bims-crepig 2025-11-09 papers

bims-crepig

Biomed News

on Chromatin regulation and epigenetics in cell fate and cancer

Issue of 2025–11–09
ten papers selected by
Connor Rogerson, University of Cambridge

Mapping chromatin structure at base-pair resolution unveils a unified model of cis-regulatory element interactions.
Comprehensive evaluation of diverse massively parallel reporter assays to functionally characterize human enhancers genome-wide.
A mechanism of synergistic Mediator recruitment in RNA polymerase II transcription activation revealed by single-molecule fluorescence.
Cross-platform motif discovery and benchmarking to explore binding specificities of poorly studied human transcription factors.
Transcription factor ZNF263 primes human embryonic stem cells for pluripotency dissolution and lineage commitment.
CRISPR live-cell imaging reveals chromatin dynamics and enhancer interactions at multiple non-repetitive loci.
Dissecting Oct4 enhancer function in pluripotent stem cells and mouse embryogenesis.
HIRA defines early replication initiation zones independently of their genome compartment.
DiffHiChIP: Identifying differential chromatin contacts from HiChIP data.
Nucleosome context regulates chromatin reader preference.

Cell. 2025 Nov 05. pii: S0092-8674(25)01178-X. [Epub ahead of print]

Mapping chromatin structure at base-pair resolution unveils a unified model of cis-regulatory element interactions.

Hangpeng Li, James L T Dalgleish, George Lister, Maria Julia Maristany, Jan Huertas, Ana M Dopico-Fernandez, Joseph C Hamley, Nicholas Denny, Gianna Bloye, Weijiao Zhang, Lance Hentges, Roman Doll, Ye Wei, Michela Maresca, Emilia Dimitrova, Lior Pytowski, Edward A J Tunnacliffe, Mira Kassouf, Doug Higgs, Elzo de Wit, Robert J Klose, Lothar Schermelleh, Rosana Collepardo-Guevara, Thomas A Milne, James O J Davies.

  Chromatin structure is a key determinant of gene expression in eukaryotes, but it has not been possible to define the structure of cis-regulatory elements at the scale of the proteins that bind them. Here, we generate multidimensional chromosome conformation capture (3C) maps at base-pair resolution using Micro Capture-C ultra (MCCu). This can resolve contacts between individual transcription factor motifs within cis-regulatory elements. Using degron systems, we show that removal of Mediator complex components alters fine-scale promoter structure and that nucleosome depletion plays a key role in transcription factor-driven enhancer-promoter contacts. We observe that chromatin is partitioned into nanoscale domains by nucleosome-depleted regions. This structural conformation is reproduced by chemically specific coarse-grained molecular dynamics simulations of the physicochemical properties of chromatin. Combining MCCu with molecular dynamics simulations and super-resolution microscopy allows us to propose a unified model in which the biophysical properties of chromatin orchestrate contacts between cis-regulatory elements.

Keywords:  chromatin structure; chromosome conformation capture; cis-regulatory elements; enhancer-promoter interactions; gene regulation; mediator complex; molecular dynamics simulations; nuclear organization; transcription factors

DOI:  https://doi.org/10.1016/j.cell.2025.10.013
Genome Biol. 2025 Nov 03. 26(1): 378

Comprehensive evaluation of diverse massively parallel reporter assays to functionally characterize human enhancers genome-wide.

Junke Zhang, Alden King-Yung Leung, Yutong Zhu, Li Yao, Avery Willis, Xiuqi Pan, Abdullah Ozer, Zhou Zhou, Keith Siklenka, Alejandro Barrera, Jin Liang, Nathaniel D Tippens, Timothy E Reddy, John T Lis, Haiyuan Yu.

BACKGROUND: Massively parallel reporter assays (MPRAs) and self-transcribing active regulatory region sequencing (STARR-seq) have revolutionized enhancer characterization by enabling high-throughput functional assessment of regulatory sequences.
RESULTS: Here, we systematically evaluate six MPRA and STARR-seq datasets generated in the human K562 cell line and find substantial inconsistencies in enhancer calls from different labs that are primarily due to technical variations in data processing and experimental workflows. To address these variations, we implement a uniform enhancer call pipeline, which significantly improve cross-assay agreement. While increasing sequence overlap thresholds enhanced concordance in STARR-seq assays, cross-assay consistency in LentiMPRA is strongly influenced by assay-specific factors. Functional validation using candidate cis-regulatory elements (cCREs) confirms that epigenomic features such as chromatin accessibility and histone modifications are strong predictors of enhancer activity. Importantly, our study validates transcription as a critical hallmark of active enhancers, demonstrating that highly transcribed regions exhibit significantly higher active rates across assays. Furthermore, we show that transcription enhances the predictive power of epigenomic features, enabling more accurate and refined enhancer annotation.
CONCLUSIONS: Our study provides a comprehensive framework for integrating different enhancer datasets and underscores the importance of accounting for assay-specific biases when interpreting enhancer activity. These findings refine enhancer identification using massively parallel reporter assays and improve the functional annotation of the human genome.

DOI: https://doi.org/10.1186/s13059-025-03828-8
Mol Cell. 2025 Nov 06. pii: S1097-2765(25)00826-3. [Epub ahead of print]85(21): 3965-3981.e10

A mechanism of synergistic Mediator recruitment in RNA polymerase II transcription activation revealed by single-molecule fluorescence.

Daniel H Zhou, Jongcheol Jeon, Nida Farheen, Larry J Friedman, Jane Kondev, Stephen Buratowski, Jeff Gelles.

  Transcription activators trigger transcript production by RNA polymerase II (RNAPII) via the Mediator coactivator complex. Here, the dynamics of activator, Mediator, and RNAPII binding at promoter DNA were analyzed using multi-wavelength single-molecule microscopy of fluorescently labeled proteins in budding yeast nuclear extract. Binding of Mediator and RNAPII to the template required an activator and an upstream activating sequence (UAS) but not a core promoter. While Mediator and RNAPII sometimes bind as a pre-formed complex, more commonly Mediator binds first and subsequently recruits RNAPII to form a pre-initiation complex precursor (pre-PIC) tethered to activators on the UAS. Interestingly, Mediator occupancy has a highly non-linear response to activator concentration, and fluorescence intensity measurements show that Mediator preferentially associates with templates having at least two activators bound. Statistical mechanical modeling suggests this "synergy" is not due to cooperative binding between activators but instead occurs when multiple DNA-bound activator molecules simultaneously interact with a single Mediator.

Keywords:  CoSMoS; TIRF; intrinsically disordered region; statistical mechanics; synergy; transcription activation domain

DOI:  https://doi.org/10.1016/j.molcel.2025.10.009
Commun Biol. 2025 Nov 07. 8(1): 1545

Cross-platform motif discovery and benchmarking to explore binding specificities of poorly studied human transcription factors.

Codebook/GRECO-BIT Consortium

A sequence motif representing the DNA-binding specificity of a transcription factor (TF) is commonly modelled with a positional weight matrix (PWM). Focusing on understudied human TFs, we processed results of 4,237 experiments for 394 TFs, assayed using five different experimental platforms. By human curation, we approved a subset of experiments that yielded consistent motifs across platforms and replicates, and evaluated quantitatively the cross-platform performance of PWMs obtained with ten motif discovery tools. Notably, nucleotide composition and information content are not correlated with motif performance and do not help in detecting underperformers, while motifs with low information content, in many cases, describe well the binding specificity assessed across different experimental platforms. By combining multiple PMWs into a random forest, we demonstrate the potential of accounting for multiple modes of TF binding. Finally, we present the Codebook Motif Explorer ( https://mex.autosome.org ), cataloguing motifs, benchmarking results, and the underlying experimental data.

DOI: https://doi.org/10.1038/s42003-025-08909-9
Nat Commun. 2025 Nov 05. 16(1): 9757

Transcription factor ZNF263 primes human embryonic stem cells for pluripotency dissolution and lineage commitment.

Qianqian Yin, Jing Huang, Hongduo Sun, Shuhan Zhou, Linjie Ruan, Yi Zeng, Yana Li, Zhaohui Gong, Peiyu Chen, Xinbo Yuan, Jiafang Wan, Qingzhong Wang, Yuangao Wang, Haipeng Li, Naihe Jing, Xin Liu, Zhen Shao.

Conventional human embryonic stem cells (hESCs) are capable of self-renewal and simultaneously poised for differentiation. But the mechanisms underlying this primed pluripotent state, which endows them with elevated responsiveness to differentiation cues, remain largely underexplored. Especially, little is known about the pivotal transcription factors (TFs) that orchestrate hESCs towards primed pluripotency. Here, we report a function of TF ZNF263 in pluripotency priming. Genetic and functional assays reveal that ZNF263 directly initiates the incipient expression of early differentiation genes and concurrently dampens the core pluripotency circuitry in hESCs, greatly tilting the balance from pluripotency maintenance to lineage priming. Importantly, ZNF263 deficiency markedly impairs pluripotency dissolution and multi-lineage differentiation in hESCs, particularly toward ectoderm. Moreover, single-cell transcriptomic profiling reveals that ZNF263 promotes the priming of cell fate commitment in hESCs, suggesting its indispensable requirement for pluripotency priming and lineage commitment continuum. Together, we demonstrate the role of ZNF263 in establishing the primed pluripotent state in hESCs and facilitating their differentiation into primary germ layer lineages.

DOI: https://doi.org/10.1038/s41467-025-64290-5
Nat Biotechnol. 2025 Nov 06.

CRISPR live-cell imaging reveals chromatin dynamics and enhancer interactions at multiple non-repetitive loci.

Meishuo Liu, Keyun Huang, Jie Zhang, Qingyang Li, Xinming Wang, Buming Gu, Hao Tang, Zhenhai Du, Liangjun Hu, Shutao Qi, Yu Ma, Hongtao Yu, Wei Xie, Xianyang Fang, Haifeng Wang.

Existing methods to visualize dynamic changes in the three-dimensional genome, promoter-enhancer interactions and the influence of epigenetic modifications in non-repetitive loci are limited. Here we introduce CRISPR PRO-LiveFISH (Pooled gRNAs with Orthogonal bases LiveFISH), which combines orthogonal bases from expanded genetic alphabet technology and rational single guide RNA (sgRNA) design to efficiently label multiple non-repetitive loci in living cells. The optimized method allows simultaneous imaging of up to six genomic loci and uses as few as 10 sgRNAs for non-repetitive loci imaging without signal amplification. We demonstrate the method in diverse cell types, including primary cells, and apply it to reveal enhancer-promoter dynamics and a correlation between genomic dynamics and epigenetic states. We also show that PCDHα-enhancer interactions may persist despite spatial mobility and that BRD4 maintains super-enhancer contacts regulating MYC oncogene expression in cancer cells. CRISPR PRO-LiveFISH can be applied to diverse studies of chromatin dynamics and genome organization in living cells.

DOI: https://doi.org/10.1038/s41587-025-02887-3
Stem Cell Reports. 2025 Nov 06. pii: S2213-6711(25)00310-8. [Epub ahead of print] 102706

Dissecting Oct4 enhancer function in pluripotent stem cells and mouse embryogenesis.

Daniel A Schmitz, Daiji Okamura, Masahiro Sakurai, Yi Ding, Seiya Oura, Emily Ballard, Yulei Wei, Leqian Yu, Yingying Hu, Jun Wu.

  OCT4 is a master regulator of pluripotency, with expression restricted to pluripotent and germ cells. Its expression is controlled by two cis-regulatory elements: the distal (DE) and proximal (PE) enhancers. Although widely used as markers for pluripotent stem cells (PSCs), their biological roles have remained incompletely defined. Here, we generated PSC lines and mouse models with targeted deletions of the Oct4 DE and PE. Our findings reveal that the DE is dispensable for sustaining the primed pluripotent state but required for the naive state, whereas the PE is necessary for the primed state but not for the naive state. Notably, PE-deficient naive mouse PSCs retained the capacity to differentiate into somatic lineages in vitro and to contribute chimeras. In contrast, deletion of either enhancer in vivo resulted in early embryonic lethality. These models offer powerful genetic tools to dissect the regulation of Oct4 expression during pluripotency and early development.

Keywords:  OCT4; POU5F1; chimeras; enhancer; formative pluripotency; naive pluripotency; pluripotent stem cells; primed pluripotency

DOI:  https://doi.org/10.1016/j.stemcr.2025.102706
Nat Commun. 2025 Nov 06. 16(1): 9715

HIRA defines early replication initiation zones independently of their genome compartment.

Tina Karagyozova, Alberto Gatto, Audrey Forest, Jean-Pierre Quivy, Rocío Nunez-Vazquez, Marc A Martí-Renom, Leonid A Mirny, Geneviève Almouzni.

Chromatin states and 3D architecture have been used as proxy to identify replication initiation zones (IZs) in mammalian cells, yet their functional interconnections remain a puzzle. Here, to dissect these relationships, we focus on the histone H3.3 chaperone HIRA recently implicated in early initiation zone (IZ) definition. We monitor 3D organisation, chromatin accessibility and histone post-translational modifications (PTMs) in wild-type and HIRA knock-out cells in parallel with early replication initiation. In the absence of HIRA, compartment A loses H3.3 enrichment and gains accessibility without changes in associated histone post-translational modifications (PTMs). Furthermore, impaired early firing at HIRA-dependent IZs does not correspond to changes in chromatin accessibility or patterns of histone H3 PTMs. Additionally, a small subset of early IZs initially in compartment A switch to B and lose early initiation in the absence of HIRA. Critically, HIRA complementation restores these early IZ, and H3.3 variant enrichment, without substantial compartment reversal. Thus, while HIRA contributes to compartment A features, its role in regulating early replication initiation can be uncoupled from accessibility, histone marks and compartment organisation.

DOI: https://doi.org/10.1038/s41467-025-65130-2
Cell Rep Methods. 2025 Nov 03. pii: S2667-2375(25)00250-4. [Epub ahead of print] 101214

DiffHiChIP: Identifying differential chromatin contacts from HiChIP data.

Sourya Bhattacharyya, Daniela Salgado Figueroa, Katia Georgopoulos, Ferhat Ay.

  Chromosome conformation capture (3C) assays such as HiChIP are widely used to study interactions between cis-regulatory and structural elements. However, robust methods for detecting condition-specific loops remain limited. We introduce DiffHiChIP, the first comprehensive framework to call differential loops from HiChIP and similar 3C protocols. DiffHiChIP supports DESeq2 and edgeR using either a complete contact map or a subset of contacts for background estimation, incorporates edgeR with generalized linear model (GLM) using either quasi-likelihood F test or likelihood ratio test, and implements independent hypothesis weighting (IHW) as well as a distance stratification technique for modeling distance decay of contacts in estimating statistical significance. Our results on five datasets suggest that edgeR GLM-based models with IHW correction reliably capture differential interactions, including long-range interactions, that are supported by published Hi-C data and reference studies. As HiChIP data become increasingly used for modeling chromatin regulation, DiffHiChIP promises to have a broad impact and utility.

Keywords:  3D genome organization; CP: computational biology; CP: genetics; HiChIP; chromatin looping; differential analysis; differential loops

DOI:  https://doi.org/10.1016/j.crmeth.2025.101214
Nucleic Acids Res. 2025 Oct 28. pii: gkaf1061. [Epub ahead of print]53(20):

Nucleosome context regulates chromatin reader preference.

Matthew R Marunde, Irina K Popova, Nathan W Hall, Anup Vaidya, James R Bone, Brandon A Boone, Peter J Brown, Ryan J Ezell, Tessa M Firestone, Harrison A Fuchs, Elisa Gibson, Zachary B Gillespie, Susan L Gloor, Allison R Hickman, Sarah A Howard, Natalia Ledo Husby, Victoria T Hsiung, Andrea L Johnstone, Laiba F Khan, Krzysztof Krajewski, Alexander S Lee, Eileen T McAnarney, Keith E Maier, Danielle N Maryanski, Jamie L McCuiston, Kelsey E Noll, Katherine Novitzky, Emily F Patteson, Keli L Rodriguez, Julio C Sanchez, Luis F Schachner, Catherine E Smith, Lu Sun, Hailey F Taylor, Rachel Watson, Hannah E Willis, Catherine A Musselman, Bryan J Venters, Marcus A Cheek, Matthew J Meiners, Zu-Wen Sun, Neil L Kelleher, Martis W Cowles, Ellen N Weinzapfel, Michael-Christopher Keogh, Jonathan M Burg.

Chromatin is more than a simple genome packaging system but rather locally distinguished by histone post-translational modifications (PTMs) that can directly change nucleosome structure and/or be "read" by chromatin-associated proteins to mediate downstream events. An accurate understanding of histone PTM binding preference is vital to explain normal function and pathogenesis and has revealed multiple therapeutic opportunities. Such studies most often use histone peptides, though these cannot represent the full regulatory potential of nucleosome context. Here we apply a range of complementary and easily adoptable biochemical and genomic approaches to interrogate fully defined peptide and nucleosome targets with a diversity of mono- or multivalent chromatin readers. In the resulting data, nucleosome context consistently refined reader binding, and multivalent engagement was more often regulatory than simply additive. This included abrogating binding of the Polycomb group malignant brain tumor (MBT) protein L3MBTL1 to lysine methylated histone tails and confirmation that the CBX7 chromodomain and AT-hook-like motif (CD-ATL) tandem act as a functional unit to confer specificity for H3K27me3. These in vitro nucleosome preferences were confirmed by in vivo reader-CUT&RUN genomic mapping. Such data confirms that more representative chromatin substrates provide greater insight into biological mechanism and human disease.

DOI: https://doi.org/10.1093/nar/gkaf1061