Multiple Histone Modifications Research Articles

The CoREST complex regulates multiple histone modifications temporal-specifically in clock neurons.

Epigenetic regulation is important for circadian rhythm. In previous studies, multiple histone modifications were found at the Period (Per) locus. However, most of these studies were not conducted in clock neurons. In our screen, we found that a CoREST mutation resulted in defects in circadian rhythm by affecting Per transcription. Based on previous studies, we hypothesized that CoREST regulates circadian rhythm by regulating multiple histone modifiers at the Per locus. Genetic and physical interaction experiments supported these regulatory relationships. Moreover, through tissue-specific chromatin immunoprecipitation assays in clock neurons, we found that the CoREST mutation led to time-dependent changes in corresponding histone modifications at the Per locus. Finally, we proposed a model indicating the role of the CoREST complex in the regulation of circadian rhythm. This study revealed the dynamic changes of histone modifications at the Per locus specifically in clock neurons. Importantly, it provides insights into the role of epigenetic factors in the regulation of dynamic gene expression changes in circadian rhythm.

The CoREST complex regulates multiple histone modifications temporal-specifically in clock neurons.

Epigenetic regulation is important for circadian rhythm. In previous studies, multiple histone modifications were found at the Period (Per) locus. However, most of these studies were not conducted in clock neurons. In our screen, we found that a CoREST mutation resulted in defects in circadian rhythm by affecting Per transcription. Based on previous studies, we hypothesized that CoREST regulates circadian rhythm by regulating multiple histone modifiers at the Per locus. Genetic and physical interaction experiments supported these regulatory relationships. Moreover, through tissue-specific chromatin immunoprecipitation assays in clock neurons, we found that the CoREST mutation led to time-dependent changes in corresponding histone modifications at the Per locus. Finally, we proposed a model indicating the role of the CoREST complex in the regulation of circadian rhythm. This study revealed the dynamic changes of histone modifications at the Per locus specifically in clock neurons. Importantly, it provides insights into the role of epigenetic factors in the regulation of dynamic gene expression changes in circadian rhythm.

F.2 Comprehensively mapping transcriptionally relevant histone modifications in aggressive meningioma leads to novel biologic insights and therapeutic vulnerabilities

Background: We recently identified four molecular subgroups of meningioma with distinct biology and outcomes. While two (MG3/MG4) are associated with poor outcome, they display divergent transcriptional profiles (enriched in metabolic and cell cycling pathways, respectively) and therapeutic vulnerabilities (MG3 has no clear treatment target). We sought to understand drivers of these key differences at a chromatin level. Methods: We profiled MG3/MG4 meningiomas for common histone marks H3K27me3, H3K27Ac, H3K4me1, H3K4me3, H3K9me3, and H3K36me3. Multiple computational approaches were used to compare MG3 and MG4 tumours including superenhancer ranking, differential binding analysis, and unsupervised clustering. Results: Our cohort includes 11-20 meningiomas per histone mark. Clustering revealed striking separation of subgroups based on multiple histone marks, particularly H3K36me3. FOXC1, a known driver of the epithelial to mesenchymal transition, was identified as a recurrent superenhancer in both groups, whereas MG3-specific superenhancers mapped to immune regulatory networks. Integrated differential binding analysis confirmed an immune-rich microenvironment in MG3 tumours driven by multiple histone marks, suggesting a role for targeting novel immune checkpoint genes CD84 and CD48. Conclusions: This study is the first to apply integrated analysis of multiple histone modifications to aggressive meningioma. We further characterize MG3 tumours by identifying an epigenetically-driven immune phenotype and propose novel treatment targets.

Coregulatory effects of multiple histone modifications in key ferroptosis-related genes for lung adenocarcinoma.

Aim: The present study was designed to investigate the coregulatory effects ofmultiple histone modifications (HMs) on gene expression in lung adenocarcinoma (LUAD). Materials & methods: Ten histones forLUAD were analyzed usingChIP-seq and RNA-seq data. An innovative computational method is proposed to quantify the coregulatory effects of multiple HMs on gene expressionto identify strong coregulatory genes and regions. This method was appliedto explore the coregulatory mechanisms of key ferroptosis-related genes in LUAD. Results: Nine strong coregulatory regions were identified for six ferroptosis-related genes with diverse coregulatory patterns (CA9, PGD, CDKN2A, PML, OTUB1 and NFE2L2). Conclusion: This quantitative method could be usedto identify important HMcoregulatory genes and regions that may be epigenetic regulatory targets in cancers.

Deletion of cis-regulatory Element in FOXL2 Promoter in a Chinese Family of Type II Blepharophimosis-ptosis-epicanthus Inversus Syndrome with Polydactyly.

Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) is a relatively uncommon autosomal-dominant genetic disorder, primarily attributed to mutations in the forkhead box L2 (FOXL2) gene. Albeit the involvement of protein-coding regions of FOXL2 has been observed in the majority of BPES cases, whether deficiencies in regulatory elements lead to the pathogenesis remains poorly understood. Herein, an autosomal-dominant BPES type II family was included. Peripheral venous blood has been collected, and genomic DNA has been extracted from leukocytes. A whole exome sequencing analysis has been performed and analyzed (Deposited in NODE database: OER422653). The promoter region of FOXL2 was amplified using polymerase chain reaction (PCR). The luciferase reporter assay was performed to identify the activity of this region. In this study, we present a Chinese family diagnosed with type II BPES, characterized by the presence of small palpebral fissures, ptosis, telecanthus, and epicanthus inversus. Notably, all male individuals within the family display polydactyly. A 225-bp deletion in the 556-bp 5'-upstream to transcription start site of FOXL2 , decorated by multiple histone modifications, was identified in affected members of the family. This deletion significantly decreased FOXL2 promoter activity, as measured by the luciferase assay. Conclusively, a novel 255-bp-deletion of the FOXL2 promoter was identified in Chinese families with BPES. Our results expand the spectrum of known FOXL2 mutations and provide additional insight into the genotype-phenotype relationships of the BPES pathogenesis. In addition, this study indicates the important role of genetic screening of cis-regulatory elements in testing heritable diseases.

Melatonin ameliorates histone modification disorders in mammalian aged oocytes by neutralizing the alkylation of HDAC1

Aging-associated histone modification changes in oocytes have been sporadically reported, but the underlying mechanisms remain elusive. Here, we systematically characterize multiple histone modifications in oocytes during aging. We find that maternal and postovulatory aging markedly alter the status of histone modifications, specifically H4K12ac and H3K4me3, in both mouse and porcine oocytes. Meanwhile, we identify a substantial reduction in HDAC1 (histone deacetylase 1) protein in aged oocytes, which contributes to the changes in H4K12ac and H3K4me3. Moreover, by employing methylglyoxal (MG) and site-directed mutagenesis, we demonstrate that the elevated reactive carbonyl species (RCS) level induces HDAC1 degradation, likely through attacking the cysteine residues, thereby influences histone modification state. Importantly, supplementation of melatonin not only prevents the loss of HDAC1 protein, but also partially corrects the H4K12ac and H3K4me3 status in aged oocytes. To sum up, this study established the link between redox disequilibrium and histone modification alterations during mammalian oocyte aging.

PKM2 dictates the poised chromatin state of PFKFB3 promoter to enhance breast cancer progression.

The hypoxic milieu is a critical modulator of aerobic glycolysis, yet the regulatory mechanisms between the key glycolytic enzymes in hypoxic cancer cells are largely unchartered. In particular, the M2 isoform of pyruvate kinase (PKM2), the rate-limiting enzyme of glycolysis, is known to confer adaptive advantages under hypoxia. Herein, we report that non-canonical PKM2 mediates HIF-1α and p300 enrichment at PFKFB3 hypoxia-responsive elements (HREs), causing its upregulation. Consequently, the absence of PKM2 activates an opportunistic occupancy of HIF-2α, along with acquisition of a poised state by PFKFB3 HREs-associated chromatin. This poised nature restricts HIF-2α from inducing PFKFB3 while permitting the maintenance of its basal-level expression by harboring multiple histone modifications. In addition, the clinical relevance of the study has been investigated by demonstrating that Shikonin blocks the nuclear translocation of PKM2 to suppress PFKFB3 expression. Furthermore, TNBC patient-derived organoids and MCF7 cells-derived xenograft tumors in mice exhibited substantial growth inhibition upon shikonin treatment, highlighting the vitality of targeting PKM2. Conclusively, this work provides novel insights into the contributions of PKM2 in modulating hypoxic transcriptome and a previously unreported poised epigenetic strategy exhibited by the hypoxic breast cancer cells for ensuring the maintenance of PFKFB3 expression.

Multi-omics profiling of cholangiocytes reveals sex-specific chromatin state dynamics during hepatic cystogenesis in polycystic liver disease

Cholangiocytes transit from quiescence to hyperproliferation during cystogenesis in polycystic liver disease (PLD), the severity of which displays prominent sex differences. Epigenetic regulation plays important roles in cell state transition. We aimed to investigate the sex-specific epigenetic basis of hepatic cystogenesis and to develop therapeutic strategies targeting epigenetic modifications for PLD treatment. Normal and cystic primary cholangiocytes were isolated from wild-type and PLD mice of both sexes. Chromatin states were characterized by analyzing chromatin accessibility (ATAC sequencing) and multiple histone modifications (chromatin immunoprecipitation sequencing). Differential gene expression was determined by transcriptomic analysis (RNA sequencing). Pharmacologic inhibition of epigenetic modifying enzymes was undertaken in PLD model mice. Through genome-wide profiling of chromatin dynamics, we revealed a profound increase of global chromatin accessibility during cystogenesis in both male and female PLD cholangiocytes. We identified a switch from H3K9me3 to H3K9ac on cis-regulatory DNA elements of cyst-associated genes and showed that inhibition of H3K9ac acetyltransferase or H3K9me3 demethylase slowed cyst growth in male, but not female, PLD mice. In contrast, we found that H3K27ac was specifically increased in female PLD mice and that genes associated with H3K27ac-gained regions were enriched for cyst-related pathways. In an integrated epigenomic and transcriptomic analysis, we identified an estrogen receptor alpha-centered transcription factor network associated with the H3K27ac-regulated cystogenic gene expression program in female PLD mice. Our findings highlight the multi-layered sex-specific epigenetic dynamics underlying cholangiocyte state transition and reveal a potential epigenetic therapeutic strategy for male PLD patients. In the present study, we elucidate a sex-specific epigenetic mechanism underlying the cholangiocyte state transition during hepatic cystogenesis and identify epigenetic drugs that effectively slow cyst growth in male PLD mice. These findings underscore the importance of sex difference in the pathogenesis of PLD and may guide researchers and physicians to develop sex-specific personalized approaches for PLD treatment.

Abstract PR006: Single-molecule and single-cell epigenetics: Decoding the epigenome for cancer research and diagnostics

Abstract Genes and genomic elements are packaged by chromatin structures that regulate their activity. We developed a novel high-throughput single-molecule imaging technology to decode combinatorial modifications on millions of individual nucleosomes. We apply this technology to image nucleosomes and delineate their combinatorial epigenetic patterns, and how these patterns are deregulated in cancer. In addition, we adapt single-cell technologies based on CyTOF to profile the global levels of multiple histone modifications in single cells, thus revealing epigenetic heterogeneity in cancer. Our research focuses on pediatric gliomas harboring lysine to methionine substitution of residue 27 on histone H3 (K27M). We provide evidence for widespread effects of the H3-K27M oncohistone on multiple core epigenetic pathways, and highlight the capability of single-molecule and single-cell tools to reveal mechanisms of chromatin deregulation and heterogeneity in cancer. We also harness the single-molecule technology as a novel liquid biopsy approach, by comprehensively profiling combinatorial epigenetic marks of plasma-isolated nucleosomes. Applying this analysis to a cohort of plasma samples detect colorectal cancer at high accuracy and sensitivity, even at early stages. Finally, combining this proteomic analysis with single-molecule DNA sequencing reveals the tissue-of-origin of the tumor. Citation Format: Efrat Shema. Single-molecule and single-cell epigenetics: Decoding the epigenome for cancer research and diagnostics. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr PR006.

Genome-wide profiling of histone modifications in Plasmodium falciparum using CUT&RUN.

We recently adapted a CUT&RUN protocol for genome-wide profiling of chromatin modifications in the human malaria parasite Plasmodium Using the step-by-step protocol described below, we were able to generate high-quality profiles of multiple histone modifications using only a small fraction of the cells required for ChIP-seq. Using antibodies against two commonly profiled histone modifications, H3K4me3 and H3K9me3, we show here that CUT&RUN profiling is highly reproducible and closely recapitulates previously published ChIP-seq-based abundance profiles of histone marks. Finally, we show that CUT&RUN requires substantially lower sequencing coverage for accurate profiling compared with ChIP-seq.

Histone Modification Analysis of Low-Mappability Regions.

Posttranslational modifications of histone are intimately related to chromatin/chromosome-mediated cellular events. Among all, the roles of histone modifications including acetylation, methylation, ubiquitination, and SUMOylation of lysine or arginine residue of nucleosome core histones in gene expression have been intensively studied. Genome-wide profiles of histone modification marks revealed their combinatorial organization in the functional features of chromatin. Analysis of histone modification by chromatin immunoprecipitation (ChIP) is one of the standard assays to examine chromatin states. Although high-throughput sequencing analysis (ChIP-seq) is now widely conducted, classical ChIP-qPCR analysis has advantages in investigation of multiple histone modification marks at a target site simply through theuse ofrelatively small numbers of cells. Since ChIP-qPCR is devoid of biases caused by overamplification and inaccurate mapping of sequencing reads, it is a more reliable quantification method than genome-wide ChIP-seq especially for analyses of the low-mappability regions, which harbor many repetitive sequences and/or highly homologous segmental multiplications as found in gene clusters. We have recently analyzed histone H3 and H4 modifications of the Zscan4 family gene loci in an 880kb gene cluster and found that the atypical enhancer-like structure is formed upon derepression of Zscan4. In this chapter, we describe the detailed protocols for histone modification ChIP-assay of repeat-enriched gene cluster regions. The protocol here we applied to mouse ES cells, but the protocol is perfectly applicableto humanculturedcellsand specimens.

B-scaling: A novel nonparametric data fusion method

Very often for the same scientific question, there may exist different techniques or experiments that measure the same numerical quantity. Historically, various methods have been developed to exploit the information within each type of data independently. However, statistical data fusion methods that could effectively integrate multisource data under a unified framework are lacking. In this paper we propose a novel data fusion method, called B-scaling, for integrating multisource data. Consider K measurements that are generated from different sources but measure the same latent variable through some linear or nonlinear ways. We seek to find a representation of the latent variable, named B-mean, which captures the common information contained in the K measurements while taking into account the nonlinear mappings between them and the latent variable. We also establish the asymptotic property of the B-mean and apply the proposed method to integrate multiple histone modifications and DNA methylation levels for characterizing epigenomic landscape. Both numerical and empirical studies show that B-scaling is a powerful data fusion method with broad applications.

Thymocyte regulatory variant alters transcription factor binding and protects from type 1 diabetes in infants

We recently mapped a genetic susceptibility locus on chromosome 6q22.33 for type 1 diabetes (T1D) diagnosed below the age of 7 years between the PTPRK and thymocyte-selection-associated (THEMIS) genes. As the thymus plays a central role in shaping the T cell repertoire, we aimed to identify the most likely causal genetic factors behind this association using thymocyte genomic data. In four thymocyte populations, we identified 253 DNA sequence motifs underlying histone modifications. The G insertion allele of rs138300818, associated with protection from diabetes, created thymocyte motifs for multiple histone modifications and thymocyte types. In a parallel approach to identifying variants that alter transcription factor binding motifs, the same variant disrupted a predicted motif for Rfx7, which is abundantly expressed in the thymus. Chromatin state and RNA sequencing data suggested strong transcription overlapping rs138300818 in fetal thymus, while expression quantitative trait locus and chromatin conformation data associate the insertion with lower THEMIS expression. Extending the analysis to other T1D loci further highlighted rs66733041 affecting the GATA3 transcription factor binding in the AFF3 locus. Taken together, our results support a role for thymic THEMIS gene expression and the rs138300818 variant in promoting the development of early-onset T1D.

Multi-omics to characterize the functional relationships of R-loops with epigenetic modifications, RNAPII transcription and gene expression.

Abnormal accumulation of R-loops results in replication stress, genome instability, chromatin alterations and gene silencing. Little research has been done to characterize functional relationships among R-loops, histone marks, RNA polymerase II (RNAPII) transcription and gene regulation. We built extremely randomized trees (ETs) models to predict the genome-wide R-loops using RNAPII and multiple histone modifications chromatin immunoprecipitation (ChIP)-seq, DNase-seq, Global Run-On sequencing (GRO-seq) and R-loop profiling data. We compared the performance of ET models to multiple machine learning approaches, and the proposed ET models achieved the best and extremely robust performances. Epigenetic profiles are highly predictive of R-loops genome-widely and they are strongly associated with R-loop formation. In addition, the presence of R-loops is significantly correlated with RNAPII transcription activity, H3K4me3 and open chromatin around the transcription start site, and H3K9me1 and H3K9me3 around the transcription termination site. RNAPII pausing defects were correlated with 5'R-loops accumulation, and transcriptional termination defects and read-throughs were correlated with 3'R-loops accumulation. Furthermore, we found driver genes with 5'R-loops and RNAPII pausing defects express significantly higher and genes with 3'R-loops and read-through transcription express significantly lower than genes without R-loops. These driver genes are enriched with chromosomal instability, Hippo-Merlin signaling Dysregulation, DNA damage response and TGF-β pathways, indicating R-loops accumulating at the 5' end of genes play oncogenic roles, whereas at the 3' end of genes play tumor-suppressive roles in tumorigenesis.

Low Levels of Perfluorooctanoic Acid Exposure Activates Steroid Hormone Biosynthesis through Repressing Histone Methylation in Rats.

Perfluorooctanoic acid (PFOA) is a persistent organic pollutant, which has endocrine-disrupting properties and can interfere with the synthesis and secretion of testicular steroid hormones, but the underlying molecular mechanisms are still not fully understood. In this study, we investigated the effects of low doses of PFOA exposure on testicular steroidogenesis in rats and revealed the role of histone modifications. It was found that the serum levels of progesterone, testosterone, and estradiol were significantly increased after 0.015 and 0.15 mg/kg of PFOA exposure, and the expression of Star, a key rate-limiting gene, was up-regulated, while other steroidogenic genes Cyp11a1, Hsd3b, Cyp17a1, and Hsd17b were down-regulated. In addition, the levels of multiple histone modifications (H3K9me1/2/3 and H3K9/18/23ac) were all significantly reduced by PFOA in rat testis. Histone H3K9 methylation is associated with gene silencing, while histone acetylation leads to gene activation. ChIP analysis further showed that H3K9me1/3 was significantly decreased in the promoter region of Star, while H3K18ac levels were down-regulated in other gene promoters. Accordingly, we suggest that low-level PFOA enhances StAR expression through the repression of H3K9me1/3, which stimulates steroid hormone production in rat testis. These results are expected to shed new light on the molecular mechanisms by which low-dose PFOA disturbs male reproductive endocrine from an epigenetic aspect and may be useful for human health risk assessment regarding environmental PFOA exposure.

Mapping cis-regulatory elements in the midgestation mouse placenta

The placenta is a temporary organ that provides the developing fetus with nutrients, oxygen, and protection in utero. Defects in its development, which may be caused by misregulated gene expression, can lead to devastating outcomes for the mother and fetus. In mouse, placental defects during midgestation commonly lead to embryonic lethality. However, the regulatory mechanisms controlling expression of genes during this period have not been thoroughly investigated. Therefore, we generated and analyzed ChIP-seq data for multiple histone modifications known to mark cis-regulatory regions. We annotated active and poised promoters and enhancers, as well as regions generally associated with repressed gene expression. We found that poised promoters were associated with neuronal development genes, while active promoters were largely associated with housekeeping genes. Active and poised enhancers were associated with placental development genes, though only active enhancers were associated with genes that have placenta-specific expression. Motif analysis within active enhancers identified a large network of transcription factors, including those that have not been previously studied in the placenta and are candidates for future studies. The data generated and genomic regions annotated provide researchers with a foundation for future studies, aimed at understanding how specific genes in the midgestation mouse placenta are regulated.

Multiplexed single-cell profiling of chromatin states at genomic loci by expansion microscopy.

Proper regulation of genome architecture and activity is essential for the development and function of multicellular organisms. Histone modifications, acting in combination, specify these activity states at individual genomic loci. However, the methods used to study these modifications often require either a large number of cells or are limited to targeting one histone mark at a time. Here, we developed a new method called Single Cell Evaluation of Post-TRanslational Epigenetic Encoding (SCEPTRE) that uses Expansion Microscopy (ExM) to visualize and quantify multiple histone modifications at non-repetitive genomic regions in single cells at a spatial resolution of ∼75 nm. Using SCEPTRE, we distinguished multiple histone modifications at a single housekeeping gene, quantified histone modification levels at multiple developmentally-regulated genes in individual cells, and evaluated the relationship between histone modifications and RNA polymerase II loading at individual loci. We find extensive variability in epigenetic states between individual gene loci hidden from current population-averaged measurements. These findings establish SCEPTRE as a new technique for multiplexed detection of combinatorial chromatin states at single genomic loci in single cells.

Dynamics of transcription-mediated conversion from euchromatin to facultative heterochromatin at the Xist promoter by Tsix

The fine-scale dynamics from euchromatin (EC) to facultative heterochromatin (fHC) has remained largely unclear. Here, we focus on Xist and its silencing initiator Tsix as a paradigm of transcription-mediated conversion from EC to fHC. In mouse epiblast stem cells, induction of Tsix recapitulates the conversion at the Xist promoter. Investigating the dynamics reveals that the conversion proceeds in a stepwise manner. Initially, a transient opened chromatin structure is observed. In the second step, gene silencing is initiated and dependent on Tsix, which is reversible and accompanied by simultaneous changes in multiple histone modifications. At the last step, maintenance of silencing becomes independent of Tsix and irreversible, which correlates with occupation of the -1 position of the transcription start site by a nucleosome and initiation of DNA methylation introduction. This study highlights the hierarchy of multiple chromatin events upon stepwise gene silencing establishment.

Iterative epigenomic analyses in the same single cell.

Gene expression in individual cells is epigenetically regulated by DNA modifications, histone modifications, transcription factors, and other DNA-binding proteins. It has been shown that multiple histone modifications can predict gene expression and reflect future responses of bulk cells to extracellular cues. However, the predictive ability of epigenomic analysis is still limited for mechanistic research at a single cell level. To overcome this limitation, it would be useful to acquire reliable signals from multiple epigenetic marks in the same single cell. Here, we propose a new approach and a new method for analysis of several components of the epigenome in the same single cell. The new method allows reanalysis of the same single cell. We found that reanalysis of the same single cell is feasible, provides confirmation of the epigenetic signals, and allows application of statistical analysis to identify reproduced reads using data sets generated only from the single cell. Reanalysis of the same single cell is also useful to acquire multiple epigenetic marks from the same single cells. The method can acquire at least five epigenetic marks: H3K27ac, H3K27me3, mediator complex subunit 1, a DNA modification, and a DNA-interacting protein. We can predict active signaling pathways in K562 single cells using the epigenetic data and confirm that the predicted results strongly correlate with actual active signaling pathways identified by RNA-seq results. These results suggest that the new method provides mechanistic insights for cellular phenotypes through multilayered epigenome analysis in the same single cells.

Inferring time series chromatin states for promoter-enhancer pairs based on Hi-C data

BackgroundCo-localized combinations of histone modifications (“chromatin states”) have been shown to correlate with promoter and enhancer activity. Changes in chromatin states over multiple time points (“chromatin state trajectories”) have previously been analyzed at promoter and enhancers separately. With the advent of time series Hi-C data it is now possible to connect promoters and enhancers and to analyze chromatin state trajectories at promoter-enhancer pairs.ResultsWe present TimelessFlex, a framework for investigating chromatin state trajectories at promoters and enhancers and at promoter-enhancer pairs based on Hi-C information. TimelessFlex extends our previous approach Timeless, a Bayesian network for clustering multiple histone modification data sets at promoter and enhancer feature regions. We utilize time series ATAC-seq data measuring open chromatin to define promoters and enhancer candidates. We developed an expectation-maximization algorithm to assign promoters and enhancers to each other based on Hi-C interactions and jointly cluster their feature regions into paired chromatin state trajectories.We find jointly clustered promoter-enhancer pairs showing the same activation patterns on both sides but with a stronger trend at the enhancer side. While the promoter side remains accessible across the time series, the enhancer side becomes dynamically more open towards the gene activation time point. Promoter cluster patterns show strong correlations with gene expression signals, whereas Hi-C signals get only slightly stronger towards activation.The code of the framework is available at https://github.com/henriettemiko/TimelessFlex.ConclusionsTimelessFlex clusters time series histone modifications at promoter-enhancer pairs based on Hi-C and it can identify distinct chromatin states at promoter and enhancer feature regions and their changes over time.