bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2024–01–21
twenty-six papers selected by
Connor Rogerson, University of Cambridge
  1. Chromatin activity identifies differential gene regulation across human ancestries.
  2. Regulation of chromatin architecture by transcription factor binding.
  3. Spatial chromatin accessibility sequencing resolves high-order spatial interactions of epigenomic markers.
  4. Targeting the epigenetic reader ENL inhibits super-enhancer-driven oncogenic transcription and synergizes with BET inhibition to suppress tumor progression.
  5. MBD2 couples DNA methylation to transposable element silencing during male gametogenesis.
  6. The histone chaperone SPT2 regulates chromatin structure and function in Metazoa.
  7. The Mediator kinase module enhances polymerase activity to regulate transcriptional memory after heat stress in Arabidopsis.
  8. Regularly spaced tyrosines in EBF1 mediate BRG1 recruitment and formation of nuclear subdiffractive clusters.
  9. Inherited blood cancer predisposition through altered transcription elongation.
  10. Structure-primed embedding on the transcription factor manifold enables transparent model architectures for gene regulatory network and latent activity inference.
  11. Epigenetic repression of Cend1 by lysine-specific demethylase 1 is essential for murine heart development.
  12. Lineage regulators TFAP2C and NR5A2 function as bipotency activators in totipotent embryos.
  13. Affinity-optimizing enhancer variants disrupt development.
  14. ZFP281 controls transcriptional and epigenetic changes promoting mouse pluripotent state transitions via DNMT3 and TET1.
  15. Molecular switching in transcription through splicing and proline-isomerization regulates stress responses in plants.
  16. Deep molecular learning of transcriptional control of a synthetic CRE enhancer and its variants.
  17. Transcription factor interactions explain the context-dependent activity of CRX binding sites.
  18. VGLL1 cooperates with TEAD4 to control human trophectoderm lineage specification.
  19. COOLAIR and PRC2 function in parallel to silence FLC during vernalization.
  20. Chromatin remodeler CHD8 is required for spermatogonial proliferation and early meiotic progression.
  21. Acetyl-CoA production by Mediator-bound 2-ketoacid dehydrogenases boosts de novo histone acetylation and is regulated by nitric oxide.
  22. Boundary stacking interactions enable cross-TAD enhancer-promoter communication during limb development.
  23. Leveraging single-cell ATAC-seq and RNA-seq to identify disease-critical fetal and adult brain cell types.
  24. Sox9 regulates alternative splicing and pancreatic beta cell function.
  25. Haplotype-specific assembly of shattered chromosomes in esophageal adenocarcinomas.
  26. Compact CRISPR genetic screens enabled by improved guide RNA library cloning.
  1. Genome Biol. 2024 Jan 15. 25(1): 21
    Chromatin activity identifies differential gene regulation across human ancestries.
    Kade P Pettie, Maxwell Mumbach, Amanda J Lea, Julien Ayroles, Howard Y Chang, Maya Kasowski, Hunter B Fraser.
       BACKGROUND: Current evidence suggests that cis-regulatory elements controlling gene expression may be the predominant target of natural selection in humans and other species. Detecting selection acting on these elements is critical to understanding evolution but remains challenging because we do not know which mutations will affect gene regulation.
    RESULTS: To address this, we devise an approach to search for lineage-specific selection on three critical steps in transcriptional regulation: chromatin activity, transcription factor binding, and chromosomal looping. Applying this approach to lymphoblastoid cells from 831 individuals of either European or African descent, we find strong signals of differential chromatin activity linked to gene expression differences between ancestries in numerous contexts, but no evidence of functional differences in chromosomal looping. Moreover, we show that enhancers rather than promoters display the strongest signs of selection associated with sites of differential transcription factor binding.
    CONCLUSIONS: Overall, our study indicates that some cis-regulatory adaptation may be more easily detected at the level of chromatin than DNA sequence. This work provides a vast resource of genomic interaction data from diverse human populations and establishes a novel selection test that will benefit future study of regulatory evolution in humans and other species.
    Keywords:  Activity-by-Contact; Cis-regulation; Directional selection; Enhancer; Gene expression; HiChIP; Human; LCL; bQTL
    DOI:  https://doi.org/10.1186/s13059-024-03165-2
  2. Elife. 2024 Jan 19. pii: RP91320. [Epub ahead of print]12
    Regulation of chromatin architecture by transcription factor binding.
    Stephanie Portillo-Ledesma, Suckwoo Chung, Jill Hoffman, Tamar Schlick.
      Transcription factors (TF) bind to chromatin and regulate the expression of genes. The pair Myc:Max binds to E-box regulatory DNA elements throughout the genome to control the transcription of a large group of specific genes. We introduce an implicit modeling protocol for Myc:Max binding to mesoscale chromatin fibers at nucleosome resolution to determine TF effect on chromatin architecture and shed light into its mechanism of gene regulation. We first bind Myc:Max to different chromatin locations and show how it can direct fiber folding and formation of microdomains, and how this depends on the linker DNA length. Second, by simulating increasing concentrations of Myc:Max binding to fibers that differ in the DNA linker length, linker histone density, and acetylation levels, we assess the interplay between Myc:Max and other chromatin internal parameters. Third, we study the mechanism of gene silencing by Myc:Max binding to the Eed gene loci. Overall, our results show how chromatin architecture can be regulated by TF binding. The position of TF binding dictates the formation of microdomains that appear visible only at the ensemble level. At the same time, the level of linker histone and tail acetylation, or different linker DNA lengths, regulates the concentration-dependent effect of TF binding. Furthermore, we show how TF binding can repress gene expression by increasing fiber folding motifs that help compact and occlude the promoter region. Importantly, this effect can be reversed by increasing linker histone density. Overall, these results shed light on the epigenetic control of the genome dictated by TF binding.
    Keywords:  chromatin; chromosomes; gene expression; mesoscale modeling; none; transcription factors
    DOI:  https://doi.org/10.7554/eLife.91320
  3. Elife. 2024 Jan 18. pii: RP87868. [Epub ahead of print]12
    Spatial chromatin accessibility sequencing resolves high-order spatial interactions of epigenomic markers.
    Yeming Xie, Fengying Ruan, Yaning Li, Meng Luo, Chen Zhang, Zhichao Chen, Zhe Xie, Zhe Weng, Weitian Chen, Wenfang Chen, Yitong Fang, Yuxin Sun, Mei Guo, Juan Wang, Shouping Xu, Hongqi Wang, Chong Tang.
      As the genome is organized into a three-dimensional structure in intracellular space, epigenomic information also has a complex spatial arrangement. However, most epigenetic studies describe locations of methylation marks, chromatin accessibility regions, and histone modifications in the horizontal dimension. Proper spatial epigenomic information has rarely been obtained. In this study, we designed spatial chromatin accessibility sequencing (SCA-seq) to resolve the genome conformation by capturing the epigenetic information in single-molecular resolution while simultaneously resolving the genome conformation. Using SCA-seq, we are able to examine the spatial interaction of chromatin accessibility (e.g. enhancer-promoter contacts), CpG island methylation, and spatial insulating functions of the CCCTC-binding factor. We demonstrate that SCA-seq paves the way to explore the mechanism of epigenetic interactions and extends our knowledge in 3D packaging of DNA in the nucleus.
    Keywords:  3D genome; DNA methylation; GpC methyltransferase; Hi-C; chromatin accessibility; chromosomes; gene expression; human; pore-C
    DOI:  https://doi.org/10.7554/eLife.87868
  4. Cancer Res. 2024 Jan 19.
    Targeting the epigenetic reader ENL inhibits super-enhancer-driven oncogenic transcription and synergizes with BET inhibition to suppress tumor progression.
    Yongheng Chen, Ying Ying, Wenlong Ma, Hongchao Ma, Liang Shi, Xuefeng Gao, Min Jia, Meiqi Li, Xiaoman Song, Weixiao Kong, Wei Chen, Xiangyi Zheng, Tobias Achu Muluh, Xiaobin Wang, Maolin Wang, Xing-Sheng Shu.
      Epigenetic alterations at cis-regulatory elements (CREs) fine-tune transcriptional output. Epigenetic readers interact with CREs and can cooperate with other chromatin regulators to drive oncogene transcription. Here, we found that the YEATS domain-containing histone acetylation reader ENL (eleven-nineteen leukemia) acts as a key regulator of super-enhancers (SEs), which are highly active distal CREs, across cancer types. ENL occupied the majority of SEs with substantially higher preference over typical-enhancers, and the enrichment of ENL at SEs depended on its ability to bind acetylated histones. Rapid depletion of ENL by auxin-inducible degron tagging severely repressed the transcription of SE-controlled oncogenes, such as MYC, by inducing the decommissioning of their SEs, and restoring ENL protein expression largely reversed these effects. Additionally, ENL was indispensable for the rapid activation of SE-regulated immediate early genes in response to growth factor stimulation. Furthermore, ENL interacted with the histone chaperone FACT complex and was required for the deposition of FACT over CREs, which mediates nucleosome reorganization required for transcription initiation and elongation. Proper control of transcription by ENL and ENL-associated FACT was regulated by the histone reader BRD4. ENL was overexpressed in colorectal cancer (CRC) and functionally contributed to CRC growth and metastasis. ENL degradation or inhibition synergized with BET inhibitors that target BRD4 in restraining CRC progression. These findings establish the essential role of epigenetic reader ENL in governing SE-driven oncogenic transcription and uncover the potential of ENL intervention to increase sensitivity to BET inhibition.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-23-1836
  5. Nat Plants. 2024 Jan 15.
    MBD2 couples DNA methylation to transposable element silencing during male gametogenesis.
    Shuya Wang, Ming Wang, Lucia Ichino, Brandon A Boone, Zhenhui Zhong, Ranjith K Papareddy, Evan K Lin, Jaewon Yun, Suhua Feng, Steven E Jacobsen.
      DNA methylation is an essential component of transposable element (TE) silencing, yet the mechanism by which methylation causes transcriptional repression remains poorly understood1-5. Here we study the Arabidopsis thaliana Methyl-CpG Binding Domain (MBD) proteins MBD1, MBD2 and MBD4 and show that MBD2 acts as a TE repressor during male gametogenesis. MBD2 bound chromatin regions containing high levels of CG methylation, and MBD2 was capable of silencing the FWA gene when tethered to its promoter. MBD2 loss caused activation at a small subset of TEs in the vegetative cell of mature pollen without affecting DNA methylation levels, demonstrating that MBD2-mediated silencing acts strictly downstream of DNA methylation. TE activation in mbd2 became more significant in the mbd5 mbd6 and adcp1 mutant backgrounds, suggesting that MBD2 acts redundantly with other silencing pathways to repress TEs. Overall, our study identifies MBD2 as a methyl reader that acts downstream of DNA methylation to silence TEs during male gametogenesis.
    DOI:  https://doi.org/10.1038/s41477-023-01599-3
  6. Nat Struct Mol Biol. 2024 Jan 18.
    The histone chaperone SPT2 regulates chromatin structure and function in Metazoa.
    Giulia Saredi, Francesco N Carelli, Stéphane G M Rolland, Giulia Furlan, Sandra Piquet, Alex Appert, Luis Sanchez-Pulido, Jonathan L Price, Pablo Alcon, Lisa Lampersberger, Anne-Cécile Déclais, Navin B Ramakrishna, Rachel Toth, Thomas Macartney, Constance Alabert, Chris P Ponting, Sophie E Polo, Eric A Miska, Anton Gartner, Julie Ahringer, John Rouse.
      Histone chaperones control nucleosome density and chromatin structure. In yeast, the H3-H4 chaperone Spt2 controls histone deposition at active genes but its roles in metazoan chromatin structure and organismal physiology are not known. Here we identify the Caenorhabditis elegans ortholog of SPT2 (CeSPT-2) and show that its ability to bind histones H3-H4 is important for germline development and transgenerational epigenetic gene silencing, and that spt-2 null mutants display signatures of a global stress response. Genome-wide profiling showed that CeSPT-2 binds to a range of highly expressed genes, and we find that spt-2 mutants have increased chromatin accessibility at a subset of these loci. We also show that SPT2 influences chromatin structure and controls the levels of soluble and chromatin-bound H3.3 in human cells. Our work reveals roles for SPT2 in controlling chromatin structure and function in Metazoa.
    DOI:  https://doi.org/10.1038/s41594-023-01204-3
  7. EMBO J. 2024 Jan 16.
    The Mediator kinase module enhances polymerase activity to regulate transcriptional memory after heat stress in Arabidopsis.
    Tim Crawford, Lara Siebler, Aleksandra Sulkowska, Bryan Nowack, Li Jiang, Yufeng Pan, Jörn Lämke, Christian Kappel, Isabel Bäurle.
      Plants are often exposed to recurring adverse environmental conditions in the wild. Acclimation to high temperatures entails transcriptional responses, which prime plants to better withstand subsequent stress events. Heat stress (HS)-induced transcriptional memory results in more efficient re-induction of transcription upon recurrence of heat stress. Here, we identified CDK8 and MED12, two subunits of the kinase module of the transcription co-regulator complex, Mediator, as promoters of heat stress memory and associated histone modifications in Arabidopsis. CDK8 is recruited to heat-stress memory genes by HEAT SHOCK TRANSCRIPTION FACTOR A2 (HSFA2). Like HSFA2, CDK8 is largely dispensable for the initial gene induction upon HS, and its function in transcriptional memory is thus independent of primary gene activation. In addition to the promoter and transcriptional start region of target genes, CDK8 also binds their 3'-region, where it may promote elongation, termination, or rapid re-initiation of RNA polymerase II (Pol II) complexes during transcriptional memory bursts. Our work presents a complex role for the Mediator kinase module during transcriptional memory in multicellular eukaryotes, through interactions with transcription factors, chromatin modifications, and promotion of Pol II efficiency.
    Keywords:  CDK8; Heat Stress; Mediator Kinase Module; RNA Polymerase II; Transcriptional Memory
    DOI:  https://doi.org/10.1038/s44318-023-00024-x
  8. Genes Dev. 2024 Jan 17.
    Regularly spaced tyrosines in EBF1 mediate BRG1 recruitment and formation of nuclear subdiffractive clusters.
    Nikolay Zolotarev, Yuanting Wang, Manyu Du, Marc Bayer, Anna Grosschedl, Ibrahim Cisse, Rudolf Grosschedl.
      B lineage priming by pioneer transcription factor EBF1 requires the function of an intrinsically disordered region (IDR). Here, we examine the role of regularly spaced tyrosines in the IDR as potential determinants of IDR function and activity of EBF1. We found that four Y > A mutations in EBF1 reduced the formation of condensates in vitro and subdiffractive clusters in vivo. Notably, Y > A mutant EBF1 was inefficient in promoting B cell differentiation and showed impaired chromatin binding, recruitment of BRG1, and activation of specific target genes. Thus, regularly spaced tyrosines in the IDR contribute to the biophysical and functional properties of EBF1.
    Keywords:  B cell differentiation; BRG1; EBF1; IDR; chromatin; condensates; phase separation
    DOI:  https://doi.org/10.1101/gad.350828.123
  9. Cell. 2024 Jan 04. pii: S0092-8674(23)01348-X. [Epub ahead of print]
    Inherited blood cancer predisposition through altered transcription elongation.
    Jiawei Zhao, Liam D Cato, Uma P Arora, Erik L Bao, Samuel C Bryant, Nicholas Williams, Yuemeng Jia, Seth R Goldman, Jyoti Nangalia, Michael A Erb, Seychelle M Vos, Scott A Armstrong, Vijay G Sankaran.
      Despite advances in defining diverse somatic mutations that cause myeloid malignancies, a significant heritable component for these cancers remains largely unexplained. Here, we perform rare variant association studies in a large population cohort to identify inherited predisposition genes for these blood cancers. CTR9, which encodes a key component of the PAF1 transcription elongation complex, is among the significant genes identified. The risk variants found in the cases cause loss of function and result in a ∼10-fold increased odds of acquiring a myeloid malignancy. Partial CTR9 loss of function expands human hematopoietic stem cells (HSCs) by increased super elongation complex-mediated transcriptional activity, which thereby increases the expression of key regulators of HSC self-renewal. By following up on insights from a human genetic study examining inherited predisposition to the myeloid malignancies, we define a previously unknown antagonistic interaction between the PAF1 and super elongation complexes. These insights could enable targeted approaches for blood cancer prevention.
    Keywords:  CTR9; PAF1 complex; cancer predisposition; hematopoiesis; hematopoietic stem cells; myeloid malignancies; self-renewal; super elongation complex; transcription elongation
    DOI:  https://doi.org/10.1016/j.cell.2023.12.016
  10. Genome Biol. 2024 Jan 18. 25(1): 24
    Structure-primed embedding on the transcription factor manifold enables transparent model architectures for gene regulatory network and latent activity inference.
    Andreas Tjärnberg, Maggie Beheler-Amass, Christopher A Jackson, Lionel A Christiaen, David Gresham, Richard Bonneau.
       BACKGROUND: Modeling of gene regulatory networks (GRNs) is limited due to a lack of direct measurements of genome-wide transcription factor activity (TFA) making it difficult to separate covariance and regulatory interactions. Inference of regulatory interactions and TFA requires aggregation of complementary evidence. Estimating TFA explicitly is problematic as it disconnects GRN inference and TFA estimation and is unable to account for, for example, contextual transcription factor-transcription factor interactions, and other higher order features. Deep-learning offers a potential solution, as it can model complex interactions and higher-order latent features, although does not provide interpretable models and latent features.
    RESULTS: We propose a novel autoencoder-based framework, StrUcture Primed Inference of Regulation using latent Factor ACTivity (SupirFactor) for modeling, and a metric, explained relative variance (ERV), for interpretation of GRNs. We evaluate SupirFactor with ERV in a wide set of contexts. Compared to current state-of-the-art GRN inference methods, SupirFactor performs favorably. We evaluate latent feature activity as an estimate of TFA and biological function in S. cerevisiae as well as in peripheral blood mononuclear cells (PBMC).
    CONCLUSION: Here we present a framework for structure-primed inference and interpretation of GRNs, SupirFactor, demonstrating interpretability using ERV in multiple biological and experimental settings. SupirFactor enables TFA estimation and pathway analysis using latent factor activity, demonstrated here on two large-scale single-cell datasets, modeling S. cerevisiae and PBMC. We find that the SupirFactor model facilitates biological analysis acquiring novel functional and regulatory insight.
    DOI:  https://doi.org/10.1186/s13059-023-03134-1
  11. iScience. 2024 Jan 19. 27(1): 108722
    Epigenetic repression of Cend1 by lysine-specific demethylase 1 is essential for murine heart development.
    Huahua Liu, Rui Zhou, Shanshan Li, Jinling Dong, Yuan Fang, Yuru Luo, Hongyu Su, Baochang Lai, Lingli Liang, Donghong Zhang, Yanmin Zhang, John Y-J Shyy, Bin Zhou, Zuyi Yuan, Yidong Wang.
      Epigenetic regulation of heart development remains incompletely understood. Here we show that LSD1, a histone demethylase, plays a crucial role in regulating cardiomyocyte proliferation during heart development. Cardiomyocyte-specific deletion of Lsd1 in mice inhibited cardiomyocyte proliferation, causing severe growth defect of embryonic and neonatal heart. In vivo RNA-seq and in vitro functional studies identified Cend1 as a target suppressed by LSD1. Lsd1 loss resulted in elevated Cend1 transcription associated with increased active histone mark H3K4me2 at Cend1 promoter. Cend1 knockdown relieved the cell-cycle arrest and proliferation defect caused by LSD1 inhibition in primary rat cardiomyocytes. Moreover, genetic deletion of Cend1 rescued cardiomyocyte proliferation defect and embryonic lethality in Lsd1 null embryos. Consistently, LSD1 promoted the cell cycle of cardiomyocytes derived from human-induced pluripotent stem cells by repressing CEND1. Together, these findings reveal an epigenetic regulatory mechanism involving the LSD1-CEND1 axis that controls cardiomyocyte proliferation essential for murine heart development.
    Keywords:  Biological sciences; Developmental biology; Natural sciences
    DOI:  https://doi.org/10.1016/j.isci.2023.108722
  12. Nat Struct Mol Biol. 2024 Jan 19.
    Lineage regulators TFAP2C and NR5A2 function as bipotency activators in totipotent embryos.
    Lijia Li, Fangnong Lai, Ling Liu, Xukun Lu, Xiaoyu Hu, Bofeng Liu, Zili Lin, Qiang Fan, Feng Kong, Qianhua Xu, Wei Xie.
      During the first lineage segregation, a mammalian totipotent embryo differentiates into the inner cell mass (ICM) and trophectoderm (TE). However, how transcription factors (TFs) regulate this earliest cell-fate decision in vivo remains elusive, with their regulomes primarily inferred from cultured cells. Here, we investigated the TF regulomes during the first lineage specification in early mouse embryos, spanning the pre-initiation, initiation, commitment, and maintenance phases. Unexpectedly, we found that TFAP2C, a trophoblast regulator, bound and activated both early TE and inner cell mass (ICM) genes at the totipotent (two- to eight-cell) stages ('bipotency activation'). Tfap2c deficiency caused downregulation of early ICM genes, including Nanog, Nr5a2, and Tdgf1, and early TE genes, including Tfeb and Itgb5, in eight-cell embryos. Transcription defects in both ICM and TE lineages were also found in blastocysts, accompanied by increased apoptosis and reduced cell numbers in ICMs. Upon trophoblast commitment, TFAP2C left early ICM genes but acquired binding to late TE genes in blastocysts, where it co-bound with CDX2, and later to extra-embryonic ectoderm (ExE) genes, where it cooperatively co-occupied with the former ICM regulator SOX2. Finally, 'bipotency activation' in totipotent embryos also applied to a pluripotency regulator NR5A2, which similarly bound and activated both ICM and TE lineage genes at the eight-cell stage. These data reveal a unique transcription circuity of totipotency underpinned by highly adaptable lineage regulators.
    DOI:  https://doi.org/10.1038/s41594-023-01199-x
  13. Nature. 2024 Jan 17.
    Affinity-optimizing enhancer variants disrupt development.
    Fabian Lim, Joe J Solvason, Genevieve E Ryan, Sophia H Le, Granton A Jindal, Paige Steffen, Simran K Jandu, Emma K Farley.
      Enhancers control the location and timing of gene expression and contain the majority of variants associated with disease1-3. The ZRS is arguably the most well-studied vertebrate enhancer and mediates the expression of Shh in the developing limb4. Thirty-one human single-nucleotide variants (SNVs) within the ZRS are associated with polydactyly4-6. However, how this enhancer encodes tissue-specific activity, and the mechanisms by which SNVs alter the number of digits, are poorly understood. Here we show that the ETS sites within the ZRS are low affinity, and identify a functional ETS site, ETS-A, with extremely low affinity. Two human SNVs and a synthetic variant optimize the binding affinity of ETS-A subtly from 15% to around 25% relative to the strongest ETS binding sequence, and cause polydactyly with the same penetrance and severity. A greater increase in affinity results in phenotypes that are more penetrant and more severe. Affinity-optimizing SNVs in other ETS sites in the ZRS, as well as in ETS, interferon regulatory factor (IRF), HOX and activator protein 1 (AP-1) sites within a wide variety of enhancers, cause gain-of-function gene expression. The prevalence of binding sites with suboptimal affinity in enhancers creates a vulnerability in genomes whereby SNVs that optimize affinity, even slightly, can be pathogenic. Searching for affinity-optimizing SNVs in genomes could provide a mechanistic approach to identify causal variants that underlie enhanceropathies.
    DOI:  https://doi.org/10.1038/s41586-023-06922-8
  14. Dev Cell. 2024 Jan 16. pii: S1534-5807(23)00698-6. [Epub ahead of print]
    ZFP281 controls transcriptional and epigenetic changes promoting mouse pluripotent state transitions via DNMT3 and TET1.
    Xin Huang, Sophie Balmer, Cong Lyu, Yunlong Xiang, Vikas Malik, Hailin Wang, Yu Zhang, Bishuang Cai, Wei Xie, Anna-Katerina Hadjantonakis, Hongwei Zhou, Jianlong Wang.
      The progression from naive through formative to primed in vitro pluripotent stem cell states recapitulates epiblast development in vivo during the peri-implantation period of mouse embryo development. Activation of the de novo DNA methyltransferases and reorganization of transcriptional and epigenetic landscapes are key events that occur during these pluripotent state transitions. However, the upstream regulators that coordinate these events are relatively underexplored. Here, using Zfp281 knockout mouse and degron knockin cell models, we identify the direct transcriptional activation of Dnmt3a/3b by ZFP281 in pluripotent stem cells. Chromatin co-occupancy of ZFP281 and DNA hydroxylase TET1, which is dependent on the formation of R-loops in ZFP281-targeted gene promoters, undergoes a "high-low-high" bimodal pattern regulating dynamic DNA methylation and gene expression during the naive-formative-primed transitions. ZFP281 also safeguards DNA methylation in maintaining primed pluripotency. Our study demonstrates a previously unappreciated role for ZFP281 in coordinating DNMT3A/3B and TET1 functions to promote pluripotent state transitions.
    Keywords:  DNA methylation; DNMT3; R-loop; TET1; ZFP281; formative; naive; pluripotency; primed
    DOI:  https://doi.org/10.1016/j.devcel.2023.12.018
  15. Nat Commun. 2024 Jan 18. 15(1): 592
    Molecular switching in transcription through splicing and proline-isomerization regulates stress responses in plants.
    Frederik Friis Theisen, Andreas Prestel, Steffie Elkjær, Yannick H A Leurs, Nicholas Morffy, Lucia C Strader, Charlotte O'Shea, Kaare Teilum, Birthe B Kragelund, Karen Skriver.
      The Arabidopsis thaliana DREB2A transcription factor interacts with the negative regulator RCD1 and the ACID domain of subunit 25 of the transcriptional co-regulator mediator (Med25) to integrate stress signals for gene expression, with elusive molecular interplay. Using biophysical and structural analyses together with high-throughput screening, we reveal a bivalent binding switch in DREB2A containing an ACID-binding motif (ABS) and the known RCD1-binding motif (RIM). The RIM is lacking in a stress-induced DREB2A splice variant with retained transcriptional activity. ABS and RIM bind to separate sites on Med25-ACID, and NMR analyses show a structurally heterogeneous complex deriving from a DREB2A-ABS proline residue populating cis- and trans-isomers with remote impact on the RIM. The cis-isomer stabilizes an α-helix, while the trans-isomer may introduce energetic frustration facilitating rapid exchange between activators and repressors. Thus, DREB2A uses a post-transcriptionally and post-translationally modulated switch for transcriptional regulation.
    DOI:  https://doi.org/10.1038/s41467-024-44859-2
  16. iScience. 2024 Jan 19. 27(1): 108747
    Deep molecular learning of transcriptional control of a synthetic CRE enhancer and its variants.
    Chan-Koo Kang, Ah-Ram Kim.
      Massively parallel reporter assay measures transcriptional activities of various cis-regulatory modules (CRMs) in a single experiment. We developed a thermodynamic computational model framework that calculates quantitative levels of gene expression directly from regulatory DNA sequences. Using the framework, we investigated the molecular mechanisms of cis-regulatory mutations of a synthetic enhancer that cause abnormal gene expression. We found that, in a human cell line, competitive binding between family transcription factors (TFs) with slightly different binding preferences significantly increases the accuracy of recapitulating the transcriptional effects of thousands of single- or multi-mutations. We also discovered that even if various harmful mutations occurred in an activator binding site, CRM could stably maintain or even increase gene expression through a certain form of competitive binding between family TFs. These findings enhance understanding the effect of SNPs and indels on CRMs and would help building robust custom-designed CRMs for biologics production and gene therapy.
    Keywords:  Artificial intelligence; Experimental models in systems biology; Molecular mechanism of gene regulation
    DOI:  https://doi.org/10.1016/j.isci.2023.108747
  17. PLoS Comput Biol. 2024 Jan 16. 20(1): e1011802
    Transcription factor interactions explain the context-dependent activity of CRX binding sites.
    Kaiser J Loell, Ryan Z Friedman, Connie A Myers, Joseph C Corbo, Barak A Cohen, Michael A White.
      The effects of transcription factor binding sites (TFBSs) on the activity of a cis-regulatory element (CRE) depend on the local sequence context. In rod photoreceptors, binding sites for the transcription factor (TF) Cone-rod homeobox (CRX) occur in both enhancers and silencers, but the sequence context that determines whether CRX binding sites contribute to activation or repression of transcription is not understood. To investigate the context-dependent activity of CRX sites, we fit neural network-based models to the activities of synthetic CREs composed of photoreceptor TFBSs. The models revealed that CRX binding sites consistently make positive, independent contributions to CRE activity, while negative homotypic interactions between sites cause CREs composed of multiple CRX sites to function as silencers. The effects of negative homotypic interactions can be overcome by the presence of other TFBSs that either interact cooperatively with CRX sites or make independent positive contributions to activity. The context-dependent activity of CRX sites is thus determined by the balance between positive heterotypic interactions, independent contributions of TFBSs, and negative homotypic interactions. Our findings explain observed patterns of activity among genomic CRX-bound enhancers and silencers, and suggest that enhancers may require diverse TFBSs to overcome negative homotypic interactions between TFBSs.
    DOI:  https://doi.org/10.1371/journal.pcbi.1011802
  18. Nat Commun. 2024 Jan 17. 15(1): 583
    VGLL1 cooperates with TEAD4 to control human trophectoderm lineage specification.
    Yueli Yang, Wenqi Jia, Zhiwei Luo, Yunpan Li, Hao Liu, Lixin Fu, Jinxiu Li, Yu Jiang, Junjian Lai, Haiwei Li, Babangida Jabir Saeed, Yi Zou, Yuan Lv, Liang Wu, Ting Zhou, Yongli Shan, Chuanyu Liu, Yiwei Lai, Longqi Liu, Andrew P Hutchins, Miguel A Esteban, Md Abdul Mazid, Wenjuan Li.
      In contrast to rodents, the mechanisms underlying human trophectoderm and early placenta specification are understudied due to ethical barriers and the scarcity of embryos. Recent reports have shown that human pluripotent stem cells (PSCs) can differentiate into trophectoderm (TE)-like cells (TELCs) and trophoblast stem cells (TSCs), offering a valuable in vitro model to study early placenta specification. Here, we demonstrate that the VGLL1 (vestigial-like family member 1), which is highly expressed during human and non-human primate TE specification in vivo but is negligibly expressed in mouse, is a critical regulator of cell fate determination and self-renewal in human TELCs and TSCs derived from naïve PSCs. Mechanistically, VGLL1 partners with the transcription factor TEAD4 (TEA domain transcription factor 4) to regulate chromatin accessibility at target gene loci through histone acetylation and acts in cooperation with GATA3 and TFAP2C. Our work is relevant to understand primate early embryogenesis and how it differs from other mammalian species.
    DOI:  https://doi.org/10.1038/s41467-024-44780-8
  19. Proc Natl Acad Sci U S A. 2024 Jan 23. 121(4): e2311474121
    COOLAIR and PRC2 function in parallel to silence FLC during vernalization.
    Mathias Nielsen, Govind Menon, Yusheng Zhao, Eduardo Mateo-Bonmati, Philip Wolff, Shaoli Zhou, Martin Howard, Caroline Dean.
      Noncoding transcription induces chromatin changes that can mediate environmental responsiveness, but the causes and consequences of these mechanisms are still unclear. Here, we investigate how antisense transcription (termed COOLAIR) interfaces with Polycomb Repressive Complex 2 (PRC2) silencing during winter-induced epigenetic regulation of Arabidopsis FLOWERING LOCUS C (FLC). We use genetic and chromatin analyses on lines ineffective or hyperactive for the antisense pathway in combination with computational modeling to define the mechanisms underlying FLC repression. Our results show that FLC is silenced through pathways that function with different dynamics: a COOLAIR transcription-mediated pathway capable of fast response and in parallel a slow PRC2 switching mechanism that maintains each allele in an epigenetically silenced state. Components of both the COOLAIR and PRC2 pathways are regulated by a common transcriptional regulator (NTL8), which accumulates by reduced dilution due to slow growth at low temperature. The parallel activities of the regulatory steps, and their control by temperature-dependent growth dynamics, create a flexible system for registering widely fluctuating natural temperature conditions that change year on year, and yet ensure robust epigenetic silencing of FLC.
    Keywords:  Arabidopsis; FLC; Polycomb Repressive Complex 2; antisense transcription
    DOI:  https://doi.org/10.1073/pnas.2311474121
  20. Nucleic Acids Res. 2024 Jan 16. pii: gkad1256. [Epub ahead of print]
    Chromatin remodeler CHD8 is required for spermatogonial proliferation and early meiotic progression.
    Kenta Nitahara, Atsuki Kawamura, Yuka Kitamura, Kiyoko Kato, Satoshi H Namekawa, Masaaki Nishiyama.
      Meiosis is a key step during germ cell differentiation, accompanied by the activation of thousands of genes through germline-specific chromatin reorganization. The chromatin remodeling mechanisms underpinning early meiotic stages remain poorly understood. Here we focus on the function of one of the major autism genes, CHD8, in spermatogenesis, based on the epidemiological association between autism and low fertility rates. Specific ablation of Chd8 in germ cells results in gradual depletion of undifferentiated spermatogonia and the failure of meiotic double-strand break (DSB) formation, leading to meiotic prophase I arrest and cell death. Transcriptional analyses demonstrate that CHD8 is required for extensive activation of spermatogenic genes in spermatogonia, necessary for spermatogonial proliferation and meiosis. CHD8 directly binds and regulates genes crucial for meiosis, including H3K4me3 histone methyltransferase genes, meiotic cohesin genes, HORMA domain-containing genes, synaptonemal complex genes, and DNA damage response genes. We infer that CHD8 contributes to meiotic DSB formation and subsequent meiotic progression through combined regulation of these meiosis-related genes. Our study uncovers an essential role of CHD8 in the proliferation of undifferentiated spermatogonia and the successful progression of meiotic prophase I.
    DOI:  https://doi.org/10.1093/nar/gkad1256
  21. Mol Cell. 2024 Jan 10. pii: S1097-2765(23)01079-1. [Epub ahead of print]
    Acetyl-CoA production by Mediator-bound 2-ketoacid dehydrogenases boosts de novo histone acetylation and is regulated by nitric oxide.
    Marta Russo, Francesco Gualdrini, Veronica Vallelonga, Elena Prosperini, Roberta Noberini, Silvia Pedretti, Carolina Borriero, Pierluigi Di Chiaro, Sara Polletti, Gabriele Imperato, Mattia Marenda, Chiara Ghirardi, Fabio Bedin, Alessandro Cuomo, Simona Rodighiero, Tiziana Bonaldi, Nico Mitro, Serena Ghisletti, Gioacchino Natoli.
      Histone-modifying enzymes depend on the availability of cofactors, with acetyl-coenzyme A (CoA) being required for histone acetyltransferase (HAT) activity. The discovery that mitochondrial acyl-CoA-producing enzymes translocate to the nucleus suggests that high concentrations of locally synthesized metabolites may impact acylation of histones and other nuclear substrates, thereby controlling gene expression. Here, we show that 2-ketoacid dehydrogenases are stably associated with the Mediator complex, thus providing a local supply of acetyl-CoA and increasing the generation of hyper-acetylated histone tails. Nitric oxide (NO), which is produced in large amounts in lipopolysaccharide-stimulated macrophages, inhibited the activity of Mediator-associated 2-ketoacid dehydrogenases. Elevation of NO levels and the disruption of Mediator complex integrity both affected de novo histone acetylation within a shared set of genomic regions. Our findings indicate that the local supply of acetyl-CoA generated by 2-ketoacid dehydrogenases bound to Mediator is required to maximize acetylation of histone tails at sites of elevated HAT activity.
    Keywords:  2-ketoacid dehydrogenases; LPS; LPS tolerance; Mediator; acetylation; chromatin; lipopolysaccharide; macrophages; nitric oxide; pyruvate dehydrogenase
    DOI:  https://doi.org/10.1016/j.molcel.2023.12.033
  22. Nat Genet. 2024 Jan 18.
    Boundary stacking interactions enable cross-TAD enhancer-promoter communication during limb development.
    Tzu-Chiao Hung, David M Kingsley, Alistair N Boettiger.
      Although promoters and their enhancers are frequently contained within a topologically associating domain (TAD), some developmentally important genes have their promoter and enhancers within different TADs. Hypotheses about molecular mechanisms enabling cross-TAD interactions remain to be assessed. To test these hypotheses, we used optical reconstruction of chromatin architecture to characterize the conformations of the Pitx1 locus on single chromosomes in developing mouse limbs. Our data support a model in which neighboring boundaries are stacked as a result of loop extrusion, bringing boundary-proximal cis-elements into contact. This stacking interaction also contributes to the appearance of architectural stripes in the population average maps. Through molecular dynamics simulations, we found that increasing boundary strengths facilitates the formation of the stacked boundary conformation, counter-intuitively facilitating border bypass. This work provides a revised view of the TAD borders' function, both facilitating and preventing cis-regulatory interactions, and introduces a framework to distinguish border-crossing from border-respecting enhancer-promoter pairs.
    DOI:  https://doi.org/10.1038/s41588-023-01641-2
  23. Nat Commun. 2024 Jan 17. 15(1): 563
    Leveraging single-cell ATAC-seq and RNA-seq to identify disease-critical fetal and adult brain cell types.
    Samuel S Kim, Buu Truong, Karthik Jagadeesh, Kushal K Dey, Amber Z Shen, Soumya Raychaudhuri, Manolis Kellis, Alkes L Price.
      Prioritizing disease-critical cell types by integrating genome-wide association studies (GWAS) with functional data is a fundamental goal. Single-cell chromatin accessibility (scATAC-seq) and gene expression (scRNA-seq) have characterized cell types at high resolution, and studies integrating GWAS with scRNA-seq have shown promise, but studies integrating GWAS with scATAC-seq have been limited. Here, we identify disease-critical fetal and adult brain cell types by integrating GWAS summary statistics from 28 brain-related diseases/traits (average N = 298 K) with 3.2 million scATAC-seq and scRNA-seq profiles from 83 cell types. We identified disease-critical fetal (respectively adult) brain cell types for 22 (respectively 23) of 28 traits using scATAC-seq, and for 8 (respectively 17) of 28 traits using scRNA-seq. Significant scATAC-seq enrichments included fetal photoreceptor cells for major depressive disorder, fetal ganglion cells for BMI, fetal astrocytes for ADHD, and adult VGLUT2 excitatory neurons for schizophrenia. Our findings improve our understanding of brain-related diseases/traits and inform future analyses.
    DOI:  https://doi.org/10.1038/s41467-024-44742-0
  24. Nat Commun. 2024 Jan 18. 15(1): 588
    Sox9 regulates alternative splicing and pancreatic beta cell function.
    Sapna Puri, Hasna Maachi, Gopika Nair, Holger A Russ, Richard Chen, Pamela Pulimeno, Zachary Cutts, Vasilis Ntranos, Matthias Hebrok.
      Despite significant research, mechanisms underlying the failure of islet beta cells that result in type 2 diabetes (T2D) are still under investigation. Here, we report that Sox9, a transcriptional regulator of pancreas development, also functions in mature beta cells. Our results show that Sox9-depleted rodent beta cells have defective insulin secretion, and aging animals develop glucose intolerance, mimicking the progressive degeneration observed in T2D. Using genome editing in human stem cells, we show that beta cells lacking SOX9 have stunted first-phase insulin secretion. In human and rodent cells, loss of Sox9 disrupts alternative splicing and triggers accumulation of non-functional isoforms of genes with key roles in beta cell function. Sox9 depletion reduces expression of protein-coding splice variants of the serine-rich splicing factor arginine SRSF5, a major splicing enhancer that regulates alternative splicing. Our data highlight the role of SOX9 as a regulator of alternative splicing in mature beta cell function.
    DOI:  https://doi.org/10.1038/s41467-023-44384-8
  25. Cell Genom. 2024 Jan 12. pii: S2666-979X(23)00336-1. [Epub ahead of print] 100484
    Haplotype-specific assembly of shattered chromosomes in esophageal adenocarcinomas.
    Jannat Ijaz, Edward Harry, Keiran Raine, Andrew Menzies, Kathryn Beal, Michael A Quail, Sonia Zumalave, Hyunchul Jung, Tim H H Coorens, Andrew R J Lawson, Daniel Leongamornlert, Hayley E Francies, Mathew J Garnett, Zemin Ning, Peter J Campbell.
      The epigenetic landscape of cancer is regulated by many factors, but primarily it derives from the underlying genome sequence. Chromothripsis is a catastrophic localized genome shattering event that drives, and often initiates, cancer evolution. We characterized five esophageal adenocarcinoma organoids with chromothripsis using long-read sequencing and transcriptome and epigenome profiling. Complex structural variation and subclonal variants meant that haplotype-aware de novo methods were required to generate contiguous cancer genome assemblies. Chromosomes were assembled separately and scaffolded using haplotype-resolved Hi-C reads, producing accurate assemblies even with up to 900 structural rearrangements. There were widespread differences between the chromothriptic and wild-type copies of chromosomes in topologically associated domains, chromatin accessibility, histone modifications, and gene expression. Differential epigenome peaks were most enriched within 10 kb of chromothriptic structural variants. Alterations in transcriptome and higher-order chromosome organization frequently occurred near differential epigenetic marks. Overall, chromothripsis reshapes gene regulation, causing coordinated changes in epigenetic landscape, transcription, and chromosome conformation.
    DOI:  https://doi.org/10.1016/j.xgen.2023.100484
  26. Genome Biol. 2024 Jan 19. 25(1): 25
    Compact CRISPR genetic screens enabled by improved guide RNA library cloning.
    Seok-Jin Heo, Lauren D Enriquez, Scot Federman, Amy Y Chang, Rachel Mace, Kaivalya Shevade, Phuong Nguyen, Adam J Litterman, Shawn Shafer, Laralynne Przybyla, Eric D Chow.
      CRISPR genome editing approaches theoretically enable researchers to define the function of each human gene in specific cell types, but challenges remain to efficiently perform genetic perturbations in relevant models. In this work, we develop a library cloning protocol that increases sgRNA uniformity and greatly reduces bias in existing genome-wide libraries. We demonstrate that our libraries can achieve equivalent or better statistical power compared to previously reported screens using an order of magnitude fewer cells. This improved cloning protocol enables genome-scale CRISPR screens in technically challenging cell models and screen formats.
    Keywords:  CRISPR screening; Guide library; Library cloning; Primary cell CRISPR screening; iPSC-derived CRISPR screening
    DOI:  https://doi.org/10.1186/s13059-023-03132-3
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