bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2024–08–25
24 papers selected by
Connor Rogerson, University of Cambridge



  1. Elife. 2024 Aug 23. pii: RP94407. [Epub ahead of print]13
      MYC family oncoproteins regulate the expression of a large number of genes and broadly stimulate elongation by RNA polymerase II (RNAPII). While the factors that control the chromatin association of MYC proteins are well understood, much less is known about how interacting proteins mediate MYC's effects on transcription. Here, we show that TFIIIC, an architectural protein complex that controls the three-dimensional chromatin organisation at its target sites, binds directly to the amino-terminal transcriptional regulatory domain of MYCN. Surprisingly, TFIIIC has no discernible role in MYCN-dependent gene expression and transcription elongation. Instead, MYCN and TFIIIC preferentially bind to promoters with paused RNAPII and globally limit the accumulation of non-phosphorylated RNAPII at promoters. Consistent with its ubiquitous role in transcription, MYCN broadly participates in hubs of active promoters. Depletion of TFIIIC further increases MYCN localisation to these hubs. This increase correlates with a failure of the nuclear exosome and BRCA1, both of which are involved in nascent RNA degradation, to localise to active promoters. Our data suggest that MYCN and TFIIIC exert an censoring function in early transcription that limits promoter accumulation of inactive RNAPII and facilitates promoter-proximal degradation of nascent RNA.
    Keywords:  MYCN; TFIIIC; cancer biology; chromosomes; gene expression; human; neuroblastoma; nuclear exosome; promoter hub
    DOI:  https://doi.org/10.7554/eLife.94407
  2. Nucleic Acids Res. 2024 Aug 21. pii: gkae706. [Epub ahead of print]
      Transcription factor (TF)-cofactor (COF) interactions define dynamic, cell-specific networks that govern gene expression; however, these networks are understudied due to a lack of methods for high-throughput profiling of DNA-bound TF-COF complexes. Here, we describe the Cofactor Recruitment (CoRec) method for rapid profiling of cell-specific TF-COF complexes. We define a lysine acetyltransferase (KAT)-TF network in resting and stimulated T cells. We find promiscuous recruitment of KATs for many TFs and that 35% of KAT-TF interactions are condition specific. KAT-TF interactions identify NF-κB as a primary regulator of acutely induced histone 3 lysine 27 acetylation (H3K27ac). Finally, we find that heterotypic clustering of CBP/P300-recruiting TFs is a strong predictor of total promoter H3K27ac. Our data support clustering of TF sites that broadly recruit KATs as a mechanism for widespread co-occurring histone acetylation marks. CoRec can be readily applied to different cell systems and provides a powerful approach to define TF-COF networks impacting chromatin state and gene regulation.
    DOI:  https://doi.org/10.1093/nar/gkae706
  3. EMBO J. 2024 Aug 22.
      Tumor cell heterogeneity defines therapy responsiveness in neuroblastoma (NB), a cancer derived from neural crest cells. NB consists of two primary subtypes: adrenergic and mesenchymal. Adrenergic traits predominate in NB tumors, while mesenchymal features becomes enriched post-chemotherapy or after relapse. The interconversion between these subtypes contributes to NB lineage plasticity, but the underlying mechanisms driving this phenotypic switching remain unclear. Here, we demonstrate that SWI/SNF chromatin remodeling complex ATPases are essential in establishing an mesenchymal gene-permissive chromatin state in adrenergic-type NB, facilitating lineage plasticity. Targeting SWI/SNF ATPases with SMARCA2/4 dual degraders effectively inhibits NB cell proliferation, invasion, and notably, cellular plasticity, thereby preventing chemotherapy resistance. Mechanistically, depletion of SWI/SNF ATPases compacts cis-regulatory elements, diminishes enhancer activity, and displaces core transcription factors (MYCN, HAND2, PHOX2B, and GATA3) from DNA, thereby suppressing transcriptional programs associated with plasticity. These findings underscore the pivotal role of SWI/SNF ATPases in driving intrinsic plasticity and therapy resistance in neuroblastoma, highlighting an epigenetic target for combinational treatments in this cancer.
    Keywords:  Cancer Cell Plasticity; Core Transcription Factors; Epigenetic Plasticity; Neuroblastoma; SWI/SNF Complexes
    DOI:  https://doi.org/10.1038/s44318-024-00206-1
  4. EMBO J. 2024 Aug 19.
      The ring-shaped cohesin complex topologically entraps two DNA molecules to establish sister chromatid cohesion. Cohesin also shapes the interphase chromatin landscape with wide-ranging implications for gene regulation, and cohesin is thought to achieve this by actively extruding DNA loops without topologically entrapping DNA. The 'loop extrusion' hypothesis finds motivation from in vitro observations-whether this process underlies in vivo chromatin loop formation remains untested. Here, using the budding yeast S. cerevisiae, we generate cohesin variants that have lost their ability to extrude DNA loops but retain their ability to topologically entrap DNA. Analysis of these variants suggests that in vivo chromatin loops form independently of loop extrusion. Instead, we find that transcription promotes loop formation, and acts as an extrinsic motor that expands these loops and defines their ultimate positions. Our results necessitate a re-evaluation of the loop extrusion hypothesis. We propose that cohesin, akin to sister chromatid cohesion establishment at replication forks, forms chromatin loops by DNA-DNA capture at places of transcription, thus unifying cohesin's two roles in chromosome segregation and interphase genome organisation.
    Keywords:  Cohesin; Loop Capture; Loop Extrusion; SMC Complexes; Transcription
    DOI:  https://doi.org/10.1038/s44318-024-00202-5
  5. Nat Struct Mol Biol. 2024 Aug 16.
      DNA supercoiling is a biophysical feature of the double helix with a pivotal role in biological processes. However, understanding of DNA supercoiling in the chromatin remains limited. Here, we developed azide-trimethylpsoralen sequencing (ATMP-seq), a DNA supercoiling assay offering quantitative accuracy while minimizing genomic bias and background noise. Using ATMP-seq, we directly visualized transcription-dependent negative and positive twin-supercoiled domains around genes and mapped kilobase-resolution DNA supercoiling throughout the human genome. Remarkably, we discovered megabase-scale supercoiling domains (SDs) across all chromosomes that are modulated mainly by topoisomerases I and IIβ. Transcription activities, but not the consequent supercoiling accumulation in the local region, contribute to SD formation, indicating the long-range propagation of transcription-generated supercoiling. Genome-wide SDs colocalize with A/B compartments in both human and Drosophila cells but are distinct from topologically associating domains (TADs), with negative supercoiling accumulation at TAD boundaries. Furthermore, genome-wide DNA supercoiling varies between cell states and types and regulates human gene expression, underscoring the importance of supercoiling dynamics in chromatin regulation and function.
    DOI:  https://doi.org/10.1038/s41594-024-01377-5
  6. Cell Rep. 2024 Aug 19. pii: S2211-1247(24)00990-2. [Epub ahead of print]43(8): 114640
      Functional enhancer annotation is critical for understanding tissue-specific transcriptional regulation and prioritizing disease-associated non-coding variants. However, unbiased enhancer discovery in disease-relevant contexts remains challenging. To identify enhancers pertinent to diabetes, we conducted a CRISPR interference (CRISPRi) screen in the human pluripotent stem cell (hPSC) pancreatic differentiation system. Among the enhancers identified, we focused on an enhancer we named ONECUT1e-664kb, ∼664 kb from the ONECUT1 promoter. Previous studies have linked ONECUT1 coding mutations to pancreatic hypoplasia and neonatal diabetes. We found that homozygous deletion of ONECUT1e-664kb in hPSCs leads to a near-complete loss of ONECUT1 expression and impaired pancreatic differentiation. ONECUT1e-664kb contains a type 2 diabetes-associated variant (rs528350911) disrupting a GATA motif. Introducing the risk variant into hPSCs reduced binding of key pancreatic transcription factors (GATA4, GATA6, and FOXA2), supporting its causal role in diabetes. This work highlights the utility of unbiased enhancer discovery in disease-relevant settings for understanding monogenic and complex disease.
    Keywords:  CP: Developmental biology; CP: Molecular biology; CRISPRi screen; ONECUT1; T2D; VUS; enhancer; neonatal diabetes; non-coding variant; pancreas development; type 2 diabetes; variant of uncertain significance
    DOI:  https://doi.org/10.1016/j.celrep.2024.114640
  7. Nucleic Acids Res. 2024 Aug 21. pii: gkae714. [Epub ahead of print]
      Chromatin changes in response to estrogen and progesterone are well established in cultured cells, but how they control gene expression under physiological conditions is largely unknown. To address this question, we examined in vivo estrous cycle dynamics of mouse uterus hormone receptor occupancy, chromatin accessibility and chromatin structure by combining RNA-seq, ATAC-seq, HiC-seq and ChIP-seq. Two estrous cycle stages were chosen for these analyses, diestrus (highest estrogen) and estrus (highest progesterone). Unexpectedly, rather than alternating with each other, estrogen receptor alpha (ERα) and progesterone receptor (PGR) were co-bound during diestrus and lost during estrus. Motif analysis of open chromatin followed by hypoxia inducible factor 2A (HIF2A) ChIP-seq and conditional uterine deletion of this transcription factor revealed a novel role for HIF2A in regulating diestrus gene expression patterns that were independent of either ERα or PGR binding. Proteins in complex with ERα included PGR and cohesin, only during diestrus. Combined with HiC-seq analyses, we demonstrate that complex chromatin architecture changes including enhancer switching are coordinated with ERα and PGR co-binding during diestrus and non-hormone receptor transcription factors such as HIF2A during estrus to regulate most differential gene expression across the estrous cycle.
    DOI:  https://doi.org/10.1093/nar/gkae714
  8. Nat Struct Mol Biol. 2024 Aug 16.
      Three-dimensional (3D) genome folding has a fundamental role in the regulation of developmental genes by facilitating or constraining chromatin interactions between cis-regulatory elements (CREs). Polycomb response elements (PREs) are a specific kind of CRE involved in the memory of transcriptional states in Drosophila melanogaster. PREs act as nucleation sites for Polycomb group (PcG) proteins, which deposit the repressive histone mark H3K27me3, leading to the formation of a class of topologically associating domain (TAD) called a Polycomb domain. PREs can establish looping contacts that stabilize the gene repression of key developmental genes during development. However, the mechanism by which PRE loops fine-tune gene expression is unknown. Using clustered regularly interspaced short palindromic repeats and Cas9 genome engineering, we specifically perturbed PRE contacts or enhancer function and used complementary approaches including 4C-seq, Hi-C and Hi-M to analyze how chromatin architecture perturbation affects gene expression. Our results suggest that the PRE loop at the dac gene locus acts as a constitutive 3D chromatin scaffold during Drosophila development that forms independently of gene expression states and has a versatile function; it restricts enhancer-promoter communication and contributes to enhancer specificity.
    DOI:  https://doi.org/10.1038/s41594-024-01375-7
  9. Nat Commun. 2024 Aug 17. 15(1): 7091
      The integration of extrinsic signaling with cell-intrinsic transcription factors can direct progenitor cells to differentiate into distinct cell fates. In the developing Drosophila eye, differentiation of photoreceptors R1-R7 requires EGFR signaling mediated by the transcription factor Pointed, and our single-cell RNA-Seq analysis shows that the same photoreceptors require the eye-specific transcription factor Glass. We find that ectopic expression of Glass and activation of EGFR signaling synergistically induce neuronal gene expression in the wing disc in a Pointed-dependent manner. Targeted DamID reveals that Glass and Pointed share many binding sites in the genome of developing photoreceptors. Comparison with transcriptomic data shows that Pointed and Glass induce photoreceptor differentiation through intermediate transcription factors, including the redundant homologs Scratch and Scrape, as well as directly activating neuronal effector genes. Our data reveal synergistic activation of a multi-layered transcriptional network as the mechanism by which EGFR signaling induces neuronal identity in Glass-expressing cells.
    DOI:  https://doi.org/10.1038/s41467-024-51429-z
  10. Genome Res. 2024 Aug 16. pii: gr.278601.123. [Epub ahead of print]
      Transcription Factors (TFs) regulate gene expression by facilitating or disrupting the formation of transcription initiation machinery at particular genomic loci. Since TF occupancy is driven in part by recognition of DNA sequence, genetic variation can influence TF-DNA associations and gene regulation. To identify variants that impact TF binding in human brain tissues, we assessed allele specific binding (ASB) at heterozygous variants for 94 TFs in 9 brain regions from two donors. Leveraging graph genomes constructed from phased genomic sequence data, we compared ChIP-seq signals between alleles at heterozygous variants within each brain region and identified thousands of variants exhibiting ASB for at least one TF. ASB reproducibility was measured by comparisons between independent experiments both within and between donors. We found that rarer alleles in the general population more frequently led to reduced TF binding, whereas common variation had an equal likelihood of increasing or decreasing binding. Motif analysis revealed TF-specific effects, with ASB variants for certain TFs displaying a greater incidence of motif alterations, as well as enrichments for variants under purifying selection. Notably, neuron-specific cis-regulatory elements (cCREs) showed depletion for ASB variants. We identified 2,670 ASB variants with prior evidence of allele-specific gene expression in the brain from GTEx data and observed increasing eQTL effect direction concordance as ASB significance increases. These results provide a valuable and unique resource for mechanistic analysis of cis-regulatory variation in human brain tissue.
    DOI:  https://doi.org/10.1101/gr.278601.123
  11. Nat Commun. 2024 Aug 22. 15(1): 7222
      CRISPR/Cas-based transcriptional activators can be enhanced by intrinsically disordered regions (IDRs). However, the underlying mechanisms are still debatable. Here, we examine 12 well-known IDRs by fusing them to the dCas9-VP64 activator, of which only seven can augment activation, albeit independently of their phase separation capabilities. Moreover, modular domains (MDs), another class of multivalent molecules, though ineffective in enhancing dCas9-VP64 activity on their own, show substantial enhancement in transcriptional activation when combined with dCas9-VP64-IDR. By varying the number of gRNA binding sites and fusing dCas9-VP64 with different IDRs/MDs, we uncover that optimal, rather than maximal, cis-trans cooperativity enables the most robust activation. Finally, targeting promoter-enhancer pairs yields synergistic effects, which can be further amplified via enhancing chromatin interactions. Overall, our study develops a versatile platform for efficient gene activation and sheds important insights into CRIPSR-based transcriptional activators enhanced with multivalent molecules.
    DOI:  https://doi.org/10.1038/s41467-024-51694-y
  12. Nat Immunol. 2024 Aug 21.
      Transcriptional condensates play a crucial role in gene expression and regulation, yet their assembly mechanisms remain poorly understood. Here, we report a multi-layered mechanism for condensate assembly by autoimmune regulator (Aire), an essential transcriptional regulator that orchestrates gene expression reprogramming for central T cell tolerance. Aire condensates assemble on enhancers, stimulating local transcriptional activities and connecting disparate inter-chromosomal loci. This functional condensate formation hinges upon the coordination between three Aire domains: polymerization domain caspase activation recruitment domain (CARD), histone-binding domain (first plant homeodomain (PHD1)), and C-terminal tail (CTT). Specifically, CTT binds coactivators CBP/p300, recruiting Aire to CBP/p300-rich enhancers and promoting CARD-mediated condensate assembly. Conversely, PHD1 binds to the ubiquitous histone mark H3K4me0, keeping Aire dispersed throughout the genome until Aire nucleates on enhancers. Our findings showed that the balance between PHD1-mediated suppression and CTT-mediated stimulation of Aire polymerization is crucial to form transcriptionally active condensates at target sites, providing new insights into controlled polymerization of transcriptional regulators.
    DOI:  https://doi.org/10.1038/s41590-024-01922-w
  13. Mol Cell. 2024 Aug 19. pii: S1097-2765(24)00661-0. [Epub ahead of print]
      Partitioning of repressive from actively transcribed chromatin in mammalian cells fosters cell-type-specific gene expression patterns. While this partitioning is reconstructed during differentiation, the chromatin occupancy of the key insulator, CCCTC-binding factor (CTCF), is unchanged at the developmentally important Hox clusters. Thus, dynamic changes in chromatin boundaries must entail other activities. Given its requirement for chromatin loop formation, we examined cohesin-based chromatin occupancy without known insulators, CTCF and Myc-associated zinc-finger protein (MAZ), and identified a family of zinc-finger proteins (ZNFs), some of which exhibit tissue-specific expression. Two such ZNFs foster chromatin boundaries at the Hox clusters that are distinct from each other and from MAZ. PATZ1 was critical to the thoracolumbar boundary in differentiating motor neurons and mouse skeleton, while ZNF263 contributed to cervicothoracic boundaries. We propose that these insulating activities act with cohesin, alone or combinatorially, with or without CTCF, to implement precise positional identity and cell fate during development.
    Keywords:  CTCF; Hox genes in development; MAZ; PATZ1; chromatin domains; cohesin; gene regulation; genome organization; insulation; motor neurons; zinc-finger proteins
    DOI:  https://doi.org/10.1016/j.molcel.2024.08.007
  14. Mol Cell. 2024 Aug 19. pii: S1097-2765(24)00630-0. [Epub ahead of print]
      Topoisomerase I (TOP1) is an essential enzyme that relaxes DNA to prevent and dissipate torsional stress during transcription. However, the mechanisms underlying the regulation of TOP1 activity remain elusive. Using enhanced cross-linking and immunoprecipitation (eCLIP) and ultraviolet-cross-linked RNA immunoprecipitation followed by total RNA sequencing (UV-RIP-seq) in human colon cancer cells along with RNA electrophoretic mobility shift assays (EMSAs), biolayer interferometry (BLI), and in vitro RNA-binding assays, we identify TOP1 as an RNA-binding protein (RBP). We show that TOP1 directly binds RNA in vitro and in cells and that most RNAs bound by TOP1 are mRNAs. Using a TOP1 RNA-binding mutant and topoisomerase cleavage complex sequencing (TOP1cc-seq) to map TOP1 catalytic activity, we reveal that RNA opposes TOP1 activity as RNA polymerase II (RNAPII) commences transcription of active genes. We further demonstrate the inhibitory role of RNA in regulating TOP1 activity by employing DNA supercoiling assays and magnetic tweezers. These findings provide insight into the coordinated actions of RNA and TOP1 in regulating DNA topological stress intrinsic to RNAPII-dependent transcription.
    Keywords:  DNA supercoiling; RNA-binding protein; TOP1; TOP1 interactome; magnetic tweezers; topology; transcription
    DOI:  https://doi.org/10.1016/j.molcel.2024.07.032
  15. Sci Adv. 2024 Aug 23. 10(34): eadp5753
      Mutations of the SNF2 family ATPase HELLS and its activator CDCA7 cause immunodeficiency, centromeric instability, and facial anomalies syndrome, characterized by DNA hypomethylation at heterochromatin. It remains unclear why CDCA7-HELLS is the sole nucleosome remodeling complex whose deficiency abrogates the maintenance of DNA methylation. We here identify the unique zinc-finger domain of CDCA7 as an evolutionarily conserved hemimethylation-sensing zinc finger (HMZF) domain. Cryo-electron microscopy structural analysis of the CDCA7-nucleosome complex reveals that the HMZF domain can recognize hemimethylated CpG in the outward-facing DNA major groove within the nucleosome core particle, whereas UHRF1, the critical activator of the maintenance methyltransferase DNMT1, cannot. CDCA7 recruits HELLS to hemimethylated chromatin and facilitates UHRF1-mediated H3 ubiquitylation associated with replication-uncoupled maintenance DNA methylation. We propose that the CDCA7-HELLS nucleosome remodeling complex assists the maintenance of DNA methylation on chromatin by sensing hemimethylated CpG that is otherwise inaccessible to UHRF1 and DNMT1.
    DOI:  https://doi.org/10.1126/sciadv.adp5753
  16. Elife. 2024 Aug 19. pii: RP96216. [Epub ahead of print]13
      Although the role of G-quadruplex (G4) DNA structures has been suggested in chromosomal looping this was not tested directly. Here, to test causal function, an array of G4s, or control sequence that does not form G4s, were inserted within chromatin in cells. In vivo G4 formation of the inserted G4 sequence array, and not the control sequence, was confirmed using G4-selective antibody. Compared to the control insert, we observed a remarkable increase in the number of 3D chromatin looping interactions from the inserted G4 array. This was evident within the immediate topologically associated domain (TAD) and throughout the genome. Locally, recruitment of enhancer histone marks and the transcriptional coactivator p300/Acetylated-p300 increased in the G4-array, but not in the control insertion. Resulting promoter-enhancer interactions and gene activation were clear up to 5 Mb away from the insertion site. Together, these show the causal role of G4s in enhancer function and long-range chromatin interactions. Mechanisms of 3D topology are primarily based on DNA-bound architectural proteins that induce/stabilize long-range interactions. Involvement of the underlying intrinsic DNA sequence/structure in 3D looping shown here therefore throws new light on how long-range chromosomal interactions might be induced or maintained.
    Keywords:  G-quadruplex; chromatin looping; chromosomes; enhancer; gene expression; human
    DOI:  https://doi.org/10.7554/eLife.96216
  17. BMC Bioinformatics. 2024 Aug 22. 25(1): 274
       BACKGROUND: Growing evidence suggests that distal regulatory elements are essential for cellular function and states. The sequences within these distal elements, especially motifs for transcription factor binding, provide critical information about the underlying regulatory programs. However, cooperativities between transcription factors that recognize these motifs are nonlinear and multiplexed, rendering traditional modeling methods insufficient to capture the underlying mechanisms. Recent development of attention mechanism, which exhibit superior performance in capturing dependencies across input sequences, makes them well-suited to uncover and decipher intricate dependencies between regulatory elements.
    RESULT: We present Transcription factors cooperativity Inference Analysis with Neural Attention (TIANA), a deep learning framework that focuses on interpretability. In this study, we demonstrated that TIANA could discover biologically relevant insights into co-occurring pairs of transcription factor motifs. Compared with existing tools, TIANA showed superior interpretability and robust performance in identifying putative transcription factor cooperativities from co-occurring motifs.
    CONCLUSION: Our results suggest that TIANA can be an effective tool to decipher transcription factor cooperativities from distal sequence data. TIANA can be accessed through: https://github.com/rzzli/TIANA .
    Keywords:  Bioinformatics; Deep Learning; Integrated gradients; Self-attention; Transcription Factors
    DOI:  https://doi.org/10.1186/s12859-024-05852-0
  18. Nat Commun. 2024 Aug 21. 15(1): 7152
      For accurate mitotic cell division, replicated chromatin must be assembled into chromosomes and faithfully segregated into daughter cells. While protein factors like condensin play key roles in this process, it is unclear how chromosome assembly proceeds as molecular events of nucleosomes in living cells and how condensins act on nucleosomes to organize chromosomes. To approach these questions, we investigate nucleosome behavior during mitosis of living human cells using single-nucleosome tracking, combined with rapid-protein depletion technology and computational modeling. Our results show that local nucleosome motion becomes increasingly constrained during mitotic chromosome assembly, which is functionally distinct from condensed apoptotic chromatin. Condensins act as molecular crosslinkers, locally constraining nucleosomes to organize chromosomes. Additionally, nucleosome-nucleosome interactions via histone tails constrain and compact whole chromosomes. Our findings elucidate the physical nature of the chromosome assembly process during mitosis.
    DOI:  https://doi.org/10.1038/s41467-024-51454-y
  19. Genome Biol. 2024 Aug 16. 25(1): 225
       BACKGROUND: Single-cell chromatin accessibility assays, such as scATAC-seq, are increasingly employed in individual and joint multi-omic profiling of single cells. As the accumulation of scATAC-seq and multi-omics datasets continue, challenges in analyzing such sparse, noisy, and high-dimensional data become pressing. Specifically, one challenge relates to optimizing the processing of chromatin-level measurements and efficiently extracting information to discern cellular heterogeneity. This is of critical importance, since the identification of cell types is a fundamental step in current single-cell data analysis practices.
    RESULTS: We benchmark 8 feature engineering pipelines derived from 5 recent methods to assess their ability to discover and discriminate cell types. By using 10 metrics calculated at the cell embedding, shared nearest neighbor graph, or partition levels, we evaluate the performance of each method at different data processing stages. This comprehensive approach allows us to thoroughly understand the strengths and weaknesses of each method and the influence of parameter selection.
    CONCLUSIONS: Our analysis provides guidelines for choosing analysis methods for different datasets. Overall, feature aggregation, SnapATAC, and SnapATAC2 outperform latent semantic indexing-based methods. For datasets with complex cell-type structures, SnapATAC and SnapATAC2 are preferred. With large datasets, SnapATAC2 and ArchR are most scalable.
    Keywords:  Benchmark; Clustering; Dimensional reduction; Feature engineering; ScATAC-seq
    DOI:  https://doi.org/10.1186/s13059-024-03356-x
  20. Nat Struct Mol Biol. 2024 Aug 20.
      Methyl-CpG-binding protein 2 (MeCP2) is an essential chromatin-binding protein whose mutations cause Rett syndrome (RTT), a severe neurological disorder that primarily affects young females. The canonical view of MeCP2 as a DNA methylation-dependent transcriptional repressor has proven insufficient to describe its dynamic interaction with chromatin and multifaceted roles in genome organization and gene expression. Here we used single-molecule correlative force and fluorescence microscopy to directly visualize the dynamics of wild-type and RTT-causing mutant MeCP2 on DNA. We discovered that MeCP2 exhibits distinct one-dimensional diffusion kinetics when bound to unmethylated versus CpG methylated DNA, enabling methylation-specific activities such as co-repressor recruitment. We further found that, on chromatinized DNA, MeCP2 preferentially localizes to nucleosomes and stabilizes them from mechanical perturbation. Our results reveal the multimodal behavior of MeCP2 on chromatin that underlies its DNA methylation- and nucleosome-dependent functions and provide a biophysical framework for dissecting the molecular pathology of RTT mutations.
    DOI:  https://doi.org/10.1038/s41594-024-01373-9
  21. Nucleic Acids Res. 2024 Aug 20. pii: gkae696. [Epub ahead of print]
      Enhancers, critical regulatory elements within the human genome, are often transcribed into enhancer RNAs. The dysregulation of enhancers leads to diseases collectively termed enhanceropathies. While it is known that enhancers play a role in diseases by regulating gene expression, the specific mechanisms by which individual enhancers cause diseases are not well understood. Studies of individual enhancers are needed to fill this gap. This study delves into the role of APOE-activating noncoding RNA, AANCR, in the central nervous system, elucidating its function as a genetic modifier in Alzheimer's Disease. We employed RNA interference, RNaseH-mediated degradation, and single-molecule RNA fluorescence in situ hybridization to demonstrate that mere transcription of AANCR is insufficient; rather, its transcripts are crucial for promoting APOE expression. Our findings revealed that AANCR is induced by ATM-mediated ERK phosphorylation and subsequent AP-1 transcription factor activation. Once activated, AANCR enhances APOE expression, which in turn imparts an inflammatory phenotype to astrocytes. These findings demonstrate that AANCR is a key enhancer RNA in some cell types within the nervous system, pivotal for regulating APOE expression and influencing inflammatory responses, underscoring its potential as a therapeutic target in neurodegenerative diseases.
    DOI:  https://doi.org/10.1093/nar/gkae696
  22. Proc Natl Acad Sci U S A. 2024 Aug 27. 121(35): e2401861121
      Insect developmental transitions are precisely coordinated by ecdysone and juvenile hormone (JH). We previously revealed that accumulated H3K27 trimethylation (H3K27me3) at the locus encoding JH signal transducer Hairy is involved in the larval-pupal transition in insects, but the underlying mechanism remains to be fully defined. Here, we show in Drosophila and Bombyx that Rpd3-mediated H3K27 deacetylation in the prothoracic gland during the last larval instar promotes ecdysone biosynthesis and the larval-pupal transition by enabling H3K27me3 accumulation at the Hairy locus to induce its transcriptional repression. Importantly, we find that the homeodomain transcription factor Schlank acts to switch active H3K27 acetylation (H3K27ac) to repressive H3K27me3 at the Hairy locus by directly binding to the Hairy promoter and then recruiting the histone deacetylase Rpd3 and the histone methyltransferase PRC2 component Su(z)12 through physical interactions. Moreover, Schlank inhibits Hairy transcription to facilitate the larval-pupal transition, and the Schlank signaling cascade is suppressed by JH but regulated in a positive feedback manner by ecdysone. Together, our data uncover that Schlank mediates epigenetic reprogramming of H3K27 modifications in hormone actions during insect developmental transition.
    Keywords:  H3K27 modifications; Schlank; developmental transition; epigenetic switch; hormone actions
    DOI:  https://doi.org/10.1073/pnas.2401861121
  23. Sci Adv. 2024 Aug 23. 10(34): eadq3087
      RNA polymerase IV (Pol IV) forms a complex with RNA-directed RNA polymerase 2 (RDR2) to produce double-stranded RNA (dsRNA) precursors essential for plant gene silencing. In the "backtracking-triggered RNA channeling" model, Pol IV backtracks and delivers its transcript's 3' terminus to RDR2, which synthesizes dsRNA. However, the mechanisms underlying Pol IV backtracking and RNA protection from cleavage are unclear. Here, we determined cryo-electron microscopy structures of Pol IV elongation complexes at four states of its nucleotide addition cycle (NAC): posttranslocation, guanosine triphosphate-bound, pretranslocation, and backtracked states. The structures reveal that Pol IV maintains an open DNA cleft and kinked bridge helix in all NAC states, loosely interacts with the nucleoside triphosphate substrate, and barely contacts proximal backtracked nucleotides. Biochemical data indicate that Pol IV is inefficient in forward translocation and RNA cleavage. These findings suggest that Pol IV transcription elongation is prone to backtracking and incapable of RNA hydrolysis, ensuring efficient dsRNA production by Pol IV-RDR2.
    DOI:  https://doi.org/10.1126/sciadv.adq3087
  24. Mol Cell. 2024 Aug 08. pii: S1097-2765(24)00620-8. [Epub ahead of print]
      The individualization of chromosomes during early mitosis and their clustering upon exit from cell division are two key transitions that ensure efficient segregation of eukaryotic chromosomes. Both processes are regulated by the surfactant-like protein Ki-67, but how Ki-67 achieves these diametric functions has remained unknown. Here, we report that Ki-67 radically switches from a chromosome repellent to a chromosome attractant during anaphase in human cells. We show that Ki-67 dephosphorylation during mitotic exit and the simultaneous exposure of a conserved basic patch induce the RNA-dependent formation of a liquid-like condensed phase on the chromosome surface. Experiments and coarse-grained simulations support a model in which the coalescence of chromosome surfaces, driven by co-condensation of Ki-67 and RNA, promotes clustering of chromosomes. Our study reveals how the switch of Ki-67 from a surfactant to a liquid-like condensed phase can generate mechanical forces during genome segregation that are required for re-establishing nuclear-cytoplasmic compartmentalization after mitosis.
    Keywords:  Ki-67; RNA; anaphase; capillary forces; chromosome; mitosis; phase separation; phosphorylation; surface condensation; surfactant
    DOI:  https://doi.org/10.1016/j.molcel.2024.07.022