Histone Transcription Research Articles

DNA supercoiling modulates eukaryotic transcription in a gene-orientation dependent manner.

Transcription introduces torsional stress in the DNA fiber causing it to transition from a relaxed to a supercoiled state that can propagate across several kilobases and modulate the binding and activity of DNA-associated proteins. As a result, transcription at one locus has the potential to impact nearby transcription events. In this study, we asked how DNA supercoiling affects histone modifications and transcription of neighboring genes in the multicellular eukaryote Caenorhabditis elegans . We acutely depleted the two major topoisomerases and measured nascent transcription by Global Run-on sequencing (GRO-seq), RNA Polymerase II occupancy by ChIP-seq, gene expression by RNA-seq and four transcription-associated histone modifications by Cut & Tag. Depletion of topoisomerases I and II led to genome-wide changes in transcription dynamics, with minor disruptions to the histone modification landscape. Our results showed that C. elegans topoisomerase I is required for transcription elongation and is partially redundant with topoisomerase II. Analysis of transcription changes with respect to neighboring genes suggest that negative supercoiling promotes the transcription of genes with a divergent neighbor and positive supercoiling suppresses transcription of convergent genes. Additionally, topoisomerase depletion caused coordinated changes in the expression of divergent gene pairs, suggesting that negative supercoiling drives their synchronized expression. Conversely, the coordinated expression of convergent genes was disrupted, suggesting that excessive positive supercoiling inhibits transcription. Overall, our data supports a model in which DNA supercoiling generated by transcription at one site propagates along the eukaryotic chromatin fiber, influencing nearby transcription in an orientation-dependent manner.

MaizeCODE reveals bi-directionally expressed enhancers that harbor molecular signatures of maize domestication

AbstractModern maize (Zea mays ssp. mays) was domesticated from Teosinte parviglumis (Zea mays ssp. parviglumis), with subsequent introgressions from Teosinte mexicana (Zea mays ssp. mexicana), yielding increased kernel row number, loss of the hard fruit case and dissociation from the cob upon maturity, as well as fewer tillers. Molecular approaches have identified transcription factors controlling these traits, yet revealed that a complex regulatory network is at play. MaizeCODE deploys ENCODE strategies to catalog regulatory regions in the maize genome, generating histone modification and transcription factor ChIP-seq in parallel with transcriptomics datasets in 5 tissues of 3 inbred lines which span the phenotypic diversity of maize, as well as the teosinte inbred TIL11. Transcriptomic analysis reveals that pollen grains share features with endosperm, and express dozens of “proto-miRNAs” potential vestiges of gene drive and hybrid incompatibility. Integrated analysis with chromatin modifications results in the identification of a comprehensive set of regulatory regions in each tissue of each inbred, and notably of distal enhancers expressing non-coding enhancer RNAs bi-directionally, reminiscent of “super enhancers” in animal genomes. Furthermore, the morphological traits selected during domestication are recapitulated, both in gene expression and within regulatory regions containing enhancer RNAs, while highlighting the conflict between enhancer activity and silencing of the neighboring transposable elements.

Histone Modification-Dependent Transcriptional Regulation of Defence Genes in Early Response of Arabidopsis to Spodoptera litura Attack.

Histone modification is a cellular process for transcriptional regulation. In herbivore-damaged plants, activation of genes involved in defence responses is required for antiherbivore properties, but little is known about how the chromatin remodelling system is involved. In Arabidopsis (Arabidopsis thaliana) plants responding to Spodoptera litura larvae, HAC1 and HDA6, a histone acetyltransferase and a histone deacetylase, respectively, were found here to be involved in histone H3 (Lys9; H3K9) acetylation/deacetylation at the promoter region of the plant defensin gene PDF1.2 and the gene body of ethylene response factor 13 (ERF13) as early as 2 h after the onset of herbivore attack. The H3K9 acetylation was responsible for the robust upregulation of PDF1.2 later, at 24 h, and ERF13 even earlier, at 1 h. TOPLESS (TPL) and TOPLESS-related (TPR) corepressors interacted with HDA6 to deacetylate H3K9 at PDF1.2 and ERF13, while negatively regulating the expression of PDF1.2 but not ERF13. Furthermore, TPL also interacted with ERF13, resulting in ERF13-mediated regulation of PDF1.2. Taken together, these data suggest a model of promoter-restricted, TPL/TPR-directed histone deacetylation and transcription factor repression in healthyArabidopsis plants for the feedback regulation of the antiherbivore response.

KPNA3 regulates histone locus body formation by modulating condensation and nuclear import of NPAT.

The histone locus body (HLB) is a membraneless organelle that determines the transcription of replication-dependent histones. However, the mechanisms underlying the appropriate formation of the HLB in the nucleus but not in the cytoplasm remain unknown. HLB formation is dependent on the scaffold protein NPAT. We identify KPNA3 as a specific importin that drives the nuclear import of NPAT by binding to the nuclear localization signal (NLS) sequence. NPAT undergoes phase separation, which is inhibited by KPNA3-mediated impairment of self-association. In this, a C-terminal self-interaction facilitator (C-SIF) motif, proximal to the NLS, binds the middle 431-1,030 sequence to mediate the self-association of NPAT. Mechanistically, the anchoring of KPNA3 to the NPAT-NLS sterically blocks C-SIF motif-dependent NPAT self-association. This leads to the suppression of aberrant NPAT condensation in the cytoplasm. Collectively, our study reveals a previously unappreciated role of KPNA3 in modulating HLB formation and delineates a steric hindrance mechanism that prevents inappropriate cytoplasmic NPAT condensation.

Mutations in the DNA processivity factor POL30 predisposes the FLO11 locus to epigenetic instability in S. cerevisiae.

The FLO genes in Saccharomyces cerevisiae are repressed by heterochromatin formation, involving histone deacetylases, transcription factors and non-coding RNAs. Here, we report that mutations in the processivity factor POL30 (PCNA) that show transient derepression at the subtelomeres and the mating-type loci do not derepress FLO loci. However, deletions of the replisome stability factors RRM3 and TOF1 along with pol30 mutations induced flocculation phenotypes. The phenotypes correlated with increased expression of reporter proteins driven by the FLO11 promoter, the frequency of silent to active conversions of FLO11, and reduced expression of the regulatory long non-coding RNAs ICR1 and PWR1. Alterations in the local replication landscape of FLO11 indicate a link between defects in the fork protection complex and the stability of gene silencing. Analyses of these mutants at the subtelomeres and the HMLα locus showed a similar derepression phenotype and suggest transient instability of both active and silent states of FLO11. We conclude that RRM3 and TOF1 interact differentially with the pol30 mutations to promote transient derepression or complete epigenetic conversions of FLO11. We suggest that the interaction between POL30, RRM3 and TOF1 is essential to maintain epigenetic stability at the studied loci.

Abstract PR002: Hypoxia-induced epigenetic changes coordinate transcription start site selection and translational control through remodeling of 5’UTRs

Abstract Adaptation to hypoxia enhances cancer cell malignancy and negatively impacts patient outcomes. Reprogramming of mRNA translation is an essential component of the hypoxia-adaptive response. During hypoxia, cells undergo a global decrease in protein synthesis. However, translation of subsets of transcripts encoding stress-response, survival and stemness factors is increased. This selective regulation is thought to be governed largely via the interactions between specific mRNA features and remodeling of the translational apparatus. However, our understanding of the mechanisms that govern translational perturbations under hypoxia remains incomplete. Here, we interrogated the effects of hypoxia on global changes in histone methylation and transcription start site (TSS) selection, coupled with monitoring corresponding alterations in total mRNA levels and translation efficiencies on a genome-wide scale in T47D breast cancer cells and H9 human embryonic stem cells. This revealed widespread epigenetic alterations leading to TSS switching and extensive remodeling of 5’UTRs, and allowed us to map the impact of specific 5’UTR features on translation efficiency under hypoxia. Pyruvate Dehydrogenase Kinase 1 (PDK1), a key enzyme in orchestrating metabolic adaption to hypoxia, undergoes TSS switching leading to increased availability of efficiently translated 5’UTR isoforms, and we show that this effect is coordinated at an epigenetic level. Therefore, our findings uncover a mechanism driving translational reprogramming under hypoxia, whereby alterations in translational apparatus are orchestrated with epigenetic perturbations that result in 5’UTR remodeling of the transcriptome. Citation Format: Kathleen Watt, Bianca Dauber, Krzysztof J Szkop, Laura Lee, Predrag Jovanovic, Shan Chen, Laia Masvidal, Kristofersson Tandoc, Ranveer Palia, Ivan Topisirovic, Ola Larsson, Lynne-Marie Postovit. Hypoxia-induced epigenetic changes coordinate transcription start site selection and translational control through remodeling of 5’UTRs [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr PR002.

ChIPmentation for Epigenomic Analysis in Fission Yeast.

Histone modifications and transcription factor-DNA interactions regulate vital processes such as transcription, recombination, repair, and accurate chromosome segregation. Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) has been instrumental in studying genome-wide distribution of DNA-bound or chromatin-associated factors and histone posttranslational modifications (PTMs). Here, we describe a ChIPmentation protocol adapted for fission yeast, Schizosaccharomyces pombe. This method merges Tn5 mediated tagmentation with existing ChIP protocols, resulting in lower sample input requirements with significant reduction in hands-on time and sample preparation costs.

ScNanoSeq-CUT&Tag: a single-cell long-read CUT&Tag sequencing method for efficient chromatin modification profiling within individual cells.

Chromatin modifications are fundamental epigenetic marks that determine genome functions, but it remains challenging to profile those of repetitive elements and complex genomic regions. Here, we develop scNanoSeq-CUT&Tag, a streamlined method, by adapting modified cleavage under targets and tagmentation (CUT&Tag) to the nanopore sequencing platform for genome-wide chromatin modification profiling within individual cells. We show that scNanoSeq-CUT&Tag can accurately profile histone marks and transcription factor occupancy patterns at single-cell resolution as well as distinguish different cell types. scNanoSeq-CUT&Tag efficiently maps the allele-specific chromatin modifications and allows analysis of their neighboring region co-occupancy patterns within individual cells. Moreover, scNanoSeq-CUT&Tag can accurately detect chromatin modifications for individual copies of repetitive elements in both human and mouse genomes. Overall, we prove that scNanoSeq-CUT&Tag is a valuable single-cell tool for efficiently profiling histone marks and transcription factor occupancies, especially for previously poorly studied complex genomic regions and blacklist genomic regions.

Transcriptional and methylation outcomes of didehydro-cortistatin A use in HIV-1-infected CD4+ T cells.

Ongoing viral transcription from the reservoir of HIV-1 infected long-lived memory CD4+ T cells presents a barrier to cure and associates with poorer health outcomes for people living with HIV, including chronic immune activation and inflammation. We previously reported that didehydro-cortistatin A (dCA), an HIV-1 Tat inhibitor, blocks HIV-1 transcription. Here, we examine the impact of dCA on host immune CD4+ T-cell transcriptional and epigenetic states. We performed a comprehensive analysis of genome-wide transcriptomic and DNA methylation profiles upon long-term dCA treatment of primary human memory CD4+ T cells. dCA prompted specific transcriptional and DNA methylation changes in cell cycle, histone, interferon-response, and T-cell lineage transcription factor genes, through inhibition of both HIV-1 and Mediator kinases. These alterations establish a tolerogenic Treg/Th2 phenotype, reducing viral gene expression and mitigating inflammation in primary CD4+ T cells during HIV-1 infection. In addition, dCA suppresses the expression of lineage-defining transcription factors for Th17 and Th1 cells, critical HIV-1 targets, and reservoirs. dCA's benefits thus extend beyond viral transcription inhibition, modulating the immune cell landscape to limit HIV-1 acquisition and inflammatory environment linked to HIV infection.

The Drosophila maternal-effect gene abnormal oocyte (ao) does not repress histone gene expression.

The abnormal oocyte (ao) gene of Drosophila melanogaster is a maternal-effect lethal gene previously identified as encoding a transcriptional regulator of core histones. However, background genetic mutations in existing ao mutant strains could compromise their utility in manipulating histone levels. To distinguish the true ao phenotype from background effects, we created two new ao reagents: a CRISPR/Cas9-mediated knockout of the ao allele for genetic and molecular analyses and an epitope-tagged ao allele for cytological experiments. Using these reagents, we confirm previous findings that ao exhibits maternal-effect lethality, which can be rescued by either a decrease in the histone gene copy number or by Y chromosome heterochromatin. We also confirm that the Ao protein localizes to the histone locus bodies in ovaries. Our data also suggest that ao genetically interacts with the histone genes and heterochromatin, as previously suggested. However, contrary to prior findings, we find that ao does not repress core histone transcript levels. Thus, the molecular basis for ao-associated maternal-effect lethality remains unknown.

Decoupled transcript and protein concentrations ensure histone homeostasis in different nutrients

To maintain protein homeostasis in changing nutrient environments, cells must precisely control the amount of their proteins, despite the accompanying changes in cell growth and biosynthetic capacity. As nutrients are major regulators of cell cycle length and progression, a particular challenge arises for the nutrient-dependent regulation of ‘cell cycle genes’, which are periodically expressed during the cell cycle. One important example are histones, which are needed at a constant histone-to-DNA stoichiometry. Here we show that budding yeast achieves histone homeostasis in different nutrients through a decoupling of transcript and protein abundance. We find that cells downregulate histone transcripts in poor nutrients to avoid toxic histone overexpression, but produce constant amounts of histone proteins through nutrient-specific regulation of translation efficiency. Our findings suggest that this allows cells to balance the need for rapid histone production under fast growth conditions with the tight regulation required to avoid toxic overexpression in poor nutrients.

AChIP is an efficient and sensitive ChIP-seq technique for economically important plant organs.

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is crucial for profiling histone modifications and transcription factor binding throughout the genome. However, its application in economically important plant organs (EIPOs) such as seeds, fruits and flowers is challenging due to their sturdy cell walls and complex constituents. Here we present advanced ChIP (aChIP), an optimized method that efficiently isolates chromatin from plant tissues while simultaneously removing cell walls and cellular constituents. aChIP precisely profiles histone modifications in all 14 tested EIPOs and identifies transcription factor and chromatin-modifying enzyme binding sites. In addition, aChIP enhances ChIP efficiency, revealing numerous novel modified sites compared with previous methods in vegetative tissues. aChIP reveals the histone modification landscape for rapeseed dry seeds, highlighting the intricate roles of chromatin dynamics during seed dormancy and germination. Altogether, aChIP is a powerful, efficient and sensitive approach for comprehensive chromatin profiling in virtually all plant tissues, especially in EIPOs.

The MYC-MAF-SAGA axis drives oncogenic gene expression in multiple myeloma.

The SAGA complex is an evolutionarily conserved histone acetyltransferase complex and transcription coactivator essential for development and disease. Dysregulation of SAGA is implicated in various human diseases, including cancer. In this issue of Genes & Development, Chen et al. (doi:10.1101/gad.351789.124) uncover a critical role for SAGA in multiple myeloma wherein SAGA's ADA2B component is required for the expression of mTORC1 pathway genes and targets of the MYC, E2F, and MAF (musculoaponeurotic fibrosarcoma) transcription factors. SAGA cooperates with MYC and MAF to sustain oncogenic gene expression programs vital for multiple myeloma survival and thus may serve as a therapeutic target for future cancer therapies.

Mapping the nuclear landscape with multiplexed super-resolution fluorescence microscopy.

The nucleus coordinates many different processes. Visualizing how these are spatially organized requires imaging protein complexes, epigenetic marks, and DNA across scales from single molecules to the whole nucleus. To accomplish this, we developed a multiplexed imaging protocol to localize 13 different nuclear targets with nanometer precision in single cells. We show that nuclear specification into active and repressive states exists along a spectrum of length scales, emerging below one micron and becoming strengthened at the nanoscale with unique organizational principles in both heterochromatin and euchromatin. HP1-α was positively correlated with DNA at the microscale but uncorrelated at the nanoscale. RNA Polymerase II, p300, and CDK9 were positively correlated at the microscale but became partitioned below 300 nm. Perturbing histone acetylation or transcription disrupted nanoscale organization but had less effect at the microscale. We envision that our imaging and analysis pipeline will be useful to reveal the organizational principles not only of the cell nucleus but also other cellular compartments.

Nuclear pyruvate dehydrogenase complex regulates histone acetylation and transcriptional regulation in the ethylene response.

Ethylene plays its essential roles in plant development, growth, and defense responses by controlling the transcriptional reprograming, in which EIN2-C-directed regulation of histone acetylation is the first key step for chromatin to perceive ethylene signaling. But how the nuclear acetyl coenzyme A (acetyl CoA) is produced to ensure the ethylene-mediated histone acetylation is unknown. Here we report that ethylene triggers the accumulation of the pyruvate dehydrogenase complex (PDC) in the nucleus to synthesize nuclear acetyl CoA to regulate ethylene response. PDC is identified as an EIN2-C nuclear partner, and ethylene triggers its nuclear accumulation. Mutations in PDC lead to an ethylene hyposensitivity that results from the reduction of histone acetylation and transcription activation. Enzymatically active nuclear PDC synthesizes nuclear acetyl CoA for EIN2-C-directed histone acetylation and transcription regulation. These findings uncover a mechanism by which PDC-EIN2 converges the mitochondrial enzyme-mediated nuclear acetyl CoA synthesis with epigenetic and transcriptional regulation for plant hormone response.

GENet: A Graph-Based Model Leveraging Histone Marks and Transcription Factors for Enhanced Gene Expression Prediction.

Understanding the regulatory mechanisms of gene expression is a crucial objective in genomics. Although the DNA sequence near the transcription start site (TSS) offers valuable insights, recent methods suggest that analyzing only the surrounding DNA may not suffice to accurately predict gene expression levels. We developed GENet (Gene Expression Network from Histone and Transcription Factor Integration), a novel approach that integrates essential regulatory signals from transcription factors and histone modifications into a graph-based model. GENet extends beyond simple DNA sequence analysis by incorporating additional layers of genetic control, which are vital for determining gene expression. Our method markedly enhances the prediction of mRNA levels compared to previous models that depend solely on DNA sequence data. The results underscore the significance of including comprehensive regulatory information in gene expression studies. GENet emerges as a promising tool for researchers, with potential applications extending from fundamental biological research to the development of medical therapies.

SpikeFlow: automated and flexible analysis of ChIP-Seq data with spike-in control.

ChIP with reference exogenous genome (ChIP-Rx) is widely used to study histone modification changes across different biological conditions. A key step in the bioinformatics analysis of this data is calculating the normalization factors, which vary from the standard ChIP-seq pipelines. Choosing and applying the appropriate normalization method is crucial for interpreting the biological results. However, a comprehensive pipeline for complete ChIP-Rx data analysis is lacking. To address these challenges, we introduce SpikeFlow, an integrated Snakemake workflow that combines features from various existing tools to streamline ChIP-Rx data processing and enhance usability. SpikeFlow automates spike-in data scaling and provides multiple normalization options. It also performs peak calling and differential analysis with distinct modalities, enabling the detection of enrichment regions for histone modifications and transcription factor binding. Our workflow runs in-depth quality control at all the processing steps and generates an analysis report with tables and graphs to facilitate results interpretation. We validated the pipeline by performing a comparative analysis with DiffBind and SpikChIP, demonstrating robust performances in various biological models. By combining diverse functionalities into a single platform, SpikeFlow aims to simplify ChIP-Rx data analysis for the research community.

Deciphering the Nuclear Role of Heme Oxygenase-1 in Prostate Cancer: Transcriptional Reprogramming and Neuroendocrine Differentiation

PURPOSE This study probes the role of Heme Oxygenase-1 (HO-1) and its nuclear interactors in prostate cancer (PCa). Previous results, demonstrated the anti-tumoral role of HO-1 in PCa and its nuclear translocation, hinting at its potential regulatory roles within the nucleus. Thus, we postulate that HO-1, alongside its interactors, can reprogram PCa cells to a less aggressive phenotype. METHODS From the 11 HO-1 nuclear interacting proteins previously identified, 4 were transcription factors Using ChIP Atlas we evaluated the binding affinity of these factors to their downstream gene targets. Gene ontology and KEGG pathway analyses were then performed to associate these targets with specific biological processes and pathologies. Additionally, RNA-seq data from patient-derived xenografts (PDXs) at MD Anderson Cancer Center (MDA PCa PDXs) were analyzed to evaluate the clinical relevance of these genes in the context of PCa heterogeneity. These PDXs offer a clinically relevant model due to their representation of the disease's heterogeneity. We scrutinized the expression of the gene clusters of interest in PDXs, including their association with neuroendocrine (NE) phenotypes. RESULTS The transcription factors among the HO-1 interactors previously described, were found to bind genes enriched in processes related to histone modification, acetylation, and transcription termination, implicating their role in gene expression regulation. KEGG pathways linked these genes to neurodegenerative diseases (including Parkinson's, Huntington's, and Alzheimer's disease), mirroring the neuroendocrine (NE) phenotype in advanced PCa, which typically emerges in advanced stages and is characterized by the transdifferentiating of epithelial cells into NE cells lacking androgen receptor expression. The unsupervised clustering of gene expression from PDXs showed that high expression of these genes is associated with an AR-negative NE histopathology, suggesting a potential for these interactors in prostate cancer subtyping. CONCLUSION HO-1 nuclear interactors, especially transcription factors, appear to influence gene transcription associated with NE differentiation and cancer aggressiveness. The therapeutic relevance of modulating HO-1's interaction with its nuclear partners warrants further investigation to ascertain its role in mitigating the NE of PCa.

Calcineurin/NFATc1 pathway represses cellular cytotoxicity by modulating histone H3 expression

Excess amounts of histones in the cell induce mitotic chromosome loss and genomic instability, and are therefore detrimental to cell survival. In yeast, excess histones are degraded by the proteasome mediated via the DNA damage response factor Rad53. Histone expression, therefore, is tightly regulated at the protein level. Our understanding of the transcriptional regulation of histone genes is far from complete. In this study, we found that calcineurin inhibitor treatment increased histone protein levels, and that the transcription factor NFATc1 (nuclear factor of activated T cells 1) repressed histone transcription and acts downstream of the calcineurin. We further revealed that NFATc1 binds to the promoter regions of many histone genes and that histone transcription is downregulated in a manner dependent on intracellular calcium levels. Indeed, overexpression of histone H3 markedly inhibited cell proliferation. Taken together, these findings suggest that NFATc1 prevents the detrimental effects of histone H3 accumulation by inhibiting expression of histone at the transcriptional level.

Minor Spliceosomal 65K/RNPC3 Interacts with ANKRD11 and Mediates HDAC3-Regulated Histone Deacetylation and Transcription.

RNA splicing is crucial in the multilayer regulatory networks for gene expression, making functional interactions with DNA- and other RNA-processing machineries in the nucleus. However, these established couplings are all major spliceosome-related; whether the minor spliceosome is involved remains unclear. Here, through affinity purification using Drosophila lysates, an interaction is identified between the minor spliceosomal 65K/RNPC3 and ANKRD11, a cofactor of histone deacetylase 3 (HDAC3). Using a CRISPR/Cas9 system, Deletion strains are constructed and found that both Dm65KΔ/Δ and Dmankrd11Δ/Δ mutants have reduced histone deacetylation at Lys9 of histone H3 (H3K9) and Lys5 of histone H4 (H4K5) in their heads, exhibiting various neural-related defects. The 65K-ANKRD11 interaction is also conserved in human cells, and the HsANKRD11 middle-uncharacterized domain mediates Hs65K association with HDAC3. Cleavage under targets and tagmentation (CUT&Tag) assays revealed that HsANKRD11 is a bridging factor, which facilitates the synergistic common chromatin-binding of HDAC3 and Hs65K. Knockdown (KD) of HsANKRD11 simultaneously decreased their common binding, resulting in reduced deacetylation of nearby H3K9. Ultimately, this study demonstrates that expression changes of many genes caused by HsANKRD11-KD are due to the decreased common chromatin-binding of HDAC3 and Hs65K and subsequently reduced deacetylation of H3K9, illustrating a novel and conserved coupling mechanism that links the histone deacetylation with minor spliceosome for the regulation of gene expression.