bims-crepig 2024-12-29 papers

bims-crepig

Biomed News

on Chromatin regulation and epigenetics in cell fate and cancer

Issue of 2024–12–29
twenty-two papers selected by
Connor Rogerson, University of Cambridge

Nuclear microRNA 9 mediates G-quadruplex formation and 3D genome organization during TGF-β-induced transcription.
Single-cell analysis of bidirectional reprogramming between early embryonic states identify mechanisms of differential lineage plasticities in mice.
TurboCas: A method for locus-specific labeling of genomic regions and isolating their associated protein interactome.
LDB1 establishes multi-enhancer networks to regulate gene expression.
Atlas-scale single-cell DNA methylation profiling with sciMETv3.
ZNF143 is a transcriptional regulator of nuclear-encoded mitochondrial genes that acts independently of looping and CTCF.
Putative looping factor ZNF143/ZFP143 is an essential transcriptional regulator with no looping function.
Increased nuclear factor I-mediated chromatin access drives transition to androgen receptor splice variant dependence in prostate cancer.
HDI-STARR-seq: Condition-specific enhancer discovery in mouse liver in vivo.
Contributing factors to the oxidation-induced mutational landscape in human cells.
3D chromatin hubs as regulatory units of identity and survival in human acute leukemia.
Defining gene ends: RNA polymerase II CTD threonine 4 phosphorylation marks transcription termination regions genome-wide.
Inter-chromosomal transcription hubs shape the 3D genome architecture of African trypanosomes.
The Role of Med15 Sequence Features in Transcription Factor Interactions.
Dual-nuclease single-cell lineage tracing by Cas9 and Cas12a.
Comprehensive mapping of genetic variation at Epromoters reveals pleiotropic association with multiple disease traits.
A novel human pluripotent stem cell gene activation system identifies IGFBP2 as a mediator in the production of haematopoietic progenitors in vitro.
ID3 promotes erythroid differentiation and is repressed by a TAL1/PRMT6 complex.
A negatively charged region within carboxy-terminal domain maintains proper CTCF DNA binding.
Deciphering single-cell 3D chromatin structure using scCTG.
Epigenomic and 3D genomic mapping reveals developmental dynamics and subgenomic asymmetry of transcriptional regulatory architecture in allotetraploid cotton.
DEMETER DNA demethylase reshapes the global DNA methylation landscape and controls cell identity transition during plant regeneration.

Nat Commun. 2024 Dec 20. 15(1): 10711

Nuclear microRNA 9 mediates G-quadruplex formation and 3D genome organization during TGF-β-induced transcription.

Julio Cordero, Guruprasadh Swaminathan, Diana G Rogel-Ayala, Karla Rubio, Adel Elsherbiny, Samina Mahmood, Witold Szymanski, Johannes Graumann, Thomas Braun, Stefan Günther, Gergana Dobreva, Guillermo Barreto.

The dynamics of three-dimensional (3D) genome organization are essential to transcriptional regulation. While enhancers regulate spatiotemporal gene expression, chromatin looping is a means for enhancer-promoter interactions yielding cell-type-specific gene expression. Further, non-canonical DNA secondary structures, such as G-quadruplexes (G4s), are related to increased gene expression. However, the role of G4s in promoter-distal regulatory elements, such as super-enhancers (SE), and in chromatin looping has remained elusive. Here we show that mature microRNA 9 (miR-9) is enriched at promoters and SE of genes that are inducible by transforming growth factor beta 1 (TGFB1) signaling. Moreover, we find that miR-9 is required for formation of G4s, promoter-super-enhancer looping and broad domains of the euchromatin histone mark H3K4me3 at TGFB1-responsive genes. Our study places miR-9 in the same functional context with G4s and promoter-enhancer interactions during 3D genome organization and transcriptional activation induced by TGFB1 signaling, a critical signaling pathway in cancer and fibrosis.

DOI: https://doi.org/10.1038/s41467-024-54740-x
Dev Cell. 2024 Dec 19. pii: S1534-5807(24)00722-6. [Epub ahead of print]

Single-cell analysis of bidirectional reprogramming between early embryonic states identify mechanisms of differential lineage plasticities in mice.

Vidur Garg, Yang Yang, Sonja Nowotschin, Manu Setty, Eralda Salataj, Ying-Yi Kuo, Dylan Murphy, Roshan Sharma, Amy Jang, Alexander Polyzos, Dana Pe'er, Effie Apostolou, Anna-Katerina Hadjantonakis.

  Two distinct lineages, pluripotent epiblast (EPI) and primitive (extra-embryonic) endoderm (PrE), arise from common inner cell mass (ICM) progenitors in mammalian embryos. To study how these sister identities are forged, we leveraged mouse embryonic stem (ES) cells and extra-embryonic endoderm (XEN) stem cells-in vitro counterparts of the EPI and PrE. Bidirectional reprogramming between ES and XEN coupled with single-cell RNA and ATAC-seq analyses showed distinct rates, efficiencies, and trajectories of state conversions, identifying drivers and roadblocks of reciprocal conversions. While GATA4-mediated ES-to-iXEN conversion was rapid and nearly deterministic, OCT4-, KLF4-, and SOX2-induced XEN-to-induced pluripotent stem (iPS) reprogramming progressed with diminished efficiency and kinetics. A dominant PrE transcriptional program, safeguarded by GATA4, alongside elevated chromatin accessibility and reduced DNA methylation of the EPI underscored the differential plasticities of the two states. Mapping in vitro to embryo trajectories tracked reprogramming cells in either direction along EPI and PrE in vivo states, without transitioning through the ICM.

Keywords:  ES cells; XEN cells; blastocyst; epiblast; extra-embryonic endoderm; lineage plasticity; pluripotency; primitive endoderm; reprogramming; single-cell analysis

DOI:  https://doi.org/10.1016/j.devcel.2024.11.022
Mol Cell. 2024 Dec 19. pii: S1097-2765(24)00912-2. [Epub ahead of print]84(24): 4929-4944.e8

TurboCas: A method for locus-specific labeling of genomic regions and isolating their associated protein interactome.

Bercin K Cenik, Yuki Aoi, Marta Iwanaszko, Benjamin C Howard, Marc A Morgan, Grant D Andersen, Elizabeth T Bartom, Ali Shilatifard.

  Regulation of gene expression during development and stress response requires the concerted action of transcription factors and chromatin-binding proteins. Because this process is cell-type specific and varies with cellular conditions, mapping of chromatin factors at individual regulatory loci is crucial for understanding cis-regulatory control. Previous methods only characterize static protein binding. We present "TurboCas," a method combining a proximity-labeling (PL) enzyme, miniTurbo, with CRISPR-dCas9 that allows for efficient and site-specific labeling of chromatin factors in mammalian cells. Validating TurboCas at the FOS promoter, we identify proteins recruited upon heat shock, cross-validated via RNA polymerase II and P-TEFb immunoprecipitation. These methodologies reveal canonical and uncharacterized factors that function to activate expression of heat-shock-responsive genes. Applying TurboCas to the MYC promoter, we identify two P-TEFb coactivators, the super elongation complex (SEC) and BRD4, as MYC co-regulators. TurboCas provides a genome-specific targeting PL, with the potential to deepen our molecular understanding of transcriptional regulatory pathways in development and stress response.

Keywords:  BRD4; FOS; FUBP3; MYC; chromatin-binding proteins; dCas9; gene regulation; heat shock; proximity labeling

DOI:  https://doi.org/10.1016/j.molcel.2024.11.007
Mol Cell. 2024 Dec 18. pii: S1097-2765(24)00992-4. [Epub ahead of print]

LDB1 establishes multi-enhancer networks to regulate gene expression.

Nicholas G Aboreden, Jessica C Lam, Viraat Y Goel, Siqing Wang, Xiaokang Wang, Susannah C Midla, Alma Quijano, Cheryl A Keller, Belinda M Giardine, Ross C Hardison, Haoyue Zhang, Anders S Hansen, Gerd A Blobel.

  How specific enhancer-promoter pairing is established remains mostly unclear. Besides the CTCF/cohesin machinery, few nuclear factors have been studied for a direct role in physically connecting regulatory elements. Using a murine erythroid cell model, we show via acute degradation experiments that LDB1 directly and broadly promotes connectivity among regulatory elements. Most LDB1-mediated contacts, even those spanning hundreds of kb, can form in the absence of CTCF, cohesin, or YY1 as determined using multiple degron systems. Moreover, an engineered LDB1-driven chromatin loop is cohesin independent. Cohesin-driven loop extrusion does not stall at LDB1-occupied sites but aids the formation of a subset of LDB1-anchored loops. Leveraging the dynamic reorganization of nuclear architecture during the transition from mitosis to G1 phase, we observe that loop formation and de novo LDB1 occupancy correlate and can occur independently of structural loops. Tri-C and Region Capture Micro-C reveal that LDB1 organizes multi-enhancer networks to activate transcription. These findings establish LDB1 as a driver of spatial connectivity.

Keywords:  chromatin architecture, enhancer, LDB1, looping, CTCF, cohesin, YY1, cell cycle, hub, LMO2

DOI:  https://doi.org/10.1016/j.molcel.2024.11.037
Cell Genom. 2024 Dec 17. pii: S2666-979X(24)00355-0. [Epub ahead of print] 100726

Atlas-scale single-cell DNA methylation profiling with sciMETv3.

Ruth V Nichols, Lauren E Rylaarsdam, Brendan L O'Connell, Zohar Shipony, Nika Iremadze, Sonia N Acharya, Andrew C Adey.

  Single-cell methods to assess DNA methylation have not achieved the same level of cell throughput per experiment compared to other modalities, with large-scale datasets requiring extensive automation, time, and other resources. Here, we describe sciMETv3, a combinatorial indexing-based technique that enables atlas-scale libraries to be produced in a single experiment. To reduce the sequencing burden, we demonstrate the compatibility of sciMETv3 with capture techniques to enrich regulatory regions, as well as the ability to leverage enzymatic conversion, which can yield higher library diversity. We showcase the throughput of sciMETv3 by producing a >140,000 cell library from human middle frontal gyrus split across four multiplexed individuals using both Illumina and Ultima sequencing instrumentation. Finally, we introduce sciMET+ATAC to enable high-throughput exploration of the interplay between chromatin accessibility and DNA methylation within the same cell.

Keywords:  DNA methylation; epigenetics; neuroscience; single cell

DOI:  https://doi.org/10.1016/j.xgen.2024.100726
Mol Cell. 2024 Dec 12. pii: S1097-2765(24)00956-0. [Epub ahead of print]

ZNF143 is a transcriptional regulator of nuclear-encoded mitochondrial genes that acts independently of looping and CTCF.

Mikhail D Magnitov, Michela Maresca, Noemí Alonso Saiz, Hans Teunissen, Jinhong Dong, Kizhakke M Sathyan, Luca Braccioli, Michael J Guertin, Elzo de Wit.

  Gene expression is orchestrated by transcription factors, which function within the context of a three-dimensional genome. Zinc-finger protein 143 (ZNF143/ZFP143) is a transcription factor that has been implicated in both gene activation and chromatin looping. To study the direct consequences of ZNF143/ZFP143 loss, we generated a ZNF143/ZFP143 depletion system in mouse embryonic stem cells. Our results show that ZNF143/ZFP143 degradation has no effect on chromatin looping. Systematic analysis of ZNF143/ZFP143 occupancy data revealed that a commonly used antibody cross-reacts with CTCF, leading to its incorrect association with chromatin loops. Nevertheless, ZNF143/ZFP143 specifically activates nuclear-encoded mitochondrial genes, and its loss leads to severe mitochondrial dysfunction. Using an in vitro embryo model, we find that ZNF143/ZFP143 is an essential regulator of organismal development. Our results establish ZNF143/ZFP143 as a conserved transcriptional regulator of cell proliferation and differentiation by safeguarding mitochondrial activity.

Keywords:  3D genome; chromatin looping; development; differentiation; gene regulation; mitochondria; transcription

DOI:  https://doi.org/10.1016/j.molcel.2024.11.031
Mol Cell. 2024 Dec 12. pii: S1097-2765(24)00957-2. [Epub ahead of print]

Putative looping factor ZNF143/ZFP143 is an essential transcriptional regulator with no looping function.

Domenic N Narducci, Anders S Hansen.

  Interactions between distal loci, including those involving enhancers and promoters, are a central mechanism of gene regulation in mammals, yet the protein regulators of these interactions remain largely undetermined. The zinc-finger transcription factor (TF) ZNF143/ZFP143 has been strongly implicated as a regulator of chromatin interactions, functioning either with or without CTCF. However, how ZNF143/ZFP143 functions as a looping factor is not well understood. Here, we tagged both CTCF and ZNF143/ZFP143 with dual-purpose degron/imaging tags to combinatorially assess their looping function and effect on each other. We find that ZNF143/ZFP143, contrary to prior reports, possesses no general looping function in mouse and human cells and that it largely functions independently of CTCF. Instead, ZNF143/ZFP143 is an essential and highly conserved transcription factor that largely binds promoters proximally, exhibits an extremely stable chromatin dwell time (>20 min), and regulates an important subset of mitochondrial and ribosomal genes.

Keywords:  3D genome; CTCF; FRAP; Micro-C; ZFP143; ZNF143; chromatin loop; gene regulation; single-molecule imaging; transcription factor

DOI:  https://doi.org/10.1016/j.molcel.2024.11.032
Cell Rep. 2024 Dec 21. pii: S2211-1247(24)01440-2. [Epub ahead of print]44(1): 115089

Increased nuclear factor I-mediated chromatin access drives transition to androgen receptor splice variant dependence in prostate cancer.

Larysa Poluben, Mannan Nouri, Jiaqian Liang, Shaoyong Chen, Andreas Varkaris, Betul Ersoy-Fazlioglu, Olga Voznesensky, Irene I Lee, Xintao Qiu, Laura Cato, Ji-Heui Seo, Matthew L Freedman, Adam G Sowalsky, Nathan A Lack, Eva Corey, Peter S Nelson, Myles Brown, Henry W Long, Joshua W Russo, Steven P Balk.

  Androgen receptor (AR) splice variants, of which ARv7 is the most common, are increased in castration-resistant prostate cancer, but the extent to which they drive AR activity is unclear. We generated a subline of VCaP cells (VCaP16) that is resistant to the AR inhibitor enzalutamide (ENZ). AR activity in VCaP16 is driven by ARv7, independently of full-length AR (ARfl), and its cistrome and transcriptome mirror those of ARfl in VCaP cells. ARv7 expression increases rapidly in response to ENZ, but there is a delay in gaining chromatin binding and transcriptional activity, which is associated with increased chromatin accessibility. AR and nuclear factor I (NFI) motifs are most enriched at more accessible sites, and NFIB/X knockdown greatly diminishes ARv7 function. These findings indicate that ARv7 can drive the AR program but that its activity is dependent on adaptations that increase chromatin accessibility to enhance its intrinsically weak chromatin binding.

Keywords:  CP: Cancer; CP: Molecular biology; FOXA1; androgen receptor; chromatin; epigenetics; nuclear factor I; prostate cancer; transcription

DOI:  https://doi.org/10.1016/j.celrep.2024.115089
BMC Genomics. 2024 Dec 24. 25(1): 1240

HDI-STARR-seq: Condition-specific enhancer discovery in mouse liver in vivo.

Ting-Ya Chang, David J Waxman.

   BACKGROUND: STARR-seq and other massively-parallel reporter assays are widely used to discover functional enhancers in transfected cell models, which can be confounded by plasmid vector-induced type-I interferon immune responses and lack the multicellular environment and endogenous chromatin state of complex mammalian tissues.
RESULTS: We describe HDI-STARR-seq, which combines STARR-seq plasmid library delivery to the liver, by hydrodynamic tail vein injection (HDI), with reporter RNA transcriptional initiation driven by a minimal Albumin promoter, which we show is essential for mouse liver STARR-seq enhancer activity assayed 7 days after HDI. Importantly, little or no vector-induced innate type-I interferon responses were observed. Comparisons of HDI-STARR-seq activity between male and female mouse livers and in livers from males treated with an activating ligand of the transcription factor (TF) CAR (Nr1i3) identified many condition-dependent enhancers linked to condition-specific gene expression. Further, thousands of active liver enhancers were identified using a high complexity STARR-seq library comprised of ~ 50,000 genomic regions released by DNase-I digestion of mouse liver nuclei. When compared to stringently inactive library sequences, the active enhancer sequences identified were highly enriched for liver open chromatin regions with activating histone marks (H3K27ac, H3K4me1, H3K4me3), were significantly closer to gene transcriptional start sites, and were significantly depleted of repressive (H3K27me3, H3K9me3) and transcribed region histone marks (H3K36me3).
CONCLUSION: HDI-STARR-seq offers substantial improvements over current methodologies for large scale, functional profiling of enhancers, including condition-dependent enhancers, in liver tissue in vivo, and can be adapted to characterize enhancer activities in a variety of species and tissues by selecting suitable tissue- and species-specific promoter sequences.

Keywords:  Chromatin accessibility; DNase-seq; Hydrodynamic tail vein injection; MPRA

DOI:  https://doi.org/10.1186/s12864-024-11162-9
Nat Commun. 2024 Dec 23. 15(1): 10722

Contributing factors to the oxidation-induced mutational landscape in human cells.

Cameron Cordero, Kavi P M Mehta, Tyler M Weaver, Justin A Ling, Bret D Freudenthal, David Cortez, Steven A Roberts.

8-oxoguanine (8-oxoG) is a common oxidative DNA lesion that causes G > T substitutions. Determinants of local and regional differences in 8-oxoG-induced mutability across genomes are currently unknown. Here, we show DNA oxidation induces G > T substitutions and insertion/deletion (INDEL) mutations in human cells and cancers. Potassium bromate (KBrO3)-induced 8-oxoGs occur with similar sequence preferences as their derived substitutions, indicating that the reactivity of specific oxidants dictates mutation sequence specificity. While 8-oxoG occurs uniformly across chromatin, 8-oxoG-induced mutations are elevated in compact genomic regions, within nucleosomes, and at inward facing guanines within strongly positioned nucleosomes. Cryo-electron microscopy structures of OGG1-nucleosome complexes indicate that these effects originate from OGG1's ability to flip outward positioned 8-oxoG lesions into the catalytic pocket while inward facing lesions are occluded by the histone octamer. Mutation spectra from human cells with DNA repair deficiencies reveals contributions of a DNA repair network limiting 8-oxoG mutagenesis, where OGG1- and MUTYH-mediated base excision repair is supplemented by the replication-associated factors Pol η and HMCES. Transcriptional asymmetry of KBrO3-induced mutations in OGG1- and Pol η-deficient cells also demonstrates transcription-coupled repair can prevent 8-oxoG-induced mutation. Thus, oxidant chemistry, chromatin structures, and DNA repair processes combine to dictate the oxidative mutational landscape in human genomes.

DOI: https://doi.org/10.1038/s41467-024-55497-z
Mol Cell. 2024 Dec 17. pii: S1097-2765(24)00995-X. [Epub ahead of print]

3D chromatin hubs as regulatory units of identity and survival in human acute leukemia.

Giovanni Gambi, Francesco Boccalatte, Javier Rodriguez Hernaez, Ziyan Lin, Bettina Nadorp, Alexander Polyzos, Jimin Tan, Kleopatra Avrampou, Giorgio Inghirami, Alex Kentsis, Effie Apostolou, Iannis Aifantis, Aristotelis Tsirigos.

  Cancer progression involves genetic and epigenetic changes that disrupt chromatin 3D organization, affecting enhancer-promoter interactions and promoting growth. Here, we provide an integrative approach, combining chromatin conformation, accessibility, and transcription analysis, validated by in silico and CRISPR-interference screens, to identify relevant 3D topologies in pediatric T cell leukemia (T-ALL and ETP-ALL). We characterize 3D hubs as regulatory centers for oncogenes and disease markers, linking them to biological processes like cell division, inflammation, and stress response. Single-cell mapping reveals heterogeneous gene activation in discrete epigenetic clones, aiding in patient stratification for relapse risk after chemotherapy. Finally, we identify MYB as a 3D hub regulator in leukemia cells and show that the targeting of key regulators leads to hub dissolution, thereby providing a novel and effective anti-leukemic strategy. Overall, our work demonstrates the relevance of studying oncogenic 3D hubs to better understand cancer biology and tumor heterogeneity and to propose novel therapeutic strategies.

Keywords:  HiChIP; MYB; chromatin structure; heterogeneity; hubs; leukemia; modules; scATAC

DOI:  https://doi.org/10.1016/j.molcel.2024.11.040
Nucleic Acids Res. 2024 Dec 24. pii: gkae1240. [Epub ahead of print]

Defining gene ends: RNA polymerase II CTD threonine 4 phosphorylation marks transcription termination regions genome-wide.

Magda Kopczyńska, Upasana Saha, Anastasiia Romanenko, Takayuki Nojima, Michał R Gdula, Kinga Kamieniarz-Gdula.

Defining the beginning of a eukaryotic protein-coding gene is relatively simple. It corresponds to the first ribonucleotide incorporated by RNA polymerase II (Pol II) into the nascent RNA molecule. This nucleotide is protected by capping and maintained in the mature messenger RNA (mRNA). However, in higher eukaryotes, the end of mRNA is separated from the sites of transcription termination by hundreds to thousands of base pairs. Currently used genomic annotations only take account of the end of the mature transcript - the sites where pre-mRNA cleavage occurs, while the regions in which transcription terminates are unannotated. Here, we describe the evidence for a marker of transcription termination, which could be widely applicable in genomic studies. Pol II termination regions can be determined genome-wide by detecting Pol II phosphorylated on threonine 4 of its C-terminal domain (Pol II CTD-T4ph). Pol II in this state pauses before leaving the DNA template. Up to date this potent mark has been underused because the evidence for its place and role in termination is scattered across multiple publications. We summarize the observations regarding Pol II CTD-T4ph in termination regions and present bioinformatic analyses that further support Pol II CTD-T4ph as a global termination mark in animals.

DOI: https://doi.org/10.1093/nar/gkae1240
Nat Commun. 2024 Dec 23. 15(1): 10716

Inter-chromosomal transcription hubs shape the 3D genome architecture of African trypanosomes.

Claudia Rabuffo, Markus R Schmidt, Prateek Yadav, Pin Tong, Roberta Carloni, Anna Barcons-Simon, Raúl O Cosentino, Stefan Krebs, Keith R Matthews, Robin C Allshire, T Nicolai Siegel.

The eukaryotic nucleus exhibits a highly organized 3D genome architecture, with RNA transcription and processing confined to specific nuclear structures. While intra-chromosomal interactions, such as promoter-enhancer dynamics, are well-studied, the role of inter-chromosomal interactions remains poorly understood. Investigating these interactions in mammalian cells is challenging due to large genome sizes and the need for deep sequencing. Additionally, transcription-dependent 3D topologies in mixed cell populations further complicate analyses. To address these challenges, we used high-resolution DNA-DNA contact mapping (Micro-C) in Trypanosoma brucei, a parasite with continuous RNA polymerase II (RNAPII) transcription and polycistronic transcription units (PTUs). With approximately 300 transcription start sites (TSSs), this genome organization simplifies data interpretation. To minimize scaffolding artifacts, we also generated a highly contiguous phased genome assembly using ultra-long sequencing reads. Our Micro-C analysis revealed an intricate 3D genome organization. While the T. brucei genome displays features resembling chromosome territories, its chromosomes are arranged around polymerase-specific transcription hubs. RNAPI-transcribed genes cluster, as expected from their localization to the nucleolus. However, we also found that RNAPII TSSs form distinct inter-chromosomal transcription hubs with other RNAPII TSSs. These findings highlight the evolutionary significance of inter-chromosomal transcription hubs and provide new insights into genome organization in T. brucei.

DOI: https://doi.org/10.1038/s41467-024-55285-9
Mol Cell Biol. 2024 Dec 24. 1-20

The Role of Med15 Sequence Features in Transcription Factor Interactions.

David G Cooper, Shulin Liu, Emma Grunkemeyer, Jan S Fassler.

  Med15 is a general transcriptional regulator and tail module subunit within the RNA Pol II mediator complex. The Saccharomyces cerevisiae Med15 protein has a well-structured N-terminal KIX domain, three activator binding domains (ABDs) and several naturally variable polyglutamine (poly-Q) tracts (Q1, Q2, Q3) embedded in an intrinsically disordered central region, and a C-terminal mediator association domain (MAD). We investigated how the presence of ABDs and changes in length and composition of poly-Q tracts influences Med15 activity using phenotypic, gene expression, transcription factor interaction and phase separation assays of truncation, deletion, and synthetic alleles. We found that individual Med15 activities were influenced by the number of activator binding domains (ABDs) and adjacent polyglutamine tract composition. Robust Med15 activity required at least the Q1 tract and the length of that tract modulated activity in a context-dependent manner. Reduced Msn2-dependent transcriptional activation due to Med15 Q1 tract variation correlated with reduced Msn2:Med15 interaction strength, but interaction strength did not always mirror phase separation propensity. We also observed that distant glutamine tracts and Med15 phosphorylation affected the activities of the KIX domain, and interaction studies revealed that intramolecular interactions may affect some Med15-transcription factor interactions.

Keywords:  Med15; Mediator complex; Msn2; liquid-liquid phase separation; polyglutamine

DOI:  https://doi.org/10.1080/10985549.2024.2436672
Cell Rep. 2024 Dec 24. pii: S2211-1247(24)01456-6. [Epub ahead of print]44(1): 115105

Dual-nuclease single-cell lineage tracing by Cas9 and Cas12a.

Cheng Chen, Yuanxin Liao, Miao Zhu, Li Wang, Xinran Yu, Meishi Li, Guangdun Peng.

  Single-cell lineage tracing based on CRISPR-Cas9 gene editing enables the simultaneous linkage of cell states and lineage history at a high resolution. Despite its immense potential in resolving the cell fate determination and genealogy within an organism, existing implementations of this technology suffer from limitations in recording capabilities and considerable barcode dropout. Here, we introduce DuTracer, a versatile tool that utilizes two orthogonal gene editing systems to record cell lineage history at single-cell resolution in an inducible manner. DuTracer shows the ability to enhance lineage recording with minimized target dropouts and potentially deeper tree depths. Applying DuTracer in mouse embryoid bodies and neuromesodermal organoids illustrates the lineage relationship of different cell types and proposes potential lineage-biased molecular drivers, showcased by identifying transcription factor Foxb1 as a modulator in the cell fate determination of neuromesodermal progenitors. Collectively, DuTracer facilitates the precise and regulatory interrogation of cellular lineages of complex biological processes.

Keywords:  CP: Genomics; Cas12a; Cas9; entropy; inter-site deletion; single-cell lineage tracing

DOI:  https://doi.org/10.1016/j.celrep.2024.115105
Nucleic Acids Res. 2024 Dec 27. pii: gkae1270. [Epub ahead of print]

Comprehensive mapping of genetic variation at Epromoters reveals pleiotropic association with multiple disease traits.

Jing Wan, Antoinette van Ouwerkerk, Jean-Christophe Mouren, Carla Heredia, Lydie Pradel, Benoit Ballester, Jean-Christophe Andrau, Salvatore Spicuglia.

There is growing evidence that a wide range of human diseases and physiological traits are influenced by genetic variation of cis-regulatory elements. We and others have shown that a subset of promoter elements, termed Epromoters, also function as enhancer regulators of distal genes. This opens a paradigm in the study of regulatory variants, as single nucleotide polymorphisms (SNPs) within Epromoters might influence the expression of several (distal) genes at the same time, which could disentangle the identification of disease-associated genes. Here, we built a comprehensive resource of human Epromoters using newly generated and publicly available high-throughput reporter assays. We showed that Epromoters display intrinsic and epigenetic features that distinguish them from typical promoters. By integrating Genome-Wide Association Studies (GWAS), expression Quantitative Trait Loci (eQTLs) and 3D chromatin interactions, we found that regulatory variants at Epromoters are concurrently associated with more disease and physiological traits, as compared with typical promoters. To dissect the regulatory impact of Epromoter variants, we evaluated their impact on regulatory activity by analyzing allelic-specific high-throughput reporter assays and provided reliable examples of pleiotropic Epromoters. In summary, our study represents a comprehensive resource of regulatory variants supporting the pleiotropic role of Epromoters.

DOI: https://doi.org/10.1093/nar/gkae1270
Elife. 2024 Dec 23. pii: RP94884. [Epub ahead of print]13

A novel human pluripotent stem cell gene activation system identifies IGFBP2 as a mediator in the production of haematopoietic progenitors in vitro.

Paolo Petazzi, Telma Ventura, Francesca Paola Luongo, Heather McClafferty, Alisha May, Helen Alice Taylor, Michael J Shipston, Nicola Romanò, Lesley M Forrester, Pablo Menendez, Antonella Fidanza.

  A major challenge in the stem cell biology field is the ability to produce fully functional cells from induced pluripotent stem cells (iPSCs) that are a valuable resource for cell therapy, drug screening, and disease modelling. Here, we developed a novel inducible CRISPR-mediated activation strategy (iCRISPRa) to drive the expression of multiple endogenous transcription factors (TFs) important for in vitro cell fate and differentiation of iPSCs to haematopoietic progenitor cells. This work has identified a key role for IGFBP2 in developing haematopoietic progenitors. We first identified nine candidate TFs that we predicted to be involved in blood cell emergence during development, then generated tagged gRNAs directed to the transcriptional start site of these TFs that could also be detected during single-cell RNA sequencing (scRNAseq). iCRISPRa activation of these endogenous TFs resulted in a significant expansion of arterial-fated endothelial cells expressing high levels of IGFBP2, and our analysis indicated that IGFBP2 is involved in the remodelling of metabolic activity during in vitro endothelial to haematopoietic transition. As well as providing fundamental new insights into the mechanisms of haematopoietic differentiation, the broader applicability of iCRISPRa provides a valuable tool for studying dynamic processes in development and for recapitulating abnormal phenotypes characterised by ectopic activation of specific endogenous gene expression in a wide range of systems.

Keywords:  CRISPR activation; developmental biology; haematopoiesis; human; pluripotent stem cells; regenerative medicine; stem cells

DOI:  https://doi.org/10.7554/eLife.94884
J Biol Chem. 2024 Dec 21. pii: S0021-9258(24)02621-8. [Epub ahead of print] 108119

ID3 promotes erythroid differentiation and is repressed by a TAL1/PRMT6 complex.

Vivien Heller, Lei Wang, Edith Schneider, Mirjam Gerstner, Luana Bajer, Robin Decker, Halvard Boenig, Joern Lausen.

Erythropoiesis is controlled by transcription factors that recruit epigenetic cofactors to establish and maintain erythrocyte-specific gene expression patterns while repressing alternative lineage commitment. The transcription factor TAL1 is critical for establishing erythroid gene expression. It acts as an activator or repressor of genes, depending on associated epigenetic cofactors. Understanding the epigenetic function of TAL1 during erythropoiesis is key to improving in vitro erythroid differentiation and understanding pathological erythropoiesis. Therefore, the regulatory mechanisms that control the function of TAL1 during erythropoiesis are under intense investigation. Here we show that TAL1 interacts with PRMT6 on the ID3 gene in K562 and hCD34+ cells. The ID protein family is a critical transcriptional regulator of hematopoietic cell differentiation. We show that TAL1 and PRMT6 are present at the ID3 promoter, and that TAL1 is involved in the recruitment of PRMT6. Here, PRMT6 epigenetically regulates ID3 expression by mediating H3R2me2a. Thus, TAL1/PRMT6 epigenetically represses ID3 expression in progenitors, which is relieved upon erythroid differentiation, leading to increased ID3 expression. Overexpression of ID3 in primary hCD34+ cells enhances erythropoiesis. Our results show that a TAL1/PRMT6 complex regulates genes important for erythropoiesis, such as ID3. Manipulation of ID3 expression may be a way to promote in vitro differentiation of hCD34+ cells into erythrocytes.

DOI: https://doi.org/10.1016/j.jbc.2024.108119
iScience. 2024 Dec 20. 27(12): 111452

A negatively charged region within carboxy-terminal domain maintains proper CTCF DNA binding.

Lian Liu, Yuanxiao Tang, Yan Zhang, Qiang Wu.

  As an essential regulator of higher-order chromatin structures, CCCTC-binding factor (CTCF) is a highly conserved protein with a central DNA-binding domain of 11 tandem zinc fingers (ZFs), which are flanked by amino (N-) and carboxy (C-) terminal domains of intrinsically disordered regions. Here we report that CRISPR deletion of the entire C-terminal domain of alternating charge blocks decreases CTCF DNA binding but deletion of the C-terminal fragment of 116 amino acids results in increased CTCF DNA binding and aberrant gene regulation. Through a series of genetic targeting experiments, in conjunction with electrophoretic mobility shift assay (EMSA), circularized chromosome conformation capture (4C), qPCR, chromatin immunoprecipitation with sequencing (ChIP-seq), and assay for transposase-accessible chromatin with sequencing (ATAC-seq), we uncovered a negatively charged region (NCR) responsible for weakening CTCF DNA binding and chromatin accessibility. AlphaFold prediction suggests an autoinhibitory mechanism of CTCF via NCR as a flexible DNA mimic domain, possibly competing with DNA binding for the positively charged ZF surface area. Thus, the unstructured C-terminal domain plays an intricate role in maintaining proper CTCF-DNA interactions and 3D genome organization.

Keywords:  Experimental systems for structural biology; Molecular Structure; Molecular physiology; Properties of biomolecules

DOI:  https://doi.org/10.1016/j.isci.2024.111452
J Chem Phys. 2024 Dec 28. pii: 245101. [Epub ahead of print]161(24):

Deciphering single-cell 3D chromatin structure using scCTG.

Ran Jiang, Yue Xue, Yanyi Huang, Yi Qin Gao.

Sequencing-based Hi-C technology has been widely used to study the three-dimensional structure of chromatin. More recently, the development of single-cell Hi-C technology has enabled the study of chromatin structural variations between individual cells. However, single-cell Hi-C data are often highly sparse, necessitating the use of imputation algorithms to address insufficient sampling. Current methods encounter challenges such as significant discrepancies from real structural features, limited reproducibility, slower computational speeds, or reliance on large amounts of training data, which hinder their broader applicability. In this study, we improved the previously published CTG (Hi-C To Geometry) algorithm to introduce the single-cell CTG (scCTG) algorithm, which combines convolution and diffusion processes to yield the spatial distance matrix for various types of single-cell chromatin structure data. scCTG algorithm shows a good performance in terms of computational efficiency, robustness, and correlation with physical spatial distances. The scCTG algorithm can be applied to effectively identify compartments and insulation strength for each locus, providing deeper insights into the relationship between chromatin structure and gene expression at the single-cell level.

DOI: https://doi.org/10.1063/5.0241334
Nat Commun. 2024 Dec 27. 15(1): 10721

Epigenomic and 3D genomic mapping reveals developmental dynamics and subgenomic asymmetry of transcriptional regulatory architecture in allotetraploid cotton.

Xianhui Huang, Yuejin Wang, Sainan Zhang, Liuling Pei, Jiaqi You, Yuexuan Long, Jianying Li, Xianlong Zhang, Longfu Zhu, Maojun Wang.

Although epigenetic modification has long been recognized as a vital force influencing gene regulation in plants, the dynamics of chromatin structure implicated in the intertwined transcriptional regulation of duplicated genes in polyploids have yet to be understood. Here, we document the dynamic organization of chromatin structure in two subgenomes of allotetraploid cotton (Gossypium hirsutum) by generating 3D genomic, epigenomic and transcriptomic datasets from 12 major tissues/developmental stages covering the life cycle. We systematically identify a subset of genes that are closely associated with specific tissue functions. Interestingly, these genes exhibit not only higher tissue specificity but also a more pronounced homoeologous bias. We comprehensively elucidate the intricate process of subgenomic collaboration and divergence across various tissues. A comparison among subgenomes in the 12 tissues reveals widespread differences in the reorganization of 3D genome structures, with the Dt subgenome exhibiting a higher extent of dynamic chromatin status than the At subgenome. Moreover, we construct a comprehensive atlas of putative functional genome elements and discover that 37 cis-regulatory elements (CREs) have selection signals acquired during domestication and improvement. These data and analyses are publicly available to the research community through a web portal. In summary, this study provides abundant resources and depicts the regulatory architecture of the genome, which thereby facilitates the understanding of biological processes and guides cotton breeding.

DOI: https://doi.org/10.1038/s41467-024-55309-4
BMC Genomics. 2024 Dec 23. 25(1): 1234

DEMETER DNA demethylase reshapes the global DNA methylation landscape and controls cell identity transition during plant regeneration.

Seunga Lee, Soon Hyung Bae, Yunji Jeon, Pil Joon Seo, Yeonhee Choi.

   BACKGROUND: Plants possess a high potential for somatic cell reprogramming, enabling the transition from differentiated tissue to pluripotent callus, followed by the formation of de novo shoots during plant regeneration. Despite extensive studies on the molecular network and key genetic factors involved in this process, the underlying epigenetic landscape remains incompletely understood.
RESULTS: Here, we explored the dynamics of the methylome and transcriptome during the two-step plant regeneration process. During the leaf-to-callus transition in Arabidopsis Ler, CG methylation shifted across genic regions, exhibiting a similar number of differentially methylated regions (DMRs) for both hypo- and hypermethylation. Pericentromeric regions underwent substantial CG and extensive CHH hypomethylation, alongside some CHG hypermethylation. Upon shoot regeneration from callus, genic regions displayed extensive reconfiguration of CG methylation, while pericentromeric methylation levels highly increased across all cytosine contexts, coinciding with the activation of the RNA-directed DNA methylation (RdDM) pathway. However, mutation in DEMETER (DME) DNA demethylase gene resulted in significant genic CG redistribution and global non-CG hypomethylation in pericentromeric regions, particularly during shoot regeneration. This non-CG hypomethylation observed in dme-2 mutants was, at least partly, due to RdDM downregulation. The dme-2 mutants affected gene expression involved in pluripotency and shoot meristem development, resulting in enhanced shoot regeneration through a reprogrammed state established by pericentromeric hypomethylation compared to wild type.
CONCLUSION: Our study demonstrates epigenetic changes, accompanied by transcriptome alterations, during pluripotency acquisition (leaf-to-callus) and regeneration (callus-to-de novo shoot). Additionally, it highlights the functions of the DME demethylase, particularly its close association with the RdDM pathway, which underlies pericentromeric non-CG methylation maintenance. These results provide important insights into the epigenetic reconfiguration associated with cell identity transition during somatic cell reprogramming.

Keywords:   Arabidopsis ; DEMETER (DME) DNA demethylase; DNA methylation; In vitro plant regeneration; RNA-directed DNA methylation (RdDM); Somatic cell reprogramming

DOI:  https://doi.org/10.1186/s12864-024-11144-x