bims-crepig 2025-02-16 papers

bims-crepig

Biomed News

on Chromatin regulation and epigenetics in cell fate and cancer

Issue of 2025–02–16
23 papers selected by
Connor Rogerson, University of Cambridge

Cryo-EM structures of the BAF-Lamin A/C complex bound to nucleosomes.
Promoter-proximal RNA polymerase II termination regulates transcription during human cell type transition.
Allovalent scavenging of activation domains in the transcription factor ANAC013 gears transcriptional regulation.
Comprehensive dissection of cis-regulatory elements in a 2.8 Mb topologically associated domain in six human cancers.
RNA polymerase II at histone genes predicts outcome in human cancer.
Biomolecular condensation of human IDRs initiates endogenous transcription via intrachromosomal looping or high-density promoter localization.
Modeling and designing enhancers by introducing and harnessing transcription factor binding units.
Lactate controls cancer stemness and plasticity through epigenetic regulation.
The phenylalanine-and-glycine repeats of NUP98 oncofusions form condensates that selectively partition transcriptional coactivators.
Multivalent nucleosome scaffolding by bromodomain and extraterminal domain tandem bromodomains.
Acetylation at lysine 27 on maternal H3.3 regulates minor zygotic genome activation.
The epigenetic landscape shapes smoking-induced mutagenesis by modulating DNA damage susceptibility and repair efficiency.
A systematic survey of TF function in E. coli suggests RNAP stabilization is a prevalent strategy for both repressors and activators.
Dynamic changes in 3D chromatin structure during male gametogenesis in Arabidopsis thaliana.
Multifaceted roles of H2B mono-ubiquitylation in D-loop metabolism during homologous recombination repair.
Histone H3 lysine 4 methylation recruits DNA demethylases to enforce gene expression in Arabidopsis.
Scalable co-sequencing of RNA and DNA from individual nuclei.
An AT-hook transcription factor promotes transcription of histone, spliced-leader, and piRNA clusters.
Converging mechanism of UM171 and KBTBD4 neomorphic cancer mutations.
Macrophage memory emerges from coordinated transcription factor and chromatin dynamics.
reg-eQTL: Integrating transcription factor effects to unveil regulatory variants.
Simultaneous profiling of chromatin-associated RNA at targeted DNA loci and RNA-RNA Interactions through TaDRIM-seq.
Active repression of cell fate plasticity by PROX1 safeguards hepatocyte identity and prevents liver tumorigenesis.

Nat Commun. 2025 Feb 10. 16(1): 1495

Cryo-EM structures of the BAF-Lamin A/C complex bound to nucleosomes.

Naoki Horikoshi, Ryosuke Miyake, Chizuru Sogawa-Fujiwara, Mitsuo Ogasawara, Yoshimasa Takizawa, Hitoshi Kurumizaka.

Barrier-to-autointegration factor (BAF) associates with mitotic chromosomes and promotes nuclear envelope assembly by recruiting proteins, such as Lamins, required for the reconstruction of the nuclear envelope and lamina. BAF also mediates chromatin anchoring to the nuclear lamina via Lamin A/C. However, the mechanism by which BAF and Lamin A/C bind chromatin and affect the chromatin organization remains elusive. Here we report the cryo-electron microscopy structures of BAF-Lamin A/C-nucleosome complexes. We find that the BAF dimer complexed with the Lamin A/C IgF domain occupies the nucleosomal dyad position, forming a tripartite nucleosomal DNA binding structure. We also show that the Lamin A/C Lys486 and His506 residues, which are reportedly mutated in lipodystrophy patients, directly contact the DNA at the nucleosomal dyad. Excess BAF-Lamin A/C complexes symmetrically bind other nucleosomal DNA sites and connect two BAF-Lamin A/C-nucleosome complexes. Although the linker histone H1 competes with BAF-Lamin A/C binding at the nucleosomal dyad region, the two BAF-Lamin A/C molecules still bridge two nucleosomes. These findings provide insights into the mechanism by which BAF, Lamin A/C, and/or histone H1 bind nucleosomes and influence chromatin organization within the nucleus.

DOI: https://doi.org/10.1038/s41467-025-56823-9
Nat Struct Mol Biol. 2025 Feb 11.

Promoter-proximal RNA polymerase II termination regulates transcription during human cell type transition.

Kseniia Lysakovskaia, Arjun Devadas, Björn Schwalb, Michael Lidschreiber, Patrick Cramer.

Metazoan gene transcription by RNA polymerase II (Pol II) is regulated in the promoter-proximal region. Pol II can undergo termination in the promoter-proximal region but whether this can contribute to transcription regulation in cells remains unclear. Here we extend our previous multiomics analysis to quantify changes in transcription kinetics during a human cell type transition event. We observe that upregulation of transcription involves an increase in initiation frequency and, at a set of genes, a decrease in promoter-proximal termination. In turn, downregulation of transcription involves a decrease in initiation frequency and an increase in promoter-proximal termination. Thus, promoter-proximal termination of Pol II contributes to the regulation of human gene transcription.

DOI: https://doi.org/10.1038/s41594-025-01486-9
Nucleic Acids Res. 2025 Feb 08. pii: gkaf065. [Epub ahead of print]53(4):

Allovalent scavenging of activation domains in the transcription factor ANAC013 gears transcriptional regulation.

Elise Delaforge, Amanda D Due, Frederik Friis Theisen, Nicolas Morffy, Charlotte O'Shea, Martin Blackledge, Lucia C Strader, Karen Skriver, Birthe B Kragelund.

Transcriptional regulation involves interactions between transcription factors, coregulators, and DNA. Intrinsic disorder is a major player in this regulation, but mechanisms driven by disorder remain elusive. Here, we address molecular communication within the stress-regulating Arabidopsis thaliana transcription factor ANAC013. Through high-throughput screening of ANAC013 for transcriptional activation activity, we identify three activation domains within its C-terminal intrinsically disordered region. Two of these overlap with acidic islands and form dynamic interactions with the DNA-binding domain and are released, not only upon binding of target promoter DNA, but also by nonspecific DNA. We show that independently of DNA binding, the RST (RCD--SRO--TAF4) domain of the negative regulator RCD1 (Radical-induced Cell Death1) scavenges the two acidic activation domains positioned vis-à-vis through allovalent binding, leading to dynamic occupation at enhanced affinity. We propose an allovalency model for transcriptional regulation, where sequentially close activation domains in both DNA-bound and DNA-free states allow for efficient regulation. The model is likely relevant for many transcription factor systems, explaining the functional advantage of carrying sequentially close activation domains.

DOI: https://doi.org/10.1093/nar/gkaf065
Nat Commun. 2025 Feb 13. 16(1): 1611

Comprehensive dissection of cis-regulatory elements in a 2.8 Mb topologically associated domain in six human cancers.

Christina M Caragine, Victoria T Le, Meer Mustafa, Bianca Jay Diaz, John A Morris, Simon Müller, Alejandro Mendez-Mancilla, Evan Geller, Noa Liscovitch-Brauer, Neville E Sanjana.

Cis-regulatory elements (CREs), such as enhancers and promoters, are fundamental regulators of gene expression and, across different cell types, the MYC locus utilizes a diverse regulatory architecture driven by multiple CREs. To better understand differences in CRE function, we perform pooled CRISPR inhibition (CRISPRi) screens to comprehensively probe the 2.8 Mb topologically-associated domain containing MYC in 6 human cancer cell lines with nucleotide resolution. We map 32 CREs where inhibition leads to changes in cell growth, including 8 that overlap previously identified enhancers. Targeting specific CREs decreases MYC expression by as much as 60%, and cell growth by as much as 50%. Using 3-D enhancer contact mapping, we find that these CREs almost always contact MYC but less than 10% of total MYC contacts impact growth when silenced, highlighting the utility of our approach to identify phenotypically-relevant CREs. We also detect an enrichment of lineage-specific transcription factors (TFs) at MYC CREs and, for some of these TFs, find a strong, tumor-specific correlation between TF and MYC expression not found in normal tissue. Taken together, these CREs represent systematically identified, functional regulatory regions and demonstrate how the same region of the human genome can give rise to complex, tissue-specific gene regulation.

DOI: https://doi.org/10.1038/s41467-025-56568-5
Science. 2025 Jan 02. 387(6735): 737-743

RNA polymerase II at histone genes predicts outcome in human cancer.

Steven Henikoff, Ye Zheng, Ronald M Paranal, Yiling Xu, Jacob E Greene, Jorja G Henikoff, Zachary R Russell, Frank Szulzewsky, H Nayanga Thirimanne, Sita Kugel, Eric C Holland, Kami Ahmad.

Genome-wide hypertranscription is common in human cancer and predicts poor prognosis. To understand how hypertranscription might drive cancer, we applied our formalin-fixed paraffin-embedded (FFPE)-cleavage under targeted accessible chromatin method for mapping RNA polymerase II (RNAPII) genome-wide in FFPE sections. We demonstrate global RNAPII elevations in mouse gliomas and assorted human tumors in small clinical samples and discover regional elevations corresponding to de novo HER2 amplifications punctuated by likely selective sweeps. RNAPII occupancy at S-phase-dependent histone genes correlated with WHO grade in meningiomas, accurately predicted rapid recurrence, and corresponded to whole-arm chromosome losses. Elevated RNAPII at histone genes in meningiomas and diverse breast cancers is consistent with histone production being rate-limiting for S-phase progression and histone gene hypertranscription driving overproliferation and aneuploidy in cancer, with general implications for precision oncology.

DOI: https://doi.org/10.1126/science.ads2169
Nucleic Acids Res. 2025 Feb 08. pii: gkaf056. [Epub ahead of print]53(4):

Biomolecular condensation of human IDRs initiates endogenous transcription via intrachromosomal looping or high-density promoter localization.

Jing Li, Shizhe Liu, Sunghwan Kim, Jacob Goell, Zachary Allen Drum, John Patrick Flores, Alex J Ma, Barun Mahata, Mario Escobar, Alex Raterink, Jeong Hyun Ahn, Erik R Terán, Rosa Selenia Guerra-Resendez, Yuhao Zhou, Bo Yu, Michael R Diehl, Gang Greg Wang, Anna-Karin Gustavsson, Douglas H Phanstiel, Isaac B Hilton.

Protein intrinsically disordered regions (IDRs) are critical gene-regulatory components and aberrant fusions between IDRs and DNA-binding/chromatin-associating domains cause diverse human cancers. Despite this importance, how IDRs influence gene expression, and how aberrant IDR fusion proteins provoke oncogenesis, remains incompletely understood. Here we develop a series of synthetic dCas9-IDR fusions to establish that locus-specific recruitment of IDRs can be sufficient to stimulate endogenous gene expression. Using dCas9 fused to the paradigmatic leukemogenic NUP98 IDR, we also demonstrate that IDRs can activate transcription via localized biomolecular condensation and in a manner that is dependent upon overall IDR concentration, local binding density, and amino acid composition. To better clarify the oncogenic role of IDRs, we construct clinically observed NUP98 IDR fusions and show that, while generally non-overlapping, oncogenic NUP98-IDR fusions convergently drive a core leukemogenic gene expression program in donor-derived human hematopoietic stem cells. Interestingly, we find that this leukemic program arises through differing mechanistic routes based upon IDR fusion partner; either distributed intragenic binding and intrachromosomal looping, or dense binding at promoters. Altogether, our studies clarify the gene-regulatory roles of IDRs and, for the NUP98 IDR, connect this capacity to pathological cellular programs, creating potential opportunities for generalized and mechanistically tailored therapies.

DOI: https://doi.org/10.1093/nar/gkaf056
Nat Commun. 2025 Feb 08. 16(1): 1469

Modeling and designing enhancers by introducing and harnessing transcription factor binding units.

Jiaqi Li, Pengcheng Zhang, Xi Xi, Liyang Liu, Lei Wei, Xiaowo Wang.

Enhancers serve as pivotal regulators of gene expression throughout various biological processes by interacting with transcription factors (TFs). While transcription factor binding sites (TFBSs) are widely acknowledged as key determinants of TF binding and enhancer activity, the significant role of their surrounding context sequences remains to be quantitatively characterized. Here we propose the concept of transcription factor binding unit (TFBU) to modularly model enhancers by quantifying the impact of context sequences surrounding TFBSs using deep learning models. Based on this concept, we develop DeepTFBU, a comprehensive toolkit for enhancer design. We demonstrate that designing TFBS context sequences can significantly modulate enhancer activities and produce cell type-specific responses. DeepTFBU is also highly efficient in the de novo design of enhancers containing multiple TFBSs. Furthermore, DeepTFBU enables flexible decoupling and optimization of generalized enhancers. We prove that TFBU is a crucial concept, and DeepTFBU is highly effective for rational enhancer design.

DOI: https://doi.org/10.1038/s41467-025-56749-2
Cell Metab. 2025 Feb 04. pii: S1550-4131(25)00002-6. [Epub ahead of print]

Lactate controls cancer stemness and plasticity through epigenetic regulation.

Nguyen T B Nguyen, Sira Gevers, Rutger N U Kok, Lotte M Burgering, Hannah Neikes, Ninouk Akkerman, Max A Betjes, Marlies C Ludikhuize, Can Gulersonmez, Edwin C A Stigter, Yvonne Vercoulen, Jarno Drost, Hans Clevers, Michiel Vermeulen, Jeroen S van Zon, Sander J Tans, Boudewijn M T Burgering, Maria J Rodríguez Colman.

  Tumors arise from uncontrolled cell proliferation driven by mutations in genes that regulate stem cell renewal and differentiation. Intestinal tumors, however, retain some hierarchical organization, maintaining both cancer stem cells (CSCs) and cancer differentiated cells (CDCs). This heterogeneity, coupled with cellular plasticity enabling CDCs to revert to CSCs, contributes to therapy resistance and relapse. Using genetically encoded fluorescent reporters in human tumor organoids, combined with our machine-learning-based cell tracker, CellPhenTracker, we simultaneously traced cell-type specification, metabolic changes, and reconstructed cell lineage trajectories during tumor organoid development. Our findings reveal distinctive metabolic phenotypes in CSCs and CDCs. We find that lactate regulates tumor dynamics, suppressing CSC differentiation and inducing dedifferentiation into a proliferative CSC state. Mechanistically, lactate increases histone acetylation, epigenetically activating MYC. Given that lactate's regulation of MYC depends on the bromodomain-containing protein 4 (BRD4), targeting cancer metabolism and BRD4 inhibitors emerge as a promising strategy to prevent tumor relapse.

Keywords:  cancer metabolism; cell plasticity; cell types; cell-cell interactions; differentiation; heterogeneity; live imaging; organoids; single-cell tracking; stem cells

DOI:  https://doi.org/10.1016/j.cmet.2025.01.002
Mol Cell. 2025 Feb 04. pii: S1097-2765(24)01067-0. [Epub ahead of print]

The phenylalanine-and-glycine repeats of NUP98 oncofusions form condensates that selectively partition transcriptional coactivators.

Jeong Hyun Ahn, Yiran Guo, Heankel Lyons, Samuel G Mackintosh, Benjamin K Lau, Ricky D Edmondson, Stephanie D Byrum, Aaron J Storey, Alan J Tackett, Ling Cai, Benjamin R Sabari, Gang Greg Wang.

  Recurrent cancer-causing fusions of NUP98 produce higher-order assemblies known as condensates. How NUP98 oncofusion-driven condensates activate oncogenes remains poorly understood. Here, we investigate NUP98-PHF23, a leukemogenic chimera of the disordered phenylalanine-and-glycine (FG)-repeat-rich region of NUP98 and the H3K4me3/2-binding plant homeodomain (PHD) finger domain of PHF23. Our integrated analyses using mutagenesis, proteomics, genomics, and condensate reconstitution demonstrate that the PHD domain targets condensate to the H3K4me3/2-demarcated developmental genes, while FG repeats determine the condensate composition and gene activation. FG repeats are necessary to form condensates that partition a specific set of transcriptional regulators, notably the KMT2/MLL H3K4 methyltransferases, histone acetyltransferases, and BRD4. FG repeats are sufficient to partition transcriptional regulators and activate a reporter when tethered to a genomic locus. NUP98-PHF23 assembles the chromatin-bound condensates that partition multiple positive regulators, initiating a feedforward loop of reading-and-writing the active histone modifications. This network of interactions enforces an open chromatin landscape at proto-oncogenes, thereby driving cancerous transcriptional programs.

Keywords:  MLL; NUP98; WDR5; biomolecular condensate; coactivator; fusion protein; leukemia; phase separation; selective partitioning; transcriptional regulation

DOI:  https://doi.org/10.1016/j.molcel.2024.12.026
J Biol Chem. 2025 Feb 10. pii: S0021-9258(25)00137-1. [Epub ahead of print] 108289

Multivalent nucleosome scaffolding by bromodomain and extraterminal domain tandem bromodomains.

Michael D Olp, Karina L Bursch, Sarah L Wynia-Smith, Raymundo Nuñez, Christopher J Goetz, Vaughn Jackson, Brian C Smith.

  Promoter-promoter and enhancer-promoter interactions are enriched in histone acetylation and central to chromatin organization in active genetic regions. Bromodomains are epigenetic 'readers' that recognize and bind histone acetylation. Bromodomains often exist in tandem or with other reader domains. Cellular knockdown of the bromodomain and extraterminal domain (BET) protein family disrupts chromatin organization, but the mechanisms through which BET proteins preserve chromatin structure are largely unknown. We hypothesize that BET proteins maintain overall chromatin structure by employing their tandem bromodomains to multivalently scaffold acetylated nucleosomes in an intra- or internucleosomal manner. To test this hypothesis biophysically, we used small-angle X-ray scattering, electron paramagnetic resonance, and Rosetta protein modeling to show that a disordered linker separates BET tandem bromodomain acetylation binding sites by 15-157 Å. Most of these modeled distances are sufficient to span the length of a nucleosome (>57 Å). Focusing on the BET family member BRD4, we employed bioluminescence resonance energy transfer and isothermal titration calorimetry to show that BRD4 bromodomain binding of multiple acetylation sites on a histone tail does not increase BRD4-histone tail affinity, suggesting that BET bromodomain intranucleosome binding is not biologically relevant. Using sucrose gradients and amplified luminescent proximity homogeneous (AlphaScreen) assays, we provide the first direct biophysical evidence that BET bromodomains can scaffold multiple acetylated nucleosomes. Taken together, our results demonstrate that BET bromodomains are capable of multivalent internucleosome scaffolding in vitro. The knowledge gained provides implications for how BET bromodomain-mediated acetylated internucleosome scaffolding may maintain cellular chromatin interactions in active genetic regions.

Keywords:  amplified luminescent proximity homogeneous assay; bioluminescence resonance energy transfer; bromodomain-containing protein 4; chromatin; computational biology; epigenetics; histone acetylation; isothermal titration calorimetry; nucleosome; sucrose gradients

DOI:  https://doi.org/10.1016/j.jbc.2025.108289
Cell Rep. 2025 Jan 28. pii: S2211-1247(24)01499-2. [Epub ahead of print]44(1): 115148

Acetylation at lysine 27 on maternal H3.3 regulates minor zygotic genome activation.

Jiaming Zhang, Xuanwen Li, Qi Zhao, Jingzhang Ji, Hongdi Cui, Weibo Hou, Xinyu Wang, Entong Song, Songling Xiao, Shukuan Ling, Shaorong Gao, Xiaoyu Liu, Duancheng Wen, Qingran Kong.

  Zygotic genome activation (ZGA) initiates transcription in early embryogenesis and requires extensive chromatin remodeling, including rapid incorporation of the histone variant H3.3. The distinct sources of H3.3 from paternal and maternal alleles (paH3.3 and maH3.3) complicate tracking their individual contributions. Here, using an H3.3B-hemagglutinin (HA)-tagged mouse model, we profile the temporal dynamics of paH3.3 and maH3.3, revealing a unique pattern of maH3.3 enrichment at the promoter regions from zygotes to 2-cell embryos, highlighting the crucial role of maternally stored H3.3 mRNAs and proteins (mH3.3) in pre-implantation development. Knockdown of mH3.3 compromises cleavage and minor ZGA. Mechanistically, mH3.3 facilitates minor ZGA through H3.3S31ph-dependent H3K27ac deposition. Profiling of H3.3 landscape in parthenogenetic (PG) and androgenetic (AG) embryos highlights the role of mH3.3 in remodeling the paternal genome by establishing H3K27ac. These findings demonstrate that mH3.3-mediated parental chromatin reprogramming is essential for orchestrating minor ZGA.

Keywords:  CP: Developmental biology; CP: Molecular biology; early embryonic development; epigenetic reprogramming; histone variants; maternal H3.3; zygotic genome activation

DOI:  https://doi.org/10.1016/j.celrep.2024.115148
Nucleic Acids Res. 2025 Feb 08. pii: gkaf048. [Epub ahead of print]53(4):

The epigenetic landscape shapes smoking-induced mutagenesis by modulating DNA damage susceptibility and repair efficiency.

Elisheva E Heilbrun, Dana Tseitline, Hana Wasserman, Ayala Kirshenbaum, Yuval Cohen, Raluca Gordan, Sheera Adar.

Lung cancer sequencing efforts have uncovered mutational signatures that are attributed to exposure to the cigarette smoke carcinogen benzo[a]pyrene. Benzo[a]pyrene metabolizes in cells to benzo[a]pyrene diol epoxide (BPDE) and reacts with guanine nucleotides to form bulky BPDE adducts. These DNA adducts block transcription and replication, compromising cell function and survival, and are repaired in human cells by the nucleotide excision repair pathway. Here, we applied high-resolution genomic assays to measure BPDE-induced damage formation and mutagenesis in human cells. We integrated the new damage and mutagenesis data with previous repair, DNA methylation, RNA expression, DNA replication, and chromatin component measurements in the same cell lines, along with lung cancer mutagenesis data. BPDE damage formation is significantly enhanced by DNA methylation and in accessible chromatin regions, including transcribed and early-replicating regions. Binding of transcription factors is associated primarily with reduced, but also enhanced damage formation, depending on the factor. While DNA methylation does not appear to influence repair efficiency, this repair was significantly elevated in accessible chromatin regions, which accumulated fewer mutations. Thus, when damage and repair drive mutagenesis in opposing directions, the final mutational patterns appear to be dictated by the efficiency of repair rather than the frequency of underlying damages.

DOI: https://doi.org/10.1093/nar/gkaf048
Nucleic Acids Res. 2025 Feb 08. pii: gkaf058. [Epub ahead of print]53(4):

A systematic survey of TF function in E. coli suggests RNAP stabilization is a prevalent strategy for both repressors and activators.

Sunil Guharajan, Vinuselvi Parisutham, Robert C Brewster.

Transcription factors (TFs) are often classified as activators or repressors, yet these context-dependent labels are inadequate to predict quantitative profiles that emerge across different promoters. A mechanistic understanding of how different regulatory sequences shape TF function is challenging due to the lack of systematic genetic control in endogenous genes. To address this, we use a library of Escherichia coli strains with precise control of TF copy number, measuring the quantitative regulatory input-output function of 90 TFs on synthetic promoters that isolate the contributions of TF binding sequence, location, and basal promoter strength to gene expression. We interpret the measured regulation of these TFs using a thermodynamic model of gene expression and uncover stabilization of RNA polymerase as a pervasive regulatory mechanism, common to both activating and repressing TFs. This property suggests ways to tune the dynamic range of gene expression through the interplay of stabilizing TF function and RNA polymerase basal occupancy, a phenomenon we confirm by measuring fold change for stabilizing TFs across synthetic promoter sequences spanning over 100-fold basal expression. Our work deconstructs TF function at a mechanistic level, providing foundational principles on how gene expression is realized across different promoter contexts, with implications for decoding the relationship between sequence and gene expression.

DOI: https://doi.org/10.1093/nar/gkaf058
Genome Biol. 2025 Feb 10. 26(1): 27

Dynamic changes in 3D chromatin structure during male gametogenesis in Arabidopsis thaliana.

Zhihan Song, Qimin Xia, Minqi Yang, Tingting Yang, Yali Liu, Dingyue Wang, Jiayue Shu, Zhiyuan Liu, Yi Chi, Heming Xu, Dong Xing, Yue Zhou.

BACKGROUND: Chromatin higher-order structure plays an important role in genome stability maintenance and gene transcriptional regulation; however, the dynamics of the three-dimensional (3D) chromatin in male gametophytes during the two rounds of mitosis remains elusive.
RESULTS: Here, we use the optimized single-nucleus and low-input Hi-C methods to investigate changes in 3D chromatin structure in four types of male gametophyte nucleus at different stages. The reconstructed genome structures show that microspore nuclei develop towards two different directions. Although the 3D chromatin organization in generative nuclei is similar to that in microspore nuclei, vegetative nuclei lose chromosome territories, display dispersed centromeres, and switched A/B compartments, which are associated with vegetative specific gene expression. Additionally, we find that there is an active transcriptional center in sperm nuclei, emphasizing the transcription in Arabidopsis sperm is not completely inhibited despite the chromosomes being condensed.
CONCLUSIONS: Our data suggest that the special 3D structures of vegetative and sperm nuclei contribute to cell type-specific expression patterns.

DOI: https://doi.org/10.1186/s13059-025-03496-8
Nucleic Acids Res. 2025 Feb 08. pii: gkaf081. [Epub ahead of print]53(4):

Multifaceted roles of H2B mono-ubiquitylation in D-loop metabolism during homologous recombination repair.

Shih-Hsun Hung, Yuan Liang, Wolf-Dietrich Heyer.

Repairing DNA double-strand breaks is crucial for maintaining genome integrity, which occurs primarily through homologous recombination (HR) in Saccharomyces cerevisiae. Nucleosomes, composed of DNA wrapped around a histone octamer, present a natural barrier to end resection to initiate HR, but the impact on the downstream HR steps of homology search, DNA strand invasion, and repair synthesis remain to be determined. Displacement loops (D-loops) play a pivotal role in HR, yet the influence of chromatin dynamics on D-loop metabolism remains unclear. Using the physical D-loop capture and D-loop extension (DLE) assays to track HR intermediates, we employed genetic analysis to reveal that H2B mono-ubiquitylation (H2Bubi) affects multiple steps during HR repair. We infer that H2Bubi modulates chromatin structure, not only promoting histone degradation for nascent D-loop formation but also stabilizing extended D-loops through nucleosome assembly. Furthermore, H2Bubi regulates DNA resection via Rad9 recruitment to suppress a feedback control mechanism that dampens D-loop formation and DLE at hyper-resected ends. Through physical and genetic assays to determine repair outcomes, we demonstrate that H2Bubi plays a crucial role in preventing break-induced replication and thus promoting genomic stability.

DOI: https://doi.org/10.1093/nar/gkaf081
Nat Plants. 2025 Feb 11.

Histone H3 lysine 4 methylation recruits DNA demethylases to enforce gene expression in Arabidopsis.

Ming Wang, Yan He, Zhenhui Zhong, Ashot Papikian, Shuya Wang, Jason Gardiner, Basudev Ghoshal, Suhua Feng, Yasaman Jami-Alahmadi, James A Wohlschlegel, Steven E Jacobsen.

Patterning of DNA methylation in eukaryotic genomes is controlled by de novo methylation, maintenance mechanisms and demethylation pathways. In Arabidopsis thaliana, DNA demethylation enzymes are clearly important for shaping methylation patterns, but how they are regulated is poorly understood. Here we show that the targeting of histone H3 lysine four trimethylation (H3K4me3) with the catalytic domain of the SDG2 histone methyltransferase potently erased DNA methylation and gene silencing at FWA and also erased CG DNA methylation in many other regions of the Arabidopsis genome. This methylation erasure was completely blocked in the ros1 dml2 dml3 triple mutant lacking DNA demethylation enzymes, showing that H3K4me3 promotes the active removal of DNA methylation. Conversely, we found that the targeted removal of H3K4me3 increased the efficiency of targeted DNA methylation. These results highlight H3K4me3 as a potent anti-DNA methylation mark and also pave the way for development of more powerful epigenome engineering tools.

DOI: https://doi.org/10.1038/s41477-025-01924-y
Nat Methods. 2025 Feb 12.

Scalable co-sequencing of RNA and DNA from individual nuclei.

Timothy R Olsen, Pranay Talla, Romella K Sagatelian, Julia Furnari, Jeffrey N Bruce, Peter Canoll, Shan Zha, Peter A Sims.

The ideal technology for directly investigating the relationship between genotype and phenotype would analyze both RNA and DNA genome-wide and with single-cell resolution; however, existing tools lack the throughput required for comprehensive analysis of complex tumors and tissues. We introduce a highly scalable method for jointly profiling DNA and expression following nucleosome depletion (DEFND-seq). In DEFND-seq, nuclei are nucleosome-depleted, tagmented and separated into individual droplets for messenger RNA and genomic DNA barcoding. Once nuclei have been depleted of nucleosomes, subsequent steps can be performed using the widely available 10x Genomics droplet microfluidic technology and commercial kits. We demonstrate the production of high-complexity mRNA and gDNA sequencing libraries from thousands of individual nuclei from cell lines, fresh and archived surgical specimens for associating gene expression with both copy number and single-nucleotide variants.

DOI: https://doi.org/10.1038/s41592-024-02579-x
Nucleic Acids Res. 2025 Feb 08. pii: gkaf079. [Epub ahead of print]53(4):

An AT-hook transcription factor promotes transcription of histone, spliced-leader, and piRNA clusters.

Yi-Hui Wang, Hannah L Hertz, Benjamin Pastore, Wen Tang.

In all three domains of life, genes with related functions can be organized into specific genomic regions known as gene clusters. In eukaryotes, histone, piRNA (Piwi-interacting RNA), and rDNA (ribosomal DNA) clusters are among the most notable clusters which play fundamental roles in chromatin formation, genome integrity, and translation, respectively. These clusters have long been thought to be regulated by distinct transcriptional mechanisms. In this study, using Caenorhabditis elegans as a model system we identify ATTF-6, a member of the AT-hook family, as a key factor for the expression of histone, piRNA, and 5S rDNA-SL1 (spliced leader 1) clusters. ATTF-6 is essential for C. elegans viability. It forms distinct nuclear foci at both piRNA and 5S rDNA-SL1 clusters. Loss of ATTF-6 leads to a depletion of histone mRNAs, SL1 transcripts, and piRNAs. Additionally, we demonstrate that ATTF-6 is required for the recruitment of USTC (Upstream Sequence Transcription Complex) to piRNA clusters, which is necessary for piRNA production. Collectively, our findings reveal a unifying role for an AT-hook transcription factor in promoting the expression of fundamental gene clusters.

DOI: https://doi.org/10.1093/nar/gkaf079
Nature. 2025 Feb 12.

Converging mechanism of UM171 and KBTBD4 neomorphic cancer mutations.

Xiaowen Xie, Olivia Zhang, Megan J R Yeo, Ceejay Lee, Ran Tao, Stefan A Harry, N Connor Payne, Eunju Nam, Leena Paul, Yiran Li, Hui Si Kwok, Hanjie Jiang, Haibin Mao, Jennifer L Hadley, Hong Lin, Melissa Batts, Pallavi M Gosavi, Vincenzo D'Angiolella, Philip A Cole, Ralph Mazitschek, Paul A Northcott, Ning Zheng, Brian B Liau.

Cancer mutations can create neomorphic protein-protein interactions to drive aberrant function1,2. As a substrate receptor of the CULLIN3-RING E3 ubiquitin ligase complex, KBTBD4 is recurrently mutated in medulloblastoma3, the most common embryonal brain tumour in children4. These mutations impart gain-of-function to KBTBD4 to induce aberrant degradation of the transcriptional corepressor CoREST5. However, their mechanism remains unresolved. Here we establish that KBTBD4 mutations promote CoREST degradation through engaging HDAC1/2 as the direct target of the mutant substrate receptor. Using deep mutational scanning, we chart the mutational landscape of the KBTBD4 cancer hotspot, revealing distinct preferences by which insertions and substitutions can promote gain-of-function and the critical residues involved in the hotspot interaction. Cryo-electron microscopy analysis of two distinct KBTBD4 cancer mutants bound to LSD1-HDAC1-CoREST reveals that a KBTBD4 homodimer asymmetrically engages HDAC1 with two KELCH-repeat β-propeller domains. The interface between HDAC1 and one of the KBTBD4 β-propellers is stabilized by the medulloblastoma mutations, which insert a bulky side chain into the HDAC1 active site pocket. Our structural and mutational analyses inform how this hotspot E3-neosubstrate interface can be chemically modulated. First, we unveil a converging shape-complementarity-based mechanism between gain-of-function E3 mutations and a molecular glue degrader, UM171. Second, we demonstrate that HDAC1/2 inhibitors can block the mutant KBTBD4-HDAC1 interface and proliferation of KBTBD4-mutant medulloblastoma cells. Altogether, our work reveals the structural and mechanistic basis of cancer mutation-driven neomorphic protein-protein interactions.

DOI: https://doi.org/10.1038/s41586-024-08533-3
Cell Syst. 2025 Feb 06. pii: S2405-4712(25)00004-3. [Epub ahead of print] 101171

Macrophage memory emerges from coordinated transcription factor and chromatin dynamics.

Andrew G Wang, Minjun Son, Aleksandr Gorin, Emma Kenna, Abinash Padhi, Bijentimala Keisham, Adam Schauer, Alexander Hoffmann, Savaş Tay.

  Cells of the immune system operate in dynamic microenvironments where the timing, concentration, and order of signaling molecules constantly change. Despite this complexity, immune cells manage to communicate accurately and control inflammation and infection. It is unclear how these dynamic signals are encoded and decoded and if individual cells retain the memory of past exposure to inflammatory molecules. Here, we use live-cell analysis, ATAC sequencing, and an in vivo model of sepsis to show that sequential inflammatory signals induce memory in individual macrophages through reprogramming the nuclear factor κB (NF-κB) network and the chromatin accessibility landscape. We use transcriptomic profiling and deep learning to show that transcription factor and chromatin dynamics coordinate fine-tuned macrophage responses to new inflammatory signals. This work demonstrates how macrophages retain the memory of previous signals despite single-cell variability and elucidates the mechanisms of signal-induced memory in dynamic inflammatory conditions like sepsis.

Keywords:  NF-κB; Toll-like receptors; dynamics; inflammatory signaling; innate immune memory; modeling; sepsis; signaling dynamics; systems biology

DOI:  https://doi.org/10.1016/j.cels.2025.101171
Am J Hum Genet. 2025 Jan 31. pii: S0002-9297(25)00015-1. [Epub ahead of print]

reg-eQTL: Integrating transcription factor effects to unveil regulatory variants.

Rekha Mudappathi, Tatiana Patton, Hai Chen, Ping Yang, Zhifu Sun, Panwen Wang, Chang-Xin Shi, Junwen Wang, Li Liu.

  Regulatory single-nucleotide variants (rSNVs) in noncoding regions of the genome play a crucial role in gene transcription by altering transcription factor (TF) binding, chromatin states, and other epigenetic modifications. Existing expression quantitative trait locus (eQTL) methods identify genomic loci associated with gene-expression changes, but they often fall short in pinpointing causal variants. We introduce reg-eQTL, a computational method that incorporates TF effects and interactions with genetic variants into eQTL analysis. This approach provides deeper insights into the regulatory mechanisms, bringing us one step closer to identifying potential causal variants by uncovering how TFs interact with SNVs to influence gene expression. This method defines a trio consisting of a genetic variant, a target gene, and a TF and tests its impact on gene transcription. In comprehensive simulations, reg-eQTL shows improved power of detecting rSNVs with low population frequency, weak effects, and synergetic interaction with TF as compared to traditional eQTL methods. Application of reg-eQTL to GTEx data from lung, brain, and whole-blood tissues uncovered regulatory trios that include eQTLs and increased the number of eQTLs shared across tissue types. Regulatory networks constructed on the basis of these trios reveal intricate gene regulation across tissue types.

Keywords:  TF-SNV interaction; bioinformatics; eQTL analysis; rare SNVs; regulatory trios; tissue-specific eQTLs; transcription factors

DOI:  https://doi.org/10.1016/j.ajhg.2025.01.015
Nat Commun. 2025 Feb 10. 16(1): 1500

Simultaneous profiling of chromatin-associated RNA at targeted DNA loci and RNA-RNA Interactions through TaDRIM-seq.

Cheng Ding, Guoting Chen, Shiping Luan, Runxin Gao, Yudong Fan, Ying Zhang, Xiaoting Wang, Guoliang Li, Mohamed F Foda, Jiapei Yan, Xingwang Li.

Eukaryotic genomes are extensively transcribed into various types of RNAs, many of which are physically associated with chromatin in cis at their transcription sites or in trans to other genomic loci. Emerging roles have been uncovered for these chromatin-associated RNAs (caRNAs) in gene regulation and genome organization, yet they remain challenging to interrogate. Here, we present TaDRIM-seq, a technique employing Protein G (PG)-Tn5-targeted DNA elements and in situ proximity ligation to concurrently probe caRNAs across diverse genomic regions as well as global RNA-RNA interactions within intact nuclei. Notably, this approach diminishes required cell inputs, minimizes hands-on time compared to established methodologies, and is compatible in both mammalian cells and plants. Using this technique, we identify extensive caRNAs at DNA anchor regions associated with chromatin loops and reveal diurnal variation in RNA-DNA and RNA-RNA connectivity networks within rice.

DOI: https://doi.org/10.1038/s41467-024-53534-5
Nat Genet. 2025 Feb 13.

Active repression of cell fate plasticity by PROX1 safeguards hepatocyte identity and prevents liver tumorigenesis.

Bryce Lim, Aryan Kamal, Borja Gomez Ramos, Juan M Adrian Segarra, Ignacio L Ibarra, Lennart Dignas, Tim Kindinger, Kai Volz, Mohammad Rahbari, Nuh Rahbari, Eric Poisel, Kanela Kafetzopoulou, Lio Böse, Marco Breinig, Danijela Heide, Suchira Gallage, Jose E Barragan Avila, Hendrik Wiethoff, Ivan Berest, Sarah Schnabellehner, Martin Schneider, Jonas Becker, Dominic Helm, Dirk Grimm, Taija Mäkinen, Darjus F Tschaharganeh, Mathias Heikenwalder, Judith B Zaugg, Moritz Mall.

Cell fate plasticity enables development, yet unlocked plasticity is a cancer hallmark. While transcription master regulators induce lineage-specific genes to restrict plasticity, it remains unclear whether plasticity is actively suppressed by lineage-specific repressors. Here we computationally predict so-called safeguard repressors for 18 cell types that block phenotypic plasticity lifelong. We validated hepatocyte-specific candidates using reprogramming, revealing that prospero homeobox protein 1 (PROX1) enhanced hepatocyte identity by direct repression of alternative fate master regulators. In mice, Prox1 was required for efficient hepatocyte regeneration after injury and was sufficient to prevent liver tumorigenesis. In line with patient data, Prox1 depletion caused hepatocyte fate loss in vivo and enabled the transition of hepatocellular carcinoma to cholangiocarcinoma. Conversely, overexpression promoted cholangiocarcinoma to hepatocellular carcinoma transdifferentiation. Our findings provide evidence for PROX1 as a hepatocyte-specific safeguard and support a model where cell-type-specific repressors actively suppress plasticity throughout life to safeguard lineage identity and thus prevent disease.

DOI: https://doi.org/10.1038/s41588-025-02081-w