bims-crepig 2024-09-01 papers

bims-crepig

Biomed News

on Chromatin regulation and epigenetics in cell fate and cancer

Issue of 2024–09–01
24 papers selected by
Connor Rogerson, University of Cambridge

Cooperative insulation of regulatory domains by CTCF-dependent physical insulation and promoter competition.
Revisiting the model for coactivator recruitment: Med15 can select its target sites independent of promoter-bound transcription factors.
RNA polymerases reshape chromatin architecture and couple transcription on individual fibers.
PHF6 cooperates with SWI/SNF complexes to facilitate transcriptional progression.
EWS-WT1 fusion isoforms establish oncogenic programs and therapeutic vulnerabilities in desmoplastic small round cell tumors.
The impact of the embryonic DNA methylation program on CTCF-mediated genome regulation.
Cancer-associated DNA hypermethylation of Polycomb targets requires DNMT3A dual recognition of histone H2AK119 ubiquitination and the nucleosome acidic patch.
MEF2B C-terminal mutations enhance transcriptional activity and stability to drive B cell lymphomagenesis.
Developmental DNA demethylation is a determinant of neural stem cell identity and gliogenic competence.
IVEA: an integrative variational Bayesian inference method for predicting enhancer-gene regulatory interactions.
A repressive H3K36me2 reader mediates Polycomb silencing.
A genome-wide CRISPR screen identifies BRD4 as a regulator of cardiomyocyte differentiation.
An integrated transcription factor framework for Treg identity and diversity.
Phase separation of phospho-HDAC6 drives aberrant chromatin architecture in triple-negative breast cancer.
CREB activates the MafA promoter through proximal E-boxes and a CCAAT motif in pancreatic β-cells.
Heterochromatin protein 1 alpha (HP1α) undergoes a monomer to dimer transition that opens and compacts live cell genome architecture.
Next-generation mapping of the salicylic acid signaling hub and transcriptional cascade.
G-quadruplexes as pivotal components of cis-regulatory elements in the human genome.
ΔNp63 bookmarks and creates an accessible epigenetic environment for TGFβ-induced cancer cell stemness and invasiveness.
Histone variant macroH2A1 regulates synchronous firing of replication origins in the inactive X chromosome.
An atlas of transcribed human cardiac promoters and enhancers reveals an important role of regulatory elements in heart failure.
Activity-dependent transcriptional programs in memory regulate motor recovery after stroke.
Massively parallel measurement of protein-protein interactions by sequencing using MP3-seq.
FRA1 controls acinar cell plasticity during murine KrasG12D-induced pancreatic acinar to ductal metaplasia.

Nat Commun. 2024 Aug 23. 15(1): 7258

Cooperative insulation of regulatory domains by CTCF-dependent physical insulation and promoter competition.

Thais Ealo, Victor Sanchez-Gaya, Patricia Respuela, María Muñoz-San Martín, Elva Martin-Batista, Endika Haro, Alvaro Rada-Iglesias.

The specificity of gene expression during development requires the insulation of regulatory domains to avoid inappropriate enhancer-gene interactions. In vertebrates, this insulator function is mostly attributed to clusters of CTCF sites located at topologically associating domain (TAD) boundaries. However, TAD boundaries allow some physical crosstalk across regulatory domains, which is at odds with the specific and precise expression of developmental genes. Here we show that developmental genes and nearby clusters of CTCF sites cooperatively foster the robust insulation of regulatory domains. By genetically dissecting a couple of representative loci in mouse embryonic stem cells, we show that CTCF sites prevent undesirable enhancer-gene contacts (i.e. physical insulation), while developmental genes preferentially contribute to regulatory insulation through non-structural mechanisms involving promoter competition rather than enhancer blocking. Overall, our work provides important insights into the insulation of regulatory domains, which in turn might help interpreting the pathological consequences of certain structural variants.

DOI: https://doi.org/10.1038/s41467-024-51602-4
Nucleic Acids Res. 2024 Aug 27. pii: gkae718. [Epub ahead of print]

Revisiting the model for coactivator recruitment: Med15 can select its target sites independent of promoter-bound transcription factors.

Vladimir Mindel, Sagie Brodsky, Hadas Yung, Wajd Manadre, Naama Barkai.

Activation domains (ADs) within transcription factors (TFs) induce gene expression by recruiting coactivators such as the Mediator complex. Coactivators lack DNA binding domains (DBDs) and are assumed to passively follow their recruiting TFs. This is supported by direct AD-coactivator interactions seen in vitro but has not yet been tested in living cells. To examine that, we targeted two Med15-recruiting ADs to a range of budding yeast promoters through fusion with different DBDs. The DBD-AD fusions localized to hundreds of genomic sites but recruited Med15 and induced transcription in only a subset of bound promoters, characterized by a fuzzy-nucleosome architecture. Direct DBD-Med15 fusions shifted DBD localization towards fuzzy-nucleosome promoters, including promoters devoid of the endogenous Mediator. We propose that Med15, and perhaps other coactivators, possess inherent promoter preference and thus actively contribute to the selection of TF-induced genes.

DOI: https://doi.org/10.1093/nar/gkae718
Mol Cell. 2024 Aug 23. pii: S1097-2765(24)00667-1. [Epub ahead of print]

RNA polymerases reshape chromatin architecture and couple transcription on individual fibers.

Thomas W Tullius, R Stefan Isaac, Danilo Dubocanin, Jane Ranchalis, L Stirling Churchman, Andrew B Stergachis.

  RNA polymerases must initiate and pause within a complex chromatin environment, surrounded by nucleosomes and other transcriptional machinery. This environment creates a spatial arrangement along individual chromatin fibers ripe for both competition and coordination, yet these relationships remain largely unknown owing to the inherent limitations of traditional structural and sequencing methodologies. To address this, we employed long-read chromatin fiber sequencing (Fiber-seq) in Drosophila to visualize RNA polymerase (Pol) within its native chromatin context with single-molecule precision along up to 30 kb fibers. We demonstrate that Fiber-seq enables the identification of individual Pol II, nucleosome, and transcription factor footprints, revealing Pol II pausing-driven destabilization of downstream nucleosomes. Furthermore, we demonstrate pervasive direct distance-dependent transcriptional coupling between nearby Pol II genes, Pol III genes, and transcribed enhancers, modulated by local chromatin architecture. Overall, transcription initiation reshapes surrounding nucleosome architecture and couples nearby transcriptional machinery along individual chromatin fibers.

Keywords:  Fiber-seq; RNA Pol II; RNA Pol III; chromatin; nucleosome; promoter proximal pausing; single-molecule; transcription initiation

DOI:  https://doi.org/10.1016/j.molcel.2024.08.013
Nat Commun. 2024 Aug 24. 15(1): 7303

PHF6 cooperates with SWI/SNF complexes to facilitate transcriptional progression.

Priya Mittal, Jacquelyn A Myers, Raymond D Carter, Sandi Radko-Juettner, Hayden A Malone, Wojciech Rosikiewicz, Alexis N Robertson, Zhexin Zhu, Ishwarya V Narayanan, Baranda S Hansen, Meadow Parrish, Natarajan V Bhanu, Robert J Mobley, Jerold E Rehg, Beisi Xu, Yiannis Drosos, Shondra M Pruett-Miller, Mats Ljungman, Benjamin A Garcia, Gang Wu, Janet F Partridge, Charles W M Roberts.

Genes encoding subunits of SWI/SNF (BAF) chromatin remodeling complexes are mutated in nearly 25% of cancers. To gain insight into the mechanisms by which SWI/SNF mutations drive cancer, we contributed ten rhabdoid tumor (RT) cell lines mutant for SWI/SNF subunit SMARCB1 to a genome-scale CRISPR-Cas9 depletion screen performed across 896 cell lines. We identify PHF6 as specifically essential for RT cell survival and demonstrate that dependency on Phf6 extends to Smarcb1-deficient cancers in vivo. As mutations in either SWI/SNF or PHF6 can cause the neurodevelopmental disorder Coffin-Siris syndrome, our findings of a dependency suggest a previously unrecognized functional link. We demonstrate that PHF6 co-localizes with SWI/SNF complexes at promoters, where it is essential for maintenance of an active chromatin state. We show that in the absence of SMARCB1, PHF6 loss disrupts the recruitment and stability of residual SWI/SNF complex members, collectively resulting in the loss of active chromatin at promoters and stalling of RNA Polymerase II progression. Our work establishes a mechanistic basis for the shared syndromic features of SWI/SNF and PHF6 mutations in CSS and the basis for selective dependency on PHF6 in SMARCB1-mutant cancers.

DOI: https://doi.org/10.1038/s41467-024-51566-5
Nat Commun. 2024 Aug 28. 15(1): 7460

EWS-WT1 fusion isoforms establish oncogenic programs and therapeutic vulnerabilities in desmoplastic small round cell tumors.

Gaylor Boulay, Liliane C Broye, Rui Dong, Sowmya Iyer, Rajendran Sanalkumar, Yu-Hang Xing, Rémi Buisson, Shruthi Rengarajan, Beverly Naigles, Benoît Duc, Angela Volorio, Mary E Awad, Raffaele Renella, Ivan Chebib, G Petur Nielsen, Edwin Choy, Gregory M Cote, Lee Zou, Igor Letovanec, Ivan Stamenkovic, Miguel N Rivera, Nicolò Riggi.

EWS fusion oncoproteins underlie several human malignancies including Desmoplastic Small Round Cell Tumor (DSRCT), an aggressive cancer driven by EWS-WT1 fusion proteins. Here we combine chromatin occupancy and 3D profiles to identify EWS-WT1-dependent gene regulation networks and target genes. We show that EWS-WT1 is a powerful chromatin activator controlling an oncogenic gene expression program that characterizes primary tumors. Similar to wild type WT1, EWS-WT1 has two isoforms that differ in their DNA binding domain and we find that they have distinct DNA binding profiles and are both required to generate viable tumors that resemble primary DSRCT. Finally, we identify candidate EWS-WT1 target genes with potential therapeutic implications, including CCND1, whose inhibition by the clinically-approved drug Palbociclib leads to marked tumor burden decrease in DSRCT PDXs in vivo. Taken together, our studies identify gene regulation programs and therapeutic vulnerabilities in DSRCT and provide a mechanistic understanding of the complex oncogenic activity of EWS-WT1.

DOI: https://doi.org/10.1038/s41467-024-51851-3
Nucleic Acids Res. 2024 Aug 24. pii: gkae724. [Epub ahead of print]

The impact of the embryonic DNA methylation program on CTCF-mediated genome regulation.

Ana Monteagudo-Sánchez, Julien Richard Albert, Margherita Scarpa, Daan Noordermeer, Maxim V C Greenberg.

During mammalian embryogenesis, both the 5-cytosine DNA methylation (5meC) landscape and three dimensional (3D) chromatin architecture are profoundly remodeled during a process known as 'epigenetic reprogramming.' An understudied aspect of epigenetic reprogramming is how the 5meC flux, per se, affects the 3D genome. This is pertinent given the 5meC-sensitivity of DNA binding for a key regulator of chromosome folding: CTCF. We profiled the CTCF binding landscape using a mouse embryonic stem cell (ESC) differentiation protocol that models embryonic 5meC dynamics. Mouse ESCs lacking DNA methylation machinery are able to exit naive pluripotency, thus allowing for dissection of subtle effects of CTCF on gene expression. We performed CTCF HiChIP in both wild-type and mutant conditions to assess gained CTCF-CTCF contacts in the absence of 5meC. We performed H3K27ac HiChIP to determine the impact that ectopic CTCF binding has on cis-regulatory contacts. Using 5meC epigenome editing, we demonstrated that the methyl-mark is able to impair CTCF binding at select loci. Finally, a detailed dissection of the imprinted Zdbf2 locus showed how 5meC-antagonism of CTCF allows for proper gene regulation during differentiation. This work provides a comprehensive overview of how 5meC impacts the 3D genome in a relevant model for early embryonic events.

DOI: https://doi.org/10.1093/nar/gkae724
Sci Adv. 2024 Aug 30. 10(35): eadp0975

Cancer-associated DNA hypermethylation of Polycomb targets requires DNMT3A dual recognition of histone H2AK119 ubiquitination and the nucleosome acidic patch.

Kristjan H Gretarsson, Stephen Abini-Agbomson, Susan L Gloor, Daniel N Weinberg, Jamie L McCuiston, Vishnu Udayakumar Sunitha Kumary, Allison R Hickman, Varun Sahu, Rachel Lee, Xinjing Xu, Natalie Lipieta, Samuel Flashner, Oluwatobi A Adeleke, Irina K Popova, Hailey F Taylor, Kelsey Noll, Carolina Lin Windham, Danielle N Maryanski, Bryan J Venters, Hiroshi Nakagawa, Michael-Christopher Keogh, Karim-Jean Armache, Chao Lu.

During tumor development, promoter CpG islands that are normally silenced by Polycomb repressive complexes (PRCs) become DNA-hypermethylated. The molecular mechanism by which de novo DNA methyltransferase(s) [DNMT(s)] catalyze CpG methylation at PRC-regulated regions remains unclear. Here, we report a cryo-electron microscopy structure of the DNMT3A long isoform (DNMT3A1) amino-terminal region in complex with a nucleosome carrying PRC1-mediated histone H2A lysine-119 monoubiquitination (H2AK119Ub). We identify regions within the DNMT3A1 amino terminus that bind H2AK119Ub and the nucleosome acidic patch. This bidentate interaction is required for effective DNMT3A1 engagement with H2AK119Ub-modified chromatin in cells. Further, aberrant redistribution of DNMT3A1 to Polycomb target genes recapitulates the cancer-associated DNA hypermethylation signature and inhibits their transcriptional activation during cell differentiation. This effect is rescued by disruption of the DNMT3A1-acidic patch interaction. Together, our analyses reveal a binding interface critical for mediating promoter CpG island DNA hypermethylation, a major molecular hallmark of cancer.

DOI: https://doi.org/10.1126/sciadv.adp0975
Nat Commun. 2024 Aug 21. 15(1): 7195

MEF2B C-terminal mutations enhance transcriptional activity and stability to drive B cell lymphomagenesis.

Chuanjiang Yu, Qiong Shen, Antony B Holmes, Tongwei Mo, Anna Tosato, Rajesh Kumar Soni, Clarissa Corinaldesi, Sanjay Koul, Laura Pasqualucci, Shafinaz Hussein, Farhad Forouhar, Riccardo Dalla-Favera, Katia Basso.

The myocyte enhancer factor 2B (MEF2B) transcription factor is frequently mutated in germinal center (GC)-derived B-cell lymphomas. Its ammino (N)-terminal mutations drive lymphomagenesis by escaping interaction with transcriptional repressors, while the function of carboxy (C)-terminal mutations remains to be elucidated. Here, we show that MEF2B C-tail is physiologically phosphorylated at specific residues and phosphorylation at serine (S)324 is impaired by lymphoma-associated mutations. Lack of phosphorylation at S324 enhances the interaction of MEF2B with the SWI/SNF chromatin remodeling complex, leading to higher transcriptional activity. In addition, these mutants show an increased protein stability due to impaired interaction with the CUL3/KLHL12 ubiquitin complex. Mice expressing a phosphorylation-deficient lymphoma-associated MEF2B mutant display GC enlargement and develop GC-derived lymphomas, when crossed with Bcl2 transgenic mice. These results unveil converging mechanisms of action for a diverse spectrum of MEF2B mutations, all leading to its dysregulation and GC B-cell lymphomagenesis.

DOI: https://doi.org/10.1038/s41467-024-51644-8
Sci Adv. 2024 Aug 30. 10(35): eado5424

Developmental DNA demethylation is a determinant of neural stem cell identity and gliogenic competence.

Ian C MacArthur, Liyang Ma, Cheng-Yen Huang, Hrutvik Bhavsar, Masako Suzuki, Meelad M Dawlaty.

DNA methylation is extensively reconfigured during development, but the functional significance and cell type-specific dependencies of DNA demethylation in lineage specification remain poorly understood. Here, we demonstrate that developmental DNA demethylation, driven by ten-eleven translocation 1/2/3 (TET1/2/3) enzymes, is essential for establishment of neural stem cell (NSC) identity and gliogenic potential. We find that loss of all three TETs during NSC specification is dispensable for neural induction and neuronal differentiation but critical for astrocyte and oligodendrocyte formation, demonstrating a selective loss of glial competence. Mechanistically, TET-mediated demethylation was essential for commissioning neural-specific enhancers in proximity to master neurodevelopmental and glial transcription factor genes and for induction of these genes. Consistently, loss of all three TETs in embryonic NSCs in mice compromised glial gene expression and corticogenesis. Thus, TET-dependent developmental demethylation is an essential regulatory mechanism for neural enhancer commissioning during NSC specification and is a cell-intrinsic determinant of NSC identity and gliogenic potential.

DOI: https://doi.org/10.1126/sciadv.ado5424
Bioinform Adv. 2024 ;4(1): vbae118

IVEA: an integrative variational Bayesian inference method for predicting enhancer-gene regulatory interactions.

Yasumasa Kimura, Yoshimasa Ono, Kotoe Katayama, Seiya Imoto.

Motivation: Enhancers play critical roles in cell-type-specific transcriptional control. Despite the identification of thousands of candidate enhancers, unravelling their regulatory relationships with their target genes remains challenging. Therefore, computational approaches are needed to accurately infer enhancer-gene regulatory relationships.
Results: In this study, we propose a new method, IVEA, that predicts enhancer-gene regulatory interactions by estimating promoter and enhancer activities. Its statistical model is based on the gene regulatory mechanism of transcriptional bursting, which is characterized by burst size and frequency controlled by promoters and enhancers, respectively. Using transcriptional readouts, chromatin accessibility, and chromatin contact data as inputs, promoter and enhancer activities were estimated using variational Bayesian inference, and the contribution of each enhancer-promoter pair to target gene transcription was calculated. Our analysis demonstrates that the proposed method can achieve high prediction accuracy and provide biologically relevant enhancer-gene regulatory interactions.
Availability and implementation: The IVEA code is available on GitHub at https://github.com/yasumasak/ivea. The publicly available datasets used in this study are described in Supplementary Table S4.

DOI: https://doi.org/10.1093/bioadv/vbae118
Nat Commun. 2024 Aug 24. 15(1): 7287

A repressive H3K36me2 reader mediates Polycomb silencing.

Mengting Xu, Qi Zhang, Huanbin Shi, Zhongling Wu, Wei Zhou, Fucheng Lin, Yanjun Kou, Zeng Tao.

In animals, evolutionarily conserved Polycomb repressive complex 2 (PRC2) catalyzes histone H3 lysine 27 trimethylation (H3K27me3) and PRC1 functions in recruitment and transcriptional repression. However, the mechanisms underlying H3K27me3-mediated stable transcriptional silencing are largely unknown, as PRC1 subunits are poorly characterized in fungi. Here, we report that in the filamentous fungus Magnaporthe oryzae, the N-terminal chromodomain and C-terminal MRG domain of Eaf3 play key roles in facultative heterochromatin formation and transcriptional silencing. Eaf3 physically interacts with Ash1, Eed, and Sin3, encoding an H3K36 methyltransferase, the core subunit of PRC2, and a histone deacetylation co-suppressor, respectively. Eaf3 co-localizes with a set of repressive Ash1-H3K36me2 and H3K27me3 loci and mediates their transcriptional silencing. Furthermore, Eaf3 acts as a histone reader for the repressive H3K36me2 and H3K27me3 marks. Eaf3-occupied regions are associated with increased nucleosome occupancy, contributing to transcriptional silencing in M. oryzae. Together, these findings reveal that Eaf3 is a repressive H3K36me2 reader and plays a vital role in Polycomb gene silencing and the formation of facultative heterochromatin in fungi.

DOI: https://doi.org/10.1038/s41467-024-51789-6
Nat Cardiovasc Res. 2024 Mar;3(3): 317-331

A genome-wide CRISPR screen identifies BRD4 as a regulator of cardiomyocyte differentiation.

Arun Padmanabhan, T Yvanka de Soysa, Angelo Pelonero, Valerie Sapp, Parisha P Shah, Qiaohong Wang, Li Li, Clara Youngna Lee, Nandhini Sadagopan, Tomohiro Nishino, Lin Ye, Rachel Yang, Ashley Karnay, Andrey Poleshko, Nikhita Bolar, Ricardo Linares-Saldana, Sanjeev S Ranade, Michael Alexanian, Sarah U Morton, Mohit Jain, Saptarsi M Haldar, Deepak Srivastava, Rajan Jain.

Human induced pluripotent stem cell (hiPSC) to cardiomyocyte (CM) differentiation has reshaped approaches to studying cardiac development and disease. In this study, we employed a genome-wide CRISPR screen in a hiPSC to CM differentiation system and reveal here that BRD4, a member of the bromodomain and extraterminal (BET) family, regulates CM differentiation. Chemical inhibition of BET proteins in mouse embryonic stem cell (mESC)-derived or hiPSC-derived cardiac progenitor cells (CPCs) results in decreased CM differentiation and persistence of cells expressing progenitor markers. In vivo, BRD4 deletion in second heart field (SHF) CPCs results in embryonic or early postnatal lethality, with mutants demonstrating myocardial hypoplasia and an increase in CPCs. Single-cell transcriptomics identified a subpopulation of SHF CPCs that is sensitive to BRD4 loss and associated with attenuated CM lineage-specific gene programs. These results highlight a previously unrecognized role for BRD4 in CM fate determination during development and a heterogenous requirement for BRD4 among SHF CPCs.

DOI: https://doi.org/10.1038/s44161-024-00431-1
Proc Natl Acad Sci U S A. 2024 Sep 03. 121(36): e2411301121

An integrated transcription factor framework for Treg identity and diversity.

Kaitavjeet Chowdhary, Juliette Léon, Diane Mathis, Christophe Benoist.

  Vertebrate cell identity depends on the combined activity of scores of transcription factors (TF). While TFs have often been studied in isolation, a systematic perspective on their integration has been missing. Focusing on FoxP3+ regulatory T cells (Tregs), key guardians of immune tolerance, we combined single-cell chromatin accessibility, machine learning, and high-density genetic variation, to resolve a validated framework of diverse Treg chromatin programs, each shaped by multi-TF inputs. This framework identified previously unrecognized Treg controllers (Smarcc1) and illuminated the mechanism of action of FoxP3, which amplified a pre-existing Treg identity, diversely activating or repressing distinct programs, dependent on different regulatory partners. Treg subpopulations in the colon relied variably on FoxP3, Helios+ Tregs being completely dependent, but RORγ+ Tregs largely independent. These differences were rooted in intrinsic biases decoded by the integrated framework. Moving beyond master regulators, this work unravels how overlapping TF activities coalesce into Treg identity and diversity.

Keywords:  autoimmunity; gene expression; immunoregulation

DOI:  https://doi.org/10.1073/pnas.2411301121
Nat Cancer. 2024 Aug 28.

Phase separation of phospho-HDAC6 drives aberrant chromatin architecture in triple-negative breast cancer.

Bing Lu, Ru Qiu, Jiatian Wei, Li Wang, Qinkai Zhang, Mingsen Li, Xiudan Zhan, Jian Chen, I-Yun Hsieh, Ciqiu Yang, Jing Zhang, Zicheng Sun, Yifan Zhu, Tao Jiang, Han Zhu, Jie Li, Wei Zhao.

How dysregulated liquid-liquid phase separation (LLPS) contributes to the oncogenesis of female triple-negative breast cancer (TNBC) remains unknown. Here we demonstrate that phosphorylated histone deacetylase 6 (phospho-HDAC6) forms LLPS condensates in the nuclei of TNBC cells that are essential for establishing aberrant chromatin architecture. The disordered N-terminal domain and phosphorylated residue of HDAC6 facilitate effective LLPS, whereas nuclear export regions exert a negative dominant effect. Through phase-separation-based screening, we identified Nexturastat A as a specific disruptor of phospho-HDAC6 condensates, which effectively suppresses tumor growth. Mechanistically, importin-β interacts with phospho-HDAC6, promoting its translocation to the nucleus, where 14-3-3θ mediates the condensate formation. Disruption of phospho-HDAC6 LLPS re-established chromatin compartments and topologically associating domain boundaries, leading to disturbed chromatin loops. The phospho-HDAC6-induced aberrant chromatin architecture affects chromatin accessibility, histone acetylation, RNA polymerase II elongation and transcriptional profiles in TNBC. This study demonstrates phospho-HDAC6 LLPS as an emerging mechanism underlying the dysregulation of chromatin architecture in TNBC.

DOI: https://doi.org/10.1038/s43018-024-00816-y
J Mol Endocrinol. 2024 Aug 01. pii: JME-24-0023. [Epub ahead of print]

CREB activates the MafA promoter through proximal E-boxes and a CCAAT motif in pancreatic β-cells.

Yuki Aida, Kohsuke Kataoka.

MafA is a key transcriptional regulator of pancreatic islet β-cell function. Its target genes include those encoding preproinsulin and the glucose transporter Glut2 (Slc2a2); thus MafA function is essential for glucose-stimulated insulin secretion. Expression levels of MafA are reduced in β-cells of diabetic mouse models and human subjects, suggesting that β-cell dysfunction associated with type 2 diabetes is attributable to loss of MafA. On the other hand, MafA is transcriptionally upregulated by incretin hormones through activation of CREB and its co-activator CRTC2. β-cell-specific expression of MafA relies on a distal enhancer element. However, the precise mechanism by which CREB-CRTC2 regulates the enhancer and proximal promoter regions of MafA remains unclear. In this report, we analyzed previously published ChIP-seq data and found that CREB and NeuroD1, a β-cell-enriched transactivator, bound to both the promoter and enhancer regions of human MAFA. A series of reporter assays revealed that CREB activated the enhancer through a conserved cAMP-responsive element (CRE), but stimulated MAFA promoter activity even when the putative CRE was deleted. Two E-box elements and a CCAAT motif which bind NeuroD1 and ubiquitous NF-Y transcription factors, respectively, were necessary for transcriptional activation of the MAFA promoter by CREB. Genome-wide analysis of CREB-bound loci in β-cells revealed that they were enriched with CCAAT motifs. Furthermore, promoter analysis of Isl1 gene encoding a β-cell-enriched transcription factor revealed that a CRE-like element and two CCAAT-motifs, but not E-box, were necessary for activation by CREB. There results provide clues to elucidate the detailed mechanism by which CREB regulates MafA as well as β-cell-specific genes.

DOI: https://doi.org/10.1530/JME-24-0023
Nucleic Acids Res. 2024 Aug 28. pii: gkae720. [Epub ahead of print]

Heterochromatin protein 1 alpha (HP1α) undergoes a monomer to dimer transition that opens and compacts live cell genome architecture.

Jieqiong Lou, Qiji Deng, Xiaomeng Zhang, Charles C Bell, Andrew B Das, Naiara G Bediaga, Courtney O Zlatic, Timothy M Johanson, Rhys S Allan, Michael D W Griffin, PrasadN Paradkar, Kieran F Harvey, Mark A Dawson, Elizabeth Hinde.

Our understanding of heterochromatin nanostructure and its capacity to mediate gene silencing in a living cell has been prevented by the diffraction limit of optical microscopy. Thus, here to overcome this technical hurdle, and directly measure the nucleosome arrangement that underpins this dense chromatin state, we coupled fluorescence lifetime imaging microscopy (FLIM) of Förster resonance energy transfer (FRET) between histones core to the nucleosome, with molecular editing of heterochromatin protein 1 alpha (HP1α). Intriguingly, this super-resolved readout of nanoscale chromatin structure, alongside fluorescence fluctuation spectroscopy (FFS) and FLIM-FRET analysis of HP1α protein-protein interaction, revealed nucleosome arrangement to be differentially regulated by HP1α oligomeric state. Specifically, we found HP1α monomers to impart a previously undescribed global nucleosome spacing throughout genome architecture that is mediated by trimethylation on lysine 9 of histone H3 (H3K9me3) and locally reduced upon HP1α dimerisation. Collectively, these results demonstrate HP1α to impart a dual action on chromatin that increases the dynamic range of nucleosome proximity. We anticipate that this finding will have important implications for our understanding of how live cell heterochromatin structure regulates genome function.

DOI: https://doi.org/10.1093/nar/gkae720
Mol Plant. 2024 Aug 22. pii: S1674-2052(24)00263-6. [Epub ahead of print]

Next-generation mapping of the salicylic acid signaling hub and transcriptional cascade.

Jordan Powers, Xing Zhang, Andres V Reyes, Raul Zavaliev, Roni Ochakovski, Shou-Ling Xu, Xinnian Dong.

  For over 60 years, salicylic acid (SA) has been known as a plant immune signal required for basal and systemic acquired resistance (SAR). SA activates these immune responses by reprogramming ∼20% of the transcriptome through the function of NPR1. However, components in the NPR1-signaling hub, which appears as nuclear condensates, and the NPR1-signaling cascade remained elusive due to difficulties in studying this transcriptional cofactor whose chromatin association is indirect and likely transient. To overcome this challenge, we applied TurboID to divulge the NPR1-proxiome, which detected almost all known NPR1-interactors as well as new components of transcription-related complexes. Testing of new components showed that chromatin remodeling and histone demethylation contribute to SA-induced resistance. Globally, NPR1-proxiome shares a striking similarity to GBPL3-proxiome involved in SA synthesis, except associated transcription factors (TFs), suggesting that common regulatory modules are recruited to reprogram specific transcriptomes by transcriptional cofactors, like NPR1, through binding to unique TFs. Stepwise greenCUT&RUN analyses showed that, upon SA-induction, NPR1 initiates the transcriptional cascade primarily through association with TGA TFs to induce expression of secondary TFs, predominantly WRKYs. WRKY54 and WRKY70 then play a major role in inducing immune-output genes without interacting with NPR1 at the chromatin. Moreover, loss of NPR1 condensate formation decreases the protein's chromatin-association and transcriptional activity, indicating the importance of condensates in organizing the NPR1-signaling hub and initiating the transcriptional cascade. This study demonstrates how combinatorial applications of TurboID and stepwise greenCUT&RUN transcend traditional genetic methods to globally map signaling hubs and transcriptional cascades for in-depth explorations.

Keywords:  NPR1; TGA TFs and WRKY TFs; TurboID; biomolecular condensate; greenCUT&RUN; salicylic acid-induced transcription

DOI:  https://doi.org/10.1016/j.molp.2024.08.008
BMC Biol. 2024 Aug 26. 22(1): 177

G-quadruplexes as pivotal components of cis-regulatory elements in the human genome.

Rongxin Zhang, Yuqi Wang, Cheng Wang, Xiao Sun, Jean-Louis Mergny.

   BACKGROUND: Cis-regulatory elements (CREs) are crucial for regulating gene expression, and G-quadruplexes (G4s), as prototypal non-canonical DNA structures, may play a role in this regulation. However, the relationship between G4s and CREs, especially with non-promoter-like functional elements, requires further systematic investigation. We aimed to investigate the associations between G4s and human cCREs (candidate CREs) inferred from the Encyclopedia of DNA Elements (ENCODE) data.
RESULTS: We found that G4s are prominently enriched in most types of cCREs, especially those with promoter-like signatures (PLS). The co-occurrence of CTCF signals with H3K4me3 or H3K27ac signals strengthens the association between cCREs and G4s. Genetic variants in G4s, particularly within their G-runs, exhibit higher regulatory potential and deleterious effects compared to cCREs. The G-runs within G4s near transcriptional start sites (TSSs) are more evolutionarily constrained compared to G-runs in cCREs, while those far from the TSS are relatively less conserved. The presence of G4s is often linked to a more favorable local chromatin environment for the activation and execution of regulatory function of cCREs, potentially attributable to the formation of G4 secondary structures. Finally, we discovered that G4-associated cCREs exhibit widespread activation in a variety of cancers.
CONCLUSIONS: Our study suggests that G4s are integral components of human cis-regulatory elements, extending beyond their potential role in promoters. The G4 primary sequences are associated with the localization of CREs, while the G4 structures are linked to the activation of these elements. Therefore, we propose defining G4s as pivotal regulatory elements in the human genome.

Keywords:   Cis-regulatory elements; CRE; G-quadruplex; G4; Genomic function; Non-coding elements

DOI:  https://doi.org/10.1186/s12915-024-01971-5
Cell Commun Signal. 2024 Aug 23. 22(1): 411

ΔNp63 bookmarks and creates an accessible epigenetic environment for TGFβ-induced cancer cell stemness and invasiveness.

Eleftheria Vasilaki, Yu Bai, Mohamad Moustafa Ali, Anders Sundqvist, Aristidis Moustakas, Carl-Henrik Heldin.

   BACKGROUND: p63 is a transcription factor with intrinsic pioneer factor activity and pleiotropic functions. Transforming growth factor β (TGFβ) signaling via activation and cooperative action of canonical, SMAD, and non-canonical, MAP-kinase (MAPK) pathways, elicits both anti- and pro-tumorigenic properties, including cell stemness and invasiveness. TGFβ activates the ΔNp63 transcriptional program in cancer cells; however, the link between TGFβ and p63 in unmasking the epigenetic landscape during tumor progression allowing chromatin accessibility and gene transcription, is not yet reported.
METHODS: Small molecule inhibitors, including protein kinase inhibitors and RNA-silencing, provided loss of function analyses. Sphere formation assays in cancer cells, chromatin immunoprecipitation and mRNA expression assays were utilized in order to gain mechanistic evidence. Mass spectrometry analysis coupled to co-immunoprecipitation assays revealed novel p63 interactors and their involvement in p63-dependent transcription.
RESULTS: The sphere-forming capacity of breast cancer cells was enhanced upon TGFβ stimulation and significantly decreased upon ΔNp63 depletion. Activation of TGFβ signaling via p38 MAPK signaling induced ΔNp63 phosphorylation at Ser 66/68 resulting in stabilized ΔNp63 protein with enhanced DNA binding properties. TGFβ stimulation altered the ratio of H3K27ac and H3K27me3 histone modification marks, pointing towards higher H3K27ac and increased p300 acetyltransferase recruitment to chromatin. By silencing the expression of ΔNp63, the TGFβ effect on chromatin remodeling was abrogated. Inhibition of H3K27me3, revealed the important role of TGFβ as the upstream signal for guiding ΔNp63 to the TGFβ/SMAD gene loci, as well as the indispensable role of ΔNp63 in recruiting histone modifying enzymes, such as p300, to these genomic regions, regulating chromatin accessibility and gene transcription. Mechanistically, TGFβ through SMAD activation induced dissociation of ΔNp63 from NURD or NCOR/SMRT histone deacetylation complexes, while promoted the assembly of ΔNp63-p300 complexes, affecting the levels of histone acetylation and the outcome of ΔNp63-dependent transcription.
CONCLUSIONS: ΔNp63, phosphorylated and recruited by TGFβ to the TGFβ/SMAD/ΔNp63 gene loci, promotes chromatin accessibility and transcription of target genes related to stemness and cell invasion.

Keywords:  Chromatin accessibility; Protein-protein interaction; Signal transduction; Transcription; Transforming growth factor β (TGFβ); p63

DOI:  https://doi.org/10.1186/s12964-024-01794-5
Nucleic Acids Res. 2024 Aug 27. pii: gkae734. [Epub ahead of print]

Histone variant macroH2A1 regulates synchronous firing of replication origins in the inactive X chromosome.

Maria Arroyo, Corella S Casas-Delucchi, Maruthi K Pabba, Paulina Prorok, Sunil K Pradhan, Cathia Rausch, Anne Lehmkuhl, Andreas Maiser, Marcus Buschbeck, Vincent Pasque, Emily Bernstein, Katja Luck, M Cristina Cardoso.

MacroH2A has been linked to transcriptional silencing, cell identity, and is a hallmark of the inactive X chromosome (Xi). However, it remains unclear whether macroH2A plays a role in DNA replication. Using knockdown/knockout cells for each macroH2A isoform, we show that macroH2A-containing nucleosomes slow down replication progression rate in the Xi reflecting the higher nucleosome stability. Moreover, macroH2A1, but not macroH2A2, regulates the number of nano replication foci in the Xi, and macroH2A1 downregulation increases DNA loop sizes corresponding to replicons. This relates to macroH2A1 regulating replicative helicase loading during G1 by interacting with it. We mapped this interaction to a phenylalanine in macroH2A1 that is not conserved in macroH2A2 and the C-terminus of Mcm3 helicase subunit. We propose that macroH2A1 enhances the licensing of pre-replication complexes via DNA helicase interaction and loading onto the Xi.

DOI: https://doi.org/10.1093/nar/gkae734
Nat Cardiovasc Res. 2023 Jan;2(1): 58-75

An atlas of transcribed human cardiac promoters and enhancers reveals an important role of regulatory elements in heart failure.

Ruslan M Deviatiiarov, Anna Gams, Ivan V Kulakovskiy, Andrey Buyan, Georgy Meshcheryakov, Roman Syunyaev, Ramesh Singh, Palak Shah, Tatiana V Tatarinova, Oleg Gusev, Igor R Efimov.

A deeper knowledge of the dynamic transcriptional activity of promoters and enhancers is needed to improve mechanistic understanding of the pathogenesis of heart failure and heart diseases. In this study, we used cap analysis of gene expression (CAGE) to identify and quantify the activity of transcribed regulatory elements (TREs) in the four cardiac chambers of 21 healthy and ten failing adult human hearts. We identified 17,668 promoters and 14,920 enhancers associated with the expression of 14,519 genes. We showed how these regulatory elements are alternatively transcribed in different heart regions, in healthy versus failing hearts and in ischemic versus non-ischemic heart failure samples. Cardiac-disease-related single-nucleotide polymorphisms (SNPs) appeared to be enriched in TREs, potentially affecting the allele-specific transcription factor binding. To conclude, our open-source heart CAGE atlas will serve the cardiovascular community in improving the understanding of the role of the cardiac gene regulatory networks in cardiovascular disease and therapy.

DOI: https://doi.org/10.1038/s44161-022-00182-x
Commun Biol. 2024 Aug 25. 7(1): 1048

Activity-dependent transcriptional programs in memory regulate motor recovery after stroke.

Mary T Joy, S Thomas Carmichael.

Stroke causes death of brain tissue leading to long-term deficits. Behavioral evidence from neurorehabilitative therapies suggest learning-induced neuroplasticity can lead to beneficial outcomes. However, molecular and cellular mechanisms that link learning and stroke recovery are unknown. We show that in a mouse model of stroke, which exhibits enhanced recovery of function due to genetic perturbations of learning and memory genes, animals display activity-dependent transcriptional programs that are normally active during formation or storage of new memories. The expression of neuronal activity-dependent genes are predictive of recovery and occupy a molecular latent space unique to motor recovery. With motor recovery, networks of activity-dependent genes are co-expressed with their transcription factor targets forming gene regulatory networks that support activity-dependent transcription, that are normally diminished after stroke. Neuronal activity-dependent changes at the circuit level are influenced by interactions with microglia. At the molecular level, we show that enrichment of activity-dependent programs in neurons lead to transcriptional changes in microglia where they differentially interact to support intercellular signaling pathways for axon guidance, growth and synaptogenesis. Together, these studies identify activity-dependent transcriptional programs as a fundamental mechanism for neural repair post-stroke.

DOI: https://doi.org/10.1038/s42003-024-06723-3
Nat Chem Biol. 2024 Aug 27.

Massively parallel measurement of protein-protein interactions by sequencing using MP3-seq.

Alexandr Baryshev, Alyssa La Fleur, Benjamin Groves, Cirstyn Michel, David Baker, Ajasja Ljubetič, Georg Seelig.

Protein-protein interactions (PPIs) regulate many cellular processes and engineered PPIs have cell and gene therapy applications. Here, we introduce massively parallel PPI measurement by sequencing (MP3-seq), an easy-to-use and highly scalable yeast two-hybrid approach for measuring PPIs. In MP3-seq, DNA barcodes are associated with specific protein pairs and barcode enrichment can be read by sequencing to provide a direct measure of interaction strength. We show that MP3-seq is highly quantitative and scales to over 100,000 interactions. We apply MP3-seq to characterize interactions between families of rationally designed heterodimers and to investigate elements conferring specificity to coiled-coil interactions. Lastly, we predict coiled heterodimer structures using AlphaFold-Multimer (AF-M) and train linear models on physics-based energy terms to predict MP3-seq values. We find that AF-M-based models could be valuable for prescreening interactions but experimentally measuring interactions remains necessary to rank their strengths quantitatively.

DOI: https://doi.org/10.1038/s41589-024-01718-x
Dev Cell. 2024 Aug 22. pii: S1534-5807(24)00483-0. [Epub ahead of print]

FRA1 controls acinar cell plasticity during murine KrasG12D-induced pancreatic acinar to ductal metaplasia.

Alina L Li, Kensuke Sugiura, Noriyuki Nishiwaki, Kensuke Suzuki, Dorsay Sadeghian, Jun Zhao, Anirban Maitra, David Falvo, Rohit Chandwani, Jason R Pitarresi, Peter A Sims, Anil K Rustgi.

  Acinar cells have been proposed as a cell-of-origin for pancreatic ductal adenocarcinoma (PDAC) after undergoing acinar-to-ductal metaplasia (ADM). ADM can be triggered by pancreatitis, causing acinar cells to de-differentiate to a ductal-like state. We identify FRA1 (gene name Fosl1) as the most active transcription factor during KrasG12D acute pancreatitis-mediated injury, and we have elucidated a functional role of FRA1 by generating an acinar-specific Fosl1 knockout mouse expressing KrasG12D. Using a gene regulatory network and pseudotime trajectory inferred from single-nuclei ATAC-seq and bulk RNA sequencing (RNA-seq), we hypothesized a regulatory model of the acinar-ADM-pancreatic intraepithelial neoplasia (PanIN) continuum and experimentally validated that Fosl1 knockout mice are delayed in the onset of ADM and neoplastic transformation. Our study also identifies that pro-inflammatory cytokines, such as granulocyte colony stimulating factor (G-CSF), can regulate FRA1 activity to modulate ADM. Our findings identify that FRA1 is a mediator of acinar cell plasticity and is critical for acinar cell de-differentiation and transformation.

Keywords:  FRA1; G-CSF; KRAS; PanIN; acinar-ductal metaplasia; pancreatic ductal adenocarcinoma; pancreatitis

DOI:  https://doi.org/10.1016/j.devcel.2024.07.021