bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2023–09–24
25 papers selected by
Connor Rogerson, University of Cambridge
  1. Epigenetic reprogramming of a distal developmental enhancer cluster drives SOX2 overexpression in breast and lung adenocarcinoma.
  2. Lola-I is a promoter pioneer factor that establishes de novo Pol II pausing during development.
  3. epiAneufinder identifies copy number alterations from single-cell ATAC-seq data.
  4. Epigenomic analysis of formalin-fixed paraffin-embedded samples by CUT&Tag.
  5. Hormone-induced enhancer assembly requires an optimal level of hormone receptor multivalent interactions.
  6. Context-dependent perturbations in chromatin folding and the transcriptome by cohesin and related factors.
  7. TP63 fusions drive multicomplex enhancer rewiring, lymphomagenesis, and EZH2 dependence.
  8. Short tandem repeats bind transcription factors to tune eukaryotic gene expression.
  9. Contributions of histone tail clipping and acetylation in nucleosome transcription by RNA polymerase II.
  10. Bi-directional nucleosome sliding by the Chd1 chromatin remodeler integrates intrinsic sequence-dependent and ATP-dependent nucleosome positioning.
  11. Acetyl-methyllysine marks chromatin at active transcription start sites.
  12. The NCOR-HDAC3 co-repressive complex modulates the leukemogenic potential of the transcription factor ERG.
  13. Perturbations in 3D genome organization can promote acquired drug resistance.
  14. Unannotated microprotein EMBOW regulates the interactome and chromatin and mitotic functions of WDR5.
  15. Single molecule analysis of CENP-A chromatin by high-speed atomic force microscopy.
  16. SCARB2 drives hepatocellular carcinoma tumor initiating cells via enhanced MYC transcriptional activity.
  17. Optimization of Cas12a for multiplexed genome-scale transcriptional activation.
  18. Aberrant gene activation in synovial sarcoma relies on SSX specificity and increased PRC1.1 stability.
  19. Allele specific binding of histone modifications and a transcription factor does not predict allele specific expression in correlated ChIP-seq peak-exon pairs.
  20. A cell size threshold triggers commitment to stomatal fate in Arabidopsis.
  21. Identifying genetic regulatory variants that affect transcription factor activity.
  22. Conformational dynamics of cohesin/Scc2 loading complex are regulated by Smc3 acetylation and ATP binding.
  23. Epigenetic landscape analysis reveals the significance of early reduced chromatin accessibility in osteoclastogenesis.
  24. High-throughput, fluorescent-aptamer-based measurements of steady-state transcription rates for the Mycobacterium tuberculosis RNA polymerase.
  25. A bioinformatics screen reveals hox and chromatin remodeling factors at the Drosophila histone locus.
  1. Nucleic Acids Res. 2023 Sep 22. pii: gkad734. [Epub ahead of print]
    Epigenetic reprogramming of a distal developmental enhancer cluster drives SOX2 overexpression in breast and lung adenocarcinoma.
    Luis E Abatti, Patricia Lado-Fernández, Linh Huynh, Manuel Collado, Michael M Hoffman, Jennifer A Mitchell.
      Enhancer reprogramming has been proposed as a key source of transcriptional dysregulation during tumorigenesis, but the molecular mechanisms underlying this process remain unclear. Here, we identify an enhancer cluster required for normal development that is aberrantly activated in breast and lung adenocarcinoma. Deletion of the SRR124-134 cluster disrupts expression of the SOX2 oncogene, dysregulates genome-wide transcription and chromatin accessibility and reduces the ability of cancer cells to form colonies in vitro. Analysis of primary tumors reveals a correlation between chromatin accessibility at this cluster and SOX2 overexpression in breast and lung cancer patients. We demonstrate that FOXA1 is an activator and NFIB is a repressor of SRR124-134 activity and SOX2 transcription in cancer cells, revealing a co-opting of the regulatory mechanisms involved in early development. Notably, we show that the conserved SRR124 and SRR134 regions are essential during mouse development, where homozygous deletion results in the lethal failure of esophageal-tracheal separation. These findings provide insights into how developmental enhancers can be reprogrammed during tumorigenesis and underscore the importance of understanding enhancer dynamics during development and disease.
    DOI:  https://doi.org/10.1093/nar/gkad734
  2. Nat Commun. 2023 Sep 21. 14(1): 5862
    Lola-I is a promoter pioneer factor that establishes de novo Pol II pausing during development.
    Vivekanandan Ramalingam, Xinyang Yu, Brian D Slaughter, Jay R Unruh, Kaelan J Brennan, Anastasiia Onyshchenko, Jeffrey J Lange, Malini Natarajan, Michael Buck, Julia Zeitlinger.
      While the accessibility of enhancers is dynamically regulated during development, promoters tend to be constitutively accessible and poised for activation by paused Pol II. By studying Lola-I, a Drosophila zinc finger transcription factor, we show here that the promoter state can also be subject to developmental regulation independently of gene activation. Lola-I is ubiquitously expressed at the end of embryogenesis and causes its target promoters to become accessible and acquire paused Pol II throughout the embryo. This promoter transition is required but not sufficient for tissue-specific target gene activation. Lola-I mediates this function by depleting promoter nucleosomes, similar to the action of pioneer factors at enhancers. These results uncover a level of regulation for promoters that is normally found at enhancers and reveal a mechanism for the de novo establishment of paused Pol II at promoters.
    DOI:  https://doi.org/10.1038/s41467-023-41408-1
  3. Nat Commun. 2023 09 20. 14(1): 5846
    epiAneufinder identifies copy number alterations from single-cell ATAC-seq data.
    Akshaya Ramakrishnan, Aikaterini Symeonidi, Patrick Hanel, Katharina T Schmid, Maria L Richter, Michael Schubert, Maria Colomé-Tatché.
      Single-cell open chromatin profiling via scATAC-seq has become a mainstream measurement of open chromatin in single-cells. Here we present epiAneufinder, an algorithm that exploits the read count information from scATAC-seq data to extract genome-wide copy number alterations (CNAs) for individual cells, allowing the study of CNA heterogeneity present in a sample at the single-cell level. Using different cancer scATAC-seq datasets, we show that epiAneufinder can identify intratumor clonal heterogeneity in populations of single cells based on their CNA profiles. We demonstrate that these profiles are concordant with the ones inferred from single-cell whole genome sequencing data for the same samples. EpiAneufinder allows the inference of single-cell CNA information from scATAC-seq data, without the need of additional experiments, unlocking a layer of genomic variation which is otherwise unexplored.
    DOI:  https://doi.org/10.1038/s41467-023-41076-1
  4. Nat Commun. 2023 Sep 22. 14(1): 5930
    Epigenomic analysis of formalin-fixed paraffin-embedded samples by CUT&Tag.
    Steven Henikoff, Jorja G Henikoff, Kami Ahmad, Ronald M Paranal, Derek H Janssens, Zachary R Russell, Frank Szulzewsky, Sita Kugel, Eric C Holland.
      For more than a century, formalin-fixed paraffin-embedded (FFPE) sample preparation has been the preferred method for long-term preservation of biological material. However, the use of FFPE samples for epigenomic studies has been difficult because of chromatin damage from long exposure to high concentrations of formaldehyde. Previously, we introduced Cleavage Under Targeted Accessible Chromatin (CUTAC), an antibody-targeted chromatin accessibility mapping protocol based on CUT&Tag. Here we show that simple modifications of our CUTAC protocol either in single tubes or directly on slides produce high-resolution maps of paused RNA Polymerase II at enhancers and promoters using FFPE samples. We find that transcriptional regulatory element differences produced by FFPE-CUTAC distinguish between mouse brain tumors and identify and map regulatory element markers with high confidence and precision, including microRNAs not detectable by RNA-seq. Our simple workflows make possible affordable epigenomic profiling of archived biological samples for biomarker identification, clinical applications and retrospective studies.
    DOI:  https://doi.org/10.1038/s41467-023-41666-z
  5. Mol Cell. 2023 Sep 14. pii: S1097-2765(23)00690-1. [Epub ahead of print]
    Hormone-induced enhancer assembly requires an optimal level of hormone receptor multivalent interactions.
    Lizhen Chen, Zhao Zhang, Qinyu Han, Barun K Maity, Leticia Rodrigues, Emily Zboril, Rashmi Adhikari, Su-Hyuk Ko, Xin Li, Shawn R Yoshida, Pengya Xue, Emilie Smith, Kexin Xu, Qianben Wang, Tim Hui-Ming Huang, Shasha Chong, Zhijie Liu.
      Transcription factors (TFs) activate enhancers to drive cell-specific gene programs in response to signals, but our understanding of enhancer assembly during signaling events is incomplete. Here, we show that androgen receptor (AR) forms condensates through multivalent interactions mediated by its N-terminal intrinsically disordered region (IDR) to orchestrate enhancer assembly in response to androgen signaling. AR IDR can be substituted by IDRs from selective proteins for AR condensation capacity and its function on enhancers. Expansion of the poly(Q) track within AR IDR results in a higher AR condensation propensity as measured by multiple methods, including live-cell single-molecule microscopy. Either weakening or strengthening AR condensation propensity impairs its heterotypic multivalent interactions with other enhancer components and diminishes its transcriptional activity. Our work reveals the requirement of an optimal level of AR condensation in mediating enhancer assembly and suggests that alteration of the fine-tuned multivalent IDR-IDR interactions might underlie AR-related human pathologies.
    Keywords:  androgen receptor; condensate formation; condensation; enhancer; hormone-induced enhancer assembly; intrinsically disordered region; multivalent interaction; phase separation
    DOI:  https://doi.org/10.1016/j.molcel.2023.08.027
  6. Nat Commun. 2023 09 19. 14(1): 5647
    Context-dependent perturbations in chromatin folding and the transcriptome by cohesin and related factors.
    Ryuichiro Nakato, Toyonori Sakata, Jiankang Wang, Luis Augusto Eijy Nagai, Yuya Nagaoka, Gina Miku Oba, Masashige Bando, Katsuhiko Shirahige.
      Cohesin regulates gene expression through context-specific chromatin folding mechanisms such as enhancer-promoter looping and topologically associating domain (TAD) formation by cooperating with factors such as cohesin loaders and the insulation factor CTCF. We developed a computational workflow to explore how three-dimensional (3D) structure and gene expression are regulated collectively or individually by cohesin and related factors. The main component is CustardPy, by which multi-omics datasets are compared systematically. To validate our methodology, we generated 3D genome, transcriptome, and epigenome data before and after depletion of cohesin and related factors and compared the effects of depletion. We observed diverse effects on the 3D genome and transcriptome, and gene expression changes were correlated with the splitting of TADs caused by cohesin loss. We also observed variations in long-range interactions across TADs, which correlated with their epigenomic states. These computational tools and datasets will be valuable for 3D genome and epigenome studies.
    DOI:  https://doi.org/10.1038/s41467-023-41316-4
  7. Sci Transl Med. 2023 Sep 20. 15(714): eadi7244
    TP63 fusions drive multicomplex enhancer rewiring, lymphomagenesis, and EZH2 dependence.
    Gongwei Wu, Noriaki Yoshida, Jihe Liu, Xiaoyang Zhang, Yuan Xiong, Tayla B Heavican-Foral, Elisa Mandato, Huiyun Liu, Geoffrey M Nelson, Lu Yang, Renee Chen, Katherine A Donovan, Marcus K Jones, Mikhail Roshal, Yanming Zhang, Ran Xu, Ajit J Nirmal, Salvia Jain, Catharine Leahy, Kristen L Jones, Kristen E Stevenson, Natasha Galasso, Nivetha Ganesan, Tiffany Chang, Wen-Chao Wu, Abner Louissaint, Lydie Debaize, Hojong Yoon, Paola Dal Cin, Wing C Chan, Shannan J Ho Sui, Samuel Y Ng, Andrew L Feldman, Steven M Horwitz, Karen Adelman, Eric S Fischer, Chun-Wei Chen, David M Weinstock, Myles Brown.
      Gene fusions involving tumor protein p63 gene (TP63) occur in multiple T and B cell lymphomas and portend a dismal prognosis for patients. The function and mechanisms of TP63 fusions remain unclear, and there is no target therapy for patients with lymphoma harboring TP63 fusions. Here, we show that TP63 fusions act as bona fide oncogenes and are essential for fusion-positive lymphomas. Transgenic mice expressing TBL1XR1::TP63, the most common TP63 fusion, develop diverse lymphomas that recapitulate multiple human T and B cell lymphomas. Here, we identify that TP63 fusions coordinate the recruitment of two epigenetic modifying complexes, the nuclear receptor corepressor (NCoR)-histone deacetylase 3 (HDAC3) by the N-terminal TP63 fusion partner and the lysine methyltransferase 2D (KMT2D) by the C-terminal TP63 component, which are both required for fusion-dependent survival. TBL1XR1::TP63 localization at enhancers drives a unique cell state that involves up-regulation of MYC and the polycomb repressor complex 2 (PRC2) components EED and EZH2. Inhibiting EZH2 with the therapeutic agent valemetostat is highly effective at treating transgenic lymphoma murine models, xenografts, and patient-derived xenografts harboring TP63 fusions. One patient with TP63-rearranged lymphoma showed a rapid response to valemetostat treatment. In summary, TP63 fusions link partner components that, together, coordinate multiple epigenetic complexes, resulting in therapeutic vulnerability to EZH2 inhibition.
    DOI:  https://doi.org/10.1126/scitranslmed.adi7244
  8. Science. 2023 Sep 22. 381(6664): eadd1250
    Short tandem repeats bind transcription factors to tune eukaryotic gene expression.
    Connor A Horton, Amr M Alexandari, Michael G B Hayes, Emil Marklund, Julia M Schaepe, Arjun K Aditham, Nilay Shah, Peter H Suzuki, Avanti Shrikumar, Ariel Afek, William J Greenleaf, Raluca Gordân, Julia Zeitlinger, Anshul Kundaje, Polly M Fordyce.
      Short tandem repeats (STRs) are enriched in eukaryotic cis-regulatory elements and alter gene expression, yet how they regulate transcription remains unknown. We found that STRs modulate transcription factor (TF)-DNA affinities and apparent on-rates by about 70-fold by directly binding TF DNA-binding domains, with energetic impacts exceeding many consensus motif mutations. STRs maximize the number of weakly preferred microstates near target sites, thereby increasing TF density, with impacts well predicted by statistical mechanics. Confirming that STRs also affect TF binding in cells, neural networks trained only on in vivo occupancies predicted effects identical to those observed in vitro. Approximately 90% of TFs preferentially bound STRs that need not resemble known motifs, providing a cis-regulatory mechanism to target TFs to genomic sites.
    DOI:  https://doi.org/10.1126/science.add1250
  9. Nucleic Acids Res. 2023 Sep 18. pii: gkad754. [Epub ahead of print]
    Contributions of histone tail clipping and acetylation in nucleosome transcription by RNA polymerase II.
    Takumi Oishi, Suguru Hatazawa, Tomoya Kujirai, Junko Kato, Yuki Kobayashi, Mitsuo Ogasawara, Munetaka Akatsu, Haruhiko Ehara, Shun-Ichi Sekine, Gosuke Hayashi, Yoshimasa Takizawa, Hitoshi Kurumizaka.
      The N-terminal tails of histones protrude from the nucleosome core and are target sites for histone modifications, such as acetylation and methylation. Histone acetylation is considered to enhance transcription in chromatin. However, the contribution of the histone N-terminal tail to the nucleosome transcription by RNA polymerase II (RNAPII) has not been clarified. In the present study, we reconstituted nucleosomes lacking the N-terminal tail of each histone, H2A, H2B, H3 or H4, and performed RNAPII transcription assays. We found that the N-terminal tail of H3, but not H2A, H2B and H4, functions in RNAPII pausing at the SHL(-5) position of the nucleosome. Consistently, the RNAPII transcription assay also revealed that the nucleosome containing N-terminally acetylated H3 drastically alleviates RNAPII pausing at the SHL(-5) position. In addition, the H3 acetylated nucleosome produced increased amounts of the run-off transcript. These results provide important evidence that the H3 N-terminal tail plays a role in RNAPII pausing at the SHL(-5) position of the nucleosome, and its acetylation directly alleviates this nucleosome barrier.
    DOI:  https://doi.org/10.1093/nar/gkad754
  10. Nucleic Acids Res. 2023 Sep 20. pii: gkad738. [Epub ahead of print]
    Bi-directional nucleosome sliding by the Chd1 chromatin remodeler integrates intrinsic sequence-dependent and ATP-dependent nucleosome positioning.
    Sangwoo Park, Giovanni B Brandani, Taekjip Ha, Gregory D Bowman.
      Chromatin remodelers use a helicase-type ATPase motor to shift DNA around the histone core. Although not directly reading out the DNA sequence, some chromatin remodelers exhibit a sequence-dependent bias in nucleosome positioning, which presumably reflects properties of the DNA duplex. Here, we show how nucleosome positioning by the Chd1 remodeler is influenced by local DNA perturbations throughout the nucleosome footprint. Using site-specific DNA cleavage coupled with next-generation sequencing, we show that nucleosomes shifted by Chd1 can preferentially localize DNA perturbations - poly(dA:dT) tracts, DNA mismatches, and single-nucleotide insertions - about a helical turn outside the Chd1 motor domain binding site, super helix location 2 (SHL2). This phenomenon occurs with both the Widom 601 positioning sequence and the natural +1 nucleosome sequence from the Saccharomyces cerevisiae SWH1 gene. Our modeling indicates that localization of DNA perturbations about a helical turn outward from SHL2 results from back-and-forth sliding due to remodeler action on both sides of the nucleosome. Our results also show that barrier effects from DNA perturbations can be extended by the strong phasing of nucleosome positioning sequences.
    DOI:  https://doi.org/10.1093/nar/gkad738
  11. Nature. 2023 Sep 20.
    Acetyl-methyllysine marks chromatin at active transcription start sites.
    William J Lu-Culligan, Leah J Connor, Yixuan Xie, Babatunde E Ekundayo, Brendan T Rose, Martin Machyna, Andreas P Pintado-Urbanc, Joshua T Zimmer, Isaac W Vock, Natarajan V Bhanu, Megan C King, Benjamin A Garcia, Franziska Bleichert, Matthew D Simon.
      Lysine residues in histones and other proteins can be modified by post-translational modifications that encode regulatory information1. Lysine acetylation and methylation are especially important for regulating chromatin and gene expression2-4. Pathways involving these post-translational modifications are targets for clinically approved therapeutics to treat human diseases. Lysine methylation and acetylation are generally assumed to be mutually exclusive at the same residue. Here we report cellular lysine residues that are both methylated and acetylated on the same side chain to form Nε-acetyl-Nε-methyllysine (Kacme). We show that Kacme is found on histone H4 (H4Kacme) across a range of species and across mammalian tissues. Kacme is associated with marks of active chromatin, increased transcriptional initiation and is regulated in response to biological signals. H4Kacme can be installed by enzymatic acetylation of monomethyllysine peptides and is resistant to deacetylation by some HDACs in vitro. Kacme can be bound by chromatin proteins that recognize modified lysine residues, as we demonstrate with the crystal structure of acetyllysine-binding protein BRD2 bound to a histone H4Kacme peptide. These results establish Kacme as a cellular post-translational modification with the potential to encode information distinct from methylation and acetylation alone and demonstrate that Kacme has all the hallmarks of a post-translational modification with fundamental importance to chromatin biology.
    DOI:  https://doi.org/10.1038/s41586-023-06565-9
  12. Nat Commun. 2023 Sep 21. 14(1): 5871
    The NCOR-HDAC3 co-repressive complex modulates the leukemogenic potential of the transcription factor ERG.
    Eitan Kugler, Shreyas Madiwale, Darren Yong, Julie A I Thoms, Yehudit Birger, David B Sykes, Johannes Schmoellerl, Aneta Drakul, Valdemar Priebe, Muhammad Yassin, Nasma Aqaqe, Avigail Rein, Hila Fishman, Ifat Geron, Chun-Wei Chen, Brian Raught, Qiao Liu, Heather Ogana, Elisabeth Liedke, Jean-Pierre Bourquin, Johannes Zuber, Michael Milyavsky, John Pimanda, Gilbert G Privé, Shai Izraeli.
      The ERG (ETS-related gene) transcription factor is linked to various types of cancer, including leukemia. However, the specific ERG domains and co-factors contributing to leukemogenesis are poorly understood. Drug targeting a transcription factor such as ERG is challenging. Our study reveals the critical role of a conserved amino acid, proline, at position 199, located at the 3' end of the PNT (pointed) domain, in ERG's ability to induce leukemia. P199 is necessary for ERG to promote self-renewal, prevent myeloid differentiation in hematopoietic progenitor cells, and initiate leukemia in mouse models. Here we show that P199 facilitates ERG's interaction with the NCoR-HDAC3 co-repressor complex. Inhibiting HDAC3 reduces the growth of ERG-dependent leukemic and prostate cancer cells, indicating that the interaction between ERG and the NCoR-HDAC3 co-repressor complex is crucial for its oncogenic activity. Thus, targeting this interaction may offer a potential therapeutic intervention.
    DOI:  https://doi.org/10.1038/s41467-023-41067-2
  13. Cell Rep. 2023 Sep 20. pii: S2211-1247(23)01136-1. [Epub ahead of print]42(10): 113124
    Perturbations in 3D genome organization can promote acquired drug resistance.
    Anna G Manjón, Stefano Giustino Manzo, Stefan Prekovic, Leon Potgeter, Tom van Schaik, Ning Qing Liu, Koen Flach, Daniel Peric-Hupkes, Stacey Joosten, Hans Teunissen, Anoek Friskes, Mila Ilic, Dorine Hintzen, Vinícius H Franceschini-Santos, Wilbert Zwart, Elzo de Wit, Bas van Steensel, René H Medema.
      Acquired drug resistance is a major problem in the treatment of cancer. hTERT-immortalized, untransformed RPE-1 cells can acquire resistance to Taxol by derepressing the ABCB1 gene, encoding for the multidrug transporter P-gP. Here, we investigate how the ABCB1 gene is derepressed. ABCB1 activation is associated with reduced H3K9 trimethylation, increased H3K27 acetylation, and ABCB1 displacement from the nuclear lamina. While altering DNA methylation and H3K27 methylation had no major impact on ABCB1 expression, nor did it promote resistance, disrupting the nuclear lamina component Lamin B Receptor did promote the acquisition of a Taxol-resistant phenotype in a subset of cells. CRISPRa-mediated gene activation supported the notion that lamina dissociation influences ABCB1 derepression. We propose a model in which nuclear lamina dissociation of a repressed gene allows for its activation, implying that deregulation of the 3D genome topology could play an important role in tumor evolution and the acquisition of drug resistance.
    Keywords:  3D genome; CP: Molecular biology; chromatin; drug resistance; gene regulation; nuclear lamina
    DOI:  https://doi.org/10.1016/j.celrep.2023.113124
  14. Cell Rep. 2023 Sep 18. pii: S2211-1247(23)01157-9. [Epub ahead of print]42(9): 113145
    Unannotated microprotein EMBOW regulates the interactome and chromatin and mitotic functions of WDR5.
    Yanran Chen, Haomiao Su, Jianing Zhao, Zhenkun Na, Kevin Jiang, Antonella Bacchiocchi, Ken H Loh, Ruth Halaban, Zhentian Wang, Xiongwen Cao, Sarah A Slavoff.
      The conserved WD40-repeat protein WDR5 interacts with multiple proteins both inside and outside the nucleus. However, it is currently unclear whether and how the distribution of WDR5 between complexes is regulated. Here, we show that an unannotated microprotein EMBOW (endogenous microprotein binder of WDR5) dually encoded in the human SCRIB gene interacts with WDR5 and regulates its binding to multiple interaction partners, including KMT2A and KIF2A. EMBOW is cell cycle regulated, with two expression maxima at late G1 phase and G2/M phase. Loss of EMBOW decreases WDR5 interaction with KIF2A, aberrantly shortens mitotic spindle length, prolongs G2/M phase, and delays cell proliferation. In contrast, loss of EMBOW increases WDR5 interaction with KMT2A, leading to WDR5 binding to off-target genes, erroneously increasing H3K4me3 levels, and activating transcription of these genes. Together, these results implicate EMBOW as a regulator of WDR5 that regulates its interactions and prevents its off-target binding in multiple contexts.
    Keywords:  CP: Cell biology; CP: Molecular biology; WDR5; histone H3K4me3; microprotein; mitosis; transcription
    DOI:  https://doi.org/10.1016/j.celrep.2023.113145
  15. Elife. 2023 Sep 20. pii: e86709. [Epub ahead of print]12
    Single molecule analysis of CENP-A chromatin by high-speed atomic force microscopy.
    Daniël P Melters, Keir C Neuman, Reda S Bentahar, Tatini Rakshit, Yamini Dalal.
      Chromatin accessibility is modulated in a variety of ways to create open and closed chromatin states, both of which are critical for eukaryotic gene regulation. At the single molecule level, how accessibility is regulated of the chromatin fiber composed of canonical or variant nucleosomes is a fundamental question in the field. Here, we developed a single-molecule tracking method where we could analyze thousands of canonical H3 and centromeric variant nucleosomes imaged by high-speed atomic force microscopy. This approach allowed us to investigate how changes in nucleosome dynamics in vitro inform us about transcriptional potential in vivo. By high-speed atomic force microscopy, we tracked chromatin dynamics in real time and determined the mean square displacement and diffusion constant for the variant centromeric CENP-A nucleosome. Furthermore, we found that an essential kinetochore protein CENP-C reduces the diffusion constant and mobility of centromeric nucleosomes along the chromatin fiber. We subsequently interrogated how CENP-C modulates CENP-A chromatin dynamics in vivo. Overexpressing CENP-C resulted in reduced centromeric transcription and impaired loading of new CENP-A molecules. From these data, we speculate that factors altering nucleosome mobility in vitro, also correspondingly alter transcription in vivo. Subsequently, we propose a model in which variant nucleosomes encode their own diffusion kinetics and mobility, and where binding partners can suppress or enhance nucleosome mobility.
    Keywords:  chromatin; chromosomes; epigenetics; gene expression; high-speed AFM; human; none; nucleosomes; single-molecule
    DOI:  https://doi.org/10.7554/eLife.86709
  16. Nat Commun. 2023 Sep 22. 14(1): 5917
    SCARB2 drives hepatocellular carcinoma tumor initiating cells via enhanced MYC transcriptional activity.
    Feng Wang, Yang Gao, Situ Xue, Luyao Zhao, Huimin Jiang, Tingting Zhang, Yunxuan Li, Chenxi Zhao, Fan Wu, Tana Siqin, Ying Liu, Jie Wu, Yechao Yan, Jian Yuan, Jian-Dong Jiang, Ke Li.
      CSCs (Cancer stem cells) with distinct metabolic features are considered to cause HCC (hepatocellular carcinoma) initiation, metastasis and therapeutic resistance. Here, we perform a metabolic gene CRISPR/Cas9 knockout library screen in tumorspheres derived from HCC cells and find that deletion of SCARB2 suppresses the cancer stem cell-like properties of HCC cells. Knockout of Scarb2 in hepatocytes attenuates HCC initiation and progression in both MYC-driven and DEN (diethylnitrosamine)-induced HCC mouse models. Mechanistically, binding of SCARB2 with MYC promotes MYC acetylation by interfering with HDCA3-mediated MYC deacetylation on lysine 148 and subsequently enhances MYC transcriptional activity. Screening of a database of FDA (Food and Drug Administration)-approved drugs shows Polymyxin B displays high binding affinity for SCARB2 protein, disrupts the SCARB2-MYC interaction, decreases MYC activity, and reduces the tumor burden. Our study identifies SCARB2 as a functional driver of HCC and suggests Polymyxin B-based treatment as a targeted therapeutic option for HCC.
    DOI:  https://doi.org/10.1038/s41467-023-41593-z
  17. Cell Genom. 2023 Sep 13. 3(9): 100387
    Optimization of Cas12a for multiplexed genome-scale transcriptional activation.
    Audrey L Griffith, Fengyi Zheng, Abby V McGee, Nathan W Miller, Zsofia M Szegletes, Ganna Reint, Fabian Gademann, Ifunanya Nwolah, Mudra Hegde, Yanjing V Liu, Amy Goodale, John G Doench.
      Cas12a CRISPR technology, unlike Cas9, allows for facile multiplexing of guide RNAs from a single transcript, simplifying combinatorial perturbations. While Cas12a has been implemented for multiplexed knockout genetic screens, it has yet to be optimized for CRISPR activation (CRISPRa) screens in human cells. Here, we develop a new Cas12a-based transactivation domain (TAD) recruitment system using the ALFA nanobody and demonstrate simultaneous activation of up to four genes. We screen a genome-wide library to identify modulators of growth and MEK inhibition, and we compare these results with those obtained with open reading frame (ORF) overexpression and Cas9-based CRISPRa. We find that the activity of multiplexed arrays is largely predictable from the best-performing guide and provide criteria for selecting active guides. We anticipate that these results will greatly accelerate the exploration of gene function and combinatorial phenotypes at scale.
    Keywords:  CRISPR activation; Cas12a; functional genomics; genetic screens
    DOI:  https://doi.org/10.1016/j.xgen.2023.100387
  18. Nat Struct Mol Biol. 2023 Sep 21.
    Aberrant gene activation in synovial sarcoma relies on SSX specificity and increased PRC1.1 stability.
    Nezha S Benabdallah, Vineet Dalal, R Wilder Scott, Fady Marcous, Afroditi Sotiriou, Felix K F Kommoss, Anastasija Pejkovska, Ludmila Gaspar, Lena Wagner, Francisco J Sánchez-Rivera, Monica Ta, Shelby Thornton, Torsten O Nielsen, T Michael Underhill, Ana Banito.
      The SS18-SSX fusion drives oncogenic transformation in synovial sarcoma by bridging SS18, a member of the mSWI/SNF (BAF) complex, to Polycomb repressive complex 1 (PRC1) target genes. Here we show that the ability of SS18-SSX to occupy H2AK119ub1-rich regions is an intrinsic property of its SSX C terminus, which can be exploited by fusion to transcriptional regulators beyond SS18. Accordingly, SS18-SSX recruitment occurs in a manner that is independent of the core components and catalytic activity of BAF. Alternative SSX fusions are also recruited to H2AK119ub1-rich chromatin and reproduce the expression signatures of SS18-SSX by engaging with transcriptional activators. Variant Polycomb repressive complex 1.1 (PRC1.1) acts as the main depositor of H2AK119ub1 and is therefore required for SS18-SSX occupancy. Importantly, the SSX C terminus not only depends on H2AK119ub1 for localization, but also further increases it by promoting PRC1.1 complex stability. Consequently, high H2AK119ub1 levels are a feature of murine and human synovial sarcomas. These results uncover a critical role for SSX-C in mediating gene deregulation in synovial sarcoma by providing specificity to chromatin and further enabling oncofusion binding by enhancing PRC1.1 stability and H2AK119ub1 deposition.
    DOI:  https://doi.org/10.1038/s41594-023-01096-3
  19. Sci Rep. 2023 Sep 20. 13(1): 15596
    Allele specific binding of histone modifications and a transcription factor does not predict allele specific expression in correlated ChIP-seq peak-exon pairs.
    Claire P Prowse-Wilkins, Jianghui Wang, Josie B Garner, Michael E Goddard, Amanda J Chamberlain.
      Allele specific expression (ASE) is widespread in many species including cows. Therefore, regulatory regions which control gene expression should show cis-regulatory variation which mirrors this differential expression within the animal. ChIP-seq peaks for histone modifications and transcription factors measure activity at functional regions and the height of some peaks have been shown to correlate across tissues with the expression of particular genes, suggesting these peaks are putative regulatory regions. In this study we identified ASE in the bovine genome in multiple tissues and investigated whether ChIP-seq peaks for four histone modifications and the transcription factor CTCF show allele specific binding (ASB) differences in the same tissues. We then investigate whether peak height and gene expression, which correlates across tissues, also correlates within the animal by investigating whether the direction of ASB in putative regulatory regions, mirrors that of the ASE in the genes they are putatively regulating. We found that ASE and ASB were widespread in the bovine genome but vary in extent between tissues. However, even when the height of a peak was positively correlated across tissues with expression of an exon, ASE of the exon and ASB of the peak were in the same direction only half the time. A likely explanation for this finding is that the correlations between peak height and exon expression do not indicate that the height of the peak causes the extent of exon expression, at least in some cases.
    DOI:  https://doi.org/10.1038/s41598-023-42637-6
  20. Sci Adv. 2023 Sep 22. 9(38): eadf3497
    A cell size threshold triggers commitment to stomatal fate in Arabidopsis.
    Yan Gong, Renee Dale, Hannah F Fung, Gabriel O Amador, Margot E Smit, Dominique C Bergmann.
      How flexible developmental programs integrate information from internal and external factors to modulate stem cell behavior is a fundamental question in developmental biology. Cells of the Arabidopsis stomatal lineage modify the balance of stem cell proliferation and differentiation to adjust the size and cell type composition of mature leaves. Here, we report that meristemoids, one type of stomatal lineage stem cell, trigger the transition from asymmetric self-renewing divisions to commitment and terminal differentiation by crossing a critical cell size threshold. Through computational simulation, we demonstrate that this cell size-mediated transition allows robust, yet flexible termination of stem cell proliferation, and we observe adjustments in the number of divisions before the differentiation threshold under several genetic manipulations. We experimentally evaluate several mechanisms for cell size sensing, and our data suggest that this stomatal lineage transition is dependent on a nuclear factor that is sensitive to DNA content.
    DOI:  https://doi.org/10.1126/sciadv.adf3497
  21. Cell Genom. 2023 Sep 13. 3(9): 100382
    Identifying genetic regulatory variants that affect transcription factor activity.
    Xiaoting Li, Tuuli Lappalainen, Harmen J Bussemaker.
      Genetic variants affecting gene expression levels in humans have been mapped in the Genotype-Tissue Expression (GTEx) project. Trans-acting variants impacting many genes simultaneously through a shared transcription factor (TF) are of particular interest. Here, we developed a generalized linear model (GLM) to estimate protein-level TF activity levels in an individual-specific manner from GTEx RNA sequencing (RNA-seq) profiles. It uses observed differential gene expression after TF perturbation as a predictor and, by analyzing differential expression within pairs of neighboring genes, controls for the confounding effect of variation in chromatin state along the genome. We inferred genotype-specific activities for 55 TFs across 49 tissues. Subsequently performing genome-wide association analysis on this virtual trait revealed TF activity quantitative trait loci (aQTLs) that, as a set, are enriched for functional features. Altogether, the set of tools we introduce here highlights the potential of genetic association studies for cellular endophenotypes based on a network-based multi-omics approach. The transparent peer review record is available.
    Keywords:  RNA-seq data; aQTL; beta-binomial distribution; gene regulation; generalized linear model; genetic variation; genome-wide association; quantitative trait locus; trans-acting genetic variation; transcription factor activity; transcription factor perturbation signatures
    DOI:  https://doi.org/10.1016/j.xgen.2023.100382
  22. Nat Commun. 2023 Sep 22. 14(1): 5929
    Conformational dynamics of cohesin/Scc2 loading complex are regulated by Smc3 acetylation and ATP binding.
    Aditi Kaushik, Thane Than, Naomi J Petela, Menelaos Voulgaris, Charlotte Percival, Peter Daniels, John B Rafferty, Kim A Nasmyth, Bin Hu.
      The ring-shaped cohesin complex is a key player in sister chromatid cohesion, DNA repair, and gene transcription. The loading of cohesin to chromosomes requires the loader Scc2 and is regulated by ATP. This process is hindered by Smc3 acetylation. However, the molecular mechanism underlying this inhibition remains mysterious. Here, using Saccharomyces cerevisiae as a model system, we identify a novel configuration of Scc2 with pre-engaged cohesin and reveal dynamic conformations of the cohesin/Scc2 complex in the loading reaction. We demonstrate that Smc3 acetylation blocks the association of Scc2 with pre-engaged cohesin by impairing the interaction of Scc2 with Smc3's head. Lastly, we show that ATP binding induces the cohesin/Scc2 complex to clamp DNA by promoting the interaction between Scc2 and Smc3 coiled coil. Our results illuminate a dynamic reconfiguration of the cohesin/Scc2 complex during loading and indicate how Smc3 acetylation and ATP regulate this process.
    DOI:  https://doi.org/10.1038/s41467-023-41596-w
  23. Bone. 2023 Sep 20. pii: S8756-3282(23)00251-X. [Epub ahead of print] 116918
    Epigenetic landscape analysis reveals the significance of early reduced chromatin accessibility in osteoclastogenesis.
    Sangyong Lee, Myoung Jun Kim, Seor I Ahn, Sung Kyung Choi, Keun Young Min, Wahn Soo Choi, Jueng Soo You.
      Recently improved techniques could provide snapshots of chromatin structure generated based on chromatin accessibility. Since chromatin accessibility determines transcriptional potential, it has been attempted in a variety of cell systems. However, there has been no genome-wide analysis of chromatin accessibility for the entire murine osteoclast (OC) differentiation process. We performed an Assay for Transposase-Accessible Chromatin (ATAC)-sequencing (seq) during RANKL-induced OC differentiation and found that global chromatin accessibility decreased, especially early in OC differentiation. The global histone H3K27Ac level, an active histone modification mark, was diminished during OC differentiation by western blot and histone extract experiments. Its genomic enrichment was also reduced based on publicly available H3K27Ac chromatin immunoprecipitation (ChIP)-seq data. ATAC-seq and H3K27Ac ChIP-seq data demonstrated that RANKL induced a less accessible chromatin state during OC differentiation. Restoration of reduced H3K27Ac, presumably representing accessible states upon acetate treatment, suppresses OC differentiation by provoking immune-related gene expression. Subsequential integrative analysis of ATAC-seq, RNA-seq after acetate treatment, and H3K27Ac ChIP-seq reveals that Irf8 and its downstream targets are the most vulnerable to chromatin accessibility changes and acetate supplementation. Taken together, our study generated chromatin accessibility maps during the whole OC differentiation and suggested perturbation of chromatin accessibility might be a potential therapeutic strategy for excessive OC diseases.
    Keywords:  ATAC-seq; Chromatin accessibility; Epigenetic landscape; H3K27Ac; Irf8; Osteoclast
    DOI:  https://doi.org/10.1016/j.bone.2023.116918
  24. Nucleic Acids Res. 2023 Sep 22. pii: gkad761. [Epub ahead of print]
    High-throughput, fluorescent-aptamer-based measurements of steady-state transcription rates for the Mycobacterium tuberculosis RNA polymerase.
    Drake Jensen, Ana Ruiz Manzano, Maxwell Rector, Eric J Tomko, M Thomas Record, Eric A Galburt.
      The first step in gene expression is the transcription of DNA sequences into RNA. Regulation at the level of transcription leads to changes in steady-state concentrations of RNA transcripts, affecting the flux of downstream functions and ultimately cellular phenotypes. Changes in transcript levels are routinely followed in cellular contexts via genome-wide sequencing techniques. However, in vitro mechanistic studies of transcription have lagged with respect to throughput. Here, we describe the use of a real-time, fluorescent-aptamer-based method to quantitate steady-state transcription rates of the Mycobacterium tuberculosis RNA polymerase. We present clear controls to show that the assay specifically reports on promoter-dependent, full-length RNA transcription rates that are in good agreement with the kinetics determined by gel-resolved, α-32P NTP incorporation experiments. We illustrate how the time-dependent changes in fluorescence can be used to measure regulatory effects of nucleotide concentrations and identity, RNAP and DNA concentrations, transcription factors, and antibiotics. Our data showcase the ability to easily perform hundreds of parallel steady-state measurements across varying conditions with high precision and reproducibility to facilitate the study of the molecular mechanisms of bacterial transcription.
    DOI:  https://doi.org/10.1093/nar/gkad761
  25. BMC Genom Data. 2023 Sep 21. 24(1): 54
    A bioinformatics screen reveals hox and chromatin remodeling factors at the Drosophila histone locus.
    Lauren J Hodkinson, Connor Smith, H Skye Comstra, Bukola A Ajani, Eric H Albanese, Kawsar Arsalan, Alvaro Perez Daisson, Katherine B Forrest, Elijah H Fox, Matthew R Guerette, Samia Khan, Madeleine P Koenig, Shivani Lam, Ava S Lewandowski, Lauren J Mahoney, Nasserallah Manai, JonCarlo Miglay, Blake A Miller, Olivia Milloway, Nhi Ngo, Vu D Ngo, Nicole F Oey, Tanya A Punjani, HaoMin SiMa, Hollis Zeng, Casey A Schmidt, Leila E Rieder.
       BACKGROUND: Cells orchestrate histone biogenesis with strict temporal and quantitative control. To efficiently regulate histone biogenesis, the repetitive Drosophila melanogaster replication-dependent histone genes are arrayed and clustered at a single locus. Regulatory factors concentrate in a nuclear body known as the histone locus body (HLB), which forms around the locus. Historically, HLB factors are largely discovered by chance, and few are known to interact directly with DNA. It is therefore unclear how the histone genes are specifically targeted for unique and coordinated regulation.
    RESULTS: To expand the list of known HLB factors, we performed a candidate-based screen by mapping 30 publicly available ChIP datasets of 27 unique factors to the Drosophila histone gene array. We identified novel transcription factor candidates, including the Drosophila Hox proteins Ultrabithorax (Ubx), Abdominal-A (Abd-A), and Abdominal-B (Abd-B), suggesting a new pathway for these factors in influencing body plan morphogenesis. Additionally, we identified six other factors that target the histone gene array: JIL-1, hormone-like receptor 78 (Hr78), the long isoform of female sterile homeotic (1) (fs(1)h) as well as the general transcription factors TBP associated factor 1 (TAF-1), Transcription Factor IIB (TFIIB), and Transcription Factor IIF (TFIIF).
    CONCLUSIONS: Our foundational screen provides several candidates for future studies into factors that may influence histone biogenesis. Further, our study emphasizes the powerful reservoir of publicly available datasets, which can be mined as a primary screening technique.
    Keywords:  ChIP-seq; Course-based Undergraduate Research Experience; Drosophila; Galaxy; Histone locus; Histone locus body; Hox factors
    DOI:  https://doi.org/10.1186/s12863-023-01147-0
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