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Histone post-translational modifications (PTMs) are critical epigenetic regulatory factors. Histone PTMs are highly dynamic and complicated, encompassing over 30 structurally diverse modifications across nearly 180 amino acid residues, which generated extensive information regarding histone marks. In proteomics-based characterization of histone PTMs, chemical derivatization and antibody-based affinity enrichment were frequently utilized to improve the identification depth. However, chemical derivatization suffered from the occurrence of side reactions, and antibody-based affinity enrichment focused on specific PTM types of interest. In this research, we developed a multi-step fractionation strategy for comprehensively unbiased detection of histone PTM sites. By combining protein-level fractionation with peptide-level alkaline and acid phase fractionation, we developed the Multidimensional Fractionation based Histone Mark Identification Technology (MudFIT) and increased PTM identification to a total of 264 histone PTM sites. To the best of our knowledge, this strategy achieved the most comprehensive characterization of histone PTM sites in a single proteomics study. Using the same starting amount of sample, MudFIT identified more Kac sites and Kac peptides than those in antibody-based acetylated peptide enrichment. Moreover, in addition to well-studied histone marks, we discovered 36 potential new histone PTM sites including H2BK116bu, H4R45me2, H1K63pr, and uncovered unknown histone PTM types like aminoadipic on lysine and nitrosylation on tyrosine. Our data provided a method and resource for in-depth characterization of histone PTM sites, facilitating further biological understanding of histone marks.

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Histone modifications, known as histone marks, are pivotal in regulating gene expression within cells. The vast array of potential combinations of histone marks presents a considerable challenge in decoding the regulatory mechanisms solely through biological experimental approaches. To overcome this challenge, we have developed a method called CatLearning. It utilizes a modified convolutional neural network architecture with a specialized adaptation Residual Network to quantitatively interpret histone marks and predict gene expression. This architecture integrates long-range histone information up to 500Kb and learns chromatin interaction features without 3D information. By using only one histone mark, CatLearning achieves a high level of accuracy. Furthermore, CatLearning predicts gene expression by simulating changes in histone modifications at enhancers and throughout the genome. These findings help comprehend the architecture of histone marks and develop diagnostic and therapeutic targets for diseases with epigenetic changes.

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A generalizable strategy with programmable site specificity for in situ profiling of histone modifications on unperturbed chromatin remains highly desirable but challenging. We herein developed a single-site-resolved multi-omics (SiTomics) strategy for systematic mapping of dynamic modifications and subsequent profiling of chromatinized proteome and genome defined by specific chromatin acylations in living cells. By leveraging the genetic code expansion strategy, our SiTomics toolkit revealed distinct crotonylation (e.g., H3K56cr) and ß-hydroxybutyrylation (e.g., H3K56bhb) upon short chain fatty acids stimulation and established linkages for chromatin acylation mark-defined proteome, genome, and functions. This led to the identification of GLYR1 as a distinct interacting protein in modulating H3K56cr's gene body localization as well as the discovery of an elevated super-enhancer repertoire underlying bhb-mediated chromatin modulations. SiTomics offers a platform technology for elucidating the "metabolites-modification-regulation" axis, which is widely applicable for multi-omics profiling and functional dissection of modifications beyond acylations and proteins beyond histones.

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Histone lysine-specific methyltransferase 2 (KMT2A-D) proteins, alternatively called mixed lineage leukemia (MLL1-4) proteins, mediate positive transcriptional memory. Acting as the catalytic subunits of human COMPASS-like complexes, KMT2A-D methylate H3K4 at promoters and enhancers. KMT2A-D contain understudied highly conserved triplets and a quartet of plant homeodomains (PHDs). Here, we show that all clustered (multiple) PHDs localize to the well-defined loci of H3K4me3 and H3 acetylation-rich active promoters and enhancers. Surprisingly, we observe little difference in binding pattern between PHDs from promoter-specific KMT2A-B and enhancer-specific KMT2C-D. Fusion of the KMT2A CXXC domain to the PHDs drastically enhances their preference for promoters over enhancers. Hence, the presence of CXXC domains in KMT2A-B, but not KMT2C-D, may explain the promoter/enhancer preferences of the full-length proteins. Importantly, targets of PHDs overlap with KMT2A targets and are enriched in genes involved in the cancer pathways. We also observe that PHDs of KMT2A-D are mutated in cancer, especially within conserved folding motifs (Cys4HisCys2Cys/His). The mutations cause a domain loss-of-function. Taken together, our data suggest that PHDs of KMT2A-D guide the full-length proteins to active promoters and enhancers, and thus play a role in positive transcriptional memory.

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Programmed cell death 1 (PD-1) is a co-inhibitory checkpoint receptor expressed in various immune cells, especially in activated T cells. Engagement of PD-1 with its ligand leads to the exhausted T cells and impaired antitumor immunity. To date, PD-1 expression and its roles have been widely reported in T cells but not well defined in innate immune cells including monocytes. In this study, expression of PD-1 was investigated in human monocytes. Here we observed that among cytokines tested, IFN-γ significantly upregulated the PD-1 expression in both THP-1 cell line and human primary monocytes in a dose- and time-dependent manner. This effect was reduced by PI3K inhibitor, suggesting that the involvement of PI3K/AKT pathway. Furthermore, enrichment of active histone mark H3K4me3 in the Pdcd1 promotor was also observed in IFN-γ-induced THP-1, indicating that epigenetic regulation also plays a role in IFN-γ-induced PD-1 expression. To investigate the biological functions of PD-1, Pdcd1 was deleted in THP-1 cell line by CRISPR/Cas9 system and the phagocytic ability was investigated. The results showed that the PD-1 deficiency in THP-1 cell line resulted in significantly poor phagocytic potency against carboxylated-modified latex beads. Moreover, the PD-1 deficiency or blocking PD-1/PD-L1 interaction by immune checkpoint inhibitor resulted in an impaired induction of IL-4-induced CD163 expression in THP-1 cell line. Taken together, these results highlighted the importance of PD-1 expression in some of key monocyte functions.

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In this paper, we describe a method for the study of colocalization effects between stretch-stretch and stretch-point genome tracks based on a set of indices varying within the (-1, +1) interval. The indices combine the distances between the centers of neighboring stretches and their lengths. The extreme boundaries of the interval correspond to the complete colocalization of the genome tracks or its complete absence. We also obtained the relevant criteria of statistical significance for such indices using the complete permutation test. The method is robust with respect to strongly inhomogeneous positioning and length distribution of the genome tracks. On the basis of this approach, we created command-line software, the Genome Track Colocalization Analyzer. The software was tested, compared with other available packages, and applied to particular problems related to gene expression. The package, Genome Track Colocalization Analyzer (GTCA), is freely available to the users. GTCA complements our previous software, the Genome Track Analyzer, intended for the search for pairwise correlations between point-like genome tracks (also freely available). The corresponding details are provided in Data Availability Statement at the end of the text.

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Chromatin immunoprecipitation coupled with sequencing (ChIP-seq) is a technique used to identify protein-DNA interaction sites through antibody pull-down, sequencing and analysis; with enrichment 'peak' calling being the most critical analytical step. Benchmarking studies have consistently shown that peak callers have distinct selectivity and specificity characteristics that are not additive and seldom completely overlap in many scenarios, even after parameter optimization. We therefore developed ChIP-AP, an integrated ChIP-seq analysis pipeline utilizing four independent peak callers, which seamlessly processes raw sequencing files to final result. This approach enables (1) better gauging of peak confidence through detection by multiple algorithms, and (2) more thoroughly surveys the binding landscape by capturing peaks not detected by individual callers. Final analysis results are then integrated into a single output table, enabling users to explore their data by applying selectivity and sensitivity thresholds that best address their biological questions, without needing any additional reprocessing. ChIP-AP therefore presents investigators with a more comprehensive coverage of the binding landscape without requiring additional wet-lab observations.

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BACKGROUND: Genome-wide association studies have identified many single nucleotide polymorphisms (SNPs) associated with increased risk for intracranial aneurysm (IA). However, how such variants affect gene expression within IA is poorly understood. We used publicly-available ChIP-Seq data to study chromatin landscapes surrounding risk loci to determine whether IA-associated SNPs affect functional elements that regulate gene expression in cell types comprising IA tissue. METHODS: We mapped 16 significant IA-associated SNPs to linkage disequilibrium (LD) blocks within human genome. Using ChIP-Seq data, we examined these regions for presence of H3K4me1, H3K27ac, and H3K9ac histone marks (typically associated with latent/active enhancers). This analysis was conducted in several cell types that are present in IA tissue (endothelial cells, smooth muscle cells, fibroblasts, macrophages, monocytes, neutrophils, T cells, B cells, NK cells). In cell types with significant histone enrichment, we used HiC data to investigate topologically associated domains (TADs) encompassing the LD blocks to identify genes that may be affected by IA-associated variants. Bioinformatics were performed to determine the biological significance of these genes. Genes within HiC-defined TADs were also compared to differentially expressed genes from RNA-seq/microarray studies of IA tissues. RESULTS: We found that endothelial cells and fibroblasts, rather than smooth muscle or immune cells, have significant enrichment for enhancer marks on IA risk haplotypes (p < 0.05). Bioinformatics demonstrated that genes within TADs subsuming these regions are associated with structural extracellular matrix components and enzymatic activity. The majority of histone marked TADs (83% fibroblasts [IMR90], 77% HUVEC) encompassed at least one differentially expressed gene from IA tissue studies. CONCLUSIONS: These findings provide evidence that genetic variants associated with IA risk act on endothelial cells and fibroblasts. There is strong circumstantial evidence that this may be mediated through altered enhancer function, as genes in TADs encompassing enhancer marks have also been shown to be differentially expressed in IA tissue. These genes are largely related to organization and regulation of the extracellular matrix. This study builds upon our previous (Poppenberg et al., BMC Med Genomics, 2019) by including a more diverse set of data from additional cell types and by identifying potential affected genes (i.e. those in TADs).

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The INhibitor of Growth family was defined in the mid-1990s by the identification of a tumour suppressor, ING1, and subsequent expansion of the family based essentially on sequence similarities. However, later work and more recent investigations demonstrate that at least a few ING proteins are actually required for normal proliferation of eukaryotic cells, from yeast to human. ING proteins are also part of a larger family of chromatin-associated factors marked by a plant homeodomain (PHD), which mediates interactions with methylated lysine residues. Herein, we discuss the role of ING proteins and their various roles in chromatin signalling in the context of cancer development and progression.

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BACKGROUND: Genetic variants in the human leukocyte antigen (HLA) locus contribute to the risk for developing scleroderma/systemic sclerosis (SSc). However, there are other replicated loci that also contribute to genetic risk for SSc, and it is unknown whether genetic risk in these non-HLA loci acts primarily on the vasculature, immune system, fibroblasts, or other relevant cell types. We used the Cistrome database to investigate the epigenetic landscapes surrounding 11 replicated SSc associated loci to determine whether SNPs in these loci may affect regulatory elements and whether they are likely to impact a specific cell type. METHODS: We mapped 11 replicated SNPs to haplotypes and sought to determine whether there was significant enrichment for H3K27ac and H3K4me1 marks, epigenetic signatures of enhancer function, on these haplotypes. We queried pathologically relevant cell types: B cells, endothelial cells, fibroblasts, monocytes, and T cells. We then identified the topologically associated domains (TADs) that encompass the SSc risk haplotypes in primary T cells to identify the full range of genes that may be influenced by SSc causal SNPs. We used gene ontology analyses of the genes within the TADs to gain insight into immunologic functions that might be affected by SSc causal SNPs. RESULTS: The SSc-associated haplotypes were enriched (p value < 0.01) for H3K4me1/H3K27ac marks in monocytes. Enrichment of one of the two histone marks was found in B cells, fibroblasts, and T cells. No enrichment was identified in endothelial cells. Ontological analyses of genes within the TADs encompassing the risk haplotypes showed enrichment for regulation of transcription, protein binding, activation of T lymphocytes, and proliferation of immune cells. CONCLUSIONS: The 11 non-HLA SSc risk haplotypes queried are highly enriched for H3K4me1/H3K27ac-marked regulatory elements in a broad range of immune cells and fibroblasts. Furthermore, in immune cells, the risk haplotypes belong to larger chromatin structures encompassing genes that regulate a wide array of immune processes associated with SSc pathogenesis. Though importance of the vasculature in the pathobiology of SSc is widely accepted, we were unable to find evidence for genetic influences on endothelial cell function in these regions.

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The development of chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing (ChIP-seq) technologies has promoted generation of large-scale epigenomics data, providing us unprecedented opportunities to explore the landscape of epigenomic profiles at scales across both histone marks and tissue types. In addition to many tools directly for data analysis, advanced computational approaches, such as deep learning, have recently become promising to deeply mine the data structures and identify important regulators from complex functional genomics data. We implemented a neural network framework, a Variational Auto-Encoder (VAE) model, to explore the epigenomic data from the Roadmap Epigenomics Project and the Encyclopedia of DNA Elements (ENCODE) project. Our model is applied to 935 reference samples, covering 28 tissues and 12 histone marks. We used the enhancer and promoter regions as the annotation features and ChIP-seq signal values in these regions as the feature values. Through a parameter sweep process, we identified the suitable hyperparameter values and built a VAE model to represent the epigenomics data and to further explore the biological regulation. The resultant Roadmap-ENCODE VAE (RE-VAE) model contained data compression and feature representation. Using the compressed data in the latent space, we found that the majority of histone marks were well clustered but not for tissues or cell types. Tissue or cell specificity was observed only in some histone marks (e.g., H3K4me3 and H3K27ac) and could be characterized when the number of tissue samples is large (e.g., blood and brain). In blood, the contributive regions and genes identified by RE-VAE model were confirmed by tissue-specificity enrichment analysis with an independent tissue expression panel. Finally, we demonstrated that RE-VAE model could detect cancer cell lines with similar epigenomics profiles. In conclusion, we introduced and implemented a VAE model to represent large-scale epigenomics data. The model could be used to explore classifications of histone modifications and tissue/cell specificity and to classify new data with unknown sources.

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The ubiquitin system regulates the DNA damage response (DDR) by modifying histone H2A at Lys15 (H2AK15ub) and triggering downstream signaling events. Here, we find that phosphorylation of ubiquitin at Thr12 (pUbT12) controls the DDR by inhibiting the function of 53BP1, a key factor for DNA double-strand break repair by non-homologous end joining (NHEJ). Detectable as a chromatin modification on H2AK15ub, pUbT12 accumulates in nuclear foci and is increased upon DNA damage. Mutating Thr12 prevents the removal of ubiquitin from H2AK15ub by USP51 deubiquitinating enzyme, leading to a pronounced accumulation of ubiquitinated chromatin. Chromatin modified by pUbT12 is inaccessible to 53BP1 but permissive to the homologous recombination (HR) proteins RNF169, RAD51, and the BRCA1/BARD1 complex. Phosphorylation of ubiquitin at Thr12 in the chromatin context is a new histone mark, H2AK15pUbT12, that regulates the DDR by hampering the activity of 53BP1 at damaged chromosomes.

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Histone H3 lysine-9 di-methylation (H3K9me2) and lysine-27 tri-methylation (H3K27me3) are linked to repression of gene expression, but the functions of repressive histone methylation dynamics during inflammatory responses remain enigmatic. Here, we report that lysine demethylases 7A (KDM7A) and 6A (UTX) play crucial roles in tumor necrosis factor (TNF)-α signaling in endothelial cells (ECs), where they are regulated by a novel TNF-α-responsive microRNA, miR-3679-5p. TNF-α rapidly induces co-occupancy of KDM7A and UTX at nuclear factor kappa-B (NF-κB)-associated elements in human ECs. KDM7A and UTX demethylate H3K9me2 and H3K27me3, respectively, and are both required for activation of NF-κB-dependent inflammatory genes. Chromosome conformation capture-based methods furthermore uncover increased interactions between TNF-α-induced super enhancers at NF-κB-relevant loci, coinciding with KDM7A and UTX recruitments. Simultaneous pharmacological inhibition of KDM7A and UTX significantly reduces leukocyte adhesion in mice, establishing the biological and potential translational relevance of this mechanism. Collectively, these findings suggest that rapid erasure of repressive histone marks by KDM7A and UTX is essential for NF-κB-dependent regulation of genes that control inflammatory responses of ECs.

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BACKGROUND: Breast cancer (BC) is a major health concern and better understanding of its biology might improve treatment decisions and patient outcomes. Histone3 Lysine27 tri-methylation (H3K27me3) is a post-translational histone modification frequently associated with altered gene expression. In BC patients, lower H3K27me3 expression has been associated with worse prognosis. We assessed H3K27me3 immunoexpression with digital imaging software assistance, in a cohort of luminal-like BC patients with long-term follow-up time and evaluated its association with clinically relevant endpoints and its clinical usefulness. METHODS: H3K27me3 immunoexpression was assessed, by means of digital-imaging system, in archival tissue samples of 160 luminal A/B-like HER2-negative invasive BC, stages I-III. Survival analysis was performed using Kaplan-Meier and Cox regression. Cases were categorized as 'low' or 'high' expression based on cut-off defined by receiver operating characteristic (ROC) curve analysis. RESULTS: The patient cohort showed a median age of 61-years, with a median follow-up time of 11.7 years. Low H3K27me3 expression (below 85% cut-off) was significantly associated with recurrence, both in univariable (HR = 1.99, 95%CI 1.066-3.724) and multivariable analysis when adjusting for grade and age (HR = 1.89, 95%CI 1.004-3.559). A trend for higher risk of death in low H3K27me3 expression BC was observed (p = 0.069), reaching statistical significance in younger patients (p = 0.021). CONCLUSIONS: H3K27me3 immunoexpression assessed by digital imaging scoring software is an independent prognosis biomarker in luminal-like BC patients and may assist in more individualized adjuvant treatment decisions, thus potentially reducing recurrences after curative-intent treatment, while sparing unnecessary toxicity.

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The role of the telomere repeat-binding factor 2 (TRF2) in telomere maintenance is well-established. However, recent findings suggest that TRF2 also functions outside telomeres, but relatively little is known about this function. Herein, using genome-wide ChIP-Seq assays of TRF2-bound chromatin from HT1080 fibrosarcoma cells, we identified thousands of TRF2-binding sites within the extra-telomeric genome. In light of this observation, we asked how TRF2 occupancy is organized within the genome. Interestingly, we found that extra-telomeric TRF2 sites throughout the genome are enriched in potential G-quadruplex-forming DNA sequences. Furthermore, we validated TRF2 occupancy at several promoter G-quadruplex motifs, which did adopt quadruplex forms in solution. TRF2 binding altered expression and the epigenetic state of several target promoters, indicated by histone modifications resulting in transcriptional repression of eight of nine genes investigated here. Furthermore, TRF2 occupancy and target gene expression were also sensitive to the well-known intracellular G-quadruplex-binding ligand 360A. Together, these results reveal an extensive genome-wide association of TRF2 outside telomeres and that it regulates gene expression in a G-quadruplex-dependent fashion.

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Genomic DNA is framed by additional layers of information, referred to as the epigenome. Epigenomic marks such as DNA methylation, histone modifications, and histone variants are concentrated on specific genomic sites, where they can both instruct and reflect gene expression. How this information is maintained, notably in the face of transcription, is not completely understood. Specifically, the extent to which modified histones themselves are retained through RNA polymerase II passage is unclear. Here, we show that several histone modifications are mislocalized when the transcription-coupled histone chaperones FACT or Spt6 are disrupted in Saccharomyces cerevisiae. In the absence of functional FACT or Spt6, transcription generates nucleosome loss, which is partially compensated for by the increased activity of non-transcription-coupled histone chaperones. The random incorporation of transcription-evicted modified histones scrambles epigenomic information. Our work highlights the importance of local recycling of modified histones by FACT and Spt6 during transcription in the maintenance of the epigenomic landscape.

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A hereditary system that is based on double-helix DNA sequences provides a stable way to store inherited traits and is favored by most life forms on Earth. However, emerging studies on the phenomenon of non-DNA sequence-based inheritance in multiple species, including mammals, urges a rethinking of the nature of hereditary information carriers and the ways in which they encode heritable traits. In this short opinion piece, we discuss how potential hereditary information carriers such as DNA-associated proteins, epigenetic marks, RNAs and organelles could function via distinct and synergistic modes of action in encoding and transmitting ancestral traits, either inherited or acquired from the environment. In particular, we discuss how the 3D structure of chromatin, in addition to its DNA sequence, may represent a higher-order carrier of hereditary information.

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Most BRAF-mutant melanoma patients experience a fulminate relapse after several months of treatment with BRAF/MEK inhibitors. To improve therapeutic efficacy, natural plant-derived compounds might be considered as potent additives. Here, we show that magnolol, a constituent of Magnolia officinalis, induced G1 arrest, apoptosis and cell death in BRAF- and NRAS-mutant melanoma cells at low concentration, with no effect in BRAF- and NRAS wild-type melanoma cells and human keratinocytes. This was confirmed in a 3D spheroid model. The apoptosis-inducing effect of magnolol was completely rescued by activating Akt suggesting a mechanism relying primarily on Akt signaling. Magnolol significantly downregulated the PI3K/Akt pathway which led to a global decrease of the active histone mark H3K4me3. Alongside, the repressive histone mark H3K9me3 was increased as a response to DNA damage. Magnolol-induced alterations of histone modifications are reversible upon activation of the Akt pathway. Magnolol-induced a synergistic effect in combination with either BRAF/MEK inhibitors dabrafenib/trametinib or docetaxel at a lower concentration than usually applied in melanoma patients. Combination of magnolol with targeted therapy or chemotherapy also led to analogous effects on histone marks, which was rescued by Akt pathway activation. Our study revealed a novel epigenetic mechanism of magnolol-induced cell death in melanoma. Magnolol might therefore be a clinically useful addition to BRAF/MEK inhibitors with enhanced efficacy delaying or preventing disease recurrence.

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Regulation of gene expression is achieved by sequence-specific transcriptional regulators, which convey the information that is contained in the sequence of DNA into RNA polymerase activity. This is achieved by the recruitment of transcriptional co-factors. One of the consequences of co-factor recruitment is the control of specific properties of nucleosomes, the basic units of chromatin, and their protein components, the core histones. The main principles are to regulate the position and the characteristics of nucleosomes. The latter includes modulating the composition of core histones and their variants that are integrated into nucleosomes, and the post-translational modification of these histones referred to as histone marks. One of these marks is the methylation of lysine 4 of the core histone H3 (H3K4). While mono-methylation of H3K4 (H3K4me1) is located preferentially at active enhancers, tri-methylation (H3K4me3) is a mark found at open and potentially active promoters. Thus, H3K4 methylation is typically associated with gene transcription. The class 2 lysine methyltransferases (KMTs) are the main enzymes that methylate H3K4. KMT2 enzymes function in complexes that contain a necessary core complex composed of WDR5, RBBP5, ASH2L, and DPY30, the so-called WRAD complex. Here we discuss recent findings that try to elucidate the important question of how KMT2 complexes are recruited to specific sites on chromatin. This is embedded into short overviews of the biological functions of KMT2 complexes and the consequences of H3K4 methylation.

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In eukaryotes, epigenetic-based mechanisms are involved in almost all the important biological processes. Amongst different epigenetic regulation pathways, the dynamic covalent modifications on histones are the most extensively investigated and characterized types. The covalent modifications on histone can be "read" by specific protein domains and then subsequently trigger downstream signaling events. Plants generally possess epigenetic regulation systems similar to animals and fungi, but also exhibit some plant-specific features. Similar to animals and fungi, plants require distinct protein domains to specifically "read" modified histones in both modification-specific and sequence-specific manners. In this review, we will focus on recent progress of the structural studies on the recognition of the epigenetic marks on histones by plant reader proteins, and further summarize the general and exceptional features of plant histone mark readers.

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