bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2023–12–24
nineteen papers selected by
Connor Rogerson, University of Cambridge
  1. Cohesin and CTCF do not assemble TADs in Xenopus sperm and male pronuclei.
  2. Super-enhancers include classical enhancers and facilitators to fully activate gene expression.
  3. FOXA1 forms biomolecular condensates that unpack condensed chromatin to function as a pioneer factor.
  4. PAX3-FOXO1 uses its activation domain to recruit CBP/P300 and shape RNA Pol2 cluster distribution.
  5. Essential transcription factors for induced neuron differentiation.
  6. Intrinsically disordered regions of the Msn2 transcription factor encode multiple functions using interwoven sequence grammars.
  7. Revisiting chromatin packaging in mouse sperm.
  8. Distinct guard cell-specific remodeling of chromatin accessibility during abscisic acid- and CO2-dependent stomatal regulation.
  9. Repetitive CREB-DNA interactions at gene loci predetermined by CBP induce activity-dependent gene expression in human cortical neurons.
  10. Nano-CUT&Tag for multimodal chromatin profiling at single-cell resolution.
  11. The ncBAF complex regulates transcription in AML through H3K27ac sensing by BRD9.
  12. Forecasting histone methylation by Polycomb complexes with minute-scale precision.
  13. BRD9 determines the cell fate of hematopoietic stem cells by regulating chromatin state.
  14. PAPerFly: Partial Assembly-based Peak Finder for ab initio binding site reconstruction.
  15. Diffusion controls local versus dispersed inheritance of histones during replication and shapes epigenomic architecture.
  16. High-throughput PRIME-editing screens identify functional DNA variants in the human genome.
  17. Transcription factor-mediated direct cellular reprogramming yields cell-type specific DNA methylation signature.
  18. Asymmetrical nucleosomal DNA signatures regulate transcriptional directionality.
  19. Somatic mutations of MLL4/COMPASS induce cytoplasmic localization providing molecular insight into cancer prognosis and treatment.
  1. Genome Res. 2023 Dec 21.
    Cohesin and CTCF do not assemble TADs in Xenopus sperm and male pronuclei.
    Gregor Jessberger, Csilla Várnai, Roman R Stocsits, Wen Tang, Georg Stary, Jan-Michael Peters.
      Paternal genomes are compacted during spermiogenesis and decompacted following fertilization. These processes are fundamental for inheritance but incompletely understood. We analyzed these processes in the frog Xenopus laevis, whose sperm can be assembled into functional pronuclei in egg extracts in vitro. In such extracts, cohesin extrudes DNA into loops, but in vivo cohesin only assembles topologically associating domains (TADs) at the mid-blastula transition (MBT). Why cohesin assembles TADs only at this stage is unknown. We first analyzed genome architecture in frog sperm and compared it to human and mouse. Our results indicate that sperm genome organization is conserved between frogs and humans and occurs without formation of TADs. TADs can be detected in mouse sperm samples, as reported, but these structures might originate from somatic chromatin contaminations. We therefore discuss the possibility that the absence of TADs might be a general feature of vertebrate sperm. To analyze sperm genome remodeling upon fertilization, we reconstituted male pronuclei in Xenopus egg extracts. In pronuclei, chromatin compartmentalization increases, but cohesin does not accumulate at CTCF sites and assemble TADs. However, if pronuclei are formed in the presence of exogenous CTCF, CTCF binds to its consensus sites, and cohesin accumulates at these and forms short-range chromatin loops, which are preferentially anchored at CTCF's N terminus. These results indicate that TADs are only assembled at MBT because before this stage CTCF sites are not occupied and cohesin only forms short-range chromatin loops.
    DOI:  https://doi.org/10.1101/gr.277865.123
  2. Cell. 2023 Dec 21. pii: S0092-8674(23)01316-8. [Epub ahead of print]186(26): 5826-5839.e18
    Super-enhancers include classical enhancers and facilitators to fully activate gene expression.
    Joseph W Blayney, Helena Francis, Alexandra Rampasekova, Brendan Camellato, Leslie Mitchell, Rosa Stolper, Lucy Cornell, Christian Babbs, Jef D Boeke, Douglas R Higgs, Mira Kassouf.
      Super-enhancers are compound regulatory elements that control expression of key cell identity genes. They recruit high levels of tissue-specific transcription factors and co-activators such as the Mediator complex and contact target gene promoters with high frequency. Most super-enhancers contain multiple constituent regulatory elements, but it is unclear whether these elements have distinct roles in activating target gene expression. Here, by rebuilding the endogenous multipartite α-globin super-enhancer, we show that it contains bioinformatically equivalent but functionally distinct element types: classical enhancers and facilitator elements. Facilitators have no intrinsic enhancer activity, yet in their absence, classical enhancers are unable to fully upregulate their target genes. Without facilitators, classical enhancers exhibit reduced Mediator recruitment, enhancer RNA transcription, and enhancer-promoter interactions. Facilitators are interchangeable but display functional hierarchy based on their position within a multipartite enhancer. Facilitators thus play an important role in potentiating the activity of classical enhancers and ensuring robust activation of target genes.
    Keywords:  alpha globin locus; cis-regulatory elements; enhancer cluster; enhancer cooperation; facilitators; gene expression; genome engineering; super-enhancers; synthetic genome; transcriptional regulation
    DOI:  https://doi.org/10.1016/j.cell.2023.11.030
  3. Mol Cell. 2023 Dec 07. pii: S1097-2765(23)00965-6. [Epub ahead of print]
    FOXA1 forms biomolecular condensates that unpack condensed chromatin to function as a pioneer factor.
    Dengyu Ji, Changrong Shao, Juan Yu, Yaoyao Hou, Xiao Gao, Yichuan Wu, Liang Wang, Ping Chen.
      Eukaryotic DNA is packaged into chromatin in the nucleus, restricting the binding of transcription factors (TFs) to their target DNA sites. FOXA1 functions as a pioneer TF to bind condensed chromatin and initiate the opening of local chromatin for gene expression. However, the principles of FOXA1 recruitment and how it subsequently unpacks the condensed chromatin remain elusive. Here, we revealed that FOXA1 intrinsically forms submicron-sized condensates through its N- and C-terminal intrinsically disordered regions (IDRs). Notably, both IDRs enable FOXA1 to dissolve the condensed chromatin. In addition, the DNA-binding capacity of FOXA1 contributes to its ability to both form condensates and dissolve condensed chromatin. Further genome-wide investigation showed that IDRs enable FOXA1 to bind and unpack the condensed chromatin to regulate the proliferation and migration of breast cancer cells. This work provides a principle of how pioneer TFs function to initiate competent chromatin states using their IDRs.
    Keywords:  FOXA1, biomolecular condensates, chromatin, pioneer factor
    DOI:  https://doi.org/10.1016/j.molcel.2023.11.020
  4. Nat Commun. 2023 Dec 15. 14(1): 8361
    PAX3-FOXO1 uses its activation domain to recruit CBP/P300 and shape RNA Pol2 cluster distribution.
    Yaw Asante, Katharina Benischke, Issra Osman, Quy A Ngo, Jakob Wurth, Dominik Laubscher, Hyunmin Kim, Bhavatharini Udhayakumar, Md Imdadul H Khan, Diana H Chin, Jadon Porch, Maharshi Chakraborty, Richard Sallari, Olivier Delattre, Sakina Zaidi, Sarah Morice, Didier Surdez, Sara G Danielli, Beat W Schäfer, Berkley E Gryder, Marco Wachtel.
      Activation of oncogenic gene expression from long-range enhancers is initiated by the assembly of DNA-binding transcription factors (TF), leading to recruitment of co-activators such as CBP/p300 to modify the local genomic context and facilitate RNA-Polymerase 2 (Pol2) binding. Yet, most TF-to-coactivator recruitment relationships remain unmapped. Here, studying the oncogenic fusion TF PAX3-FOXO1 (P3F) from alveolar rhabdomyosarcoma (aRMS), we show that a single cysteine in the activation domain (AD) of P3F is important for a small alpha helical coil that recruits CBP/p300 to chromatin. P3F driven transcription requires both this single cysteine and CBP/p300. Mutants of the cysteine reduce aRMS cell proliferation and induce cellular differentiation. Furthermore, we discover a profound dependence on CBP/p300 for clustering of Pol2 loops that connect P3F to its target genes. In the absence of CBP/p300, Pol2 long range enhancer loops collapse, Pol2 accumulates in CpG islands and fails to exit the gene body. These results reveal a potential novel axis for therapeutic interference with P3F in aRMS and clarify the molecular relationship of P3F and CBP/p300 in sustaining active Pol2 clusters essential for oncogenic transcription.
    DOI:  https://doi.org/10.1038/s41467-023-43780-4
  5. Nat Commun. 2023 Dec 15. 14(1): 8362
    Essential transcription factors for induced neuron differentiation.
    Congyi Lu, Görkem Garipler, Chao Dai, Timothy Roush, Jose Salome-Correa, Alex Martin, Noa Liscovitch-Brauer, Esteban O Mazzoni, Neville E Sanjana.
      Neurogenins are proneural transcription factors required to specify neuronal identity. Their overexpression in human pluripotent stem cells rapidly produces cortical-like neurons with spiking activity and, because of this, they have been widely adopted for human neuron disease models. However, we do not fully understand the key downstream regulatory effectors responsible for driving neural differentiation. Here, using inducible expression of NEUROG1 and NEUROG2, we identify transcription factors (TFs) required for directed neuronal differentiation by combining expression and chromatin accessibility analyses with a pooled in vitro CRISPR-Cas9 screen targeting all ~1900 TFs in the human genome. The loss of one of these essential TFs (ZBTB18) yields few MAP2-positive neurons. Differentiated ZBTB18-null cells have radically altered gene expression, leading to cytoskeletal defects and stunted neurites and spines. In addition to identifying key downstream TFs for neuronal differentiation, our work develops an integrative multi-omics and TFome-wide perturbation platform to rapidly characterize essential TFs for the differentiation of any human cell type.
    DOI:  https://doi.org/10.1038/s41467-023-43602-7
  6. Nucleic Acids Res. 2023 Dec 18. pii: gkad1191. [Epub ahead of print]
    Intrinsically disordered regions of the Msn2 transcription factor encode multiple functions using interwoven sequence grammars.
    Vladimir Mindel, Sagie Brodsky, Aileen Cohen, Wajd Manadre, Felix Jonas, Miri Carmi, Naama Barkai.
      Intrinsically disordered regions (IDRs) are abundant in eukaryotic proteins, but their sequence-function relationship remains poorly understood. IDRs of transcription factors (TFs) can direct promoter selection and recruit coactivators, as shown for the budding yeast TF Msn2. To examine how IDRs encode both these functions, we compared genomic binding specificity, coactivator recruitment, and gene induction amongst a large set of designed Msn2-IDR mutants. We find that both functions depend on multiple regions across the > 600AA IDR. Yet, transcription activity was readily disrupted by mutations that showed no effect on the Msn2 binding specificity. Our data attribute this differential sensitivity to the integration of a relaxed, composition-based code directing binding specificity with a more stringent, motif-based code controlling the recruitment of coactivators and transcription activity. Therefore, Msn2 utilizes interwoven sequence grammars for encoding multiple functions, suggesting a new IDR design paradigm of potentially general use.
    DOI:  https://doi.org/10.1093/nar/gkad1191
  7. Genome Res. 2023 Dec 21.
    Revisiting chromatin packaging in mouse sperm.
    Qiangzong Yin, Chih-Hsiang Yang, Olga S Strelkova, Jingyi Wu, Yu Sun, Sneha Gopalan, Liyan Yang, Job Dekker, Thomas G Fazzio, Xin Zhiguo Li, Johan Gibcus, Oliver J Rando.
      Mammalian sperm show an unusual and heavily compacted genomic packaging state. In addition to its role in organizing the compact and hydrodynamic sperm head, it has been proposed that sperm chromatin architecture helps to program gene expression in the early embryo. Scores of genome-wide surveys in sperm have reported patterns of chromatin accessibility, nucleosome localization, histone modification, and chromosome folding. Here, we revisit these studies in light of recent reports that sperm obtained from the mouse epididymis are contaminated with low levels of cell-free chromatin. In the absence of proper sperm lysis, we readily recapitulate multiple prominent genome-wide surveys of sperm chromatin, suggesting that these profiles primarily reflect contaminating cell-free chromatin. Removal of cell-free DNA, and appropriate lysis conditions, are together required to reveal a sperm chromatin state distinct from most previous reports. Using ATAC-seq to explore relatively accessible genomic loci, we identify a landscape of open loci associated with early development and transcriptional control. Histone modification and chromosome folding profiles also strongly support the hypothesis that prior studies suffer from contamination, but technical challenges associated with reliably preserving the architecture of the compacted sperm head prevent us from confidently assaying true localization patterns for these epigenetic marks. Together, our studies show that our knowledge of chromosome packaging in mammalian sperm remains largely incomplete, and motivate future efforts to more accurately characterize genome organization in mature sperm.
    DOI:  https://doi.org/10.1101/gr.277845.123
  8. Proc Natl Acad Sci U S A. 2023 Dec 26. 120(52): e2310670120
    Distinct guard cell-specific remodeling of chromatin accessibility during abscisic acid- and CO2-dependent stomatal regulation.
    Charles A Seller, Julian I Schroeder.
      In plants, epidermal guard cells integrate and respond to numerous environmental signals to control stomatal pore apertures, thereby regulating gas exchange. Chromatin structure controls transcription factor (TF) access to the genome, but whether large-scale chromatin remodeling occurs in guard cells during stomatal movements, and in response to the hormone abscisic acid (ABA) in general, remains unknown. Here, we isolate guard cell nuclei from Arabidopsis thaliana plants to examine whether the physiological signals, ABA and CO2 (carbon dioxide), regulate guard cell chromatin during stomatal movements. Our cell type-specific analyses uncover patterns of chromatin accessibility specific to guard cells and define cis-regulatory sequences supporting guard cell-specific gene expression. We find that ABA triggers extensive and dynamic chromatin remodeling in guard cells, roots, and mesophyll cells with clear patterns of cell type specificity. DNA motif analyses uncover binding sites for distinct TFs enriched in ABA-induced and ABA-repressed chromatin. We identify the Abscisic Acid Response Element (ABRE) Binding Factor (ABF) bZIP-type TFs that are required for ABA-triggered chromatin opening in guard cells and roots and implicate the inhibition of a clade of bHLH-type TFs in controlling ABA-repressed chromatin. Moreover, we demonstrate that ABA and CO2 induce distinct programs of chromatin remodeling, whereby elevated atmospheric CO2 had only minimal impact on chromatin dynamics. We provide insight into the control of guard cell chromatin dynamics and propose that ABA-induced chromatin remodeling primes the genome for abiotic stress resistance.
    Keywords:  chromatin; guard cells; plant hormones; stomata; transcription factors
    DOI:  https://doi.org/10.1073/pnas.2310670120
  9. Cell Rep. 2023 Dec 15. pii: S2211-1247(23)01588-7. [Epub ahead of print] 113576
    Repetitive CREB-DNA interactions at gene loci predetermined by CBP induce activity-dependent gene expression in human cortical neurons.
    Yuri Atsumi, Ryohei Iwata, Hiroshi Kimura, Pierre Vanderhaeghen, Nobuhiko Yamamoto, Noriyuki Sugo.
      Neuronal activity-dependent transcription plays a key role in plasticity and pathology in the brain. An intriguing question is how neuronal activity controls gene expression via interactions of transcription factors with DNA and chromatin modifiers in the nucleus. By utilizing single-molecule imaging in human embryonic stem cell (ESC)-derived cortical neurons, we demonstrate that neuronal activity increases repetitive emergence of cAMP response element-binding protein (CREB) at histone acetylation sites in the nucleus, where RNA polymerase II (RNAPII) accumulation and FOS expression occur rapidly. Neuronal activity also enhances co-localization of CREB and CREB-binding protein (CBP). Increased binding of a constitutively active CREB to CBP efficiently induces CREB repetitive emergence. On the other hand, the formation of histone acetylation sites is dependent on CBP histone modification via acetyltransferase (HAT) activity but is not affected by neuronal activity. Taken together, our results suggest that neuronal activity promotes repetitive CREB-CRE and CREB-CBP interactions at predetermined histone acetylation sites, leading to rapid gene expression.
    Keywords:  BRD4; CBP; CP: Molecular biology; CP: Neuroscience; CREB; RNAPII; activity-dependent gene; histone acetylation; human cortical neuron; neuronal activity; single-molecule imaging
    DOI:  https://doi.org/10.1016/j.celrep.2023.113576
  10. Nat Protoc. 2023 Dec 21.
    Nano-CUT&Tag for multimodal chromatin profiling at single-cell resolution.
    José Ramón Bárcenas-Walls, Federico Ansaloni, Bastien Hervé, Emilia Strandback, Tomas Nyman, Gonçalo Castelo-Branco, Marek Bartošovič.
      The ability to comprehensively analyze the chromatin state with single-cell resolution is crucial for understanding gene regulatory principles in heterogenous tissues or during development. Recently, we developed a nanobody-based single-cell CUT&Tag (nano-CT) protocol to simultaneously profile three epigenetic modalities-two histone marks and open chromatin state-from the same single cell. Nano-CT implements a new set of secondary nanobody-Tn5 fusion proteins to direct barcoded tagmentation by Tn5 transposase to genomic targets labeled by primary antibodies raised in different species. Such nanobody-Tn5 fusion proteins are currently not commercially available, and their in-house production and purification can be completed in 3-4 d by following our detailed protocol. The single-cell indexing in nano-CT is performed on a commercially available platform, making it widely accessible to the community. In comparison to other multimodal methods, nano-CT stands out in data complexity, low sample requirements and the flexibility to choose two of the three modalities. In addition, nano-CT works efficiently with fresh brain samples, generating multimodal epigenomic profiles for thousands of brain cells at single-cell resolution. The nano-CT protocol can be completed in just 3 d by users with basic skills in standard molecular biology and bioinformatics, although previous experience with single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) is beneficial for more in-depth data analysis. As a multimodal assay, nano-CT holds immense potential to reveal interactions of various chromatin modalities, to explore epigenetic heterogeneity and to increase our understanding of the role and interplay that chromatin dynamics has in cellular development.
    DOI:  https://doi.org/10.1038/s41596-023-00932-6
  11. Cancer Res Commun. 2023 Dec 21.
    The ncBAF complex regulates transcription in AML through H3K27ac sensing by BRD9.
    David C Klein, Santana M Lardo, Sarah J Hainer.
      The non-canonical BAF complex (ncBAF) subunit BRD9 is essential for acute myeloid leukemia (AML) cell viability but has an unclear role in leukemogenesis. Because BRD9 is required for ncBAF complex assembly through its DUF3512 domain, precise bromodomain inhibition is necessary to parse the role of BRD9 as a transcriptional regulator from that of a scaffolding protein. To understand the role of BRD9 bromodomain function in regulating AML, we selected a panel of five AML cell lines with distinct driver mutations, disease classifications, and genomic aberrations and subjected these cells to short-term BRD9 bromodomain inhibition. We examined the bromodomain-dependent growth of these cell lines, identifying a dependency in AML cell lines but not HEK293T cells. To define a mechanism through which BRD9 maintains AML cell survival, we examined nascent transcription, chromatin accessibility, and ncBAF complex binding genome-wide after bromodomain inhibition. We identified extensive regulation of transcription by BRD9 bromodomain activity, including repression of myeloid maturation factors and tumor suppressor genes, while standard AML chemotherapy targets were repressed by inhibition of the BRD9 bromodomain. BRD9 bromodomain activity maintained accessible chromatin at both gene promoters and gene-distal putative enhancer regions, in a manner that qualitatively correlated with enrichment of BRD9 binding. Furthermore, we identified reduced chromatin accessibility at GATA, ETS, and AP-1 motifs and increased chromatin accessibility at SNAIL-, HIC-, and TP53-recognized motifs after BRD9 inhibition. These data suggest a role for BRD9 in regulating AML cell differentiation through modulation of accessibility at hematopoietic transcription factor binding sites.
    DOI:  https://doi.org/10.1158/2767-9764.CRC-23-0382
  12. Sci Adv. 2023 Dec 22. 9(51): eadj8198
    Forecasting histone methylation by Polycomb complexes with minute-scale precision.
    Moa J Lundkvist, Ludvig Lizana, Yuri B Schwartz.
      Animals use the Polycomb system to epigenetically repress developmental genes. The repression requires trimethylation of lysine 27 of histone H3 (H3K27me3) by Polycomb Repressive Complex 2 (PRC2), but the dynamics of this process is poorly understood. To bridge the gap, we developed a computational model that forecasts H3K27 methylation in Drosophila with high temporal resolution and spatial accuracy of contemporary experimental techniques. Using this model, we show that pools of methylated H3K27 in dividing cells are defined by the effective concentration of PRC2 and the replication frequency. We find that the allosteric stimulation by preexisting H3K27me3 makes PRC2 better in methylating developmental genes as opposed to indiscriminate methylation throughout the genome. Applied to Drosophila development, our model argues that, in this organism, the intergenerationally inherited H3K27me3 does not "survive" rapid cycles of embryonic chromatin replication and is unlikely to transmit the memory of epigenetic repression to the offspring. Our model is adaptable to other organisms, including mice and humans.
    DOI:  https://doi.org/10.1126/sciadv.adj8198
  13. Nat Commun. 2023 Dec 15. 14(1): 8372
    BRD9 determines the cell fate of hematopoietic stem cells by regulating chromatin state.
    Muran Xiao, Shinji Kondo, Masaki Nomura, Shinichiro Kato, Koutarou Nishimura, Weijia Zang, Yifan Zhang, Tomohiro Akashi, Aaron Viny, Tsukasa Shigehiro, Tomokatsu Ikawa, Hiromi Yamazaki, Miki Fukumoto, Atsushi Tanaka, Yasutaka Hayashi, Yui Koike, Yumi Aoyama, Hiromi Ito, Hiroyoshi Nishikawa, Toshio Kitamura, Akinori Kanai, Akihiko Yokoyama, Tohru Fujiwara, Susumu Goyama, Hideki Noguchi, Stanley C Lee, Atsushi Toyoda, Kunihiko Hinohara, Omar Abdel-Wahab, Daichi Inoue.
      ATP-dependent chromatin remodeling SWI/SNF complexes exist in three subcomplexes: canonical BAF (cBAF), polybromo BAF (PBAF), and a newly described non-canonical BAF (ncBAF). While cBAF and PBAF regulate fates of multiple cell types, roles for ncBAF in hematopoietic stem cells (HSCs) have not been investigated. Motivated by recent discovery of disrupted expression of BRD9, an essential component of ncBAF, in multiple cancers, including clonal hematopoietic disorders, we evaluate here the role of BRD9 in normal and malignant HSCs. BRD9 loss enhances chromatin accessibility, promoting myeloid lineage skewing while impairing B cell development. BRD9 significantly colocalizes with CTCF, whose chromatin recruitment is augmented by BRD9 loss, leading to altered chromatin state and expression of myeloid-related genes within intact topologically associating domains. These data uncover ncBAF as critical for cell fate specification in HSCs via three-dimensional regulation of gene expression and illuminate roles for ncBAF in normal and malignant hematopoiesis.
    DOI:  https://doi.org/10.1038/s41467-023-44081-6
  14. BMC Bioinformatics. 2023 Dec 19. 24(1): 487
    PAPerFly: Partial Assembly-based Peak Finder for ab initio binding site reconstruction.
    Kateřina Faltejsková, Jiří Vondrášek.
       BACKGROUND: The specific recognition of a DNA locus by a given transcription factor is a widely studied issue. It is generally agreed that the recognition can be influenced not only by the binding motif but by the larger context of the binding site. In this work, we present a novel heuristic algorithm that can reconstruct the unique binding sites captured in a sequencing experiment without using the reference genome.
    RESULTS: We present PAPerFly, the Partial Assembly-based Peak Finder, a tool for the binding site and binding context reconstruction from the sequencing data without any prior knowledge. This tool operates without the need to know the reference genome of the respective organism. We employ algorithmic approaches that are used during genome assembly. The proposed algorithm constructs a de Bruijn graph from the sequencing data. Based on this graph, sequences and their enrichment are reconstructed using a novel heuristic algorithm. The reconstructed sequences are aligned and the peaks in the sequence enrichment are identified. Our approach was tested by processing several ChIP-seq experiments available in the ENCODE database and comparing the results of Paperfly and standard methods.
    CONCLUSIONS: We show that PAPerFly, an algorithm tailored for experiment analysis without the reference genome, yields better results than an aggregation of ChIP-seq agnostic tools. Our tool is freely available at https://github.com/Caeph/paperfly/ or on Zenodo ( https://doi.org/10.5281/zenodo.7116424 ).
    Keywords:  Algorithm; ChIP-seq; DNA recognition; Graph theory; Peak analysis; Transcription factor
    DOI:  https://doi.org/10.1186/s12859-023-05613-5
  15. PLoS Comput Biol. 2023 Dec 18. 19(12): e1011725
    Diffusion controls local versus dispersed inheritance of histones during replication and shapes epigenomic architecture.
    Archit Singh, Shaon Chakrabarti.
      The dynamics of inheritance of histones and their associated modifications across cell divisions can have major consequences on maintenance of the cellular epigenomic state. Recent experiments contradict the long-held notion that histone inheritance during replication is always local, suggesting that active and repressed regions of the genome exhibit fundamentally different histone dynamics independent of transcription-coupled turnover. Here we develop a stochastic model of histone dynamics at the replication fork and demonstrate that differential diffusivity of histones in active versus repressed chromatin is sufficient to quantitatively explain these recent experiments. Further, we use the model to predict patterns in histone mark similarity between pairs of genomic loci that should be developed as a result of diffusion, but cannot originate from either PRC2 mediated mark spreading or transcriptional processes. Interestingly, using a combination of CHIP-seq, replication timing and Hi-C datasets we demonstrate that all the computationally predicted patterns are consistently observed for both active and repressive histone marks in two different cell lines. While direct evidence for histone diffusion remains controversial, our results suggest that dislodged histones in euchromatin and facultative heterochromatin may exhibit some level of diffusion within "Diffusion-Accessible-Domains" (DADs), leading to redistribution of epigenetic marks within and across chromosomes. Preservation of the epigenomic state across cell divisions therefore might be achieved not by passing on strict positional information of histone marks, but by maintaining the marks in somewhat larger DADs of the genome.
    DOI:  https://doi.org/10.1371/journal.pcbi.1011725
  16. Mol Cell. 2023 Dec 21. pii: S1097-2765(23)00966-8. [Epub ahead of print]83(24): 4633-4645.e9
    High-throughput PRIME-editing screens identify functional DNA variants in the human genome.
    Xingjie Ren, Han Yang, Jovia L Nierenberg, Yifan Sun, Jiawen Chen, Cooper Beaman, Thu Pham, Mai Nobuhara, Maya Asami Takagi, Vivek Narayan, Yun Li, Elad Ziv, Yin Shen.
      Despite tremendous progress in detecting DNA variants associated with human disease, interpreting their functional impact in a high-throughput and single-base resolution manner remains challenging. Here, we develop a pooled prime-editing screen method, PRIME, that can be applied to characterize thousands of coding and non-coding variants in a single experiment with high reproducibility. To showcase its applications, we first identified essential nucleotides for a 716 bp MYC enhancer via PRIME-mediated single-base resolution analysis. Next, we applied PRIME to functionally characterize 1,304 genome-wide association study (GWAS)-identified non-coding variants associated with breast cancer and 3,699 variants from ClinVar. We discovered that 103 non-coding variants and 156 variants of uncertain significance are functional via affecting cell fitness. Collectively, we demonstrate that PRIME is capable of characterizing genetic variants at single-base resolution and scale, advancing accurate genome annotation for disease risk prediction, diagnosis, and therapeutic target identification.
    Keywords:  disease variants; enhancer; high-throughput screens; prime editing; single-base resolution
    DOI:  https://doi.org/10.1016/j.molcel.2023.11.021
  17. Sci Rep. 2023 Dec 15. 13(1): 22317
    Transcription factor-mediated direct cellular reprogramming yields cell-type specific DNA methylation signature.
    Kenichi Horisawa, Shizuka Miura, Hiromitsu Araki, Fumihito Miura, Takashi Ito, Atsushi Suzuki.
      Direct reprogramming, inducing the conversion of one type of somatic cell into another by the forced expression of defined transcription factors, is a technology with anticipated medical applications. However, due to the many unresolved aspects of the induction mechanisms, it is essential to thoroughly analyze the epigenomic state of the generated cells. Here, we performed comparative genome-wide DNA methylation analyses of mouse embryonic fibroblasts (MEFs) and cells composing organoids formed by intestinal stem cells (ISCs) or induced ISCs (iISCs) that were directly induced from MEFs. We found that the CpG methylation state was similar between cells forming ISC organoids and iISC organoids, while they differed widely from those in MEFs. Moreover, genomic regions that were differentially methylated between ISC organoid- and iISC organoid-forming cells did not significantly affect gene expression. These results demonstrate the accuracy and safety of iISC induction, leading to the medical applications of this technology.
    DOI:  https://doi.org/10.1038/s41598-023-49546-8
  18. Cell Rep. 2023 Dec 20. pii: S2211-1247(23)01617-0. [Epub ahead of print]43(1): 113605
    Asymmetrical nucleosomal DNA signatures regulate transcriptional directionality.
    Alicia García, Laura Durán, Mar Sánchez, Sara González, Rodrigo Santamaría, Francisco Antequera.
      Despite the symmetrical structure of nucleosomes, in vitro studies have shown that transcription proceeds with different efficiency depending on the orientation of the DNA sequence around them. However, it is unclear whether this functional asymmetry is present in vivo and whether it could regulate transcriptional directionality. Here, we report that the proximal and distal halves of nucleosomal DNA contribute differentially to nucleosome stability in the genome. In +1 nucleosomes, this asymmetry facilitates or hinders transcription depending on the orientation of its underlying DNA, and this difference is associated with an asymmetrical interaction between DNA and histones. These properties are encoded in the DNA signature of +1 nucleosomes, since its incorporation in the two orientations into downstream nucleosomes renders them asymmetrically accessible to MNase and inverts the balance between sense and antisense transcription. Altogether, our results show that nucleosomal DNA endows nucleosomes with asymmetrical properties that modulate the directionality of transcription.
    Keywords:  +1 nucleosome; CP: Molecular biology; antisense transcription; chromatin; nucleosomal signatures; nucleosomes; promoter; transcription
    DOI:  https://doi.org/10.1016/j.celrep.2023.113605
  19. Proc Natl Acad Sci U S A. 2023 Dec 26. 120(52): e2310063120
    Somatic mutations of MLL4/COMPASS induce cytoplasmic localization providing molecular insight into cancer prognosis and treatment.
    Zibo Zhao, Yuki Aoi, Cassandra N Philips, Khyati A Meghani, Sarah R Gold, Yanni Yu, Luke St John, Jun Qian, Jacob M Zeidner, Joshua J Meeks, Ali Shilatifard.
      Cancer genome sequencing consortiums have recently catalogued an abundance of somatic mutations, across a wide range of human cancers, in the chromatin-modifying enzymes that regulate gene expression. Defining the molecular mechanisms underlying the potentially oncogenic functions of these epigenetic mutations could serve as the basis for precision medicine approaches to cancer therapy. MLL4 encoded by the KMT2D gene highly mutated in a large number of human cancers, is a key histone lysine monomethyltransferase within the Complex of Proteins Associated with Set1 (COMPASS) family that regulates gene expression through enhancer function, potentially functioning as a tumor suppressor. We report that the KMT2D mutations which cause MLL4 protein truncation also alter MLL4's subcellular localization, resulting in loss-of-function in the nucleus and gain-of-function in the cytoplasm. We demonstrate that isogenic correction of KMT2D truncation mutation rescues the aberrant localization phenotype and restores multiple regulatory functions of MLL4, including COMPASS integrity/stabilization, histone H3K4 mono-methylation, enhancer activation, and therefore transcriptional regulation. Moreover, isogenic correction diminishes the sensitivity of KMT2D-mutated cancer cells to targeted metabolic inhibition. Using immunohistochemistry, we identified that cytoplasmic MLL4 is unique to the tissue of bladder cancer patients with KMT2D truncation mutations. Using a preclinical carcinogen model of bladder cancer in mouse, we demonstrate that truncated cytoplasmic MLL4 predicts response to targeted metabolic inhibition therapy for bladder cancer and could be developed as a biomarker for KMT2D-mutated cancers. We also highlight the broader potential for prognosis, patient stratification and treatment decision-making based on KMT2D mutation status in MLL4 truncation-relevant diseases, including human cancers and Kabuki Syndrome.
    Keywords:  cancer biomarker; chromatin modifiers; epigenetics; mutation; treatment
    DOI:  https://doi.org/10.1073/pnas.2310063120
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