclinical basis for assessing SMARCA4 defects as a biomarker of ATRi efficacy.
    DOI:  https://doi.org/10.1093/narcan/zcaa005
  10. STAR Protoc. 2021 Sep 17. 2(3): 100643
    FACS-based isolation of fixed mouse neuronal nuclei for ATAC-seq and Hi-C.
    Ekaterina Eremenko, Anastasia Golova, Daniel Stein, Monica Einav, Ekaterina Khrameeva, Debra Toiber.
      The organization of chromatin structure plays a crucial role in gene expression, DNA replication, and repair. Chromatin alterations influence gene expression, and modifications could be associated with genomic instability in the cells during aging or diseases. Here, we provide a modified protocol to isolate fixed neuronal nuclei from a single mouse cortex to investigate the spatial organization of chromatin structure on a genome-wide scale by ATAC-seq (the assay for transposase-accessible chromatin with high-throughput sequencing) and chromatin conformation by Hi-C (high-throughput chromosome conformation capture).
    Keywords:  Cell Biology; Cell separation/fractionation; Flow Cytometry/Mass Cytometry; Genomics; Neuroscience
    DOI:  https://doi.org/10.1016/j.xpro.2021.100643
  11. Methods Mol Biol. 2021 ;2350 31-41
    Multiplexed Imaging of Posttranslational Modifications of Endogenous Proteins in Live Cells.
    Yuko Sato, Hiroshi Kimura.
      Posttranslational histone modifications are associated with the regulation of genome function. Some modifications are quite stable to maintain epigenome states of chromatin, and others can exhibit dynamic changes in response to internal and external stimuli. To track the local and global changes in histone modifications, multiplexed imaging in living cells is beneficial. Among live cell probes for detecting histone modifications, genetically encoded modification-specific intracellular antibodies, or mintbodies, are convenient and suitable tools for this purpose. We here describe the mintbody-based methods to monitor the changes in histone modification levels induced by histone methyltransferase and deacetylase inhibitors. By measuring the nuclear to cytoplasmic intensity ratios of mintbodies in living cells, changes in histone H4 lysine 20 methylation states and the increase in histone H3 acetylation were detected.
    Keywords:  Histone modifications; Live-cell imaging; Modification-specific intracellular antibody
    DOI:  https://doi.org/10.1007/978-1-0716-1593-5_3
  12. J Cell Sci. 2021 Jul 15. pii: jcs253088. [Epub ahead of print]134(14):
    APC/CFZR-1 regulates centrosomal ZYG-1 to limit centrosome number.
    Jeffrey C Medley, Joseph R DiPanni, Luke Schira, Blake M Shaffou, Brandon M Sebou, Mi Hye Song.
      Aberrant centrosome numbers are associated with human cancers. The levels of centrosome regulators positively correlate with centrosome number. Thus, tight control of centrosome protein levels is critical. In Caenorhabditis elegans, the anaphase-promoting complex/cyclosome and its co-activator FZR-1 (APC/CFZR-1), a ubiquitin ligase, negatively regulates centrosome assembly through SAS-5 degradation. In this study, we report the C. elegans ZYG-1 (Plk4 in humans) as a potential substrate of APC/CFZR-1. Inhibiting APC/CFZR-1 or mutating a ZYG-1 destruction (D)-box leads to elevated ZYG-1 levels at centrosomes, restoring bipolar spindles and embryonic viability to zyg-1 mutants, suggesting that APC/CFZR-1 influences centrosomal ZYG-1 via the D-box motif. We also show the Slimb/βTrCP-binding (SB) motif is critical for ZYG-1 degradation, substantiating a conserved mechanism by which ZYG-1/Plk4 stability is regulated by the SKP1-CUL1-F-box (Slimb/βTrCP)-protein complex (SCFSlimb/βTrCP)-dependent proteolysis via the conserved SB motif in C. elegans. Furthermore, we show that co-mutating ZYG-1 SB and D-box motifs stabilizes ZYG-1 in an additive manner, suggesting that the APC/CFZR-1 and SCFSlimb/βTrCP ubiquitin ligases function cooperatively for timely ZYG-1 destruction in C. elegans embryos where ZYG-1 activity remains at threshold level to ensure normal centrosome number.
    Keywords:   C. elegans ; APC/C; Centrosome; FZR-1; Proteolysis; ZYG-1
    DOI:  https://doi.org/10.1242/jcs.253088
  13. DNA Repair (Amst). 2021 Jul 17. pii: S1568-7864(21)00138-5. [Epub ahead of print]106 103182
    Defining R-loop classes and their contributions to genome instability.
    Daisy Castillo-Guzman, Frédéric Chédin.
      R-loops are non-B DNA structures that form during transcription when the nascent RNA anneals to the template DNA strand forming a RNA:DNA hybrid. Understanding the genomic distribution and function of R-loops is an important goal, since R-loops have been implicated in a number of adaptive and maladaptive processes under physiological and pathological conditions. Based on R-loop mapping datasets, we propose the existence of two main classes of R-loops, each associated with unique characteristics. Promoter-paused R-loops (Class I) are short R-loops that form at high frequency during promoter-proximal pausing by RNA polymerase II. Elongation-associated R-loops (Class II) are long structures that occur throughout gene bodies at modest frequencies. We further discuss the relationships between each R-loop class with instances of genome instability and suggest that increased class I R-loops, resulting from enhanced promoter-proximal pausing, represent the main culprits for R-loop mediated genome instability under pathological conditions.
    Keywords:  DNA damage; Elongation; Genome instability; Promoter-pausing; R-loops; Transcription
    DOI:  https://doi.org/10.1016/j.dnarep.2021.103182
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