Testis-specific histone variants are crucial to promote open chromatin structure to enable nucleosome disassembly in the final stages of spermiogenesis. However, even after histone replacement, mature sperm retain a proportion of these variants, the function of which is unknown. The present study aimed to understand the functional relevance of the retained H2B and H2A variants, TH2B and TH2A. While no literature is available on the phenotype of TH2A knockouts, TH2B/TH2A double knockout male mice are reported to be infertile. In this study, ChIP-seq analysis was done for TH2B and TH2A to understand the epigenomics of the retained TH2B and TH2A, using murine caudal sperm. Distribution across genomic partitions revealed ∼35% of the TH2B peaks within ±5 kb of TSS whereas TH2A peaks distribution was sparse at TSS. Gene Ontology revealed embryo development as the most significant term associated with TH2B. Also, based on genomic regions, TH2B was observed to be associated with spindle assembly and various meiosis-specific genes, which is an important finding as TH2A/TH2B DKO mice have been reported to have defective cohesin release. A comparison of mouse and human TH2B-linked chromatin revealed 26% overlap between murine and human TH2B-associated genes. This overlap included genes crucial for embryogenesis. Most importantly, heterogeneity in the epigenetic landscape of TH2A and TH2B was seen, which is intriguing as TH2B and TH2A are well reported to be present in the same nucleosomes to promote open chromatin. Additionally, unlike TH2B, TH2A was enriched on the mitochondrial chromosome. TH2A was found to be associated with Nuclear insertion of Mitochondrial DNA sequences (NUMTs) in sperm. A comprehensive analysis of these observations indicates novel functions for the sperm-retained TH2B and TH2A. Mature sperm and round spermatids harbor the same haploid genome but different epigenomes. Developmental limitations of embryos resulting from somatic cell nuclear transfer (SCNT) and round spermatid injection (ROSI) as compared to sperm borne embryos reveal the importance of the unique sperm chromatin in efficient embryogenesis . The negNucleosome, being an octamer, contains two copies each of H2A, H2B, H3, and H4 draped by two turns of duplex DNA . The porGerm cell-specific variants of H2B and H2A, namely, TH2B (Hist1h2ba) and TH2A (Hist1h2aa), were first reported in mammalian testis . SubsequCrystal structure analysis comparing nucleosomal core particles (NCP) revealed that TH2A/TH2B containing nucleosomes have decreased hydrogen bonds and fewer DNA-histone contacts as compared to canonical counterparts. This leads to a more open chromatin in TH2A/TH2B nucleosome , 2017. IThe TH2A/TH2B duo is crucial for spermatogenesis and chromatin structure. Double knockout mouse of TH2A and TH2B show male sterility owing to loss of cohesin release during interkinesis and defects in transition protein 2 (TP2) incorporation and histone replacement . These lad libitum. For experiments, mice were euthanized via cervical dislocation.BALB/c-129-E mice (8–12 weeks old) were used in this study. Mouse husbandry and experimental procedures were carried out as per the guidelines of the National Institute for Research in Reproductive Health Institutional Animal Ethics Committee (NIRRH-IAEC). All animal experiments were approved by the NIRRH-IAEC (project no. 07/18). Mice were housed in groups of four per cage under conditions of 12-h light–dark cycle and provided with water and food g*30 min at 4°C to eliminate somatic cells, if any. Quantity and purity of sperm was determined using Neubauer chamber. On an average, sperm yield per mouse  was ∼5–6 million.Mouse sperm were isolated as per the protocol described by g for 10 min at 4°C. The pellet was washed thrice by centrifugation as above and resuspended in 250 µl of 2D lysis buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 0.1 M DTT and protease inhibitor cocktail (Roche).Western blot analysis of TH2A and TH2B was done to detect their presence in mouse caudal sperm (mature sperm). Whole testicular cell lysates were used as positive control. Towards this, after removing the tunica albuginea, the seminiferous tubules were teased out in PBS and incubated at 37°C for 1 h at 70 rpm. Following this, the testicular cell suspension was filtered through 40-µm Falcon™ Cell Strainers (Fisher scientific) and pelleted by centrifugation at 2,000g for 10 min at 4°C, pellet washed as described above, and resuspended in 150 µl of 2D lysis buffer. Caudal sperm suspension and whole testicular cell lysate were incubated on a rotary shaker overnight at 4°C. The following day, the lysates were subjected to bead beating homogenization in a Fast prep-24 homogenizer . The homogenates were incubated on ice for an hour and then centrifuged at 16,000 g for 30 min at 4°C. The supernatants containing the protein lysate were quantified for protein content using the 2-D Quant Kit .Caudal sperm (∼8 million cells) collected by the sperm swim-up procedure were initially treated with 10 mM dithiothreitol (DTT) in PBS at room temperature for 1 h to release the protamines. The DTT pre-treated sperm were then pelleted by centrifugation at 2,000 trans-blotted on nitrocellulose membrane at 100 V for 1 h Non-specific binding to the membrane was blocked with 3% NFDM in 0.1 M PBS (blocking buffer) for 1 h at room temperature. Next, these membranes were incubated with primary antibody overnight at 4°C, followed by two washes with PBS containing 0.05% Tween 20 (PBST). The membranes were subsequently incubated with HRP-conjugated secondary antibody for 1 h at RT. The unbound antibodies were eliminated by washing the membrane thrice with 0.05% PBST. Chemiluminescence detection of protein of interest was done using Western Blot Chemiluminescence HRP substrate (Takara-bio), in ChemiDoc imager (Bio-Rad).For Western analysis, 200 µg of caudal sperm for TH2A and 100 µg for TH2B and 50 µg testicular cell lysates were resuspended in Laemmli buffer containing 25% 2-mercaptoethanol, 0.01% bromophenol blue, 50% Glycerol, 20% SDS, and 0.25 M Tris-HCl, pH 6.8. Samples were heated for 5 min at 95°C and subjected to 15% SDS-PAGE at 100 V for 2 h and Primary antibodies used were rabbit anti-mouse TH2A  at a dilution of 1:250 and rabbit anti mouse TH2B Antibody (Sigma-Aldrich #07–680) at a dilution of 1:5,000. Secondary antibody used was HRP-conjugated swine anti-rabbit IgG (Dako) at a dilution of 1:1,500 in 1% blocking buffer.The protocol described by g for 10 min at 4°C to pellet down the denser protamine-bound DNA, and the supernatant containing mononucleosomal chromatin was retrieved. Preclearing of the MNase-digested chromatin was done with blocked protein-A Sepharose beads  for 1 h. The chromatin (from 12 million sperm in two replicates) was then immunoprecipitated overnight with 6 µg each of either TH2A or TH2B antibody. The following day, blocked beads were added and incubated with end-to-end mixing at 4°C for 4 h, to capture the antibody chromatin complexes.The MNase digestion was stopped by addition of 0.5 M EDTA. The digestion mixture was centrifuged at 16,000 g for 2 min at RT. The eluted (∼300 µl) immunoprecipitated chromatin DNA was treated with 6 µl of RNaseA (10 mg/ml) at 37°C for 30 min and overnight incubation at 56°C with 6 µl of proteinase K (10 mg/ml) and extracted via column-based DNA extraction. Mononucleosomal DNA (one-tenth of the IP reaction) was saved as input. DNA quality and concentration were evaluated using Qubit Fluorometric Quantification (Invitrogen Qubit™ four Fluorometer #Q33226). The entire eluate was mixed with 6× DNA loading dye  and electrophoresed on a 5% polyacrylamide gel for 45 min at 100 V with 0.1 M TBE as electrode buffer. The gel was post-stained with ethidium bromide. Mononucleosomal DNA band at ∼150 bp was excised and the mononucleosomal DNA was re-precipitated and subjected to sequencing after pooling the replicates.The antibody–bead complexes were washed by centrifugation  and the chromatin was eluted in two rounds in Tris-EDTA buffer, pH 8.0, containing 10 mM Tris-HCl, 1 mM disodium EDTA, and sodium dodecyl sulfate (1%) by centrifugation at 11,000 www.bioinformatics.babraham.ac.uk/projects/fastqc): 1) Base quality score distribution, 2) Sequence quality score distribution, 3) Average base content per read, 4) GC distribution in the reads, 5) PCR amplification issue, and 6) Checking for overrepresented sequences (Ver-0.36) (Mus Musculus (mm10) release downloaded from Sanger Institute database. Alignment was performed using BWA MEM (Ver-0.7.12)  using HOMER (annotatepeaks)  indicates a range of 100–1,000 with majority of them being approximately 100–400. We have used BedSect V3 tool for peaks classification into common and unique peaks across each sample  (Agilent). Sequencing was performed with Illumina Hiseq using 150 bp paired end sequencing. For Input and TH2B ChIP-Seq libraries and for TH2A, ∼30 million and 34 million unique paired end reads were attained, respectively. The following parameters from fastq file were determined using FastQC (equences . Based oer-0.36)  to retai-0.7.12) . The ali-0.7.12) . Statisttepeaks) . The lenh sample .https://bioviz.org/)  was the same for both TH2A and TH2B graph tracks for comparisons. Heatmaps of different BigWig coverage ChIP signals for sperm PTMs (from GSE79227) on TH2B peaks were generated using Deeptools 3.3.2, computeMatrix (computeMatrix 3.3.2), and plotHeatmap (plotHeatmap 3.3.2) function in Galaxy server   was done for the enrichment of annotated peaks over the genomic features and the distance from TSS, and visualized using the GenomicDistribution package in the LOLAweb tool, which is based on LOLA R package (LOLAweb version: 2ddd6d682) . Assessmiz.org/) . Relativ3.2.0.1) . Mouse s3.2.0.1) . All the3.2.0.1) .http://chip-atlas.org), peaks associated with primordial germ cells (PGCs), spermatogonial stem cells (SSC), round spermatids, and sperm with a threshold significance of more than 200 were viewed on IGV Ver 2.8.13  in 25 μl of total volume. Two individual 10-μl reactions were set up in CFX96 Touch Real-Time PCR Detection System (Bio-Rad). The PCR conditions were as follows: 95°C for 3 min; 95°C for 30 s, appropriate annealing/extension and plate read for 30 s for 40 cycles, and melt curve analysis 75–95°C at 0.5°C increment at 5 s/step.The enrichment of gene regions identified to be associated with TH2A/TH2B by ChIP assays were confirmed by quantitative real-time PCR in triplicate. MACS2 peak specific primers were generated for TH2A-associated genes—and Mxi1 . Input wChIP DNA enrichment was calculated as the percent of input  using the formula:100*2^ (Adjusted Input—Ct (IP))http://bejerano.stanford.edu/great/public/html/index.php) version 4.0.4 was used for functional analysis of TH2A/TH2B-associated genomic regions  for Mouse: GRCm38  (Genomic Regions Enrichment of Annotations Tool (GREAT) (c. 2011) . The defhttps://www.ncbi.nlm.nih.gov/research/HistoneDB2.0/):Protein FASTA sequences for the following canonical and testis-specific histone variants were retrieved from HistoneDB2.0 . Coloring of the residues according to the hydrophobicity was also performed using Jalview .Histone variants differ from their replication-dependent canonical counterparts in terms of protein sequences and domains. This difference in sequences may ultimately lead to recruitment of entirely new post-translational modifications (PTMs) and novel interacting chromatin modeling enzymes or modifiers and consequently an altogether new epigenetic profile as compared to that of the somatic histone. Thus, it becomes crucial to closely study the amino acid sequences of these variants. Also, since mice is an experimental model for mammalian studies, to understand the evolutionary conservation of the variants TH2B and TH2A, their homology in protein sequences was assessed by pairwise global alignment of TH2B and TH2A in human and mouse. This revealed ∼95% identity between human and mouse TH2B and ∼89% homology between amino acid sequence of human and mouse TH2A, which differ mostly at the C-terminal . Within Hist1h2aa and Hist1h2ba genes were epigenetically profiled across the murine male germ cell stages, starting from PGCs to sperm using multiple ChIP-seq data from ChIP atlas  . We therIP atlas . Only hiIP atlas . ChIP-seIP atlas . In the H2A/TH2B .H3f3b and H1fnt. A profile similar to that of TH2A/TH2B was found for H3f3b (H3.3) whereas H1fnt, which is involved in spermiogenesis, predominantly showed H4ac and H3K9ac marks in round spermatids and H3K4me3 in sperm . A flow chart depicting a brief summary of the bioinformatics analysis is shown in www.bioinformatics.babraham.ac.uk/projects/goliath/). A chromosome wise coverage map was generated  of binary alignment files in Integrated Genome Browser (IGB)  revealedenerated . Consideenerated , an occuTH2B association was noted on all autosomes and sex chromosomes except the mitochondrial (MT) chromosome. However, unlike TH2B, a staggered association of TH2A binding loci with genic regions was seen, with binding sites only on chromosomes 1, 4–8, 10–13, 17–19, MT, X, and Y. A fair enrichment was seen on MT chromosome. Locus enrichment overlap analysis was done using LOLAweb . TH2A- acis-regulatory interactions of TH2A/TH2B with their associated loci) and GOliath (gene list-based Gene Ontology) were used for Gene Ontology analyses of the genomic regions for TBAG and TAAG lists  is an endodeoxyribonuclease, important for the resolution of Holliday Junctions (HJs), which is essential for segregation of chromosomes. Yeast Slx4’s mammalian ortholog, BTBD12 mutants are sub-fertile with almost under 15% normal spermatozoa  is an important meiotic synaptonemal complex protein part of axial elements/lateral elements. Male mutants of Sycp2 have defects in meiosis with failure in axial element formation and are sterile (Hus1b) cell cycle checkpoint gene required for genomic integrity , RIKEN cDNA 1110017D15 gene (1110017D15Rik), and testis, prostate, and placenta expressed (Tepp)  . This suTgif1, Mxi1, Zfp830, Thbd, Ppard, Dlk1, and Asf1b are also involved in various aspects of embryogenesis like blastocyst growth, development and hatching, embryo implantation, and gastrulation. From their pioneer study using sperm from fertile human donors, Hammoud et al. have shown that the retention of histones is not random but on loci of developmental importance, imprinted genes, and regulatory RNAs  and IMPC data. This search yielded six TBAGs—Epas1, Epc2, Lmbrd1, Msx1, Pitx2, and Slc40a1  were referred . Four em Slc40a1 . Moreove Slc40a1 . One hun Slc40a1 . Four THe testis . These me testis .Hoxa9, Rec8, Dhh, Nectin2, Sox30, Adam1a, and Msxi1—and TAAGs—2610005L07Rik, Tcte2, Mt-tp, and Mt-co1—were cross-validated by ChIP-qPCR (A set of TBAGs—hIP-qPCR . These fhttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE79227) were used for identifying activating histone marks (H3K4me3/me2 and H3K9Ac), super enhancer modifications (H3K27Ac and H3K4me1), and repressive marks . The genome-wide BigWig coverage tracks for mm10 were retrieved using ChIP atlas and these bigWig scores were submitted to Galaxy along with the TH2B genomic interval file. Using the computeMatrix function, a matrix file was generated for signals ±2 kb around the TH2B MACS2 peak. The computed matrix was then visualized using plotHeatmap. While repressive marks H3K27me3, H3K9me3, and H3K36me3 showed no overlap with TH2B chromatin, a positive enrichment of H3K4me2 and H3K4me3 overlapping the peak sequences was observed  with expression restricted to testis, nine genes  with biased testicular tissue expression, and eight ubiquitously expressing genes  with highest expression in testis. Only three genes  were identified with broad expression across all the tissues and highest expression in testis. Intriguingly, two enhancers  controlled the expression of six genes: 4921511H03Rik, Gm9758, Gm10354, Speer4d, Gm17019, and Speer4e, which have either biased or restricted testicular expression.A mild correlation was observed between active enhancer mark H3K27Ac and TH2B marked loci. Recent chromatin analysis studies on sperm have shown that H3K4me2 and H3K4me3 marks are both enriched in sperm, with many overlapping regions marked by these PTMs . Thus, ovia sperm H3K4me3  studies , 2017. PWhile the importance of TH2A/TH2B in histone eviction is well acknowledged, the significance of their retention in mature sperm is still unclear. The present study attempted to demystify the relevance of sperm-retained TH2A/TH2B.in silico via ChIP-seq data across germ cell stages . We obsvia separases and the sister chromatin cohesion is disrupted, followed by their pulling apart to the opposite poles via spindles. In the double mutant TH2A/TH2B mice, there is an incomplete release of cohesin at interkinesis and accumulation of secondary spermatocytes  accompanying TH2B marked loci is crucial to unravel the molecular events and significance of TH2B-associated chromatin. With respect to repressive marks H3K27me3, H3K9me3, and H3K36me3, there was almost no overlap with TH2B chromatin. We observed a positive enrichment of H3K4me3 overlapping with the peak sequences. Both H3K4me3and H3K4me2 were enriched . We alsoNr4a3, Osr1, and Zpr1 are important for gastrulation and blastocyst differentiation. N4bp2l2 is crucial for embryo development (It is important to extrapolate the findings in mouse to humans as the former is an experimental model. Also, in the literature as previously stated, there is a range in the histone retention percentage in sperm. For humans, it is 4%–10%, whereas for mice, it varies from ∼1% to 10%. These differences make it important to compare the chromatin occupancy of TH2B between the orthologs. Any conservation may be important from the point of evolution. To highlight the relevance of our findings, we explored the commonality in the dynamics of TH2B between murine and human sperm. A subset of genes important for morphogenesis and embryo development were found common between the two . Genes selopment . It is eSendler et al. have found sperm RNAs to be associated with H3K4me3 but not H3K27me3 . We see in silico analysis have attributed a myriad of novel functions to sperm-retained TH2B with respect to embryo development and spermatogenesis. Our data indicate a degree of conservation between the TH2B–DNA linkage across human and mouse. Our observations also convincingly demonstrate that TH2B/TH2A are relatively independent in their genome-wide chromatin associations. The TH2A–MtDNA link observed by us needs to be explored further. In vivo, validation of these observations will be crucial to further understand TH2B dynamics.In summary, our murine TH2A and TH2B ChIP-seq data and


Senescence is a complex cellular stress response that abolishes proliferative capacity and generates a unique secretory pattern that is implicated in organismal aging and age-related disease. How a cell transitions to a senescent state is multifactorial and often requires transcriptional regulation of multiple genes. Epigenetic alterations to DNA and chromatin are powerful regulators of genome architecture and gene expression, and they play a crucial role in mediating the induction and maintenance of senescence. This review will highlight the changes in chromatin, DNA methylation, and histone alterations that establish and maintain cellular senescence, alongside the specific epigenetic regulation of the senescence-associated secretory phenotype (SASP). INK4a, a cyclin-dependent kinase inhibitor that blocks cell-cycle progression from the G1 to S phase, is rapidly induced upon wounding, and clearance of these p16+ senescent cells from wound sites significantly delays healing [Cellular senescence is characterized as a hallmark of biological and chronological aging and a potential indicator of pathological tissue states . Senesce healing ,5. Furth healing . Recent V600E or RAS, which mediate cell proliferation and are known drivers of tumor progression and metastasis [CDKN1A promoter driving translation of p21, a cyclin-dependent kinase inhibitor that prevents cell-cycle progression [Cellular senescence was first described in 1961 when Hayflick and Morehead discovered human lung fibroblasts terminally exit the cell cycle after serial passaging. This phenomenon has since been termed the Hayflick limit . This fotastasis ,11. Thistastasis . Escalatgression . In addigression . Mitochogression . Interesgression ,17,18. TGLB1, which accumulates specifically in senescent cells along with the increase in lysosomal content [Senescent cells undergo dramatic phenotypic changes . Morphol content . Impaire content . Senesce content ,22. Anot content   genes, a group of proinflammatory cytokines, chemokines, proteases, and growth factors that can drive tissue dysfunction and carcinogenesis . SASP faINK4A expression was shown to decrease stem cell regeneration across multiple tissue compartments in mice [It is well documented that senescent cells accumulate in aged tissues  and are  in mice ,43.Another feature of aged and senescent cells is a dysregulated metabolome. Recent evidence has demonstrated that nutrient metabolites play an important role in mediating epigenetic enzymatic activity, regulating aging processes, and acting as potent cell signaling agents . Their rC. elegans, and S. cerevisiae [Eukaryotic chromosomes are generally organized into two architecturally distinct domains: heterochromatin and euchromatin. Heterochromatin is characterized by its relative inaccessibility to transcription factors due to the compact structure of the chromatin. Conversely, euchromatin contains permissive and relaxed regions of DNA that allow for active transcription . Heterocrevisiae ,52,53,54revisiae ,56. In srevisiae . Ivanov revisiae .The many post-translational modifications of histones play fundamental roles in gene expression and alter the structure of chromatin. Histone acetyltransferases (HATs) catalyze the addition of a negatively charged acetyl group to positively charged lysine residues on histone tails protruding from the nucleosome. This mark destabilizes electrostatic interactions between the DNA wrapped around histone cores, allowing for transcription and increased gene expression. Conversely, removal of the acetyl group by histone deacetylases (HDAC) regenerates the stabilizing electrostatic interactions, decreasing transcription and gene expression. Methylation of histone lysine and arginine residues is another gene-regulating mark. Histone methylation is complex in that residues may be mono-, di-, or trimethylated, and these marks may activate or repress gene transcription . Indeed,Polycomb-group (PcG) proteins are highly conserved epigenetic chromatin modifiers that govern stem cell fate , are potArf, driving neoplastic transformation [Ink4a, driving p16-dependent arrest in the setting of OIS [Deletion of the polycomb repressive complex 2 (PRC2) genes in mice results in embryonic lethality , while oormation . Recentlormation . Jumonjig of OIS , suggesthTERT/Ras/E1A cells, which are optimized for senescence bypass through oncogenic transformation. Knockout of HDAC4 promoted senescence of these cells through upregulation of senescence-associated genes, as well as increased SA-β-galactosidase activity, a highly specific marker for senescence. Further, eliminating HDAC4 in leiomyosarcomas, cancers known for high expression of HDAC4, led to senescence, arrested proliferation, and enriched SASP factors. In a later study, these investigators demonstrated specific HDAC4/H3K27ac interactions at senescence super-enhancer regions via ChIP-seq [Histone deacetylase 4 (HDAC4), a deacetylase with diverse cellular functions , has rolChIP-seq , solidifChIP-seq . HoweverSenescence drives specialized regions of facultative heterochromatin, which silences proliferation-promoting genes, forming senescence-associated heterochromatin foci (SAHF) . SAHFs hBraf mouse model via inactivation of HIRA [Histone variants of the canonical histone proteins H2A, H2B, H3, and H4 have crucial roles in transcriptional regulation, chromosome segregation, DNA repair, and generation and maintenance of epigenetic states . While m of HIRA . SAHFs a of HIRA . Another of HIRA .Unlike previously mentioned variants that facilitate and maintain senescence, H2A.Z is a potent suppressor of senescence induction in proliferating cells by localizing to p53 binding sites at the p21 promoter. Upon treatment with a DNA-damaging agent, ChIP assay confirmed eviction of H2A.Z from the p21 promoter, allowing p53 binding and inducing cellular senescence. Further, depletion of H2A.Z from primary fibroblasts was sufficient to induce senescence .γ-H2AX, which denotes phosphorylation of the Ser-139 residue of the histone variant H2AX, is a histone variant that is present at double-stranded DNA breaks (DSB) where it colocalizes to the DSB, inducing a rapid epigenetic response to drive resolution of the break .Altogether, variant histones are highly abundant in senescent cells relative to their proliferating cell counterparts, stabilizing and maintaining SAHFs, as well as facilitating SASP factor gene expression, allowing for sustained cellular senescence.DNA methylation is a powerful epigenetic regulatory modification that promotes transcriptional silencing. In replicative senescence, an overall decrease in DNA methylation was observed in senescent cells compared to proliferating cells. This hypomethylation was attributed to a failure of DNA methyltransferase 1 (DNMT1) to maintain methylation over many repeated replication cycles, which drives decreased expression of DNMT1  and deloDNA methylation and subsequent oxidation of 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5hmC) are key epigenetic events regulating development and stem cell differentiation in mammals ,101,102.Tet1 deficiency reduces fertility and leads to accelerated reproductive failure (premature ovarian insufficiency) syndrome with age [TET2 mutations contribute to myeloid expansion and innate immune dysregulation with age accompanied by an increased risk of transformation to hematological malignancies, including myeloproliferative and myelodysplastic neoplasms and acute myeloid leukemia [Increasing evidence suggests that Tet enzymes contribute to aging of reproductive and hematopoietic systems. with age . Tet1-dewith age . Somaticleukemia .Hox genes and genes mediating cellular differentiation [An “epigenetic clock” has been proposed via next-generation sequencing technology that describes highly reproducible patterns of methylation that can accurately predict human chronological age . Furtherntiation . The simntiation ,113, sugntiation . Indeed,INK4A and p21CIP1, increased telomere length, and restoration of proliferation capacity. In vivo reprogramming was also used in the LAKI progeroid mouse model, which harbors a mutation in the lamin A gene. Short-term cyclic expression of OSKM restored heterochromatin maintenance marks H3K9me3 and H4K20me3 to healthy levels. Liver cells isolated from these mice harbored significantly fewer SA-β-Gal positive cells compared to control mice. Furthermore, fibroblasts harvested from these progeroid mice that underwent reprogramming demonstrated increased mitochondrial function, reduced cellular senescence evidenced by Il-6 and β-galactosidase expression, and decreased DNA damage [Recent studies further interrogated the epigenetic clock to recapitulate a juvenile epigenetic profile in aged cells. Investigators transfected aged primary human fibroblasts and endothelial cells with an mRNA cocktail expressing OSKMLN, the canonical induced pluripotent stem cell (iPSC) reprogramming factors. This specific methodology allows for epigenetic reprogramming without altering the original cell identity. After treatment, aged primary cells exhibited a rapid reversion of their epigenetic clock methylation pattern, appearing similar to their young cell counterparts. Immunofluorescence data further demonstrated a youthful epigenetic profile with increased nuclear staining of H3K9me3, HP1γ, and lamin-associated polypeptide 2α (LAP2α), a nuclear lamin support protein. Further evidence of improved cellular function included increased SIRT1 expression, decreased mitochondrial reactive oxygen species, and improved active clearance of cellular waste and macromolecules. . This moA damage . This trA damage .Altogether, alterations in DNA methylation and DNA methylation associated enzymes during aging and senescence significantly contribute to cellular dysfunction, facilitate a chronic inflammatory state via SASP activation, and may mediate certain age-related pathologies.IL8 gene locus into the SAHF was demonstrated to repress this SASP factor in HMGB2 knockdown senescent cells. Targeting this pathway may allow beneficial repression of neoplastic transformation and proliferation genes via cellular senescence while inhibiting a chronic proinflammatory and protumorigenic environment of SASP factors [High-mobility-group protein B2 (HMGB2), a multifunction protein involved in chromatin remodeling, transcription regulation, and DNA repair , binds l factors .Chromatin remodeling events during cellular senescence drive the formation of super-enhancers at SASP gene loci to facilitate transcription. Increasing H3K27 acetylation at these super-enhancer sites and recruitment of Bromodomain-containing protein 4 (BRD4) is necessary for upregulated expression of key SASP genes and inhibition of BRD4 suppressed SASP expression. Further, pharmacological inhibition of BRD4 allowed for immune escape and decreased clearance of premalignant senescent hepatocytes in a liver cancer mouse model .IL1A gene locus, driving increased transcription. Like MLL1, this phenomenon was independent of senescence-induced cell-cycle arrest, making MLL1 and DOT1L potential therapeutic targets to limit the negative consequences of SASP factors of the tissue environment without losing the antineoplastic transformation effect of senescence [The H3K4 methyltransferase, mixed-lineage leukemia 1 (MLL1), is required for SASP activation, as silencing of MLL1 with shRNA dramatically decreased expression of key SASP genes, including IL-1A, IL-8, IL-6, MMP1, and MMP3m in OIS-transformed fibroblasts and OIS primary human melanocytes. Interestingly, inhibition of MLL1 blocked the expression of SASP-like proteins in response to DNA damage that did not induce senescence, suggesting an anti-inflammatory role independent of senescence . Disruptnescence .The DDR during senescence promotes proteasomal degradation of G9a and G9a-like protein, both H3K9 histone methyltransferases. In senescence-induced cells, Cdc14B- and p21-dependent activation of APC/C ubiquitin ligase drive decreased H3K9 methylation, allowing for increased expression of IL-6 and IL-8 . JMJD3 wNAD-dependent deacetylase sirtuin-1 (SIRT-1) negatively regulates SASP genes. During cellular senescence, decreased expression of SIRT-1 allows for increased acetylation of H3K9 and H4K16 driving transcription of these cytokines, a phenomenon recapitulated in nonsenescent SIRT-1 depleted cells . InteresAs more data implicate epigenetic regulatory mechanisms of gene expression playing an integral role in the manifestation and maintenance of age-related diseases, investigation has turned toward therapies that combat these pathologies alongside promoting health and lifespan. Resveratrol is a sirtuin-1 activator that has improved health and lifespan in many species, including mice ,130,131,Drugs targeting epigenetic pathways remain an exciting field of therapeutics; however, they are currently targeted towards hematologic cancers. Continuation of research examining the histone code, epigenetic protein/nucleotide interactions, and combination therapies will improve the efficacy of these drugs, limit unintended effects, and perhaps expand their usefulness therapeutic to additional domains such as senescence and aging. As discussed, epigenetic changes in senescence are diverse and dynamic, and there is no universal phenotype of senescent cells. As epigenetic regulation of senescence is dependent on tissue and cell type, as well as a senescence induction method, there is a need for expansion of the tissue types and cells studied during senescence induction would allow better insight into how different cell types may reorganize chromatin due to various senescence-promoting stimuli.Large-scale chromatin remodeling, histone alterations, and DNA methylation activate or repress cellular senescence in response to a variety of stimuli, including DNA damage, oncogenic stress, mitochondrial dysfunction, and metabolic stress. Further, some epigenetic phenotypes and pathways may be highly specific to various senescence-inducing sources, with others having dramatic overlap. Young, healthy cells maintain an epigenome that promotes precise regulation of gene expression, thus avoiding replicative and oxidative stress. Conversely, aged cells accumulate stochastic insults over time, driving alterations in gene expression, decreased fitness and functionality, ultimately leading to cell-cycle arrest and senescent states. The reversible capacity of these tightly regulated epigenetic pathways and mechanisms serves as a potential source for therapy in myriad disease states caused by senescence.


Liquid–liquid phase‐separated (LLPS) transcriptional factor assemblies at super‐enhancers (SEs) provide a conceptual framework for underlying transcriptional control in mammal cells. However, the mechanistic understanding of LLPS in aberrant transcription driven by dysregulation of SEs in human malignancies is still elusive. By integrating SE profiling and core regulatory circuitry (CRC) calling algorithm, the CRC of metastatic and chemo‐resistant osteosarcoma is delineated. CRC components, HOXB8 and FOSL1, produce dense and dynamic phase‐separated droplets in vitro and liquid‐like puncta in cell nuclei. Disruption of CRC phase separation decreases the chromatin accessibility in SE regions and inhibits the release of RNA polymerase II from the promoter of SE‐driven genes. Importantly, absence of CRC key component causes a reduction in osteosarcoma tumor growth and metastasis. Moreover, it is shown that CRC condensates can be specifically attenuated by the H3K27 demethylase inhibitor, GSK‐J4. Pharmacological inhibition of the CRC phase separation results in metastasis suppression and re‐sensitivity to chemotherapy drugs in patient‐derived xenograft model. Taken together, this study reveals a previously unknown mechanism that CRC factors formed LLPS condensates, and provides a phase separation‐based pharmacological strategy to target undruggable CRC components for the treatment of metastatic and chemo‐resistant osteosarcoma. Core regulatory circuitry (CRC) factors forms liquid–liquid phase‐separated condensates at super‐enhancers to regulate chromatin accessibility and oncogenic transcription. Pharmacological inhibition of the CRC phase separation by GSK‐J4 suppresses metastasis and chemoresistance in osteosarcoma patient‐derived xenograft model. In‐depth analyses of the CRC elucidate putative gene regulatory mechanisms of the cell‐type‐specific gene expression. Despite the recent progress of defining SEs in tumors, the CRC in the metastatic and chemoresistant tumors remains poorly understood.Genetic alterations in cancer cells invariably cause dysregulation in transcriptional programs leading to cancer cells dependency on certain master regulators of oncogene expression, also known as transcriptional addiction.1 This It is hypothesized that formation of phase separation more likely occurred at SEs than at typical enchancers (TEs). The transcriptional coactivators BRD4 and MED1 which co‐localized with SEs are components of liquid–liquid phase‐separated (LLPS) condensates. The SE‐bound TFs in LLPS condensates typically contain large intrinsically disordered regions (IDRs), which can multivalently but weakly interact with each other. These condensates guarantee the highly concentrated transcriptional machinery at SE regions and play prominent roles in 3D genome organization to robustly activate the expression of cell identity genes. An important question related to this is whether CRC components produce LLPS condensates to control the transcriptional addiction‐associated SEs.The assembly of dynamic membrane‐free chambers by phase separation is essential for the control of many biochemical processes, including gene transcription.4 It i Moreover, metastasis and chemoresistance remain the most important fatal complication of osteosarcoma. Nearly all patients succumbed to osteosarcoma develop lung metastases with marked chemoresistance. Hence, understanding the mechanisms involving in osteosarcoma metastasis and chemoresistance is urged to develop new strategies and innovative therapies against this disease. As SEs are particularly sensitive to perturbation, agents targeting SEs could be a promising therapeutic options for osteosarcoma. Several studies have shown that inhibitors of chromatin modifiers  and components of the transcriptional machinery  diminish the proliferation of patient‐derived cancer cells and increase survival in mouse models through SE‐driven gene inhibition. The SEs are occupied by high densities of TFs and known to form condensates. We hypothesize that if these SEs can be used as scaffolds, CRC, and transcription machinery proteins will be concentrated at SEs by phase separation to promote metastasis or chemoresistance related gene activation.Osteosarcoma is the most common primary malignancy of the bone affecting children and adolescents, particularly. Until now, osteosarcoma patients are still receiving treatment that was developed in the 1970s, and the outcomes remain unimproved throughout the years. Although many efforts have been made aiming the oncogenic signaling of osteosarcoma, clinical studies on the targeted agents and immunotherapy yielded disappointing outcomes due to high genetic and pathological heterogeneity of osteosarcoma.7, 8Scheme To this end, we utilized an integrative and large‐scale approach to address how CRC formed condensate to regulate oncogenic transcription in metastatic and chemoresistant osteosarcoma. Furthermore, the insights described in this study can be translated into a better therapeutic strategy using GSK‐J4 by dampening CRC condensate‐mediated transcriptional programs in osteosarcoma with drug‐resistant and metastatic malignancy Scheme 1.22.1 embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and osteoblasts served as normal controls. We ranked all the putative SEs by increasing the H3K27ac signal and defined SEs as those that showed high H3K27ac enrichment in osteosarcoma . mEGFP‐HOXB8‐IDR and mCherry‐FOSL1‐IDR formed spherical droplets in a concentration dependent manner  revealed that both HOXB8 and FOSL1 formed puncta in 143B cells Figure . HOXB8 i2.3 Chromatin Interaction Analysis with Paired‐End‐Tag sequencing (ChIA‐PET) data suggests that SE constituents occupied by polymerase II (Pol II), HOXB8, and FOSL1 are close to each other in the 3D structure of chromatin, exampled by MAZ and MYC  demethylase inhibitor, GSK‐J4 Figure 5a. Bio‐lGSK‐J4 also inhibited the CRC condensates produced by the co‐aggregation of HOXB8‐IDR and FOSL1‐IDR Figure . PhotoblImportantly, GSK‐J4 inhibited the proliferation of various osteosarcoma cell lines at clinically achievable concentrations and showed limited efficacy against normal mesenchymal stem cell and osteoblast cell proliferation at the same dosage , lung metastasis (p < 0.0001), tumor relapse (p = 0.0067), and chemoresistance (p < 0.001) are positively correlated with HOXB8 staining  and cisplatin resistance, we generated two chemoresistant human primary cell lines (Patient‐1 and Patient‐2) derived from osteosarcoma patients Figure . In a sh3 Therefore, CRC has recently been highlighted as attractive targets for epigenetic therapy of cancer. How these master TFs of CRC aggregate and orchestrate at SEs to generate downstream outcomes is still an unanswered question. Here, we demonstrated that formation of phase‐separated condensates is an important mechanism that enables CRC‐mediated transcription addiction in metastasis and chemoresistance. In particular, we present a small molecule compound, GSK‐J4, which modulates phase separation of CRC factors, for therapeutic intervention of chemoresistant and metastatic osteosarcoma.Tumor SE‐driven master TFs in CRC are enriched at oncogenic driver genes that play prominent roles in malignancies, including metastasis and drug resistance.21 The Another study showed that a retinoid‐dependent cis‐regulatory element, distal element RARE, determined HOXB cluster gene expression, and leukemic state. It is an open question why HOXB, the important TFs during limb development, promote osteosarocma malignancy if expressed in maligant cells. Our study suggests that phase separation may play an important role in the modulation of transcription specificity of HOXB8 by driving assembly of HOXB8 with oncogenic TFs in the “oncogenic” condensate and preventing binding of HOXB8 with original development related TFs. The CRC condensates where HOXB8 is located provides a new approach for the formation of transcription addiction related to metastasis and chemoresistance.Our study clarifies the role of HOXB cluster genes, in particular HOXB8, as the master TFs of CRC in metastasis and chemoresistance. Previous studies showed that HOXB8 functions as a key TF involved in limb development and bone marrow‐derived microglia differentiation. Our study reveals that the osteosarcoma malignancy exhibits a dependence on CRC TFs ‐mediated oncogenic transcriptional programs. This type of transcriptional dependency appears to operate in many certain types of cancers. For example, recent studies have shown that HOXB cluster genes can be classified as SEs in acute myeloid leukemia, and HOXB cluster gene expression is dependent on mutant NPM1 and its aberrant cytoplasmic localization.22 AnoThe majority of Pol II‐transcribed genes are regulated at a checkpoint called promoter‐proximal pausing. In this study, we found that the inhibition of CRC could block the release from pausing during transcription, leading to transcriptional elongation suppression in metastatic osteosarcoma cells. Previous publications also demonstrated MYC hyperactivation induces transcriptional amplification by increasing mRNA synthesis. Notably, MYC functions to amplify the entire gene expression program, which is a critical step for generating transcriptional addiction in cancers. Intriguingly, the newly identified SE‐driven gene MAZ may assist MYC in metastasis and functions as another important effector of CRC in transcriptional addiction. The downstream targets of MAZ in osteosarcoma need to be further identified.We identified that H3K27me3 demethylase inhibitor, GSK‐J4, suppressed the phase separation of CRC factors. An attractive hypothesis is that GSK‐J4 reduces HOXB8 expression by inhibiting JMJD3/UTX which are putative nuclear epigenetic factors required for H3K27me3 demethylation. But the cell‐free assays showing HOXB8‐mEGFP forming little phase separated droplets after GSK‐J4 treatment makes this scenario unlikely. Therefore, we speculate that GSK‐J4 binds IDR of HOXB8 could somehow affect HOXB8 protein folding. Moreover, treatment of GSK‐J4 may further suppress the expression of SE‐driven oncogenic genes in osteosarcoma. One prime example of SE inhibitors is the small molecule inhibition of histone deacetylases  in diffuse intrinsic pontine glioma, for which clinical trials are ongoing. Our results demonstrate that, in addition to histone acetylation, the CRC phase separation may present additional avenues for therapeutic intervention in malignancies with aberrant changes in the oncogenic SEs. Notably, although GSK‐J4 targets HOXB8 to disrupt CRC phase separation, we cannot exclude the contribution of other targets to the therapeutic activity of GSK‐J4 in osteosarcoma.24In summary, we identify the chemoresistant‐ and metastatic‐specific CRC formed phase‐separated condensates at SE loci in osteosarcoma. The disruption of CRC phase separation suppressed aberrant oncogenic transcriptional programs via modulating chromatin accessibility. Moreover, a small molecule compound, GSK‐J4, can be used as a potential treatment for metastatic and chemoresistant osteosarcoma, which will greatly benefit osteosarcoma patients.4Both osteosarcoma and normal bone tissue surgical specimens were collected in the Department of Bone Tumor, the First Affiliated Hospital, Sun Yat‐sen University in accordance with institution‐approved protocols. Written informed consent was obtained from each study participant after a thorough explanation of the procedure and its risk according to the Declaration of Helsinki. All specimens were examined by a pathologist to verify tumor types and grades.2 atmosphere.The human osteosarcoma cell lines of 143B, SJSA1, U2, MNNG, ZOS, and ZOSM were cultured in DMEM medium  with 10% fetal bovine serum  at 37 °C in a humidified 5% CO2 atmosphere.For the culture of primary osteosarcoma cells and osteoblasts, surgically removed osteosarcoma specimens and normal bone tissue were washed with and minced in sterile phosphate‐buffered saline (PBS). The single‐cell suspension was obtained by pressing the minced tissues through 70‐µm cell strainers . Dissociated cells were cultured in DMEM medium supplemented with 15% FBS  at 37 °C in a humidified 5% COFemale Balb/c nude mice  were obtained from the Model Animal Research Center of Nanjing University (China). All animal experiments were carried out according to the guidance of the ethics committee of Sun Yat‐Sen University.6 osteosarcoma cells in 100 µL PBS. Thereafter, tumor size was periodically measured with calipers every 2 days by measuring the length and width. Tumor volumes were calculated according to the following formula: volume (mm3) = (length × width × width) / 2. After the mice were sacrificed, the tumor xenografts were removed, fixed in formalin, and stored at 4 °C. Animal experiments were approved by the Animal Care and Use Committee of Sun Yat‐sen University.Five‐week‐old female Balb/c athymic nude mice were housed in individually ventilated micro‐isolator cages. Each mouse was injected subcutaneously in the right flank with 106 osteosarcoma cells was injected into the right tibial medulla of chloral hydrate‐anesthetized 5‐week‐old nude mice. Five weeks later, using an in vivo imaging system , mice were imaged under isoflurane anesthesia to analyze osteosarcoma tumor growth in vivo.Single‐cell suspension (10 µL) containing 1–1.5 × 10em26Il2rgem26/Nju) mice under anesthesia with 10% chloral hydrate. Xenografts appeared at the graft site 1 to 3 months after grafting. They were subsequently transplanted from mouse to mouse. Animals were excluded if no tumors were present, and animals were randomized to control and treatment groups so that each group had equivalent distribution of initial tumor sizes.Tumor specimens were obtained from osteosarcoma patients with their informed consent. Tumor fragments were removed during surgery and saved on ice for less than 45 min. Briefly, fresh tumor fragments were grafted subcutaneously into the subcutaneous tissue upper back of NCG . 143B and SJSA1 osteosarcoma cells were infected with the obtained lentiviruses to knockdown target genes. The shRNA‐targeting sequences are listed in Table 6 to 10 × 106 cross‐linked cells, and sequencing libraries were prepared as previously described. The following antibodies were used for ChIP: rabbit anti‐H3K4me3 (Abcam), rabbit anti‐H3K27ac (Abcam), rabbit anti‐H3K27me3 (CST), and rabbit anti‐HA (CST). ChIP‐seq libraries were sequenced on the HiSeq 2000 platform at the Novogene LLC. Analysis was performed as previously described.ChIPs were performed using 2 × 10p < 0.05. Changes in splicing isoforms were analyzed by replicate multivariate analysis of transcript splicing, a Bayesian statistical framework.All treatment conditions were collected in biological duplicate. Cells were lysed in TRIzol reagent and frozen at −80 °C. Total RNA was extracted using RNeasy Mini kit  following the user's manual and quantified using Nanodrop 2000. The integrity of the RNA was analyzed using the Agilent Bioanalyzer 2100. Paired‐end reads were generated by Illumina Hi‐Seq 2500 platform and mapped to the human genome. An R package, DESeq, was applied for transcription quantification and differential expression analysis using a cutoff of Trim_galore and Fastqc were used for trimming adapters and filtering raw sequencing reads. Sequencing reads were aligned to the UCSC hg19 human genome reference using bowtie2. All unmapped reads, non‐uniquely mapped reads, and PCR duplicates were removed. ChIP‐seq peaks were then called using MACS2. Correlation of ChIP‐seq samples were calculated by R/Bioconductor package DiffBind. Chip peak motif discovery was performed using findMotifsGenome.pl. Genome coverage files for visualization were generated by igvtools using count command and default options. Chip peak browsing and representative snapshots capturing were performed using the Integrative Genomics Viewer.https://bitbucket.org/young_computation/rose). The SE was classified as a set of H3K27ac peaks (detected by MACS2) within a 12.5 kb distance, and greater than 2.5 kb distance from the transcriptional start site (TSS). Super enhancers were further defined by those demonstrating the greatest levels of H3K27 acetylation as detected by graphing an inflection plot and selecting values for which the slope of a fitted curve exceeded a value of 1. Enhancers below the point on that curve with a slope of 1 were thus defined to be typical‐enhancers.We identified SEs using the Rank Ordering of Super Enhancers (ROSE) algorithm (te (TSS).11 Suphttps://pypi.python.org/pypi/coltron) and CRCmapper (https://github.com/younglab/CRCmapper) that calculated IN and OUT degree index for SE‐regulated TFs. Briefly, for any given TF, the IN degree index was defined as the number of SE‐driven TFs with a binding at the given TF proximal SE. The OUT degree index was defined as the number of binding sites for SE‐driven TFs. An FDR cutoff of 0.01 was used to identify enriched TF binding sites. The TF in CRC was defined as the number of IN and OUT degree index was greater than 150.Osteosarcoma CRC analysis was determined using the COLTRON  and the normalized coverage of Pol II in the promoter of genes (defined as ±500 bp from the TSS).Paired‐end ATAC‐seq fragments of 180–247 bp were isolated and the average dyad density at single nucleotide resolution was plotted around FOSL1 motifs. The spacing between mono‐nucleosomes was determined by the maximum labeling density of upstream and downstream nucleotides around the FOSL1 motif.A lentiviral overexpression plasmid for HOXB8 was generated by cloning the full‐length ORF of human HOXB8 gene (NM_024016.4) into the FG‐EH‐DEST‐FLAG‐MCS vector, followed by a 6 amino acid GS linker sequence “GSGSGS” and mEGFP. FOSL1 expression plasmid was generated by cloning the full‐length ORF of human FOSL1 gene (NM_005438.5) with 5′HA tag into the FG‐EH‐DEST‐FLAG‐MCS vector followed by a 6 amino acid GS linker mentioned above and mCherry. FRAP was performed on LSM880 Airyscan microscope with 488 nm laser. The bleach spot was centered on a cluster and images were taken with 1 s interval for 100 s to measure the fluorescence recovery in the cluster. The integrated intensity of the cluster was determined as a function of time, background intensity was subtracted from a neighboring region of equal size, was corrected for overall photo‐bleaching based on a reference region within the same cell, and was normalized to pre‐bleach intensity.t‐test was performed to compare the statistical significance of all tests. Chi‐squared test, one‐way and multivariate ANOVA were performed to compare clinical data. p‐values of less than 0.05 were considered statistically significant and marked as “*”; p‐values less than 0.01 or 0.001 were marked as “**” and “***,” respectively. All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supporting Information. Additional data are available from authors upon request.All the in vitro experiments were tested at least in triplicate. All analyses were performed using GraphPad Prism version 6.0 . Data were presented as the mean ± SD. Unpaired two‐tailed The authors declare no conflict of interest.B.L. and C.Z. contributed equally to this work. B.L. performed the experiments and wrote the manuscript. C.Z. performed the xenograft experiments and IHC staining assay. M.Y. and Y.H. performed LLPS assays. J.H. conducted bioinformatic analysis of sequencing data. C.X.Z. performed the co‐IP assays. S.C. and J.Y. prepared the plasmids and lentiviral vectors. K.Y.L. and Q.C. interpreted the data and revised the manuscript. W.Z. designed the experiments, interpreted the data, wrote the manuscript, and provided supervision.Supporting InformationClick here for additional data file.