bims-crepig 2024-03-10 papers

bims-crepig

Biomed News

on Chromatin regulation and epigenetics in cell fate and cancer

Issue of 2024‒03‒10
thirty papers selected by
Connor Rogerson, University of Cambridge

MYC activity at enhancers drives prognostic transcriptional programs through an epigenetic switch.
Dissection of a CTCF topological boundary uncovers principles of enhancer-oncogene regulation.
Decoding chromatin states by proteomic profiling of nucleosome readers.
Capsid-mediated control of adeno-associated viral transcription determines host range.
Alternative splicing decouples local from global PRC2 activity.
Epigenetic profiling reveals key genes and cis-regulatory networks specific to human parathyroids.
H2AK119ub1 differentially fine-tunes gene expression by modulating canonical PRC1- and H1-dependent chromatin compaction.
Parental histone transfer caught at the replication fork.
Kdm1a safeguards the topological boundaries of PRC2-repressed genes and prevents aging-related euchromatinization in neurons.
Atlas of cardiac endothelial cell enhancer elements linking the mineralocorticoid receptor to pathological gene expression.
The transcription factor NF-κB orchestrates nucleosome remodeling during the primary response to Toll-like receptor 4 signaling.
Dynamic enhancer landscapes in human craniofacial development.
Guiding the HBO1 complex function through the JADE subunit.
Synthetic reversed sequences reveal default genomic states.
A SWI/SNF-dependent transcriptional regulation mediated by POU2AF2/C11orf53 at enhancer.
Imprinted X chromosome inactivation at the gamete-to-embryo transition.
RNAP II antagonizes mitotic chromatin folding and chromosome segregation by condensin.
Single-cell chromatin accessibility analysis reveals the epigenetic basis and signature transcription factors for the molecular subtypes of colorectal cancers.
Sister chromatid cohesion is mediated by individual cohesin complexes.
TEAD1 is crucial for developmental myelination, Remak bundles, and functional regeneration of peripheral nerves.
scAuto as a comprehensive framework for single-cell chromatin accessibility data analysis.
Determinant of m6A regional preference by transcriptional dynamics.
Predicting transcription factor activity using prior biological information.
ARID1A orchestrates SWI/SNF-mediated sequential binding of transcription factors with ARID1A loss driving pre-memory B cell fate and lymphomagenesis.
SMARCA4 is a haploinsufficient B cell lymphoma tumor suppressor that fine-tunes centrocyte cell fate decisions.
KCTD15 acts as an anti-tumor factor in colorectal cancer cells downstream of the demethylase FTO and the m6A reader YTHDF2.
Vitamin A resolves lineage plasticity to orchestrate stem cell lineage choices.
Sister chromatid cohesion halts DNA loop expansion.
Region-specific DNA hydroxymethylation along the malignant progression of IDH-mutant gliomas.
Extensive DNA methylome rearrangement during early lamprey embryogenesis.

Nat Genet. 2024 Mar 07.

MYC activity at enhancers drives prognostic transcriptional programs through an epigenetic switch.

Simon T Jakobsen, Rikke A M Jensen, Maria S Madsen, Tina Ravnsborg, Christian S Vaagenso, Majken S Siersbæk, Hjorleifur Einarsson, Robin Andersson, Ole N Jensen, Rasmus Siersbæk.

The transcription factor MYC is overexpressed in most cancers, where it drives multiple hallmarks of cancer progression. MYC is known to promote oncogenic transcription by binding to active promoters. In addition, MYC has also been shown to invade distal enhancers when expressed at oncogenic levels, but this enhancer binding has been proposed to have low gene-regulatory potential. Here, we demonstrate that MYC directly regulates enhancer activity to promote cancer type-specific gene programs predictive of poor patient prognosis. MYC induces transcription of enhancer RNA through recruitment of RNA polymerase II (RNAPII), rather than regulating RNAPII pause-release, as is the case at promoters. This process is mediated by MYC-induced H3K9 demethylation and acetylation by GCN5, leading to enhancer-specific BRD4 recruitment through its bromodomains, which facilitates RNAPII recruitment. We propose that MYC drives prognostic cancer type-specific gene programs through induction of an enhancer-specific epigenetic switch, which can be targeted by BET and GCN5 inhibitors.

DOI: https://doi.org/10.1038/s41588-024-01676-z
Mol Cell. 2024 Feb 28. pii: S1097-2765(24)00126-6. [Epub ahead of print]

Dissection of a CTCF topological boundary uncovers principles of enhancer-oncogene regulation.

Kyung Lock Kim, Gilbert J Rahme, Viraat Y Goel, Chadi A El Farran, Anders S Hansen, Bradley E Bernstein.

  Enhancer-gene communication is dependent on topologically associating domains (TADs) and boundaries enforced by the CCCTC-binding factor (CTCF) insulator, but the underlying structures and mechanisms remain controversial. Here, we investigate a boundary that typically insulates fibroblast growth factor (FGF) oncogenes but is disrupted by DNA hypermethylation in gastrointestinal stromal tumors (GISTs). The boundary contains an array of CTCF sites that enforce adjacent TADs, one containing FGF genes and the other containing ANO1 and its putative enhancers, which are specifically active in GIST and its likely cell of origin. We show that coordinate disruption of four CTCF motifs in the boundary fuses the adjacent TADs, allows the ANO1 enhancer to contact FGF3, and causes its robust induction. High-resolution micro-C maps reveal specific contact between transcription initiation sites in the ANO1 enhancer and FGF3 promoter that quantitatively scales with FGF3 induction such that modest changes in contact frequency result in strong changes in expression, consistent with a causal relationship.

Keywords:  ANO1; CTCF insulator; DNA methylation; FGF3; FGF4; SDH deficiency; chromatin; enhancer regulation; gastrointestinal stromal tumor; genome topology

DOI:  https://doi.org/10.1016/j.molcel.2024.02.007
Nature. 2024 Mar 06.

Decoding chromatin states by proteomic profiling of nucleosome readers.

Saulius Lukauskas, Andrey Tvardovskiy, Nhuong V Nguyen, Mara Stadler, Peter Faull, Tina Ravnsborg, Bihter Özdemir Aygenli, Scarlett Dornauer, Helen Flynn, Rik G H Lindeboom, Teresa K Barth, Kevin Brockers, Stefanie M Hauck, Michiel Vermeulen, Ambrosius P Snijders, Christian L Müller, Peter A DiMaggio, Ole N Jensen, Robert Schneider, Till Bartke.

DNA and histone modifications combine into characteristic patterns that demarcate functional regions of the genome1,2. While many 'readers' of individual modifications have been described3-5, how chromatin states comprising composite modification signatures, histone variants and internucleosomal linker DNA are interpreted is a major open question. Here we use a multidimensional proteomics strategy to systematically examine the interaction of around 2,000 nuclear proteins with over 80 modified dinucleosomes representing promoter, enhancer and heterochromatin states. By deconvoluting complex nucleosome-binding profiles into networks of co-regulated proteins and distinct nucleosomal features driving protein recruitment or exclusion, we show comprehensively how chromatin states are decoded by chromatin readers. We find highly distinctive binding responses to different features, many factors that recognize multiple features, and that nucleosomal modifications and linker DNA operate largely independently in regulating protein binding to chromatin. Our online resource, the Modification Atlas of Regulation by Chromatin States (MARCS), provides in-depth analysis tools to engage with our results and advance the discovery of fundamental principles of genome regulation by chromatin states.

DOI: https://doi.org/10.1038/s41586-024-07141-5
Cell Rep. 2024 Mar 02. pii: S2211-1247(24)00230-4. [Epub ahead of print]43(3): 113902

Capsid-mediated control of adeno-associated viral transcription determines host range.

Ezra J Loeb, Patrick L Havlik, Zachary C Elmore, Alan Rosales, Sophia M Fergione, Trevor J Gonzalez, Timothy J Smith, Abigail R Benkert, David N Fiflis, Aravind Asokan.

  Adeno-associated virus (AAV) is a member of the genus Dependoparvovirus, which infects a wide range of vertebrate species. Here, we observe that, unlike most primate AAV isolates, avian AAV is transcriptionally silenced in human cells. By swapping the VP1 N terminus from primate AAVs (e.g., AAV8) onto non-mammalian isolates (e.g., avian AAV), we identify a minimal component of the AAV capsid that controls viral transcription and unlocks robust transduction in both human cells and mouse tissue. This effect is accompanied by increased AAV genome chromatin accessibility and altered histone methylation. Proximity ligation analysis reveals that host factors are selectively recruited by the VP1 N terminus of AAV8 but not avian AAV. Notably, these include AAV essential factors implicated in the nuclear factor κB pathway, chromatin condensation, and histone methylation. We postulate that the AAV capsid has evolved mechanisms to recruit host factors to its genome, allowing transcriptional activation in a species-specific manner.

Keywords:  CP: Microbiology; CP: Molecular biology; Dependoparvovirus; adeno-associated virus; capsid biology; histone methylation; host range; host-virus interactions; proteomics; transcriptional regulation; viral tropism

DOI:  https://doi.org/10.1016/j.celrep.2024.113902
Mol Cell. 2024 Feb 28. pii: S1097-2765(24)00130-8. [Epub ahead of print]

Alternative splicing decouples local from global PRC2 activity.

Niccolò Arecco, Ivano Mocavini, Enrique Blanco, Cecilia Ballaré, Elina Libman, Sophie Bonnal, Manuel Irimia, Luciano Di Croce.

  The Polycomb repressive complex 2 (PRC2) mediates epigenetic maintenance of gene silencing in eukaryotes via methylation of histone H3 at lysine 27 (H3K27). Accessory factors define two distinct subtypes, PRC2.1 and PRC2.2, with different actions and chromatin-targeting mechanisms. The mechanisms orchestrating PRC2 assembly are not fully understood. Here, we report that alternative splicing (AS) of PRC2 core component SUZ12 generates an uncharacterized isoform SUZ12-S, which co-exists with the canonical SUZ12-L isoform in virtually all tissues and developmental stages. SUZ12-S drives PRC2.1 formation and favors PRC2 dimerization. While SUZ12-S is necessary and sufficient for the repression of target genes via promoter-proximal H3K27me3 deposition, SUZ12-L maintains global H3K27 methylation levels. Mouse embryonic stem cells (ESCs) lacking either isoform exit pluripotency more slowly and fail to acquire neuronal cell identity. Our findings reveal a physiological mechanism regulating PRC2 assembly and higher-order interactions in eutherians, with impacts on H3K27 methylation and gene repression.

Keywords:  H3K27me3; PRC2; PanAS; SUZ12; alternative splicing; dimerization; gene silencing; neuronal differentiation; polycomb; stem cells

DOI:  https://doi.org/10.1016/j.molcel.2024.02.011
Nat Commun. 2024 Mar 07. 15(1): 2106

Epigenetic profiling reveals key genes and cis-regulatory networks specific to human parathyroids.

Youngsook Lucy Jung, Wenping Zhao, Ian Li, Dhawal Jain, Charles B Epstein, Bradley E Bernstein, Sareh Parangi, Richard Sherwood, Cassianne Robinson-Cohen, Yi-Hsiang Hsu, Peter J Park, Michael Mannstadt.

In all terrestrial vertebrates, the parathyroid glands are critical regulators of calcium homeostasis and the sole source of parathyroid hormone (PTH). Hyperparathyroidism and hypoparathyroidism are clinically important disorders affecting multiple organs. However, our knowledge regarding regulatory mechanisms governing the parathyroids has remained limited. Here, we present the comprehensive maps of the chromatin landscape of the human parathyroid glands, identifying active regulatory elements and chromatin interactions. These data allow us to define regulatory circuits and previously unidentified genes that play crucial roles in parathyroid biology. We experimentally validate candidate parathyroid-specific enhancers and demonstrate their integration with GWAS SNPs for parathyroid-related diseases and traits. For instance, we observe reduced activity of a parathyroid-specific enhancer of the Calcium Sensing Receptor gene, which contains a risk allele associated with higher PTH levels compared to the wildtype allele. Our datasets provide a valuable resource for unraveling the mechanisms governing parathyroid gland regulation in health and disease.

DOI: https://doi.org/10.1038/s41467-024-46181-3
Mol Cell. 2024 Feb 29. pii: S1097-2765(24)00136-9. [Epub ahead of print]

H2AK119ub1 differentially fine-tunes gene expression by modulating canonical PRC1- and H1-dependent chromatin compaction.

Jicheng Zhao, Jie Lan, Min Wang, Cuifang Liu, Zheng Fang, Aoqun Song, Tiantian Zhang, Liang Wang, Bing Zhu, Ping Chen, Juan Yu, Guohong Li.

  Polycomb repressive complex 1 (PRC1) is a key transcriptional regulator in development via modulating chromatin structure and catalyzing histone H2A ubiquitination at Lys119 (H2AK119ub1). H2AK119ub1 is one of the most abundant histone modifications in mammalian cells. However, the function of H2AK119ub1 in polycomb-mediated gene silencing remains debated. In this study, we reveal that H2AK119ub1 has two distinct roles in gene expression, through differentially modulating chromatin compaction mediated by canonical PRC1 and the linker histone H1. Interestingly, we find that H2AK119ub1 plays a positive role in transcription through interfering with the binding of canonical PRC1 to nucleosomes and therefore counteracting chromatin condensation. Conversely, we demonstrate that H2AK119ub1 facilitates H1-dependent chromatin condensation and enhances the silencing of developmental genes in mouse embryonic stem cells, suggesting that H1 may be one of several possible pathways for H2AK119ub1 in repressing transcription. These results provide insights and molecular mechanisms by which H2AK119ub1 differentially fine-tunes developmental gene expression.

Keywords:  H2AK119ub1; canonical PRC1; polycomb repressive complex; the linker histone H1; transcription regulation

DOI:  https://doi.org/10.1016/j.molcel.2024.02.017
Nature. 2024 Mar 06.

Parental histone transfer caught at the replication fork.

Ningning Li, Yuan Gao, Yujie Zhang, Daqi Yu, Jianwei Lin, Jianxun Feng, Jian Li, Zhichun Xu, Yingyi Zhang, Shangyu Dang, Keda Zhou, Yang Liu, Xiang David Li, Bik Kwoon Tye, Qing Li, Ning Gao, Yuanliang Zhai.

In eukaryotes, DNA compacts into chromatin through nucleosomes1,2. Replication of the eukaryotic genome must be coupled to the transmission of the epigenome encoded in the chromatin3,4. Here we report cryo-electron microscopy structures of yeast (Saccharomyces cerevisiae) replisomes associated with the FACT (facilitates chromatin transactions) complex (comprising Spt16 and Pob3) and an evicted histone hexamer. In these structures, FACT is positioned at the front end of the replisome by engaging with the parental DNA duplex to capture the histones through the middle domain and the acidic carboxyl-terminal domain of Spt16. The H2A-H2B dimer chaperoned by the carboxyl-terminal domain of Spt16 is stably tethered to the H3-H4 tetramer, while the vacant H2A-H2B site is occupied by the histone-binding domain of Mcm2. The Mcm2 histone-binding domain wraps around the DNA-binding surface of one H3-H4 dimer and extends across the tetramerization interface of the H3-H4 tetramer to the binding site of Spt16 middle domain before becoming disordered. This arrangement leaves the remaining DNA-binding surface of the other H3-H4 dimer exposed to additional interactions for further processing. The Mcm2 histone-binding domain and its downstream linker region are nested on top of Tof1, relocating the parental histones to the replisome front for transfer to the newly synthesized lagging-strand DNA. Our findings offer crucial structural insights into the mechanism of replication-coupled histone recycling for maintaining epigenetic inheritance.

DOI: https://doi.org/10.1038/s41586-024-07152-2
Nat Commun. 2024 Mar 07. 15(1): 1781

Kdm1a safeguards the topological boundaries of PRC2-repressed genes and prevents aging-related euchromatinization in neurons.

Beatriz Del Blanco, Sergio Niñerola, Ana M Martín-González, Juan Paraíso-Luna, Minji Kim, Rafael Muñoz-Viana, Carina Racovac, Jose V Sanchez-Mut, Yijun Ruan, Ángel Barco.

Kdm1a is a histone demethylase linked to intellectual disability with essential roles during gastrulation and the terminal differentiation of specialized cell types, including neurons, that remains highly expressed in the adult brain. To explore Kdm1a's function in adult neurons, we develop inducible and forebrain-restricted Kdm1a knockouts. By applying multi-omic transcriptome, epigenome and chromatin conformation data, combined with super-resolution microscopy, we find that Kdm1a elimination causes the neuronal activation of nonneuronal genes that are silenced by the polycomb repressor complex and interspersed with active genes. Functional assays demonstrate that the N-terminus of Kdm1a contains an intrinsically disordered region that is essential to segregate Kdm1a-repressed genes from the neighboring active chromatin environment. Finally, we show that the segregation of Kdm1a-target genes is weakened in neurons during natural aging, underscoring the role of Kdm1a safeguarding neuronal genome organization and gene silencing throughout life.

DOI: https://doi.org/10.1038/s41467-024-45773-3
Sci Adv. 2024 Mar 08. 10(10): eadj5101

Atlas of cardiac endothelial cell enhancer elements linking the mineralocorticoid receptor to pathological gene expression.

Lisa Deng, Luisa Pollmeier, Rebecca Bednarz, Can Cao, Patrick Laurette, Luisa Wirth, Argen Mamazhakypov, Christine Bode, Lutz Hein, Ralf Gilsbach, Achim Lother.

Endothelial cells play crucial roles in physiology and are increasingly recognized as therapeutic targets in cardiovascular disease. Here, we analyzed the regulatory landscape of cardiac endothelial cells by assessing chromatin accessibility, histone modifications, and 3D chromatin organization and confirmed the functional relevance of enhancer-promoter interactions by CRISPRi-mediated enhancer silencing. We used this dataset to explore mechanisms of transcriptional regulation in cardiovascular disease and compared six different experimental models of heart failure, hypertension, or diabetes. Enhancers that regulate gene expression in diseased endothelial cells were enriched with binding sites for a distinct set of transcription factors, including the mineralocorticoid receptor (MR), a known drug target in heart failure and hypertension. For proof of concept, we applied endothelial cell-specific MR deletion in mice to confirm MR-dependent gene expression and predicted direct MR target genes. Overall, we have compiled here a comprehensive atlas of cardiac endothelial cell enhancer elements that provides insight into the role of transcription factors in cardiovascular disease.

DOI: https://doi.org/10.1126/sciadv.adj5101
Immunity. 2024 Feb 28. pii: S1074-7613(24)00080-3. [Epub ahead of print]

The transcription factor NF-κB orchestrates nucleosome remodeling during the primary response to Toll-like receptor 4 signaling.

An-Chieh Feng, Brandon J Thomas, Prabhat K Purbey, Filipe Menegatti de Melo, Xin Liu, Allison E Daly, Fei Sun, Jerry Hung-Hao Lo, Lijing Cheng, Michael F Carey, Philip O Scumpia, Stephen T Smale.

  Inducible nucleosome remodeling at hundreds of latent enhancers and several promoters shapes the transcriptional response to Toll-like receptor 4 (TLR4) signaling in macrophages. We aimed to define the identities of the transcription factors that promote TLR-induced remodeling. An analysis strategy based on ATAC-seq and single-cell ATAC-seq that enriched for genomic regions most likely to undergo remodeling revealed that the transcription factor nuclear factor κB (NF-κB) bound to all high-confidence peaks marking remodeling during the primary response to the TLR4 ligand, lipid A. Deletion of NF-κB subunits RelA and c-Rel resulted in the loss of remodeling at high-confidence ATAC-seq peaks, and CRISPR-Cas9 mutagenesis of NF-κB-binding motifs impaired remodeling. Remodeling selectivity at defined regions was conferred by collaboration with other inducible factors, including IRF3- and MAP-kinase-induced factors. Thus, NF-κB is unique among TLR4-activated transcription factors in its broad contribution to inducible nucleosome remodeling, alongside its ability to activate poised enhancers and promoters assembled into open chromatin.

Keywords:  IRF3; NF-κB; chromatin; macrophages; nucleosome remodeling; transcription

DOI:  https://doi.org/10.1016/j.immuni.2024.02.004
Nat Commun. 2024 Mar 06. 15(1): 2030

Dynamic enhancer landscapes in human craniofacial development.

Sudha Sunil Rajderkar, Kitt Paraiso, Maria Luisa Amaral, Michael Kosicki, Laura E Cook, Fabrice Darbellay, Cailyn H Spurrell, Marco Osterwalder, Yiwen Zhu, Han Wu, Sarah Yasmeen Afzal, Matthew J Blow, Guy Kelman, Iros Barozzi, Yoko Fukuda-Yuzawa, Jennifer A Akiyama, Veena Afzal, Stella Tran, Ingrid Plajzer-Frick, Catherine S Novak, Momoe Kato, Riana D Hunter, Kianna von Maydell, Allen Wang, Lin Lin, Sebastian Preissl, Steven Lisgo, Bing Ren, Diane E Dickel, Len A Pennacchio, Axel Visel.

The genetic basis of human facial variation and craniofacial birth defects remains poorly understood. Distant-acting transcriptional enhancers control the fine-tuned spatiotemporal expression of genes during critical stages of craniofacial development. However, a lack of accurate maps of the genomic locations and cell type-resolved activities of craniofacial enhancers prevents their systematic exploration in human genetics studies. Here, we combine histone modification, chromatin accessibility, and gene expression profiling of human craniofacial development with single-cell analyses of the developing mouse face to define the regulatory landscape of facial development at tissue- and single cell-resolution. We provide temporal activity profiles for 14,000 human developmental craniofacial enhancers. We find that 56% of human craniofacial enhancers share chromatin accessibility in the mouse and we provide cell population- and embryonic stage-resolved predictions of their in vivo activity. Taken together, our data provide an expansive resource for genetic and developmental studies of human craniofacial development.

DOI: https://doi.org/10.1038/s41467-024-46396-4
Nat Struct Mol Biol. 2024 Mar 06.

Guiding the HBO1 complex function through the JADE subunit.

Nitika Gaurav, Akinori Kanai, Catherine Lachance, Khan L Cox, Jiuyang Liu, Adrian T Grzybowski, Nehmé Saksouk, Brianna J Klein, Yosuke Komata, Shuhei Asada, Alexander J Ruthenburg, Michael G Poirier, Jacques Côté, Akihiko Yokoyama, Tatiana G Kutateladze.

JADE is a core subunit of the HBO1 acetyltransferase complex that regulates developmental and epigenetic programs and promotes gene transcription. Here we describe the mechanism by which JADE facilitates recruitment of the HBO1 complex to chromatin and mediates its enzymatic activity. Structural, genomic and complex assembly in vivo studies show that the PZP (PHD1-zinc-knuckle-PHD2) domain of JADE engages the nucleosome through binding to histone H3 and DNA and is necessary for the association with chromatin targets. Recognition of unmethylated H3K4 by PZP directs enzymatic activity of the complex toward histone H4 acetylation, whereas H3K4 hypermethylation alters histone substrate selectivity. We demonstrate that PZP contributes to leukemogenesis, augmenting transforming activity of the NUP98-JADE2 fusion. Our findings highlight biological consequences and the impact of the intact JADE subunit on genomic recruitment, enzymatic function and pathological activity of the HBO1 complex.

DOI: https://doi.org/10.1038/s41594-024-01245-2
Nature. 2024 Mar 06.

Synthetic reversed sequences reveal default genomic states.

Brendan R Camellato, Ran Brosh, Hannah J Ashe, Matthew T Maurano, Jef D Boeke.

Pervasive transcriptional activity is observed across diverse species. The genomes of extant organisms have undergone billions of years of evolution, making it unclear whether these genomic activities represent effects of selection or 'noise'1-4. Characterizing default genome states could help understand whether pervasive transcriptional activity has biological meaning. Here we addressed this question by introducing a synthetic 101-kb locus into the genomes of Saccharomyces cerevisiae and Mus musculus and characterizing genomic activity. The locus was designed by reversing but not complementing human HPRT1, including its flanking regions, thus retaining basic features of the natural sequence but ablating evolved coding or regulatory information. We observed widespread activity of both reversed and native HPRT1 loci in yeast, despite the lack of evolved yeast promoters. By contrast, the reversed locus displayed no activity at all in mouse embryonic stem cells, and instead exhibited repressive chromatin signatures. The repressive signature was alleviated in a locus variant lacking CpG dinucleotides; nevertheless, this variant was also transcriptionally inactive. These results show that synthetic genomic sequences that lack coding information are active in yeast, but inactive in mouse embryonic stem cells, consistent with a major difference in 'default genomic states' between these two divergent eukaryotic cell types, with implications for understanding pervasive transcription, horizontal transfer of genetic information and the birth of new genes.

DOI: https://doi.org/10.1038/s41586-024-07128-2
Nat Commun. 2024 Mar 07. 15(1): 2067

A SWI/SNF-dependent transcriptional regulation mediated by POU2AF2/C11orf53 at enhancer.

Aileen Szczepanski, Natsumi Tsuboyama, Huijue Lyu, Ping Wang, Oguzhan Beytullahoglu, Te Zhang, Benjamin David Singer, Feng Yue, Zibo Zhao, Lu Wang.

Recent studies have identified a previously uncharacterized protein C11orf53 (now named POU2AF2/OCA-T1), which functions as a robust co-activator of POU2F3, the master transcription factor which is critical for both normal and neoplastic tuft cell identity and viability. Here, we demonstrate that POU2AF2 dictates opposing transcriptional regulation at distal enhance elements. Loss of POU2AF2 leads to an inhibition of active enhancer nearby genes, such as tuft cell identity genes, and a derepression of Polycomb-dependent poised enhancer nearby genes, which are critical for cell viability and differentiation. Mechanistically, depletion of POU2AF2 results in a global redistribution of the chromatin occupancy of the SWI/SNF complex, leading to a significant 3D genome structure change and a subsequent transcriptional reprogramming. Our genome-wide CRISPR screen further demonstrates that POU2AF2 depletion or SWI/SNF inhibition leads to a PTEN-dependent cell growth defect, highlighting a potential role of POU2AF2-SWI/SNF axis in small cell lung cancer (SCLC) pathogenesis. Additionally, pharmacological inhibition of SWI/SNF phenocopies POU2AF2 depletion in terms of gene expression alteration and cell viability decrease in SCLC-P subtype cells. Therefore, impeding POU2AF2-mediated transcriptional regulation represents a potential therapeutic approach for human SCLC therapy.

DOI: https://doi.org/10.1038/s41467-024-46492-5
Mol Cell. 2024 Feb 29. pii: S1097-2765(24)00132-1. [Epub ahead of print]

Imprinted X chromosome inactivation at the gamete-to-embryo transition.

Chunyao Wei, Barry Kesner, Hao Yin, Jeannie T Lee.

  In mammals, dosage compensation involves two parallel processes: (1) X inactivation, which equalizes X chromosome dosage between males and females, and (2) X hyperactivation, which upregulates the active X for X-autosome balance. The field currently favors models whereby dosage compensation initiates "de novo" during mouse development. Here, we develop "So-Smart-seq" to revisit the question and interrogate a comprehensive transcriptome including noncoding genes and repeats in mice. Intriguingly, de novo silencing pertains only to a subset of Xp genes. Evolutionarily older genes and repetitive elements demonstrate constitutive Xp silencing, adopt distinct signatures, and do not require Xist to initiate silencing. We trace Xp silencing backward in developmental time to meiotic sex chromosome inactivation in the male germ line and observe that Xm hyperactivation is timed to Xp silencing on a gene-by-gene basis. Thus, during the gamete-to-embryo transition, older Xp genes are transmitted in a "pre-inactivated" state. These findings have implications for the evolution of imprinting.

Keywords:  DNA methylation; H3K27me3; LINE; Polycomb; X hyperactivation; Xist; chromatin accessibility; imprinted X inactivation; mouse preimplantation embryo; zygotic genome activation

DOI:  https://doi.org/10.1016/j.molcel.2024.02.013
Cell Rep. 2024 Mar 05. pii: S2211-1247(24)00229-8. [Epub ahead of print]43(3): 113901

RNAP II antagonizes mitotic chromatin folding and chromosome segregation by condensin.

Jérémy Lebreton, Léonard Colin, Elodie Chatre, Pascal Bernard.

  Condensin shapes mitotic chromosomes by folding chromatin into loops, but whether it does so by DNA-loop extrusion remains speculative. Although loop-extruding cohesin is stalled by transcription, the impact of transcription on condensin, which is enriched at highly expressed genes in many species, remains unclear. Using degrons of Rpb1 or the torpedo nuclease Dhp1XRN2 to either deplete or displace RNAPII on chromatin in fission yeast metaphase cells, we show that RNAPII does not load condensin on DNA. Instead, RNAPII retains condensin in cis and hinders its ability to fold mitotic chromatin and to support chromosome segregation, consistent with the stalling of a loop extruder. Transcription termination by Dhp1 limits such a hindrance. Our results shed light on the integrated functioning of condensin, and we argue that a tight control of transcription underlies mitotic chromosome assembly by loop-extruding condensin.

Keywords:  CP: Molecular biology; SMC complexes; condensin; loop extrusion; mitotic chromosome assembly; transcription; transcription-termination

DOI:  https://doi.org/10.1016/j.celrep.2024.113901
Cancer Discov. 2024 Mar 06.

Single-cell chromatin accessibility analysis reveals the epigenetic basis and signature transcription factors for the molecular subtypes of colorectal cancers.

Zhenyu Liu, Yuqiong Hu, Haoling Xie, Kexuan Chen, Lu Wen, Wei Fu, Xin Zhou, Fuchou Tang.

Colorectal cancer (CRC) is a highly heterogeneous disease, with well-characterized subtypes based on genome, DNA methylome, and transcriptome signatures. To chart the epigenetic landscape of CRCs, we generated a high-quality single-cell chromatin accessibility atlas of epithelial cells for 29 patients. Abnormal chromatin states acquired in adenomas were largely retained in CRCs, which were tightly accompanied by opposite changes of DNA methylation. Unsupervised analysis on malignant cells revealed two epigenetic subtypes, exactly matching iCMS classification, and key iCMS-specific transcription factors were identified, including HNF4A, PPARA for iCMS2 tumors, and FOXA3, MAFK for iCMS3 tumors. Notably, subtype-specific TFs bind to distinct target gene sets and contribute to both inter-patient similarities and diversities for both chromatin accessibilities and RNA expressions. Moreover, we identified CpG-island methylator phenotypes and pinpointed chromatin state signatures and TF regulators for CIMP-High subtype. Our work systematically revealed the epigenetic basis of the well-known iCMS and CIMP classifications of CRCs.

DOI: https://doi.org/10.1158/2159-8290.CD-23-1445
Science. 2024 Mar 08. 383(6687): 1122-1130

Sister chromatid cohesion is mediated by individual cohesin complexes.

Fena Ochs, Charlotte Green, Aleksander Tomasz Szczurek, Lior Pytowski, Sofia Kolesnikova, Jill Brown, Daniel Wolfram Gerlich, Veronica Buckle, Lothar Schermelleh, Kim Ashley Nasmyth.

Eukaryotic genomes are organized by loop extrusion and sister chromatid cohesion, both mediated by the multimeric cohesin protein complex. Understanding how cohesin holds sister DNAs together, and how loss of cohesion causes age-related infertility in females, requires knowledge as to cohesin's stoichiometry in vivo. Using quantitative super-resolution imaging, we identified two discrete populations of chromatin-bound cohesin in postreplicative human cells. Whereas most complexes appear dimeric, cohesin that localized to sites of sister chromatid cohesion and associated with sororin was exclusively monomeric. The monomeric stoichiometry of sororin:cohesin complexes demonstrates that sister chromatid cohesion is conferred by individual cohesin rings, a key prediction of the proposal that cohesion arises from the co-entrapment of sister DNAs.

DOI: https://doi.org/10.1126/science.adl4606
Elife. 2024 Mar 08. pii: e87394. [Epub ahead of print]13

TEAD1 is crucial for developmental myelination, Remak bundles, and functional regeneration of peripheral nerves.

Matthew Grove, Hyukmin Kim, Shuhuan Pang, Jose Paz Amaya, Guoqing Hu, Jiliang Zhou, Michel A Lemay, Young-Jin Son.

  Previously we showed that the hippo pathway transcriptional effectors, YAP and TAZ, are essential for Schwann cells (SCs) to develop, maintain and regenerate myelin (Grove et al., 2017; Grove, Lee, Zhao, & Son, 2020). Although TEAD1 has been implicated as a partner transcription factor, the mechanisms by which it mediates YAP/TAZ regulation of SC myelination are unclear. Here, using conditional and inducible knockout mice, we show that TEAD1 is crucial for SCs to develop and regenerate myelin. It promotes myelination by both positively and negatively regulating SC proliferation, enabling Krox20/Egr2 to upregulate myelin proteins, and upregulating the cholesterol biosynthetic enzymes FDPS and IDI1. We also show stage-dependent redundancy of TEAD1 and that non-myelinating SCs have a unique requirement for TEAD1 to enwrap nociceptive axons in Remak bundles. Our findings establish TEAD1 as a major partner of YAP/TAZ in developmental myelination and functional nerve regeneration and as a novel transcription factor regulating Remak bundle integrity.

Keywords:  mouse; neuroscience

DOI:  https://doi.org/10.7554/eLife.87394
Comput Biol Med. 2024 Feb 29. pii: S0010-4825(24)00314-7. [Epub ahead of print]171 108230

scAuto as a comprehensive framework for single-cell chromatin accessibility data analysis.

Meiqin Gong, Yun Yu, Zixuan Wang, Junming Zhang, Xiongyi Wang, Cheng Fu, Yongqing Zhang, Xiaodong Wang.

  Interpreting single-cell chromatin accessibility data is crucial for understanding intercellular heterogeneity regulation. Despite the progress in computational methods for analyzing this data, there is still a lack of a comprehensive analytical framework and a user-friendly online analysis tool. To fill this gap, we developed a pre-trained deep learning-based framework, single-cell auto-correlation transformers (scAuto), to overcome the challenge. Following DNABERT's methodology of pre-training and fine-tuning, scAuto learns a general understanding of DNA sequence's grammar by being pre-trained on unlabeled human genome via self-supervision; it is then transferred to the single-cell chromatin accessibility analysis task of scATAC-seq data for supervised fine-tuning. We extensively validated scAuto on the Buenrostro2018 dataset, demonstrating its superior performance on chromatin accessibility prediction, single-cell clustering, and data denoising. Based on scAuto, we further developed an interactive web server for single-cell chromatin accessibility data analysis. It integrates tutorial-style interfaces for those with limited programming skills. The platform is accessible at http://zhanglab.icaup.cn. To our knowledge, this work is expected to help analyze single-cell chromatin accessibility data and facilitate the development of precision medicine.

Keywords:  Chromatin accessibility; Data analysis tools; Deep learning; Single-cell genomics; Web server

DOI:  https://doi.org/10.1016/j.compbiomed.2024.108230
Nucleic Acids Res. 2024 Mar 07. pii: gkae169. [Epub ahead of print]

Determinant of m6A regional preference by transcriptional dynamics.

Yalan Wang, Shen Wang, Zhen Meng, Xiao-Min Liu, Yuanhui Mao.

N6-Methyladenosine (m6A) is the most abundant chemical modification occurring on eukaryotic mRNAs, and has been reported to be involved in almost all stages of mRNA metabolism. The distribution of m6A sites is notably asymmetric along mRNAs, with a strong preference toward the 3' terminus of the transcript. How m6A regional preference is shaped remains incompletely understood. In this study, by performing m6A-seq on chromatin-associated RNAs, we found that m6A regional preference arises during transcription. Nucleosome occupancy is remarkedly increased in the region downstream of m6A sites, suggesting an intricate interplay between m6A methylation and nucleosome-mediated transcriptional dynamics. Notably, we found a remarkable slowdown of Pol-II movement around m6A sites. In addition, inhibiting Pol-II movement increases nearby m6A methylation levels. By analyzing massively parallel assays for m6A, we found that RNA secondary structures inhibit m6A methylation. Remarkably, the m6A sites associated with Pol-II pausing tend to be embedded within RNA secondary structures. These results suggest that Pol-II pausing could affect the accessibility of m6A motifs to the methyltransferase complex and subsequent m6A methylation by mediating RNA secondary structure. Overall, our study reveals a crucial role of transcriptional dynamics in the formation of m6A regional preference.

DOI: https://doi.org/10.1093/nar/gkae169
iScience. 2024 Mar 15. 27(3): 109124

Predicting transcription factor activity using prior biological information.

William M Yashar, Joseph Estabrook, Hannah D Holly, Julia Somers, Olga Nikolova, Özgün Babur, Theodore P Braun, Emek Demir.

  Dysregulation of normal transcription factor activity is a common driver of disease. Therefore, the detection of aberrant transcription factor activity is important to understand disease pathogenesis. We have developed Priori, a method to predict transcription factor activity from RNA sequencing data. Priori has two key advantages over existing methods. First, Priori utilizes literature-supported regulatory information to identify transcription factor-target gene relationships. It then applies linear models to determine the impact of transcription factor regulation on the expression of its target genes. Second, results from a third-party benchmarking pipeline reveals that Priori detects aberrant activity from 124 single-gene perturbation experiments with higher sensitivity and specificity than 11 other methods. We applied Priori and other top-performing methods to predict transcription factor activity from two large primary patient datasets. Our work demonstrates that Priori uniquely discovered significant determinants of survival in breast cancer and identified mediators of drug response in leukemia.

Keywords:  Biocomputational method; Biological constraints; Gene network; Molecular mechanism of gene regulation

DOI:  https://doi.org/10.1016/j.isci.2024.109124
Cancer Cell. 2024 Mar 04. pii: S1535-6108(24)00054-0. [Epub ahead of print]

ARID1A orchestrates SWI/SNF-mediated sequential binding of transcription factors with ARID1A loss driving pre-memory B cell fate and lymphomagenesis.

Darko Barisic, Christopher R Chin, Cem Meydan, Matt Teater, Ioanna Tsialta, Coraline Mlynarczyk, Amy Chadburn, Xuehai Wang, Margot Sarkozy, Min Xia, Sandra E Carson, Santo Raggiri, Sonia Debek, Benedikt Pelzer, Ceyda Durmaz, Qing Deng, Priya Lakra, Martin Rivas, Christian Steidl, David W Scott, Andrew P Weng, Christopher E Mason, Michael R Green, Ari Melnick.

  ARID1A, a subunit of the canonical BAF nucleosome remodeling complex, is commonly mutated in lymphomas. We show that ARID1A orchestrates B cell fate during the germinal center (GC) response, facilitating cooperative and sequential binding of PU.1 and NF-kB at crucial genes for cytokine and CD40 signaling. The absence of ARID1A tilts GC cell fate toward immature IgM+CD80-PD-L2- memory B cells, known for their potential to re-enter new GCs. When combined with BCL2 oncogene, ARID1A haploinsufficiency hastens the progression of aggressive follicular lymphomas (FLs) in mice. Patients with FL with ARID1A-inactivating mutations preferentially display an immature memory B cell-like state with increased transformation risk to aggressive disease. These observations offer mechanistic understanding into the emergence of both indolent and aggressive ARID1A-mutant lymphomas through the formation of immature memory-like clonal precursors. Lastly, we demonstrate that ARID1A mutation induces synthetic lethality to SMARCA2/4 inhibition, paving the way for potential precision therapy for high-risk patients.

Keywords:  BAF complex; chromatin; chromatin remodeling; clonal precursor cells; epigenetics; humoral immunity; lymphoma; pioneer transcription factors; plasticity; precision therapy

DOI:  https://doi.org/10.1016/j.ccell.2024.02.010
Cancer Cell. 2024 Mar 01. pii: S1535-6108(24)00055-2. [Epub ahead of print]

SMARCA4 is a haploinsufficient B cell lymphoma tumor suppressor that fine-tunes centrocyte cell fate decisions.

Qing Deng, Priya Lakra, Panhong Gou, Haopeng Yang, Cem Meydan, Matthew Teater, Christopher Chin, Wenchao Zhang, Tommy Dinh, Usama Hussein, Xubin Li, Estela Rojas, Weiguang Liu, Patrick K Reville, Atish Kizhakeyil, Darko Barisic, Sydney Parsons, Ashley Wilson, Jared Henderson, Brooks Scull, Channabasavaiah Gurumurthy, Francisco Vega, Amy Chadburn, Branko Cuglievan, Nader Kim El-Mallawany, Carl Allen, Christopher Mason, Ari Melnick, Michael R Green.

  SMARCA4 encodes one of two mutually exclusive ATPase subunits in the BRG/BRM associated factor (BAF) complex that is recruited by transcription factors (TFs) to drive chromatin accessibility and transcriptional activation. SMARCA4 is among the most recurrently mutated genes in human cancer, including ∼30% of germinal center (GC)-derived Burkitt lymphomas. In mice, GC-specific Smarca4 haploinsufficiency cooperated with MYC over-expression to drive lymphomagenesis. Furthermore, monoallelic Smarca4 deletion drove GC hyperplasia with centroblast polarization via significantly increased rates of centrocyte recycling to the dark zone. Mechanistically, Smarca4 loss reduced the activity of TFs that are activated in centrocytes to drive GC-exit, including SPI1 (PU.1), IRF family, and NF-κB. Loss of activity for these factors phenocopied aberrant BCL6 activity within murine centrocytes and human Burkitt lymphoma cells. SMARCA4 therefore facilitates chromatin accessibility for TFs that shape centrocyte trajectories, and loss of fine-control of these programs biases toward centroblast cell-fate, GC hyperplasia and lymphoma.

Keywords:  B-cell; BAF; SMARCA4; SWI/SNF; epigenetics; germinal center; immunology; lymphoma; transcription

DOI:  https://doi.org/10.1016/j.ccell.2024.02.011
Commun Biol. 2024 Mar 04. 7(1): 262

KCTD15 acts as an anti-tumor factor in colorectal cancer cells downstream of the demethylase FTO and the m6A reader YTHDF2.

Fang-Yuan Zhang, Lin Wu, Tie-Ning Zhang, Huan-Huan Chen.

Potassium Channel Tetramerization Domain Containing 15 (KCTD15) participates in the carcinogenesis of several solid malignancies; however, its role in colorectal cancer (CRC) remains unclear. Here we find that KCTD15 exhibits lower expression in CRC tissues as compared to para-carcinoma tissues. Tetracycline (tet)-induced overexpression and knockdown of KCTD15 confirms KCTD15 as an anti-proliferative and pro-apoptotic factor in CRC both in vitro and in xenografted tumors. N6-methyladenosine (m6A) is known to affect the expression, stabilization, and degradation of RNAs with this modification. We demonstrate that upregulation of fat mass and obesity-associated protein (FTO), a classical m6A eraser, prevents KCTD15 mRNA degradation in CRC cells. Less KCTD15 RNA is recognized by m6A 'reader' YTH N6-Methyladenosine RNA Binding Protein F2 (YTHDF2) in FTO-overexpressed cells. Moreover, KCTD15 overexpression decreases protein expression of histone deacetylase 1 (HDAC1) but increases acetylation of critical tumor suppressor p53 at Lys373 and Lys382. Degradation of p53 is delayed in CRC cells post-KCTD15 overexpression. We further show that the regulatory effects of KCTD15 on p53 are HDAC1-dependent. Collectively, we conclude that KCTD15 functions as an anti-growth factor in CRC cells, and its expression is orchestrated by the FTO-YTHDF2 axis. Enhanced p53 protein stabilization may contribute to KCTD15's actions in CRC cells.

DOI: https://doi.org/10.1038/s42003-024-05880-9
Science. 2024 Mar 08. 383(6687): eadi7342

Vitamin A resolves lineage plasticity to orchestrate stem cell lineage choices.

Matthew T Tierney, Lisa Polak, Yihao Yang, Merve Deniz Abdusselamoglu, Inwha Baek, Katherine S Stewart, Elaine Fuchs.

Lineage plasticity-a state of dual fate expression-is required to release stem cells from their niche constraints and redirect them to tissue compartments where they are most needed. In this work, we found that without resolving lineage plasticity, skin stem cells cannot effectively generate each lineage in vitro nor regrow hair and repair wounded epidermis in vivo. A small-molecule screen unearthed retinoic acid as a critical regulator. Combining high-throughput approaches, cell culture, and in vivo mouse genetics, we dissected its roles in tissue regeneration. We found that retinoic acid is made locally in hair follicle stem cell niches, where its levels determine identity and usage. Our findings have therapeutic implications for hair growth as well as chronic wounds and cancers, where lineage plasticity is unresolved.

DOI: https://doi.org/10.1126/science.adi7342
Mol Cell. 2024 Feb 29. pii: S1097-2765(24)00100-X. [Epub ahead of print]

Sister chromatid cohesion halts DNA loop expansion.

Nathalie Bastié, Christophe Chapard, Axel Cournac, Sanae Nejmi, Henri Mboumba, Olivier Gadal, Agnès Thierry, Frederic Beckouët, Romain Koszul.

  Eukaryotic genomes are folded into DNA loops mediated by structural maintenance of chromosomes (SMC) complexes such as cohesin, condensin, and Smc5/6. This organization regulates different DNA-related processes along the cell cycle, such as transcription, recombination, segregation, and DNA repair. During the G2 stage, SMC-mediated DNA loops coexist with cohesin complexes involved in sister chromatid cohesion (SCC). However, the articulation between the establishment of SCC and the formation of SMC-mediated DNA loops along the chromatin remains unknown. Here, we show that SCC is indeed a barrier to cohesin-mediated DNA loop expansion along G2/M Saccharomyces cerevisiae chromosomes.

Keywords:  S. cerevisiae; SCC; SMC; chromatin loop; cohesin; loop extrusion; mitosis; roadblock; segregation; yeast

DOI:  https://doi.org/10.1016/j.molcel.2024.02.004
Cancer Sci. 2024 Mar 03.

Region-specific DNA hydroxymethylation along the malignant progression of IDH-mutant gliomas.

Taijun Hana, Akitake Mukasa, Masashi Nomura, Genta Nagae, Shogo Yamamoto, Kenji Tatsuno, Hiroki Ueda, Shiro Fukuda, Takayoshi Umeda, Shota Tanaka, Takahide Nejo, Yosuke Kitagawa, Erika Yamazawa, Satoshi Takahashi, Tsukasa Koike, Yoshihiro Kushihara, Hirokazu Takami, Shunsaku Takayanagi, Hiroyuki Aburatani, Nobuhito Saito.

  The majority of low-grade isocitrate dehydrogenase-mutant (IDHmt ) gliomas undergo malignant progression (MP), but their underlying mechanism remains unclear. IDHmt gliomas exhibit global DNA methylation, and our previous report suggested that MP could be partly attributed to passive demethylation caused by accelerated cell cycles. However, during MP, there is also active demethylation mediated by ten-eleven translocation, such as DNA hydroxymethylation. Hydroxymethylation is reported to potentially contribute to gene expression regulation, but its role in MP remains under investigation. Therefore, we conducted a comprehensive analysis of hydroxymethylation during MP of IDHmt astrocytoma. Five primary/malignantly progressed IDHmt astrocytoma pairs were analyzed with oxidative bisulfite and the Infinium EPIC methylation array, detecting 5-hydroxymethyl cytosine at over 850,000 locations for region-specific hydroxymethylation assessment. Notably, we observed significant sharing of hydroxymethylated genomic regions during MP across the samples. Hydroxymethylated CpGs were enriched in open sea and intergenic regions (p < 0.001), and genes undergoing hydroxymethylation were significantly associated with cancer-related signaling pathways. RNA sequencing data integration identified 91 genes with significant positive/negative hydroxymethylation-expression correlations. Functional analysis suggested that positively correlated genes are involved in cell-cycle promotion, while negatively correlated ones are associated with antineoplastic functions. Analyses of The Cancer Genome Atlas clinical data on glioma were in line with these findings. Motif-enrichment analysis suggested the potential involvement of the transcription factor KLF4 in hydroxymethylation-based gene regulation. Our findings shed light on the significance of region-specific DNA hydroxymethylation in glioma MP and suggest its potential role in cancer-related gene expression and IDHmt glioma malignancy.

Keywords:  5-hydroxymethyl cytosine (5hmC); glioma; hydroxymethylation; isocitrate dehydrogenase (IDH); malignant progression

DOI:  https://doi.org/10.1111/cas.16127
Nat Commun. 2024 Mar 04. 15(1): 1977

Extensive DNA methylome rearrangement during early lamprey embryogenesis.

Allegra Angeloni, Skye Fissette, Deniz Kaya, Jillian M Hammond, Hasindu Gamaarachchi, Ira W Deveson, Robert J Klose, Weiming Li, Xiaotian Zhang, Ozren Bogdanovic.

DNA methylation (5mC) is a repressive gene regulatory mark widespread in vertebrate genomes, yet the developmental dynamics in which 5mC patterns are established vary across species. While mammals undergo two rounds of global 5mC erasure, teleosts, for example, exhibit localized maternal-to-paternal 5mC remodeling. Here, we studied 5mC dynamics during the embryonic development of sea lamprey, a jawless vertebrate which occupies a critical phylogenetic position as the sister group of the jawed vertebrates. We employed 5mC quantification in lamprey embryos and tissues, and discovered large-scale maternal-to-paternal epigenome remodeling that affects ~30% of the embryonic genome and is predominantly associated with partially methylated domains. We further demonstrate that sequences eliminated during programmed genome rearrangement (PGR), are hypermethylated in sperm prior to the onset of PGR. Our study thus unveils important insights into the evolutionary origins of vertebrate 5mC reprogramming, and how this process might participate in diverse developmental strategies.

DOI: https://doi.org/10.1038/s41467-024-46085-2