bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2024–12–22
25 papers selected by
Connor Rogerson, University of Cambridge
  1. Integrative analysis of the 3D genome and epigenome in mouse embryonic tissues.
  2. Nucleosome fibre topology guides transcription factor binding to enhancers.
  3. Rapid degradation of histone deacetylase 1 (HDAC1) reveals essential roles in both gene repression and active transcription.
  4. Abo1 ATPase facilitates the dissociation of FACT from chromatin.
  5. BRD8 Guards the Pluripotent State by Sensing and Maintaining Histone Acetylation.
  6. ATAC and SAGA histone acetyltransferase modules facilitate transcription factor binding to nucleosomes independent of their acetylation activity.
  7. A genome-wide nucleosome-resolution map of promoter-centered interactions in human cells corroborates the enhancer-promoter looping model.
  8. Histone methyltransferase SETDB1 safeguards mouse fetal hematopoiesis by suppressing activation of cryptic enhancers.
  9. Transcriptional regulatory network reveals key transcription factors for regulating agronomic traits in soybean.
  10. Multiome Perturb-seq unlocks scalable discovery of integrated perturbation effects on the transcriptome and epigenome.
  11. Genome-wide single-cell and single-molecule footprinting of transcription factors with deaminase.
  12. The three-dimensional genome drives the evolution of asymmetric gene duplicates via enhancer capture-divergence.
  13. Engineered extrachromosomal oncogene amplifications promote tumorigenesis.
  14. Conformational switching of Arp5 subunit regulates INO80 chromatin remodeling.
  15. Remodeling of Individual Nucleosomes in Nucleosome Arrays.
  16. The emergence and evolution of gene expression in genome regions replete with regulatory motifs.
  17. ZNF143 binds DNA and stimulates transcription initiation to activate and repress direct target genes.
  18. PRMT1-mediated methylation regulates MLL2 stability and gene expression.
  19. Dissecting the Kaiso binding profile in clear renal cancer cells.
  20. Assessing the Effect of Chromatin-Binding Enzymes on Condensed Chromatin with Optical Tweezers.
  21. A modular toolbox for the optogenetic deactivation of transcription.
  22. EpiGePT: a pretrained transformer-based language model for context-specific human epigenomics.
  23. HiCrayon reveals distinct layers of multi-state 3D chromatin organization.
  24. HIPSD&R-seq enables scalable genomic copy number and transcriptome profiling.
  25. Progressive chromatin rewiring by ETO2::GLIS2 revealed in a human iPSC model of pediatric leukemia initiation.
  1. Nat Struct Mol Biol. 2024 Dec 16.
    Integrative analysis of the 3D genome and epigenome in mouse embryonic tissues.
    Miao Yu, Nathan R Zemke, Ziyin Chen, Ivan Juric, Rong Hu, Ramya Raviram, Armen Abnousi, Rongxin Fang, Yanxiao Zhang, David U Gorkin, Yang E Li, Yuan Zhao, Lindsay Lee, Shreya Mishra, Anthony D Schmitt, Yunjiang Qiu, Diane E Dickel, Axel Visel, Len A Pennacchio, Ming Hu, Bing Ren.
      While a rich set of putative cis-regulatory sequences involved in mouse fetal development have been annotated recently on the basis of chromatin accessibility and histone modification patterns, delineating their role in developmentally regulated gene expression continues to be challenging. To fill this gap, here we mapped chromatin contacts between gene promoters and distal sequences across the genome in seven mouse fetal tissues and across six developmental stages of the forebrain. We identified 248,620 long-range chromatin interactions centered at 14,138 protein-coding genes and characterized their tissue-to-tissue variations and developmental dynamics. Integrative analysis of the interactome with previous epigenome and transcriptome datasets from the same tissues revealed a strong correlation between the chromatin contacts and chromatin state at distal enhancers, as well as gene expression patterns at predicted target genes. We predicted target genes of 15,098 candidate enhancers and used them to annotate target genes of homologous candidate enhancers in the human genome that harbor risk variants of human diseases. We present evidence that schizophrenia and other adult disease risk variants are frequently found in fetal enhancers, providing support for the hypothesis of fetal origins of adult diseases.
    DOI:  https://doi.org/10.1038/s41594-024-01431-2
  2. Nature. 2024 Dec 18.
    Nucleosome fibre topology guides transcription factor binding to enhancers.
    Michael R O'Dwyer, Meir Azagury, Katharine Furlong, Amani Alsheikh, Elisa Hall-Ponsele, Hugo Pinto, Dmitry V Fyodorov, Mohammad Jaber, Eleni Papachristoforou, Hana Benchetrit, James Ashmore, Kirill Makedonski, Moran Rahamim, Marta Hanzevacki, Hazar Yassen, Samuel Skoda, Adi Levy, Steven M Pollard, Arthur I Skoultchi, Yosef Buganim, Abdenour Soufi.
      Cellular identity requires the concerted action of multiple transcription factors (TFs) bound together to enhancers of cell-type-specific genes. Despite TFs recognizing specific DNA motifs within accessible chromatin, this information is insufficient to explain how TFs select enhancers1. Here we compared four different TF combinations that induce different cell states, analysing TF genome occupancy, chromatin accessibility, nucleosome positioning and 3D genome organization at the nucleosome resolution. We show that motif recognition on mononucleosomes can decipher only the individual binding of TFs. When bound together, TFs act cooperatively or competitively to target nucleosome arrays with defined 3D organization, displaying motifs in particular patterns. In one combination, motif directionality funnels TF combinatorial binding along chromatin loops, before infiltrating laterally to adjacent enhancers. In other combinations, TFs assemble on motif-dense and highly interconnected loop junctions, and subsequently translocate to nearby lineage-specific sites. We propose a guided-search model in which motif grammar on nucleosome fibres acts as signpost elements, directing TF combinatorial binding to enhancers.
    DOI:  https://doi.org/10.1038/s41586-024-08333-9
  3. Nucleic Acids Res. 2024 Dec 19. pii: gkae1223. [Epub ahead of print]
    Rapid degradation of histone deacetylase 1 (HDAC1) reveals essential roles in both gene repression and active transcription.
    David M English, Samuel N Lee, Khadija A Sabat, India M Baker, Trong Khoa Pham, Mark O Collins, Shaun M Cowley.
      Histone Deacetylase 1 (HDAC1) removes acetyl groups from lysine residues on core histones, a critical step in regulating chromatin accessibility. Despite histone deacetylation being an apparently repressive activity, suppression of HDACs causes both up- and downregulation of gene expression. Here we exploited the degradation tag (dTAG) system to rapidly degrade HDAC1 in mouse embryonic stem cells (ESCs) lacking its paralog, HDAC2. The dTAG system allowed specific degradation and removal of HDAC1 in <1 h (100x faster than genetic knockouts). This rapid degradation caused increased histone acetylation in as little as 2 h, with H2BK5 and H2BK11 being the most sensitive. The majority of differentially expressed genes following 2 h of HDAC1 degradation were upregulated (275 genes up versus 15 down) with increased proportions of downregulated genes observed at 6 h (1153 up versus 443 down) and 24 h (1146 up versus 967 down), respectively. Upregulated genes showed increased H2BK5ac and H3K27ac around their transcriptional start site (TSS). In contrast, decreased acetylation and chromatin accessibility of super-enhancers was linked to the most strongly downregulated genes. These findings suggest a paradoxical role for HDAC1 in the maintenance of histone acetylation levels at critical enhancer regions required for the pluripotency-associated gene network.
    DOI:  https://doi.org/10.1093/nar/gkae1223
  4. Nucleic Acids Res. 2024 Dec 16. pii: gkae1229. [Epub ahead of print]
    Abo1 ATPase facilitates the dissociation of FACT from chromatin.
    Juwon Jang, Yujin Kang, Martin Zofall, Sangmin Woo, Soyeong An, Carol Cho, Shiv Grewal, Ja Yil Lee, Ji-Joon Song.
      The histone chaperone FAcilitates Chromatin Transcription (FACT) is a heterodimeric complex consisting of Spt16 and Pob3, crucial for preserving nucleosome integrity during transcription and DNA replication. Loss of FACT leads to cryptic transcription and heterochromatin defects. FACT was shown to interact with Abo1, an AAA + family histone chaperone involved in nucleosome dynamics. Depletion of Abo1 causes FACT to stall at transcription start sites and mimics FACT mutants, indicating a functional association between Abo1 and FACT. However, the precise role of Abo1 in FACT function remains poorly understood. Here, we reveal that Abo1 directly interacts with FACT and facilitates the dissociation of FACT from nucleosome. Specifically, the N-terminal region of Abo1 utilizes its FACT-interacting helix to bind to the N-terminal domain of Spt16. In addition, using single-molecule fluorescence imaging, we discovered that Abo1 facilitates the ATP-dependent dissociation of FACT from nucleosomes. Furthermore, we demonstrate that the interaction between Abo1 and FACT is essential for maintaining heterochromatin in fission yeast. In summary, our findings suggest that Abo1 regulates FACT turnover in an ATP-dependent manner, proposing a model of histone chaperone recycling driven by inter-chaperone interactions.
    DOI:  https://doi.org/10.1093/nar/gkae1229
  5. Adv Sci (Weinh). 2024 Dec 10. e2409160
    BRD8 Guards the Pluripotent State by Sensing and Maintaining Histone Acetylation.
    Li Sun, Xiuling Fu, Zhen Xiao, Gang Ma, Yibin Zhou, Haoqing Hu, Liyang Shi, Dongwei Li, Ralf Jauch, Andrew Paul Hutchins.
      Epigenetic control of cell fates is a critical determinant to maintain cell type stability and permit differentiation during embryonic development. However, the epigenetic control mechanisms are not well understood. Here, it is shown that the histone acetyltransferase reader protein BRD8 impairs the conversion of primed mouse EpiSCs (epiblast stem cells) to naive mouse ESCs (embryonic stem cells). BRD8 works by maintaining histone acetylation on promoters and transcribed gene bodies. BRD8 is responsible for maintaining open chromatin at somatic genes, and histone acetylation at naive-specific genes. When Brd8 expression is reduced, chromatin accessibility is unchanged at primed-specific genes, but histone acetylation is reduced. Conversely, naive-specific genes has reduced repressive chromatin marks and acquired accessible chromatin more rapidly during the cell type conversion. It is shown that this process requires active histone deacetylation to promote the conversion of primed to naive. This data supports a model for BRD8 reading histone acetylation to accurately localize the genome-wide binding of the histone acetyltransferase KAT5. Overall, this study shows how the reading of the histone acetylation state by BRD8 maintains cell type stability and both enables and impairs stem cell differentiation.
    Keywords:  chromatin; epigenetics; histone acetylation; pluripotent stem cells
    DOI:  https://doi.org/10.1002/advs.202409160
  6. Nucleic Acids Res. 2024 Dec 04. pii: gkae1120. [Epub ahead of print]
    ATAC and SAGA histone acetyltransferase modules facilitate transcription factor binding to nucleosomes independent of their acetylation activity.
    Kristin V Chesnutt, Gizem Yayli, Christine Toelzer, Mylène Damilot, Khan Cox, Gunjan Gautam, Imre Berger, László Tora, Michael G Poirier.
      Transcription initiation involves the coordination of multiple events, starting with activators binding specific DNA target sequences, which recruit transcription coactivators to open chromatin and enable binding of general transcription factors and RNA polymerase II to promoters. Two key human transcriptional coactivator complexes, ATAC (ADA-two-A-containing) and SAGA (Spt-Ada-Gcn5 acetyltransferase), containing histone acetyltransferase (HAT) activity, target genomic loci to increase promoter accessibility. To better understand the function of ATAC and SAGA HAT complexes, we used in vitro biochemical and biophysical assays to characterize human ATAC and SAGA HAT module interactions with nucleosomes and how a transcription factor (TF) coordinates these interactions. We found that ATAC and SAGA HAT modules bind nucleosomes with high affinity, independent of their HAT activity and the tested TF. ATAC and SAGA HAT modules directly interact with the VP16 activator domain and this domain enhances acetylation activity of both HAT modules. Surprisingly, ATAC and SAGA HAT modules increase TF binding to its DNA target site within the nucleosome by an order of magnitude independent of histone acetylation. Altogether, our results reveal synergistic coordination between HAT modules and a TF, where ATAC and SAGA HAT modules (i) acetylate histones to open chromatin and (ii) facilitate TF targeting within nucleosomes independently of their acetylation activity.
    DOI:  https://doi.org/10.1093/nar/gkae1120
  7. Elife. 2024 Dec 17. pii: RP91596. [Epub ahead of print]12
    A genome-wide nucleosome-resolution map of promoter-centered interactions in human cells corroborates the enhancer-promoter looping model.
    Arkadiy K Golov, Alexey A Gavrilov, Noam Kaplan, Sergey V Razin.
      The enhancer-promoter looping model, in which enhancers activate their target genes via physical contact, has long dominated the field of gene regulation. However, the ubiquity of this model has been questioned due to evidence of alternative mechanisms and the lack of its systematic validation, primarily owing to the absence of suitable experimental techniques. In this study, we present a new MNase-based proximity ligation method called MChIP-C, allowing for the measurement of protein-mediated chromatin interactions at single-nucleosome resolution on a genome-wide scale. By applying MChIP-C to study H3K4me3 promoter-centered interactions in K562 cells, we found that it had greatly improved resolution and sensitivity compared to restriction endonuclease-based C-methods. This allowed us to identify EP300 histone acetyltransferase and the SWI/SNF remodeling complex as potential candidates for establishing and/or maintaining enhancer-promoter interactions. Finally, leveraging data from published CRISPRi screens, we found that most functionally verified enhancers do physically interact with their cognate promoters, supporting the enhancer-promoter looping model.
    Keywords:  chromatin; chromosomes; gene expression; gene regulation; genetics; genome organization; genomics; human
    DOI:  https://doi.org/10.7554/eLife.91596
  8. Proc Natl Acad Sci U S A. 2024 Dec 24. 121(52): e2409656121
    Histone methyltransferase SETDB1 safeguards mouse fetal hematopoiesis by suppressing activation of cryptic enhancers.
    Maryam Kazerani, Filippo Cernilogar, Alessandra Pasquarella, Maria Hinterberger, Alexander Nuber, Ludger Klein, Gunnar Schotta.
      The H3K9me3-specific histone methyltransferase SETDB1 is critical for proper regulation of developmental processes, but the underlying mechanisms are only partially understood. Here, we show that deletion of Setdb1 in mouse fetal liver hematopoietic stem and progenitor cells (HSPCs) results in compromised stem cell function, enhanced myeloerythroid differentiation, and impaired lymphoid development. Notably, Setdb1-deficient HSPCs exhibit reduced quiescence and increased proliferation, accompanied by the acquisition of a lineage-biased transcriptional program. In Setdb1-deficient HSPCs, we identify genomic regions that are characterized by loss of H3K9me3 and increased chromatin accessibility, suggesting enhanced transcription factor (TF) activity. Interestingly, hematopoietic TFs like PU.1 bind these cryptic enhancers in wild-type HSPCs, despite the H3K9me3 status. Thus, our data indicate that SETDB1 restricts activation of nonphysiological TF binding sites which helps to ensure proper maintenance and differentiation of fetal liver HSPCs.
    Keywords:  ERV; H3K9me3; epigenetics; hematopoietic stem cells; heterochromatin
    DOI:  https://doi.org/10.1073/pnas.2409656121
  9. Genome Biol. 2024 Dec 18. 25(1): 313
    Transcriptional regulatory network reveals key transcription factors for regulating agronomic traits in soybean.
    Wu Jiao, Mangmang Wang, Yijian Guan, Wei Guo, Chang Zhang, Yuanchun Wei, Zhenwei Zhao, Hongyu Ma, Longfei Wang, Xinyu Jiang, Wenxue Ye, Dong Cao, Qingxin Song.
       BACKGROUND: Transcription factors (TFs) bind regulatory genomic regions to orchestrate spatio-temporal expression of target genes. Global dissection of the cistrome is critical for elucidating transcriptional networks underlying complex agronomic traits in crops.
    RESULTS: Here, we generate a comprehensive genome-wide binding map for 148 TFs using DNA affinity purification sequencing in soybean. We find TF binding sites (TFBSs) exhibit elevated chromatin accessibility and contain more rare alleles than other genomic regions. Intriguingly, the methylation variations at TFBSs partially contribute to expression bias among whole genome duplication paralogs. Furthermore, we construct a soybean gene regulatory network (SoyGRN) by integrating TF-target interactions with diverse datasets encompassing gene expression, TFBS motifs, chromatin accessibility, and evolutionarily conserved regulation. SoyGRN comprises 2.44 million genome-wide interactions among 3188 TFs and 51,665 target genes. We successfully identify key TFs governing seed coat color and oil content and prioritize candidate genes within quantitative trait loci associated with various agronomic traits using SoyGRN. To accelerate utilization of SoyGRN, we develop an interactive webserver ( www.soytfbase.cn ) for soybean community to explore functional TFs involved in trait regulation.
    CONCLUSIONS: Overall, our study unravels intricate landscape of TF-target interactions in soybean and provides a valuable resource for dissecting key regulators for control of agronomic traits to accelerate soybean improvement.
    Keywords:  Gene expression; Gene regulatory network; Soybean; Transcription factors
    DOI:  https://doi.org/10.1186/s13059-024-03454-w
  10. Cell Syst. 2024 Dec 14. pii: S2405-4712(24)00366-1. [Epub ahead of print]
    Multiome Perturb-seq unlocks scalable discovery of integrated perturbation effects on the transcriptome and epigenome.
    Eli Metzner, Kaden M Southard, Thomas M Norman.
      Single-cell CRISPR screens link genetic perturbations to transcriptional states, but high-throughput methods connecting these induced changes to their regulatory foundations are limited. Here, we introduce Multiome Perturb-seq, extending single-cell CRISPR screens to simultaneously measure perturbation-induced changes in gene expression and chromatin accessibility. We apply Multiome Perturb-seq in a CRISPRi screen of 13 chromatin remodelers in human RPE-1 cells, achieving efficient assignment of sgRNA identities to single nuclei via an improved method for capturing barcode transcripts from nuclear RNA. We organize expression and accessibility measurements into coherent programs describing the integrated effects of perturbations on cell state, finding that ARID1A and SUZ12 knockdowns induce programs enriched for developmental features. Modeling of perturbation-induced heterogeneity connects accessibility changes to changes in gene expression, highlighting the value of multimodal profiling. Overall, our method provides a scalable and simply implemented system to dissect the regulatory logic underpinning cell state. A record of this paper's transparent peer review process is included in the supplemental information.
    Keywords:  CRISPRi; Perturb-seq; chromatin accessibility; functional genomics; gene regulatory networks; multiome; single-cell genomics
    DOI:  https://doi.org/10.1016/j.cels.2024.12.002
  11. Proc Natl Acad Sci U S A. 2024 Dec 24. 121(52): e2423270121
    Genome-wide single-cell and single-molecule footprinting of transcription factors with deaminase.
    Runsheng He, Wenyang Dong, Zhi Wang, Chen Xie, Long Gao, Wenping Ma, Ke Shen, Dubai Li, Yuxuan Pang, Fanchong Jian, Jiankun Zhang, Yuan Yuan, Xinyao Wang, Zhen Zhang, Yinghui Zheng, Shuang Liu, Cheng Luo, Xiaoran Chai, Jun Ren, Zhanxing Zhu, Xiaoliang Sunney Xie.
      Decades of research have established that mammalian transcription factors (TFs) bind to each gene's regulatory regions and cooperatively control tissue specificity, timing, and intensity of gene transcription. Mapping the combination of TF binding sites genome wide is critically important for understanding functional genomics. Here, we report a technique to measure TFs' binding sites on the human genome with a near single-base resolution by footprinting with deaminase (FOODIE) on a single-molecule and single-cell basis. Single-molecule sequencing reads after enzymatic deamination allow detection of the TF binding fraction on a particular footprint and the binding cooperativity of any two adjacent TFs, which can be either positive or negative. As a newcomer of single-cell genomics, single-cell FOODIE enables the detection of cell-type-specific TF footprints in a pure cell population in a heterogeneous tissue, such as the brain. We found that genes carrying out a certain biological function together in a housing-keeping correlated gene module (CGM) or a tissues-specific CGM are coordinated by shared TFs in the gene's promoters and enhancers, respectively. Scalable and cost-effective, FOODIE allows us to create an open FOODIE database for cell lines, with applicability to human tissues and clinical samples.
    Keywords:  TF binding cooperativity; TF footprinting; deaminase; single-cell genomics; single-molecule sequencing
    DOI:  https://doi.org/10.1073/pnas.2423270121
  12. Sci Adv. 2024 Dec 20. 10(51): eadn6625
    The three-dimensional genome drives the evolution of asymmetric gene duplicates via enhancer capture-divergence.
    UnJin Lee, Deanna Arsala, Shengqian Xia, Cong Li, Mujahid Ali, Nicolas Svetec, Christopher B Langer, Débora R Sobreira, Ittai Eres, Dylan Sosa, Jianhai Chen, Li Zhang, Patrick Reilly, Alexander Guzzetta, J J Emerson, Peter Andolfatto, Qi Zhou, Li Zhao, Manyuan Long.
      Previous evolutionary models of duplicate gene evolution have overlooked the pivotal role of genome architecture. Here, we show that proximity-based regulatory recruitment by distally duplicated genes is an efficient mechanism for modulating tissue-specific production of preexisting proteins. By leveraging genomic asymmetries, we performed a coexpression analysis on Drosophila melanogaster tissue data to show the generality of enhancer capture-divergence (ECD) as a significant evolutionary driver of asymmetric, distally duplicated genes. We use the recently evolved gene HP6/Umbrea as an example of the ECD process. By assaying genome-wide chromosomal conformations in multiple Drosophila species, we show that HP6/Umbrea was inserted near a preexisting, long-distance three-dimensional genomic interaction. We then use this data to identify a newly found enhancer (FLEE1), buried within the coding region of the highly conserved, essential gene MFS18, that likely neofunctionalized HP6/Umbrea. Last, we demonstrate ancestral transcriptional coregulation of HP6/Umbrea's future insertion site, illustrating how enhancer capture provides a highly evolvable, one-step solution to Ohno's dilemma.
    DOI:  https://doi.org/10.1126/sciadv.adn6625
  13. Nature. 2024 Dec 18.
    Engineered extrachromosomal oncogene amplifications promote tumorigenesis.
    Davide Pradella, Minsi Zhang, Rui Gao, Melissa A Yao, Katarzyna M Gluchowska, Ylenia Cendon-Florez, Tanmay Mishra, Gaspare La Rocca, Moritz Weigl, Ziqi Jiao, Hieu H M Nguyen, Marta Lisi, Mateusz M Ozimek, Chiara Mastroleo, Kevin Chen, Felix Grimm, Jens Luebeck, Shu Zhang, Andrea Alice Zolli, Eric G Sun, Bhargavi Dameracharla, Zhengqiao Zhao, Yuri Pritykin, Carlie Sigel, Howard Y Chang, Paul S Mischel, Vineet Bafna, Cristina R Antonescu, Andrea Ventura.
      Focal gene amplifications are among the most common cancer-associated mutations1 but have proven challenging to engineer in primary cells and model organisms. Here we describe a general strategy to engineer large (more than 1 Mbp) focal amplifications mediated by extrachromosomal DNAs (ecDNAs)2 in a spatiotemporally controlled manner in cells and in mice. By coupling ecDNA formation with expression of selectable markers, we track the dynamics of ecDNA-containing cells under physiological conditions and in the presence of specific selective pressures. We also apply this approach to generate mice harbouring Cre-inducible Myc- and Mdm2-containing ecDNAs analogous to those occurring in human cancers. We show that the engineered ecDNAs spontaneously accumulate in primary cells derived from these animals, promoting their proliferation, immortalization and transformation. Finally, we demonstrate the ability of Mdm2-containing ecDNAs to promote tumour formation in an autochthonous mouse model of hepatocellular carcinoma. These findings offer insights into the role of ecDNA-mediated gene amplifications in tumorigenesis. We anticipate that this approach will be valuable for investigating further unresolved aspects of ecDNA biology and for developing new preclinical immunocompetent mouse models of human cancers harbouring specific focal gene amplifications.
    DOI:  https://doi.org/10.1038/s41586-024-08318-8
  14. Nucleic Acids Res. 2024 Dec 16. pii: gkae1187. [Epub ahead of print]
    Conformational switching of Arp5 subunit regulates INO80 chromatin remodeling.
    Shagun Shukla, Somnath Paul, Jeison Garcia, Yuan Zhong, Sara Sanz Juste, Karissa Beauchemin, Blaine Bartholomew.
      The INO80 chromatin remodeler is a versatile enzyme capable of several functions, including spacing nucleosomes equal distances apart, precise positioning of nucleosomes based on DNA shape/sequence and exchanging histone dimers. Within INO80, the Arp5 subunit plays a central role in INO80 remodeling, evidenced by its interactions with the histone octamer, nucleosomal and extranucleosomal DNA, and its necessity in linking INO80's ATPase activity to nucleosome movement. We find two distinct regions of Arp5 binding near the acidic pocket of nucleosomes. One region has an arginine anchor that binds nucleosomes and is vital for INO80 mobilizing nucleosomes. The other region has a hydrophobic/acid patch of Leu and Asp that binds free histone H2A-H2B dimers. These two regions have different roles in remodeling nucleosomes as seen both in vitro and in vivo and the hydrophobic/acidic patch of Arp5 is likely needed for displacing DNA from the H2A-H2B surface and dimer exchange by INO80.
    DOI:  https://doi.org/10.1093/nar/gkae1187
  15. Methods Mol Biol. 2025 ;2881 271-291
    Remodeling of Individual Nucleosomes in Nucleosome Arrays.
    Petra Vizjak, Nicola Hepp, Felix Mueller-Planitz.
      Adenosin triphosphate (ATP)-dependent nucleosome remodeling factors sculpt the nucleosomal landscape of eukaryotic chromatin. They deposit, evict, or reposition nucleosomes along DNA in a process termed nucleosome sliding. Remodeling has traditionally been analyzed using mononucleosomes as a model substrate. In vivo, however, nucleosomes form extended arrays with regular spacing. Here we describe how regularly spaced nucleosome arrays can be reconstituted in vitro and how these arrays can be used to dissect remodeling in the test tube. We outline two assays. Both assays exploit the changes in the accessibility of DNA to restriction enzymes during the remodeling reaction. The first assay uses the restriction enzyme to cleave the restriction site as soon as it becomes accessible during remodeling. As such, this assay mostly reports the kinetic parameter of the "forward" reaction of nucleosome remodeling. In contrast, the second assay measures how fast a particular nucleosome in the array reaches its steady-state position.
    Keywords:   Chromatin reconstitution; Chromatin remodeling enzyme; Histone; Chromatin accessibility; ISWI; Nucleosome arrays; Nucleosome sliding/repositioning; Snf2-family ATPase; Adenosin triphosphate (ATP)-dependent nucleosome remodeling
    DOI:  https://doi.org/10.1007/978-1-0716-4280-1_14
  16. Elife. 2024 Dec 20. pii: RP98654. [Epub ahead of print]13
    The emergence and evolution of gene expression in genome regions replete with regulatory motifs.
    Timothy Fuqua, Yiqiao Sun, Andreas Wagner.
      Gene regulation is essential for life and controlled by regulatory DNA. Mutations can modify the activity of regulatory DNA, and also create new regulatory DNA, a process called regulatory emergence. Non-regulatory and regulatory DNA contain motifs to which transcription factors may bind. In prokaryotes, gene expression requires a stretch of DNA called a promoter, which contains two motifs called -10 and -35 boxes. However, these motifs may occur in both promoters and non-promoter DNA in multiple copies. They have been implicated in some studies to improve promoter activity, and in others to repress it. Here, we ask whether the presence of such motifs in different genetic sequences influences promoter evolution and emergence. To understand whether and how promoter motifs influence promoter emergence and evolution, we start from 50 'promoter islands', DNA sequences enriched with -10 and -35 boxes. We mutagenize these starting 'parent' sequences, and measure gene expression driven by 240,000 of the resulting mutants. We find that the probability that mutations create an active promoter varies more than 200-fold, and is not correlated with the number of promoter motifs. For parent sequences without promoter activity, mutations created over 1500 new -10 and -35 boxes at unique positions in the library, but only ~0.3% of these resulted in de-novo promoter activity. Only ~13% of all -10 and -35 boxes contribute to de-novo promoter activity. For parent sequences with promoter activity, mutations created new -10 and -35 boxes in 11 specific positions that partially overlap with preexisting ones to modulate expression. We also find that -10 and -35 boxes do not repress promoter activity. Overall, our work demonstrates how promoter motifs influence promoter emergence and evolution. It has implications for predicting and understanding regulatory evolution, de novo genes, and phenotypic evolution.
    Keywords:  E. coli; chromosomes; de-novo genes; emergence; evolution; evolutionary biology; gene expression; gene regulation; promoters; transcription
    DOI:  https://doi.org/10.7554/eLife.98654
  17. Nucleic Acids Res. 2024 Dec 16. pii: gkae1182. [Epub ahead of print]
    ZNF143 binds DNA and stimulates transcription initiation to activate and repress direct target genes.
    Jinhong Dong, Kizhakke Mattada Sathyan, Thomas G Scott, Rudradeep Mukherjee, Michael J Guertin.
      Transcription factors bind to sequence motifs and act as activators or repressors. Transcription factors interface with a constellation of accessory cofactors to regulate distinct mechanistic steps to regulate transcription. We rapidly degraded the essential and pervasively expressed transcription factor ZNF143 to determine its function in the transcription cycle. ZNF143 facilitates RNA polymerase initiation and activates gene expression. ZNF143 binds the promoter of nearly all its activated target genes. ZNF143 also binds near the site of genic transcription initiation to directly repress a subset of genes. Although ZNF143 stimulates initiation at ZNF143-repressed genes (i.e. those that increase transcription upon ZNF143 depletion), the molecular context of binding leads to cis repression. ZNF143 competes with other more efficient activators for promoter access, physically occludes transcription initiation sites and promoter-proximal sequence elements, and acts as a molecular roadblock to RNA polymerases during early elongation. The term context specific is often invoked to describe transcription factors that have both activation and repression functions. We define the context and molecular mechanisms of ZNF143-mediated cis activation and repression.
    DOI:  https://doi.org/10.1093/nar/gkae1182
  18. Nucleic Acids Res. 2024 Dec 19. pii: gkae1227. [Epub ahead of print]
    PRMT1-mediated methylation regulates MLL2 stability and gene expression.
    Dongju An, Jihyun Kim, Byul Moon, Hyoungmin Kim, Hoa Nguyen, Sunghu Park, J Eugene Lee, Jung-Ae Kim, Jaehoon Kim.
      The interplay between multiple transcription factors precisely regulates eukaryotic transcription. Here, we report that the protein methyltransferases, MLL2/KMT2B and PRMT1, interact directly and act collectively to regulate gene expression. PRMT1 binds to the N-terminal region of MLL2, considered an intrinsically disordered region, and methylates multiple arginine residues within its RGG/RG motifs. Notably, overexpression of PRMT1 decreased poly-ubiquitylation of MLL2, whereas mutations on methylation sites in MLL2 increased MLL2 poly-ubiquitylation, suggesting that PRMT1-mediated methylation stabilizes MLL2. MLL2 and PRMT1 cooperatively stimulated the expression of a chromosomal reporter gene in a PRMT1-mediated, MLL2-methylation-dependent manner. RNA-seq analysis found that MLL2 and PRMT1 jointly regulate the expression of genes involved in cell membrane and extracellular matrix functions, and depletion of either resulted in impaired cell migration and invasion. Our study provides evidence that PRMT1-mediated MLL2 methylation regulates MLL2 protein stability and the expression of their target genes.
    DOI:  https://doi.org/10.1093/nar/gkae1227
  19. Epigenetics Chromatin. 2024 Dec 19. 17(1): 38
    Dissecting the Kaiso binding profile in clear renal cancer cells.
    Alexey Starshin, Pavel Abramov, Yaroslava Lobanova, Fedor Sharko, Galina Filonova, Dmitry Kaluzhny, Daria Kaplun, Igor Deyev, Alexander Mazur, Egor Prokchortchouk, Svetlana Zhenilo.
       BACKGROUND: There has been a notable increase in interest in the transcriptional regulator Kaiso, which has been linked to the regulation of clonal hematopoiesis, myelodysplastic syndrome, and tumorigenesis. Nevertheless, there are no consistent data on the binding sites of Kaiso in vivo in the genome. Previous ChIP-seq analyses for Kaiso contradicted the accumulated data of Kaiso binding sites obtained in vitro. Here, we studied this discrepancy by characterizing the distribution profile of Kaiso binding sites in Caki-1 cells using Kaiso-deficient cells as a negative control, and compared its pattern on chromatin with that in lymphoblastoid cell lines.
    RESULTS: We employed Caki-1 kidney carcinoma cells and their derivative, which lacks the Kaiso gene, as a model system to identify the genomic targets of Kaiso. The principal binding motifs for Kaiso are CGCG and CTGCNAT, with 60% of all binding sites containing both sequences. The significance of methyl-DNA binding activity was confirmed through examination of the genomic distribution of the E535A mutant variant of Kaiso, which cannot bind methylated DNA in vitro but is able to interact with CTGCNA sequences. Our findings indicate that Kaiso is present at CpG islands with a preference for methylated ones. We identified Kaiso target genes whose methylation and transcription are dependent on its expression. Furthermore, Kaiso binding sites are enriched at CpG islands, with partial methylation at the 5' and/or 3' boundaries. We discovered CpG islands exhibiting wave-like methylation patterns, with Kaiso detected in the majority of these areas. Similar data were obtained in other cell lines.
    CONCLUSION: The present study delineates the genomic distribution of Kaiso in cancer cells, confirming its role as a factor with a complex mode of DNA binding and a strong association with CpG islands, particularly with methylated and eroded CpG islands, revealing a new potential Kaiso target gene-SQSTM1, involved in differentiation of acute myeloid leukemia cells. Furthermore, we discovered the existence of a new class of CpG islands characterized by wave-like DNA methylation.
    Keywords:  CpG islands erosion; DNA methylation; Kaiso; Methyl-DNA binding proteins; SQSTM1
    DOI:  https://doi.org/10.1186/s13072-024-00565-3
  20. Methods Mol Biol. 2025 ;2881 345-355
    Assessing the Effect of Chromatin-Binding Enzymes on Condensed Chromatin with Optical Tweezers.
    Dieter Kamp, Petra Vizjak, Johannes Stigler.
      The formation of biomolecular condensates in vitro and in vivo has become an increasingly important subject of studies. One particular area of interest is the phase separation of chromatin in the nucleus. However, the interplay of condensed chromatin and chromatin-binding enzymes has barely been studied as of now. Here, we show an optical tweezer-based assay that uses controlled fusion of two condensates to probe the effect of enzymes, such as chromatin remodeling motor proteins, on the fluidity of condensates. The assay provides a powerful tool that enables the study of if and how chromatin-enzyme interactions alter biophysical properties in these dense chromatin condensates.
    Keywords:   Chromatin; Condensates; Optical tweezers; Liquid-liquid phase separation (LLPS)
    DOI:  https://doi.org/10.1007/978-1-0716-4280-1_17
  21. Nucleic Acids Res. 2024 Dec 16. pii: gkae1237. [Epub ahead of print]
    A modular toolbox for the optogenetic deactivation of transcription.
    Philipp Muench, Matteo Fiumara, Nicholas Southern, Davide Coda, Sabine Aschenbrenner, Bruno Correia, Johannes Gräff, Dominik Niopek, Jan Mathony.
      Light-controlled transcriptional activation is a commonly used optogenetic strategy that allows researchers to regulate gene expression with high spatiotemporal precision. The vast majority of existing tools are, however, limited to light-triggered induction of gene expression. Here, we inverted this mode of action and created optogenetic systems capable of efficiently terminating transcriptional activation in response to blue light. First, we designed highly compact regulators by photo-controlling the VP16 (pcVP16) transactivation peptide. Then, applying a two-hybrid strategy, we engineered LOOMINA (light off-operated modular inductor of transcriptional activation), a versatile transcriptional control platform for mammalian cells that is compatible with various effector proteins. Leveraging the flexibility of CRISPR systems, we combined LOOMINA with dCas9 to control transcription with blue light from endogenous promoters with exceptionally high dynamic ranges in multiple cell lines. Functionally and mechanistically, the versatile LOOMINA platform and the exceptionally compact pcVP16 transactivator represent valuable additions to the optogenetic repertoire for transcriptional regulation.
    DOI:  https://doi.org/10.1093/nar/gkae1237
  22. Genome Biol. 2024 Dec 18. 25(1): 310
    EpiGePT: a pretrained transformer-based language model for context-specific human epigenomics.
    Zijing Gao, Qiao Liu, Wanwen Zeng, Rui Jiang, Wing Hung Wong.
      The inherent similarities between natural language and biological sequences have inspired the use of large language models in genomics, but current models struggle to incorporate chromatin interactions or predict in unseen cellular contexts. To address this, we propose EpiGePT, a transformer-based model designed for predicting context-specific human epigenomic signals. By incorporating transcription factor activities and 3D genome interactions, EpiGePT outperforms existing methods in epigenomic signal prediction tasks, especially in cell-type-specific long-range interaction predictions and genetic variant impacts, advancing our understanding of gene regulation. A free online prediction service is available at http://health.tsinghua.edu.cn/epigept .
    Keywords:  3D genome; Epigenomics; Gene Regulation; Language model; Transformer
    DOI:  https://doi.org/10.1186/s13059-024-03449-7
  23. NAR Genom Bioinform. 2024 Dec;6(4): lqae182
    HiCrayon reveals distinct layers of multi-state 3D chromatin organization.
    Ben Nolan, Hannah L Harris, Achyuth Kalluchi, Timothy E Reznicek, Christopher T Cummings, M Jordan Rowley.
      Chromatin contact maps are often shown as 2D heatmaps and visually compared to 1D genomic data by simple juxtaposition. While common, this strategy is imprecise, placing the onus on the reader to align features with each other. To remedy this, we developed HiCrayon, an interactive tool that facilitates the integration of 3D chromatin organization maps and 1D datasets. This visualization method integrates data from genomic assays directly into the chromatin contact map by coloring interactions according to 1D signal. HiCrayon is implemented using R shiny and python to create a graphical user interface application, available in both web and containerized format to promote accessibility. We demonstrate the utility of HiCrayon in visualizing the effectiveness of compartment calling and the relationship between ChIP-seq and various features of chromatin organization. We also demonstrate the improved visualization of other 3D genomic phenomena, such as differences between loops associated with CTCF/cohesin versus those associated with H3K27ac. We then demonstrate HiCrayon's visualization of organizational changes that occur during differentiation and use HiCrayon to detect compartment patterns that cannot be assigned to either A or B compartments, revealing a distinct third chromatin compartment.
    DOI:  https://doi.org/10.1093/nargab/lqae182
  24. Genome Biol. 2024 Dec 18. 25(1): 316
    HIPSD&R-seq enables scalable genomic copy number and transcriptome profiling.
    Jan Otoničar, Olga Lazareva, Jan-Philipp Mallm, Milena Simovic-Lorenz, George Philippos, Pooja Sant, Urja Parekh, Linda Hammann, Albert Li, Umut Yildiz, Mikael Marttinen, Judith Zaugg, Kyung Min Noh, Oliver Stegle, Aurélie Ernst.
      Single-cell DNA sequencing (scDNA-seq) enables decoding somatic cancer variation. Existing methods are hampered by low throughput or cannot be combined with transcriptome sequencing in the same cell. We propose HIPSD&R-seq (HIgh-throughPut Single-cell Dna and Rna-seq), a scalable yet simple and accessible assay to profile low-coverage DNA and RNA in thousands of cells in parallel. Our approach builds on a modification of the 10X Genomics platform for scATAC and multiome profiling. In applications to human cell models and primary tissue, we demonstrate the feasibility to detect rare clones and we combine the assay with combinatorial indexing to profile over 17,000 cells.
    Keywords:  Single Cell DNA sequencing; Single Cell copy Number profiling; Single Cell multiome
    DOI:  https://doi.org/10.1186/s13059-024-03450-0
  25. Blood. 2024 Dec 10. pii: blood.2024024505. [Epub ahead of print]
    Progressive chromatin rewiring by ETO2::GLIS2 revealed in a human iPSC model of pediatric leukemia initiation.
    Fabien Boudia, Marie Baille, Loelia Babin, Zakia Aid, Elie Robert, Julie Riviere, Klaudia Galant, Verónica Alonso-Pérez, Laura Anselmi, Brahim Arkoun, Nassera Abermil, Christophe Marzac, Salvatore Nicola Bertuccio, Alexia Regnault de Premesnil, Cecile K Lopez, Alexandre Eeckhoutte, Audrey Naimo, Betty Leite, Cyril Catelain, Christophe Metereau, Patrick Gonin, Nathalie Gaspar, Juerg Schwaller, Olivier A Bernard, Hana Raslova, Murielle Gaudry, Antonin Marchais, Hélène Lapillonne, Arnaud Petit, Francoise Pflumio, Marie-Laure Arcangeli, Erika Brunet, Thomas Mercher.
      Pediatric acute myeloid leukemia frequently harbor fusion oncogenes associated with poor prognosis, including KMT2A, NUP98 and GLIS2 rearrangements. While murine models have demonstrated their leukemogenic activities, the steps from a normal human cell to leukemic blasts remain unclear. Here, we precisely reproduced the inversion of chromosome 16 resulting in ETO2::GLIS2 fusion in human induced pluripotent stem cells (iPSC). IPSC-derived ETO2::GLIS2-expressing hematopoietic cells showed differentiation alterations in vitro and efficiently induced in vivo development of leukemia that closely phenocopied human acute megakaryoblastic leukemia (AMKL) reflected by flow cytometry and single cell transcriptomes. Comparison of iPS-derived cells with patient-derived cells revealed altered chromatin accessibility at early and later bona fide leukemia stages with aberrantly higher accessibility and expression of the osteogenic homeobox factor DLX3 that preceded increased accessibility to ETS factors. DLX3 overexpression in normal CD34+ cells increased accessibility to ETS motifs and reduced accessibility to GATA motifs. A DLX3 transcriptional module was globally enriched in both ETO2::GLIS2 AMKL and some aggressive pediatric osteosarcoma. Importantly, DLX3 knock-out abrogated leukemia initiation in this ETO2::GLIS2 iPSC model. Collectively, characterization of a novel human iPSC-derived AMKL model revealed hijacking of the osteogenic homeobox transcription factor DLX3 as an essential early step in chromatin changes and leukemogenesis driven by the ETO2::GLIS2 fusion oncogene.
    DOI:  https://doi.org/10.1182/blood.2024024505
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