Epigenetics & Chromatin

BackgroundDynamic chromatin behavior, which is related to chromatin accessibility, plays a critical role in various genome DNA functions such as RNA transcription and DNA replication/repair. Previous studies using highly synchronized cells showed that average local chromatin motion, captured by single-nucleosome imaging and tracking on a second time scale, remained almost constant throughout G1, S, and G2 phases in living human cells, although possible effects of prolonged drug treatments for cell-cycle synchronization could not be excluded. ResultsTo avoid possible effects of prolonged drug treatment, we combined single-nucleosome imaging with Fucci probes to visualize cell-cycle progression through G1, S, and G2. Using HeLa and HCT116 cells expressing H2B-HaloTag and Fucci probes, we found that local nucleosome motion remained similar on average throughout interphase, except for elevated motion in early G1. Transcription inhibition similarly increased nucleosome motion throughout interphase. Local nucleosome motion also increased following replication stress or DNA damage. ConclusionOur findings suggest that near-constant chromatin motion supports housekeeping functions under similar physical conditions during interphase. Our findings also suggest that cells can transiently change chromatin motion to perform ad hoc tasks in response to signals from inside and outside the cell, such as DNA damage.

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.

BACKGROUNDDuring spermiogenesis, histones are exchanged by protamines (PRMs) in spermatids, which results in DNA hypercondensation and protection. Rodents and primates express two PRMs (PRM1 and PRM2) in a species-specific ratio. Maintaining this ratio is necessary for functional chromatin reorganization and alteration is associated with sub- or infertility in mice and humans. Prm1 and Prm2 deficient mice are infertile, while Prm1+/- males are subfertile showing a severely altered PRM ratio. Prm2+/- males are fertile and display a protamine ratio comparable to WT. OBJECTIVESHere, we addressed the question whether loss of one allele of Prm1 and one allele of Prm2 affects fertility. MATERIAL AND METHODSDouble heterozygous (dHET) mice lacking one allele of Prm1 and one allele of Prm2 were generated and analyzed RESULTSdHET males were infertile with sperm showing retention of histones and TNPs, high levels of PRM2 precursor and decreased levels of mature PRM2. In mature sperm the PRM ratio and the total PRM content was not altered. However, CMA3 staining revealed incomplete protamination and sperm nuclei appeared more rounded and slightly bigger, suggesting impaired DNA-hypercondensation. In dHET sperm, DNA degradation was apparent, but to a lower level compared to sperm from Prm1 and Prm2 deficient males. Increased 8-OHdG levels suggested oxidative stress in the epididymis of dHET mice. However, a fraction of dHET sperm were capable of fertilization, with embryonic development up to 8-cell stage. DISCUSSION AND CONCLUSIONThese results suggest, that male factor infertility might not be reliably detected by measuring PRM1/PRM2 ratio but rather by determining the level of protamination by e.g. CMA3 analysis and pre-PRM2 retention.

Histone lactylation is a recently identified modification linked to metabolism. Genome-wide profiling has suggested that H3K18la marks tissue/cell type-specific enhancers in combination with H3K27ac; however, it remains unclear whether and how these modifications differ. Through a systematic comparison of histone mark distributions, we showed that H3K18la marks active enhancers irrespective of H3K27ac. Our analysis revealed that H3K18la predominantly localizes at non-promoter regions, and exhibits strong association with lineage-specific transcription factor binding and p300/CBP dependency. In contrast, H3K27ac-marked regions were more enriched in promoters and less sensitive to p300/CBP inhibition. Notably, massively parallel reporter assay data suggest that H3K18la-marked regions exert enhancer functions even without H3K27ac co-occupancy. Although the characteristics of H3K18la are similar to those of N-terminal acetylation of H2B (H2BNTac), a recently described enhancer signature, we found distinct genomic distributions of these marks. Collectively, our findings will advance the current understanding of how histone modifications establish cis-element landscapes.

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.

Nuclear blebs are herniations of the nucleus that occur in many human conditions including aging, heart disease, muscular dystrophy, and many cancers. Nuclear blebbing causes nuclear rupture and cellular dysfunction. However, understanding the formation, stability, and identification of nuclear blebs remains an ongoing challenge. Our previous studies reveal that nuclear blebs are best hallmarked by decreased DNA density. To determine if chromatin decompaction underlies decreased DNA density in nuclear blebs, we investigated the histone composition of nuclear blebs across multiple cell lines. Time lapse and immunofluorescence imaging revealed that global histone H2B and H3 levels are decreased in the nuclear bleb relative to the nuclear body. Next, we imaged histone modification states of euchromatin and heterochromatin, which respectively track decompact and compact states of chromatin. Overall, we find that nuclear blebs display variable histone modification state across cell lines, as euchromatin does not consistently enrich nor is heterochromatin consistently depleted. Nuclear blebs did consistently show active RNA Pol II initiation is enriched relative to elongation. Thus, we find that the local histone modification state is not an essential component of nuclear blebs while transcription initiation enrichment over elongation is reproducible across cell lines and conditions. Summary statementWe measured histones and their modification states in nuclear blebs. We find that chromatin state is variable while transcription initiation is consistently enriched relative to elongation in nuclear blebs.

4C-seq is a cost-effective 3C-based assay that measures the interactions between a single genomic element and all other genomic elements. However, 4C-seq data remains semi-quantitative because it cannot be deduplicated without UMIs. To address this, we developed an open source method, FourC, based on a Bayesian Bernoulli regression model, that overcomes the duplication problem and models spatial patterns with Gaussian processes to identify significantly enriched and differential contacts. We demonstrate the utility of FourC on 4C-seq data that profiles the local chromatin structure at key genes necessary for pancreatic differentiation and under CRISPR perturbation of enhancers.

Accurate chromosome segregation relies on proper centromere and kinetochore formation and phospho-regulation. We previously demonstrated that a pluripotent state confers a low fidelity of chromosome segregation, however it is unknown how a pluripotent state impacts centromere and kinetochore function. Here, we demonstrate that both centromere and kinetochore structural organization and phosphorylation in mitosis are developmentally regulated. CENP-A, CENP-C, and HEC1 protein abundance is reduced at mitotic centromeres and kinetochores of human pluripotent stem cells (hPSCs) compared to isogenic somatic cells; however, elevating their levels does not improve chromosome segregation fidelity. Rather, we find that reduced phosphorylation of kinetochores is responsible for their low fidelity. HEC1 is hypophosphorylated at kinetochores of hPSCs compared to isogenic somatic cells at Cyclin B/Cdk1 and Aurora kinase phospho-sites. Inhibiting PP2A phosphatase activity or differentiation increases HEC1 phosphorylation at hPSC kinetochores decreasing chromosome segregation errors. Thus, mitotic fidelity in non-transformed human cells depends on the developmental regulation of the kinase and phosphatase networks controlling kinetochore phosphorylation. SummaryGalaviz Sarmiento et al show that the developmental regulation of kinetochore phosphorylation governs mitotic fidelity. HEC1 is hypophosphorylated at kinetochores of hPSCs during mitosis contributing to their high rate of chromosome segregation errors. While differentiation increases HEC1 phosphorylation improving chromosome segregation fidelity.

Common sex chromosome aneuploidies (SCAs) often present with cognitive and cardiovascular dysfunction in humans. To address SCA effects on gene expression and DNA methylation in relevant cell types, we differentiated neural precursor cells (NPCs) and cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) with different numbers of sex chromosomes, including isogenic and independent lines. As expected, the expression of genes that escape X inactivation (escapees) mostly increases with the number of inactive X chromosomes (Xi). However, allelic analysis shows dampening of escapees specifically on the Xi in XXY compared to XX in both NPCs and CMs, revealing a novel type of dosage compensation in SCA. In contrast, Y-linked gene expression is higher in XXY versus XY NPCs, but the opposite is observed in CMs. This may explain the greater number of differentially expressed autosomal genes in NPCs versus CMs with an added Y chromosome, while effects of added X chromosomes are similar between cell types. Concordantly, changes in autosomal DNA methylation are mainly driven by the presence of a Y chromosome. These findings highlight the cell-type specificity of sex-linked and autosomal gene regulation in SCA conditions. HighlightsO_LISex chromosome aneuploidy induces cell-type specific changes in gene expression C_LIO_LIDampening of the inactive X chromosome in XXY alleviate X overexpression C_LIO_LIHigh Y-linked gene expression in XXY neuronal precursor cells but not cardiomyocytes C_LIO_LISex chromosome aneuploidy disrupts sex biases in autosomal gene expression C_LI

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)

Epimutations are changes in chromatin modifications, such as DNA methylation or histone modifications. Some of these epigenetic changes can be inherited for several generations, and they potentially contribute to evolutionary processes. Estimates of epimutation rates now exists in a few species, but the presence and function of epigenetic marks are not conserved across different species. To understand the properties of epimutations in fungi, we performed a mutation accumulation experiment with the filamentous fungus Neurospora crassa and investigated spontaneous changes in DNA methylation and trimethylation of lysine 9 on histone H3 (H3K9me3) in the mutation accumulation lines. We observed that centromeric regions are hotspots of spontaneous DNA methylation changes in N. crassa. In these hotspot regions, DNA methylation changes were transmitted across mitoses, but changes occurring in euchromatin were not maintained. The rate of DNA methylation changes was around 30 000 fold faster than the genetic mutation rate. We did not observe spontaneous changes in H3K9me3 that were transmitted across mitoses. Our results show that while spontaneous epimutations occur in this species, they occur predominantly in gene poor heterochromatic regions, so their impact for evolutionary adaptation may be limited.

Purine moieties are essential for many functions within the eukaryotic cell, including energy, signaling and nucleic acid synthesis. While purine starvation is known to induce stress resistance in eukaryotic model organism budding yeast Saccharomyces cerevisiae, it remains unclear whether the physiological response is related to disruption of synthesis pathway in particular position or it is uniform across all genetic deficiencies within the de novo adenine biosynthesis pathway. It is also not known how purine starved cells perceive purine shortage - weather they share the same signaling elements with nitrogen starvation or not. MethodsWe characterised physiology of strains with deletions in adenine biosynthesis pathway when cultivated in full or purine deficient and compared to cell physiological parameters when cultivated in nitrogen deficient media. We tested stress tolerance, carbon flux, cell cycle arrest and did transcription profiling (RNA-seq). ResultsOur findings demonstrate that purine starvation-induced stress resistance is significantly modulated by the specific step at which the pathway is interrupted. Transcriptional analysis revealed that purine starvation in many aspects phenocopies nitrogen starvation, particularly - in both starvations strong downregulation of ribosome related genes occurs. In the same time several metabolic features which differ from N- and ade- starvations: pentose phosphate pathway is specifically upregulated within ade4{Delta}-ade2{Delta} and downregulated in N-cells. Notably, the expression of stress-responsive genes such as HSP12, HSP26, and GRE1 varied between mutants, suggesting that the accumulation of pathway intermediates (e.g., AIR in ade2{Delta}) or the absence of downstream precursors (AICAR) alters the perception of starvation especially in the case of ade16{Delta}ade17{Delta} strain. ConclusionsMetabolic and stress-tolerance phenotypes of purine auxotrophs are not merely a result of purine depletion but seems that the response is signalled via the same pathways, like TOR1. The results suggest that strains having mutations within various positions of the purine pathway "perceive" purine limitation a bit differently - especially when we compare the end of the pathway with the other mutants. Different phenotypic outcomes of the occasional purine depletion might give preferences for organisms which have mutations in the beginning rather at the end of the pathway. Besides, our findings might have implications in the design of synthetic pathways and the use of auxotrophic markers in yeast research.

Trimethylation of lysine 4 of histone H3 (H3K4me3) is a post-translational modification (PTM) enriched at promoters of actively transcribed genes. H3K4me3 is removed by the human histone demethylases of the KDM5 family. KDM5 demethylases act as transcriptional repressors through their catalytic activity in addition to more complex roles that depend on their interactions with other chromatin regulators and may be independent of demethylase activity. To better understand the mechanistic differences of the closely related paralogs KDM5A and KDM5B as well as their interactions with Retinoblastoma protein (RB), we systematically analyzed and compared their demethylase activities, nucleosome engagement, and RB binding. We used recombinant nucleosome binding and demethylase activity assays, as well as an integrative structural biology approach using negative-stain electron microscopy (EM), AlphaFold predictions, and cross-linking mass spectrometry for a comprehensive in vitro analysis of these critical and largely non-redundant enzymes. KDM5A and KDM5B showed differences in enzyme kinetics using peptide substrates, as well as in nucleosome binding. Furthermore, KDM5A interacts with RB, mainly mediated by its canonical LxCxE RB binding motif. KDM5B, on the other hand, lacks an LxCxE binding motif and does not stably bind to RB under the conditions tested here. RB directly interacts with nucleosomes, and its nucleosome binding does not measurably affect KDM5A demethylase activity or nucleosome interactions. Our findings provide a biochemical framework for the differences between KDM5A and KDM5B regarding RB interactions and nucleosome engagement.

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.

DNA methylation is a key epigenetic mechanism influencing gene regulation and cellular identity. In skeletal muscle, methylation contributes to fiber-type specification, metabolic programming, and satellite cell function, with evidence of sex-specific differences. Here, we investigated whether spatial regionalization of gene expression along the proximal-distal axis of the tibialis anterior (TA) is mirrored by corresponding patterns of DNA methylation. Using MeDseq on TA sections from muscles previously analyzed by spatial transcriptomics, we profiled methylation across transcriptional start sites (TSS), gene bodies, and regulatory elements. Despite robust spatial differences in transcriptomes, methylation patterns were largely uniform along the proximal-distal axis, indicating that DNA methylation does not underlie regional gene expression in adult TA muscle. In contrast, sex emerged as the primary determinant of methylation variation. Male muscles exhibited widespread hypermethylation at TSS, gene-bodies and regulatory regions, corresponding with sex-specific transcriptional programs, including glycolytic fiber enrichment in males and oxidative fiber markers in females. Notably, chromatin- and methylation-associated regulators such as Setd7, Gsk3a, and Bmyc were upregulated in males, suggesting mechanisms linking transcriptional control to epigenetic state. These findings highlight that while spatial gene expression is transcriptionally driven, sex-specific epigenetic programs dominate adult skeletal muscle, underscoring the need to consider sex in multi-omic studies of muscle biology.

BackgroundDNA methylation can be profiled using multiple technologies that vary in resolution, coverage and cost. Yet systematic benchmarks across these methods remain scarce. MethodsWe compared six widely used technologies -- Illumina EPIC array, TWIST, Whole-Genome Enzymatic Conversion (WGEC), Reduced Representation Bisulfite Sequencing (RRBS), long-read genome sequencing (LR-GS) with Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) -- using Genome in a Bottle (GIAB) reference samples and ten samples derived of blood and fibroblast cultures of 5 individuals. We assessed CpG coverage, consistency of differentially methylated cytosine (DMC) detection and genomic annotation, with particular attention to overlapping signals across assays. ResultsDespite major differences in assay design, all technologies consistently identified DMCs enriched in promoter and intronic regions, highlighting these loci as robust hotspots of epigenetic variability. Annotation redundancy strongly influenced initial interpretations, with CpG island-related categories largely disappearing once annotations were collapsed to unique features. Sequencing-based methods (WGEC, TWIST, ONT) achieved the most comprehensive coverage, whereas EPIC arrays reproducibly captured promoter-associated differences despite limited scope. ONT sequencing enabled direct, long-read-based methylation profiling with phasing capability and showed strong concordance with short-read sequencing methods after coverage filtering, but required higher and more uniform coverage to achieve reproducible CpG-level agreement. PacBio methylation profiles showed a coverage-dependent discrepancy, with cross-platform concordance plateauing in GIAB samples despite high mean coverage, indicating residual technology-specific biases beyond simple coverage effects. ConclusionsCross-platform benchmarking yields coherent biological insights when coverage and annotation redundancies are carefully addressed. Practically, EPIC arrays remain valuable for promoter-focused cohort studies, WGEC and TWIST enable genome-wide discovery and ONT provides unique phasing and multimodal potential. This comparative framework can guide method selection and support more robust interpretation of DNA methylation data across diverse platforms.

Biological sex regulates fundamental neurobiology, as well as the etiology and prevalence of neuropsychiatric disorders. Cyclin-dependent kinase 5 (Cdk5) is a neuronally enriched kinase that regulates synaptic plasticity, neuronal homeostasis, and hippocampal-dependent memory. While Cdk5 protein activity is necessary and sufficient to promote memory in male rodents, its role in females and its gene regulation in either sex remain poorly understood. In males, Cdk5 protein inhibition impairs fear memory. We previously showed that fear conditioning activates Cdk5 gene expression and increases permissive chromatin acetylation in male, but not female hippocampus. We hypothesize that Cdk5 gene repression would impair fear memory in males. We developed an excitatory neuron-specific, CRISPR/dCas9-HDAC3 epigenetic editing tool to target histone acetylation at the endogenous Cdk5 promoter. This strategy reduced histone acetylation and decreased Cdk5 mRNA, protein, and kinase activity in both sexes. Interestingly, Cdk5 repression in hippocampal neurons impaired fear and spatial memory in both male and female mice. Targeted deacetylation also evicted the transcription factor CREB1 from the Cdk5 promoter, revealing a link between histone acetylation and Cdk5 transcriptional activation. These findings demonstrate that Cdk5 acetylation in neurons is necessary for hippocampal memory in both sexes, providing new insight into sex-specific epigenetic regulation of memory.

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

A long non-coding RNA (lncRNA) known as the X-inactivation specific transcript (XIST) plays a central role in X chromosome inactivation - a transcriptional process that silences one of the two X chromosomes in females to ensure dosage compensation between males and females. Much research has been conducted on how XIST regulates X chromosome transcription critical to embryonic development, but recent studies suggest a non-canonical role for XIST in regulating cancer stem cells and cellular plasticity. As cell adhesion and adhesome genes are integral to the regulation of cancer stemness, we explored the previously unrecognized link between XIST and the adhesome network. By performing gene expression and gene ontology analysis on XIST-knockdown ovarian cancer cells, our study showed that XIST loss altered adhesome gene expression and downstream adhesion pathways. Using Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) datasets, we identified distinct correlations between XIST lncRNA and adhesome genes across normal and cancer tissue samples, which are associated with cell stemness. Furthermore, network analysis suggests that XIST may interact with specific adhesome genes within the cell nucleus. This interaction may have significant functional implications, as demonstrated by the hazard ratio analysis of XIST and adhesome gene expression in relation to clinical outcomes. Overall, our results show that among well-annotated functional lncRNAs, XIST appears to be a modulator strongly associated with the adhesome network and cell stemness. Our findings thus support a novel link between lncRNA-mediated epigenetic regulation of cell adhesion genes, highlighting XIST as a key regulator contributing to the adhesome network. Significance StatementThis study identified that XIST, a long non-coding RNA essential for X-chromosome dosage compensation and embryonic development, plays a significant role in modulating the adhesome network. We found that XIST knockdown affected adhesion pathways in ovarian cancer cells, whereas XIST expression is strongly correlated with adhesome gene expression across all tissues. We observed that the interaction between XIST and adhesome genes changes significantly between tumors and normal tissues, and this altered interaction is associated with certain cancer outcomes. These findings reveal a possible link between lncRNA-mediated regulation and adhesome control that is associated with cell stemness signatures and the emergence of cancerous tissues.

BackgroundMaize knobs are regions of constitutive heterochromatin that are readily identified in both meiotic and somatic chromosomes. These structures have been characterized as stable throughout the cell cycle, exhibiting late replication during the S-phase, and are composed of two specific families of highly repetitive DNA sequences: K180 and TR-1. Although widely used as cytogenetic markers due to their variability in number and chromosomal position across inbred lines, hybrids, and landraces, little is known about their chromatin structure and dynamics. In this study, we analyzed chromatin accessibility of knobs using DNS-seq data across four maize tissues representing distinct developmental stages. ResultsOur results reveal that K180 knobs exhibit tissue-specific variation in chromatin accessibility, transitioning between open and closed states during development. In contrast, the TR-1 knob of chromosome 4 remained consistently inaccessible across all tissues analyzed. A knob composed of both K180, and TR-1 further supported this observation, with only the K180 region showing dynamic accessibility. To validate these findings, we also analyzed other repetitive regions such as centromeres, which showed a uniformly closed chromatin structure similar to TR-1. These results suggest a unique developmental modulation of chromatin accessibility associated with K180 repeats. While the chromatin accessibility of knobs does not reach the levels observed at Transcription Start Sites (TSS), the comparison among different classes of repetitive DNA within maize constitutive heterochromatin provides compelling evidence for sequence-specific and tissue-specific chromatin dynamics. ConclusionsOur findings uncover a previously unrecognized property of maize knobs and establish a reference for future studies on chromatin organization and epigenetic regulation of repetitive DNA in plant genomes.