Epigenetics & Chromatin

Enhancers are key epigenetic regulatory elements that orchestrate spatiotemporal gene expression and are critical in mammalian development, gene regulation, and disease. Histone modifications such as H3K4me1 (a canonical enhancer mark) and H3K27ac (which distinguishes active enhancers) remain poorly characterized during early mammalian embryogenesis. Using low-input CUT&RUN (Cleavage Under Targets and Release Using Nuclease) with input as low as 50 cells, this study profiles genome-wide H3K4me1 and H3K27ac patterns in mouse oocytes and pre-implantation embryos. Both marks are enriched in distal regions and exhibit distinct sequence preferences and reprogramming dynamics in pre-implantation embryos. H3K27ac is reprogrammed at the 2-cell stage and marks active enhancers, while H3K4me1 is remodeled at the 4-cell stage and co-localizes with H3K27ac, overlapping with accessible chromatin regions. Interestingly, the co-localization of H3K4me1 and H3K27ac is also detected in promoter regions, where they exhibit a mutually exclusive pattern with H3K4me3. Three enhancer types-active (H3K4me1/H3K27ac), primed (H3K4me1), and poised (H3K4me1/H3K27me3)-are dynamically remodeled during maternal-to-zygotic transition (MZT), with active enhancers increasing significantly after zygotic genome activation. Furthermore, genome-wide super-enhancers are identified and mainly enriched in promoters. The differences in gene expression at different stages may be related to the specific motifs enriched by super-enhancers.

DDI2 and DDI3 (DDI2/3) are duplicated genes in Saccharomyces cerevisiae that exhibit strong induction by a transcription factor Fzf1 in response to chemical treatments like cyanamide (CY) and methyl methanesulfonate (MMS). Although, like DDI2/3, SSU1, YHB1 and YNR064C also contain an Fzf1-binding consensus sequence CS2 and are coordinately regulated by Fzf1, these genes are only modestly induced by CY and MMS. To identify additional cis-acting elements in the DDI2/3 promoter, we made DDI2/3 promoter deletions in a reporter system and identified upstream repressing sequences (URS) spanning 480 nucleotides. To test a hypothesis that the chromatin structure constitutes the URS, we utilized a yeast strain capable of histone H3/H4 depletion by shifting carbon sources. Following histone depletion, DDI2/3 were strongly induced in an Fzf1 dependent manner, while YHB1 was repressed. Interestingly, under histone depletion conditions, CY or MMS treatment further increased expression of all Fzf1-regulated genes to comparable levels in an Fzf1 dependent manner. A genome-wide MNase-seq analysis showed that CY treatment reduced the nucleosome occupancy at the mapped DDI2/3 URS region in wild-type cells, but not in in fzf1{Delta} cells. These findings collectively indicate that Fzf1 plays dual roles in regulating the DDI2/3 response to CY. Firstly, it binds CS2 and serves as a transcription activator. Secondly, it is required for the chromatin remodeling at URS. This two-tier regulation at the DDI2/3 promoter helps to explain why DDI2/3 achieve much higher fold induction by CY and MMS than other Fzf1-regulated genes, suggesting Fzf1 to be a candidate pioneer transcription factor.

The eukaryotic ribosomal genes are multi-copy genes, transcribed from the rDNA, and approximately one third of them is actively transcribed in differentiated cells. A number of lncRNAs have been identified from the intergenic spacer between the rRNA genes, among those the spacer RNA and PAPAS that are involved silencing of rRNA gene copies by altering the chromatin configuration. Here, we have identified lncRNAs that are transcribed from the human rDNA loci and modulate the loci; IGS38 positively regulates rRNA gene transcription by associating to the 47S rRNA gene promoter and modulating the rRNA promoter accessibility while IGS32as associates with heterochromatin. IGS38 binds to the 47S gene promoter through the RNA pol I factors TAF1C and RRN3 as well as the Williams Syndrome Transcription Factor (WSTF), a component of the B-WICH chromatin remodelling complex. The increased accessibility of the promoter stabilises the architectural protein Upstream Binding Factor (UBF) at the rRNA promoter, thereby facilitating RNA pol I promoter escape. Furthermore, IGS38 knock down displays and increased dsRNA abundance in the cytoplasm with a weak induction of the dsRNA sensor OAS2, typically induced by interferon and viral dsRNA. Overall, the both IGS38 and IGS32as are chromatin associated lncRNAs involved in rDNA chromatin changes, and IGS38 is stimulating, together with WSTF, rRNA gene transcription in human cells. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/722362v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@14d4159org.highwire.dtl.DTLVardef@fd773forg.highwire.dtl.DTLVardef@a0030dorg.highwire.dtl.DTLVardef@1285301_HPS_FORMAT_FIGEXP M_FIG C_FIG IGS stabilises 47S rRNA transcription, disruption of IGS38 expression leads to the release of dsRNA in the cytoplasm and a weak immune activation of OAS2. Created by biorender (https://biorender.com/shortURL)

Histone lactylation is a recently identified histone post-translational modification (PTM) that links energy metabolism to chromatin regulation. Although histone lactylation has been implicated in transcriptional activation, its function in meiotic chromatin remains unclear. Previously, we identified enrichment of multiple histone lactylation marks within the meiotic karyosome, a highly condensed and transcriptionally repressive chromatin structure formed in Drosophila oocytes. Here, through an RNAi-based screen, we identified the CBP family protein dCBP as a regulator of histone lactylation in the karyosome. Germline-specific knockdown of dCBP preferentially reduced histone lactylation, including H4K8 lactylation, and caused premature disruption of the synaptonemal complex, abnormal egg chamber development with excess nurse cells, reduced egg production, and decreased embryonic viability. Corresponding histone acetylation marks were comparatively less affected than histone lactylation by dCBP knockdown. Together, our findings provide evidence that dCBP-mediated histone lactylation contributes to meiotic chromosome maintenance and suggest a potential link between energy metabolism and meiotic chromatin regulation.

Selective deployment of multiple transcription start sites is a major regulatory feature of human transcriptomes. FANTOM CAGE data exhibit a near-universal TSS deployment parsimony which is disrupted in cancers. We have recently shown that TSS deployment is sensitive to gene function, futile upstream transcription, and cellular biosynthetic states. Patterns in FANTOM CAGE data can reveal mechanisms underlying TSS co-deployments. We propose and test the possibility that some TSSs act like epromoters and act as co-varying hubs of transcriptional activities for multiple other promoters. Using deep analysis of CAGE data implemented through neural networks we show that non-cancers implement transcription co-deployments through cores of epromoter-like TSSs which are generally proximal to their start codons. These TSSs show enhancer-like TFBSs profiles. A comparison with cancer CAGE data shows that the concentrated epromoter core is disrupted in cancers with multiple distal TSSs replacing the proximal TSS cores. We provide evidence that the core TSSs are rich in YY1 and CTCF binding sites and associated with genes coding for transcription factors. Our findings show that covariance of TSS deployment is sensitive to transcriptional resource cost and a parsimonic design of TSS co-deployments depends on proximal TSSs in non-cancers, a mechanism grossly disrupted in cancers. HighlightsO_LIHeterogeneous FANTOM CAGE data contains universal patterns of TSSs co-deployments. C_LIO_LITSS co-deployments exhibit a parsimonious "core-covariant" scheme which is disrupted in cancers. C_LIO_LICore TSSs are enriched in transcription factor binding sites and gene functions which justify biological features of the samples. C_LIO_LIThe DL pipeline we present identifies the core-covariant TSS sets in an unbiased manner. C_LI

Understanding cis-regulatory elements (CREs) at the single cell level is fundamental to deciphering transcriptional changes during development, cell differentiation, and homeostasis. Recent studies have shown that arbitrary peak-calling thresholds complicate data interpretation and cross-study comparisons. Furthermore, due to the inherent sparsity of single-nuclei ATAC-seq (snATAC-seq) data, distinguishing between truly inaccessible regions and technical dropouts remains challenging. Our analysis of snATAC-seq experiments performed in a well-established cell line suggests that the dichotomy between accessible (open) or inaccessible (close) CREs is misleading. Thousands of accessible regions are present in a very small fraction of cells of the population but they are repeatedly identified, suggesting that they have a low accessibility or are only transiently accessible. However, depending on the detection threshold selected they could be considered as either genuine CREs or noise. To resolve this inconsistency, we propose a model where chromatin accessibility is treated as a continuum, defined by a probability of accessibility (Pa) for each accessible region across cell types and conditions. Through computational simulations, we demonstrate that snATAC-seq results can be explained by a simple "balls into bins" probability model, offering a theoretical framework for calculating Pa distributions from any snATAC-seq dataset. Furthermore, we examine how Pa distributions shift following activation of the TGF{beta} signaling pathway. This probabilistic approach removes the reliance on arbitrary thresholds and supports a more quantitative, and dynamic understanding of accessible regions function.

Vitamin D signaling has recognized roles in female reproductive physiology, but its effects at the chromatin level in endometrial stromal cells are still unclear. Here, we investigated how the active form of vitamin D, 1,25-dihydroxyvitamin D3, or calcitriol, influences the accessible chromatin landscape of human endometrial stromal cells. Assay for transposase-accessible chromatin using sequencing (ATAC-seq) was performed on T-HESCs treated with either a vehicle or 1,25(OH)2D3. Ligand treatment increased overall chromatin accessibility, shown by higher ATAC-seq signal intensity, while causing only minor changes in the total number of called peaks. Peak annotation revealed that accessible regions were spread across both promoter-proximal and distal genomic areas. Integrating this data with CUT&RUN and RNA sequencing showed that most vitamin D-responsive cistromic modifications and transcripts were linked to nearby open chromatin, though fewer were associated with regions that were significantly differentially accessible. These results suggest that 1,25(OH)2D3-dependent transcription mainly occurs within a permissive, pre-accessible chromatin environment. This study offers new evidence that active vitamin D influences the epigenomic landscape of human endometrial stromal cells, establishing the chromatin-based molecular response to a chemically-defined VDR ligand, 1,25(OH)2D3, relevant to stromal differentiation and preparation for decidualization. HighlightsO_LIFirst evidence suggesting the direct impact of active vitamin D, 1,25-dihydroxyvitamin D3, 1,25(OH)2D3, enhanced the signal intensity of chromatin accessibility in human endometrial stromal cells C_LIO_LIMost accessible chromatin regions were shared between vehicle and ligand-treated human endometrial stromal cells C_LIO_LI1,25(OH)2D3-responsive transcription occurs largely within pre-accessible chromatin in human endometrial stromal cells C_LIO_LIAssay for transposase-accessible chromatin sequencing (ATAC-seq) defines a chromatin-level pharmacologic response to a chemically defined VDR ligand in human endometrial stromal cells C_LI

MotivationTranscription factors (TFs) regulate gene expression by binding specific DNA sequences, which are commonly represented by motif models. Although position weight matrices (PWMs) remain the dominant motif representation, alternative models, such as Markov models, can capture interpositional dependencies and may provide higher predictive performance. However, existing motif comparison tools are designed mainly for PWMs or require motifs to be reduced to PWM/PPM representations. This creates a major bottleneck for comparing motifs represented by different model architectures. This limitation complicates the interpretation of de novo motif discovery results and hinders the systematic integration of diverse motif models into genomic analyses. ResultsWe present MIMOSA (Model-Independent Motif Similarity Assessment), a model-independent framework for direct comparison of TF binding site (TFBS) motifs regardless of their mathematical representation. MIMOSA assesses motif similarity by comparing calibrated recognition profiles produced by motifs of different models on the same DNA sequence set, rather than by comparing the motifs themselves. In a cross-database benchmark on HOCOMOCO motifs, MIMOSA achieved retrieval performance comparable to established PWM-oriented tools, including Tomtom and MACRO-APE, with MRR and Recall@k close to the best-performing methods. Pairwise ranking comparisons showed that MIMOSA captures a similarity signal consistent with existing approaches while providing a representation-independent comparison strategy. Application to de novo motifs derived from ChIP-seq data for the ATF3 TF demonstrated that recognition-profile comparison distinguished alternative spacer variants represented as separate PWMs from their integration within more flexible models such as BaMM and Slim. Thus, MIMOSA enables quantitative cross-model motif comparison and supports interpretation of motif heterogeneity in TFBS analyses. Availability and implementationMIMOSA is implemented in Python and is freely available at https://github.com/ubercomrade/mimosa.

The slowing of executive function and memory impairment are the leading hallmarks of cognitive decline with age. The exact cause of this change is unknown and is the focus of aging research. Expression levels of Vesicle Associated Membrane Protein 2 (VAMP2)/Synaptobrevin-2 (syb2) are decreased with age. Here we report results from a novel transgenic mouse model (TgV2) that overexpresses syb2. We hypothesized that overexpression of syb2 improves synaptic function during aging, thus it delays dementia. Aged TgV2 mice, which maintained syb2 levels, performed better in spatial memory tests than 2-year-old WT control mice, which had lost half of syb2 due aging. In hippocampal CA1 synapses of aged TgV2 mice, long-term potentiation was increased. These effects of maintained syb2 levels were beneficial for both males and females providing improved synaptic plasticity. These results indicate that overexpression of syb2 supports cognitive function throughout the aging process and better resist age-related synaptic dysfunction.

The relationship between age-related genetic factors and health conditions has become a pivotal focus in aging research, particularly as the World Health Organization (WHO) delineates the leading global causes of mortality. However, the direct impact of age-related genes on the leading cause of death remains poorly understood. To investigate this gene-aging relationship, we analyzed protein-protein interactions using gene set enrichment analysis (GSEA) of a set of 307 age-related genes previously curated. The results indicated significant associations with 113 diverse disease categories, while adhering to a stringent false discovery rate (FDR) threshold of less than 1 x 10-5. Due to the difficulties in aligning the disease categories with WHOs leading causes of death, we reclassified the WHO categories using the more precise nomenclature specified in the 11th Revision of the International Classification of Diseases (ICD-11). The age-related genes account for the leading causes of death, with the exceptions being two infectious communicable diseases, tuberculosis and COVID-19. They impact the cardiovascular system, brain, lungs, and the whole body, while this study could not identify death by aging, which is not a well-defined medical cause of death. Furthermore, we identified a set of 15 recurring genes shared among multiple diseases, including TNF, AKT1, IL6, CDK2A, APOE, and TP53. This gene set was enriched for several disease categories, including cancer, inflammatory diseases, metabolic disorders, and neurodegenerative diseases. Additionally, it shows significant enrichment in various biological categories, with the regulation of nitric oxide activity being the most prominent; other enriched categories include the regulation of microRNA, lipid and carbohydrate metabolism, smooth muscle cell proliferation, insulin signaling, and phosphatidylinositol-3 kinase (PI3K) signaling. The findings suggest that the recurring genes act as pleiotropic hubs, influencing multiple leading causes of death, while other genes are more specific to each disease category.

Bacteria require rapid adaptation under fluctuating environmental conditions. Commonly recognized global regulators enable bacteria to respond promptly to external changes, though they are either restricted to specific bacterial taxonomies or physiological statuses, suggesting that additional regulators are required for adaptation. DNA methylation is a reversible modification affecting bacterial gene regulation. However, conventional methods can only detect one DNA methylation form each round, leaving the understanding of DNA methylation in bacterial adaptation mostly unknown. This study aimed to identify genome-wide DNA methylation variation (N6-methyladenine, N4-methylcytosine, and 5-methylcytosine) in Escherichia coli under different culture conditions using Oxford Nanopore sequencing. DNA samples from six conditions (normal, low oxygen, low pH, high temperature, high salt, and recovery after low pH exposure) during the exponential and stationary phases were extracted. When culture conditions were compared to the normal condition, E. coli exhibited more differentially methylated sites during the exponential phase than in the stationary phase. During the exponential phase, the genes differentially methylated in all conditions were involved in cellular activities, such as cellular and metabolic processes. During the stationary phase, universally differentially methylated genes were associated with oxidation responses. Subsequent analysis found that although DNA methylation analysis was affected by batch effects, some genes (e.g. rpoS) showed consistently differential methylation across datasets. Our findings suggest that the E. coli DNA methylation profile was affected by growth phases and conditions, and DNA methylation profiling by Oxford Nanopore sequencing could be a potential approach for gene activity estimation in environmental samples. ImportanceBacterial DNA methylation is a reversible genetic modification affecting gene regulation, enabling rapid adaptation. Three major forms in bacteria are N6-methyladenine, N4-methylcytosine, and 5-methylcytosine. Using Oxford Nanopore sequencing, we characterized genome-wide variation in these methylation types in Escherichia coli under six conditions (normal, low oxygen, low pH, high temperature, high salt, and recovery after low pH exposure). DNA methylation signatures in E. coli varied with growth conditions. Using the normal condition as a baseline, E. coli during the exponential phase exhibited more differentially methylated genomic loci under stress conditions compared to the stationary phase. Under stress conditions, genes with differential methylation were associated with cellular processes or oxidative responses, depending on the growth phase. Our findings reveal that the DNA methylation signature in E. coli was affected by growth phases and conditions, and Oxford Nanopore-based DNA methylation profiling could be a potential approach for gene activity estimation in environmental samples.

BackgroundHemoglobin A1c (HbA1c), an important diagnostic biomarker for type 2 diabetes (T2D), is also associated with aging, cognitive performance, and mortality. To identify epistatic interactions, we assessed 133 known gene variants associated with HbA1c among 3,778 non-diabetic subjects of European ancestry in the Long Life Family Study (LLFS). MethodsWe applied Bayesian Imputation Based Association Mapping (BIMBAM) to identify significant pairwise epistatic interactions among genetic variants that were previously shown to be associated with levels of HbA1c. To take into account confounding effects, we adjusted age, sex, field centers, body mass index (BMI), and genetic principal components (PCs). ResultsThis analysis yielded seven pairs with log10(BF)>10; of those, six pairs were confirmed using a full-term mixed regression model. Specifically, these included significant interactions of HK1-rs17476364 with variants in GCK (rs2971670, rs4607517) or G6PC2 (rs560887), as well as between HK1-rs16926246 and the same variants (P values for each term [&le;] 7.14x10-3). All epistatic interactions between HK1 and GCK, and between HK1 and G6PC2 were replicated in two large independent studies (namely, Framingham Offspring Study, P < 0.05; Health and Retirement Study, P < 0.05). ConclusionThe present study revealed that HK1 and GCK interact to contribute to regulating levels of HbA1c and are likely to be involved in molecular mechanisms underlying healthy aging processes.

Central metabolites function as essential co-substrates for chromatin-modifying enzymes, directly linking cellular metabolism to chromatin regulation. Accordingly, whole-cell fluctuations in co-substrate availabilities have been shown to promote diverse phenotypes through chromatin-dependent mechanisms. There is emerging evidence that metabolic enzymes producing co-substrates for chromatin modifying enzymes can exist in the nucleus, suggesting that nucleus-specific metabolite availability regulates chromatin state. Here, we developed CRISPRm (CRISPR metabolite) to assess how nucleus-specific metabolic perturbations influence chromatin function. Five dCas9-metabolic enzyme fusions (i.e., dCas9-ACSS2, -NMNAT1, -MAT2A, -GDH, and -AHCY) were used to modulate nuclear levels of essential co-substrates involved in histone (de)acetylation and (de)methylation reactions. Transient expression of all dCas9 fusions in HEK293T cells induced distinct global changes in gene expression patterns, with dCas9-ACSS2 (acetyl-CoA producing) and NMNAT1 (NAD+ producing) eliciting large opposing changes in gene expression, suggesting transcriptional responses to nuclear acetyl-CoA and NAD+ production may be directly facilitated by acetylation or deacetylation reactions, respectively. Targeting dCas9-ACSS2 and -NMNAT1 to promoters of select candidate genes revealed enhanced transcriptional modulation. dCas9-ACSS2 upregulated, and dCas9-NMNAT1 downregulated genes showed basal enrichment of H3K9ac, H3K18ac, H3K27ac, H3K4me3, and p300, suggesting these genomic loci reside within epigenetic environments susceptible to fluctuations in acetyl-CoA and NAD+ availability. Of significant genes altered, dCas9-MAT2A (SAM producing) increased expression of 72% whereas dCAS9-GDH (alpha-ketoglutarate producing) decreased expression of 79%. Surprisingly, dCAS9-AHCY (SAH hydrolysis) led to down-regulation of shared genes up-regulated by dCas9-MAT2A. The observations amongst the methylation-specific enzymes revealed unexpected and unique gene-regulatory sensitivities to SAM, SAH and alpha-ketoglutarate. Together, these results demonstrate the utility of CRISPRm in studying nuclear metabolic regulation of transcription and provide strong evidence that perturbations in nuclear co-substrates do not lead to a large mass- action changes in chromatin acetylation/methylation but rather to modulation of select chromatin-modifying enzymes with targeted transcription responses. HighlightsO_LICRISPRm is a novel, modular dCas9-effector platform that enables interrogation of the metabolism-epigenome axis C_LIO_LIdCas9-ACSS2, -NMNAT1, -MAT2A, -AHCY, and -GDH induce distinct transcriptional programs. C_LIO_LITargeting CRISPRm to promoters enhances transcriptional responses. C_LIO_LIdCas9-ACSS2 and -NMNAT1 sensitive gene promoters exhibit unique enrichment of chromatin features, including H3K9ac, H3K18ac, H3K27ac, H3K4me3 and p300. C_LI

Cartilage and bone that comprise craniofacial structures as well as neurons and glia of the peripheral nervous system are derived from a multipotent population of cranial neural crest cells, that respond to both cell intrinsic and extrinsic cues to differentiate into precise cell states. Both a genetic and epigenetic regulatory network are required for each step in the differentiation process, involving transcription factors, histone modifiers and chromatin remodelers. Here, we examined the direct transcriptional targets of two histone methyltransferases, Prdm3 and Prdm16 in zebrafish neural crest cells at 48 hours post fertilization in zebrafish. Using CUT&RUN, we examined both direct DNA binding and nucleosome association. At this stage of development, CUT&RUN fragment size analysis indicated that Prdm3 and Prdm16 are largely associated with nucleosomes. We further analyzed these nucleosome peak sets to identify 6 clusters where differential binding of Prdm3 and Prdm16 and differential enrichment of gene ontology terms for target genes was observed. We validated gene expression in each cluster by in situ hybridization chain reaction (HCR) at 48 hpf demonstrating that prdm3 and prdm16 mutants exhibit corresponding changes in gene expression of the putative gene targets identified. Finally, we performed CUT&RUN-qPCR in prdm3 and prdm16 mutant zebrafish embryos and demonstrated reduced binding at putative target loci. Together these data suggest that Prdm3 and Prdm16 regulate their transcriptional targets primarily by binding nucleosomes around their putative target loci to control downstream gene expression. HighlightsPrdm3 and Prdm16 associate with nucleosomes for regulation of gene expression Gene targets are altered in prdm3 and prdm16 mutant zebrafish Reduced binding is observed in respective mutants

SummaryDNA methylation datasets from public repositories such as NCBI Gene Expression Omnibus are central to the development and evaluation of epigenetic aging clocks, yet existing resources and tools do not fully resolve the bottlenecks of dataset retrieval and metadata harmonization. Current benchmarking frameworks often rely on static curated collections, support only a subset of available Gene Expression Omnibus studies, focus on specific tissues, or require substantial manual intervention when metadata fields and supplementary files are inconsistently structured across studies. We developed MethylCurate, an agentic AI framework that addresses these limitations by automating the retrieval of DNA methylation datasets from the Gene Expression Omnibus, harmonizing heterogeneous metadata, mapping datasets to a unified format, and enabling scalable evaluation of epigenetic aging clocks through an integrated, dialogue-driven workflow. Availability and ImplementationMethylCurate is implemented in Python and combines deterministic modules for Gene Expression Omnibus dataset retrieval, quality control, and clock evaluation with large language model-assisted agents for metadata extraction, metadata harmonization, and DNA methylation data parsing. Source code, documentation, and example workflows are available at: https://github.com/Travyse/methylcurate Contacttravyse.edwards@pennmedicine.upenn.edu Supplementary InformationSupplementary data are available at Bioinformatics online. Graphical AbstractMethylCurate is an agentic-AI framework that converts user-specified NCBI Gene Expression Omnibus DNA methylation datasets into standardized metadata, beta matrices, artifacts, logs, and aging clock benchmarking outputs through automated retrieval, quality control, metadata extraction, harmonization, and evaluation workflows. Figure generated with Biorender. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=51 SRC="FIGDIR/small/723515v1_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@197c0fborg.highwire.dtl.DTLVardef@1feace4org.highwire.dtl.DTLVardef@108b0d5org.highwire.dtl.DTLVardef@191a1b8_HPS_FORMAT_FIGEXP M_FIG C_FIG Key MessagesO_LIAutomated curation of DNA methylation datasets from the Gene Expression Omnibus. C_LIO_LIStandardized preprocessing and metadata harmonization. C_LIO_LIIntegrated benchmarking of epigenetic aging clocks. C_LI

Gene-by-environment (GxE) interactions play a major role in shaping both phenotypic and molecular variation, with important implications for human health and disease. In this study, we used the Doxycycline (Dox) regulated, tetracycline-responsive (Tet-Off) promoter system to sequentially reduce or titrate gene expression levels of the essential yeast transcription factor Repressor Activator Protein 1 (RAP1) similar to a hypomorph allele series, across three distinct environments: Yeast Peptone Dextrose (YPD) media, YPD media with Heat Shock (HS), and Yeast Peptone Acetate (YPAC) media. We then performed RNA sequencing (RNA Seq) to assess global transcriptional responses to RAP1 reduction in these different growth environments. Our analysis first focused on the independent effects of varying RAP1 expression levels within and across environments. We then explored GxE interactions, revealing a subset of genes with significant consequences of reduced levels of RAP1 and environment-specific expression patterns. Notably, many genes exhibited opposite effects of RAP1 titration on gene expression when yeast were grown in YPAC media compared to YPD media and/or HS, suggesting environment-dependent regulatory architecture. This design reveals how cells integrate internal transcriptional and regulatory changes with external environmental cues, providing a deeper view of GxE architecture. Using Weighted Gene Co-expression Network Analysis (WGCNA), we identified co-regulated gene modules, and by combining this with transcription factor motif enrichment tests, our study identified candidate regulators driving their dynamics. Our findings demonstrate that gene regulatory networks can vary dramatically depending on the environmental context an organism experiences, which can then influence the specific phenotypes produced by a particular genetic perturbation. This illustrates the complexity of genotype-environment interactions and the importance of studying gene function in multiple environments to gain a truly comprehensive understanding of a genes sometimes numerous and diverse functions.

Citrullination is a charge-modifying post-translational modification whereby proteinogenic arginine is converted to the non-coded amino acid citrulline by calcium-activated protein arginine deiminases (PADs; EC 3.5.3.15). The five known PAD enzymes in humans (PADs 1, 2, 3, 4, and 6) are differentially expressed and have distinct targets, including histones. While some PAD histone citrullination sites are known, a comprehensive investigation of all histone tail arginines targeted by catalytically active PADs 1-4 is lacking. Here, we sought to identify PAD citrullination sites in histone tails, both within histone peptides and in reconstituted nucleosomes. Toward this objective, we utilized a real-time 1H-15N NMR spectroscopy-based assay. By monitoring both arginine and citrulline backbone amide peak intensities over time, we identified sites of citrullination in 15N-labeled histone tails within peptides and reconstituted nucleosome core particles. We found that PADs 1, 2, and 4 citrullinate all directly observable histone tail arginines to varying degrees. This is distinct from PAD3, which only moderately citrullinates H2A and H4 arginine residues and does not modify H3 tail arginines. Together, these data suggest a level of histone arginine specificity by each PAD. Furthermore, histone tail citrullination is altered within nucleosomes compared to isolated peptides, which we interpret to reflect changes in conformation and accessibility. We speculate that citrullination increases nucleosomal histone tail dynamics, with implications for crosstalk between sites of histone citrullination and other important sites of regulation by PTMs (including lysines) within and between tails.

Abstract/SummaryGross chromosomal rearrangements are a hallmark of many diseases and cancers. The study of their biogenesis and the mechanisms underlying their formation is greatly facilitated by the availability of genetic reporter assays in model organisms. We present here a novel GCR assay developed in fission yeast, a highly relevant model for understanding genome instability related to human biology. The reporter employs canavanine counter-selection to detect GCRs within a chromosomal context. Using this assay, we identified natural hotspots for GCRs, including inverted long terminal repeats (IR-LTRs). Structural analysis of GCR events showed that IR-LTR-induced GCRs mainly result in either terminal deletions with adjacent inverted duplications or repair via long-range break-induced replication (BIR). Deleting IR-LTRs reduces the GCR rate and reveals another hotspot driven by BIR between homeologous aldo/keto reductase genes on opposite arms of chromosome I. This is the first evidence that BIR can occur in S. pombe on long tracks reaching up to 600 kb. Besides highlighting genome rearrangement hotspots, the assay also identifies regulators of genome instability in fission yeast. Loss of Nup132, a component of the nuclear pore complex, increases IR-LTRs-induced GCRs, while the budding yeast homolog Nup133 has no effect on the stability of a structurally similar IR. In contrast, disrupting djc9, which encodes a conserved histone H3-H4 binding protein, decreases GCR rates. Overall, this sensitive GCR assay enables the identification of factors that control spontaneous and fragile motif-induced chromosomal instability, including those conserved in humans but lost through evolution in other organisms.

BackgroundGenomic stability is maintained through the coordinated regulation of DNA repair, dNTP pool balance, and histone dynamics--the three pillars of the DNA damage response. Because histones constitute the fundamental 3D physical scaffold of the genome, their precise regulation is essential for the spatial organization that dictates environmental fitness. In the radiotolerant pathogen Cryptococcus neoformans, the Rad53-Bdr1 pathway is a central DDR mediator; however, the mechanisms linking this checkpoint to histone dynamics remain poorly understood. Because conventional one-dimensional analyses cannot capture how spatial chromatin folding shapes transcriptional reprogramming, we integrated high-throughput chromosome conformation capture (Hi-C) with transcriptomic profiling to address this gap. ResultsWe demonstrate that HTA1 and HTB1, encoding H2A and H2B, are essential for viability, whereas H3 and H4 paralogs exhibit functional redundancy. Although most core histones are regulated by Rad53, HHT1 and variant HTZ1 are expressed independently of the Rad53. Notably, loss of the H3 paralog HHT2 induces growth defects under diverse stress conditions. Integrated RNA sequencing and Hi-C analyses reveal that HHT2 deletion drives transcriptional reprogramming of stress-responsive genes, coinciding with large-scale chromatin rearrangements such as A/B compartment switching and topologically associating domain boundary shifts. Furthermore, HHT2 loss impairs virulence factor formation and attenuates virulence. ConclusionOur findings identify core histones as essential architects of the 3D genome in C. neoformans. By establishing a causal link between chromatin structural collapse and transcriptional reprogramming, this study highlights 3D genome architecture as a decisive physical switch linking nucleosome-level dynamics to global transcriptional programs required for environmental survival.

Polycomb Repressive Complex 2 (PRC2) maintains repression of genes specific for other cell differentiation states. PRC2 binds RNA in vitro with a preference for G-rich sequences. UV-based crosslinking coupled with immunoprecipitation (CLIP) experiments have shown that PRC2 also binds RNA in cells. Recently, Guo et al reported that a stringent denaturing variant of CLIP called CLAP did not detect PRC2 RNA binding in cells. We present a reanalysis of CLAP data that supports direct interaction of PRC2 with RNA in cells. CLAP for Halo-tagged PRC2 subunits from mixed populations of human and mouse cells specifically enriched for RNA from the species in which the proteins were tagged. The lack of apparent PRC2 RNA binding in Guo and colleagues analysis stems from a scaling step that deflates enrichment scores for low-complexity CLAP samples. Our findings pave the way for studies seeking to determine the physiological roles of PRC2 RNA binding activity.