Hyperdiploidy is the largest genetic entity B-cell precursor acute lymphoblastic leukemia in children. The diagnostic hallmark of its two variants that will be discussed in detail herein is a chromosome count between 52 and 67, respectively. The classical HD form consists of heterozygous di-, tri-, and tetrasomies, whereas the nonclassical one  contains only disomies and tetrasomies. Despite their apparently different clinical behavior, we show that these two sub-forms can in principle be produced by the same chromosomal maldistribution mechanism. Moreover, their respective array, gene expression, and mutation patterns also indicate that they are biologically more similar than hitherto appreciated. Even though in-depth analyses of the genomic intricacies of classical HD leukemias are indispensable for the elucidation of the disease process, the ensuing results play at present surprisingly little role in treatment stratification, a fact that can be attributed to the overall good prognoses and low relapse rates of the concerned patients and, consequently, their excellent treatment outcome. Irrespective of this underutilization, however, the detailed genetic characterization of HD leukemias may, especially in planned treatment reduction trials, eventually become important for further treatment stratification, patient management, and the clinical elucidation of outcome data. It should therefore become an integral part of all upcoming treatment studies. Hyperdiploidy (HD) was first described by Fritz Lampert in 1967 when he measured the DNA content of leukemic cells by meticulously comparing their diameters with those of normal lymphocytes . , 6. 3, 6The karyotypes of the second, much rarer nonclassical HD variant, contain only disomies and tetrasomies. This unique pattern was already recognized and described in the early days of cytogenetics Fig.  17]. Ar. Ar17]. The chromosome number of near-triploid cases lies between 60 and 78, which means that at least those with a chromosome number below 67, overlap with those classical HD ones with a chromosome number above 60 Fig.  7, 8, 1–15. BecaAlthough constitutional trisomy 21 is by far the most common leukemia-predisposing factor, it is odd that classical HD forms are significantly underrepresented in this condition. To the best of our knowledge, none of the other aneuploid leukemia types have ever been reported in Down syndrome patients either , 26–28. https://pecan.stjude.cloud/proteinpaint/DYRK1A) and render both DYRK1A and FOXO1 worthwhile therapeutic targets in this specific subset of ALL [Of further interest in this context is DYRK1A, a serine/threonine kinase that is encoded on chromosome 21 and whose copy number determined overexpression is implicated in several pathologies in Down syndrome. Notably, this overexpression increases the expression and phosphorylation of two transcription factors, FOXO1 and STAT3, that are indispensable for B-cell development and therefore also contribute to the development and maintenance of BCP leukemias . These et of ALL .Nonrandom secondary events in the form of structural abnormalities include chromosome 1q duplications 10–15%), 6q deletions (5%) and isochromosomes 17q (2–5%) and 7q (1–2%) . They oc%, 6q delBCR::ABL1, a t/TCF3::PBX1 or a t/KMT2A::AFF1 [Another notable phenomenon is the co-occurrence of classical HD and an additional class-specific abnormality within the same cell clone, such as a t9;22)/2A::AFF1 , 31. Eit2A::AFF1 , 32, 33.Germline predisposition factors comprise genome-wide association study–ascertained allelic variants, which function as genetic modifiers, and distinct pathogenic gene defects Table . An HD-iARID5B, CEPBE, BMI1, and PIP4K2A genes [CEBPE encodes one of six basic leucine transcription factors. The risk-defining SNP rs2239635 in its promotor disrupts the binding of the Ikaros transcriptional repressor [CEBPE is on chromosome 14, which is incidentally also one of the most common tri- and tetrasomic chromosomes in HD. Its copy-number-linked overexpression may thus be one of the critical contributing leukemia-promoting factors [CDKN2A, which is the most frequently deleted gene in ALL. The risk-defining SNP rs11591377 is in a predicted hematopoietic stem cell enhancer and reinforces the preferential binding of the MYBL2 and p300 transcription factors [PIP4K2A encodes an enzyme that is part of the phosphoinositide signal transduction pathways that co-regulate cell proliferation, differentiation, and motility. The risk-defining SNP rs4748812 lies within a PIP4K2A regulatory element and is predicted to alter the binding of the RUNX1 transcription factor [The four most relevant susceptibility loci reside within or in close proximity to the 2A genes –41. ARID2A genes , 42, 43.2A genes , 42–46. ETV6 and the PTPN11 genes [ETV6 germline-mutated cases frequently also acquire somatic mutations in NRAS, KRAS, and PTPN11 [PTPN11 mutation became duplicated in the form of a uniparental disomy 12 [A not yet exactly determined proportion of HD ALL cases have predisposing pathogenic germline defects in genes that encode members of B-cell development, receptor tyrosine kinase/RAS (RTK/RAS), epigenetic regulatory, and DNA repair pathways Table . The two11 genes , 47–49. 11 genes , 49. ETVisomy 12 .ARID5B and ETV6, will augment the respective risk remains to be determined [Predisposing germline factors are unlikely to be directly responsible for the formation of HD precursor cells per se but rather alleviate the immediate survival of independently created cells by equipping them with essential elements that founder cells in non-predisposed individuals are forced to acquire as secondary changes . The exttermined .KRAS, NRAS, FLT3, SOS, and PTPN11, as well as in the chromatin-modifying genes CREBBP, NSD2, SUV420H1, SETD2, and EZH2 . It was therefore proposed that this copy number discrepancy may likewise unbalance the essential expression equilibrium between these genes [The methylation, gene expression, and protein structure of HD leukemias are tightly interlocked and mainly shaped by dosage effects that are exerted by the surplus chromosomes , 61–68. eletions . The traeletions , 70. By se genes . Althougse genes , 69–71. se genes , 72. TogNotably, the gene expression profiles of classical and nonclassical HD forms are virtually identical. In unsupervised hierarchical clustering and principal component analyses, monoclonal nonclassical HD, bi-clonal, and monoclonal hyperhaploid entities form a single discrete cluster that clearly separates them from the hypodiploid/near-triploid entities, which, in turn, form their own cluster . MoreoveSLC19A1 gene on chromosome 21q22.3, which encodes the predominant folate and MTX uptake transporter [Finally, we need to mention the copy-number-related overexpression of the nsporter , 75. Thensporter –80.HD leukemias evolve from a single immature BCP cell, which is transformed very early during fetal development –89. AlthThe current and intensely scrutinized view suggests that a nonclassical HD karyoytpe can only derive from the duplication of a previously generated hyperhaploid one , 8, 14. 9/l and no extramedullary disease. HD blast cells show a high expression of CD9, CD20, CD22 CD58 CD 66c, CD86, and CD123, and a low expression of CD45 [In the western world, up to 35% of childhood leukemias are hyperdiploid, but with 15–25%, they are far less prevalent in patients of Asian, African, and Native American descent –93. Chil of CD45 . Their m of CD45 –97. ThisARID5B risk allele [One thought-provoking discovery is the fact that heterozygous HLA-DPB1*0201 alleles are significantly enriched in patients with HD leukemias . HLA-DPBk allele –103.HD ALL blast cells are inherently difficult to maintain and propagate in culture, which makes it extremely difficult to perform not only cytogenetic analyses, but also any other type of research that depends on viable cells . UnderliTable https://clinicaltrials.gov/ct2/show/NCT03911128). Whether such a challenging detailed threshold definition is indeed also technically feasible to routinely achieve and therefore worthwhile to implement in future clinical studies, remains to be seen. Second, Enshaei et al. convincingly proved that the copy number assessment of four chromosomes, whose prognostic relevance was already appreciated previously is sufficient to demarcate two distinct risk groups. The low-risk group, which in their study makes up 80%, is defined by trisomies 17 and/or 18, whereas their poor risk group makes up 20% and is defined by trisomies 5 and 20 [The two central challenges that clinicians are nowadays confronted with in HD leukemias are how to identify patients with a high propensity to relapse already at diagnosis and how to reduce treatment in low-risk patients without jeopardizing their good outcome. Virtually all contemporary, ongoing, and planned treatment studies rely on the assessment of the measurable residual disease (MRD), either based on the PCR-based quantification of immunoglobulin and T-cell receptor gene rearrangements or based on the flow-cytometric assessment of the immunophenotypic criteria of blast cells. Only some of these studies also require the identification of HD cases, which is then achieved by determining the DNA content, the overall chromosome number and/or the copy numbers of selected chromosomes with fluorescence in situ hybridization (FISH) that are considered appropriate for the delineation of good risk cases. Such a chromosomal risk classification has, for instance, already been used successfully in the Children’s Oncology Group study for over 25 years Table  116–118118. O’Co5 and 20 . This po5 and 20 , it willSETD2 mutations had an inferior event-free  and overall survival than nonaffected ones [Of further note in this context is the recent observation that nonclassical HD cases with ted ones .Owing to the lack of more refined genetic discriminators, all mono- and bi-clonal hyperhaploid and nonclassical HD forms are hitherto stratified as high risk. However, Mullighan et al. showed that even in those cases MRD is the most important prognostic indicator since all cases with a negative MRD status (<0.01%) are highly curable with intensive chemotherapy alone .TCF3::PBX1- and ETV6::RUNX1-positive cases, it is definitely not adequate for all HD leukemias. As a result of relapses, their disease-free survival was only 56.6 ± 10.3% [The high cure rates of low-risk classical HD ALL cases imply that many of them are probably overtreated. The daunting challenge is now to reduce treatment intensity to diminish side effects and avoid ensuing long-term sequelae without endangering the hitherto achieved excellent overall treatment outcome. Some of the trials that have addressed this issue so far have been quite successful (even without utilizing any genetic risk score), whereas those trials which reduced the duration of maintenance therapy to 6 months were not, at least as regards HD ALL , 122–125 ± 10.3% . Neverth ± 10.3% .NR3C1/2, CREBBP, and WHSC1 for glucocorticoids; NT5C2, MSH2/6, PMS2, and PRPS1/2 for thiopurines; and FPGS for MTX [The emergence of specific mutations in disease recurrences suggests that many of them are treatment induced. These mutations comprise, for instance, those in  for MTX . Examini for MTX . They weTogether these observations confirm that an adequate length of maintenance therapy is essential for preventing early relapses in classical HD leukemias but also, that this treatment component cannot be held accountable for inducing late ones, as seems to be the case in other genetic subtypes.TP53 mutated. However, it is less clear whether the remaining TP53 wild-type ones with a similar high chromosome count should be also stratified as near-triploid or rather as classical hyperdiploid ones. Although this concerns only a few cases, their diagnostic clarification, and most appropriate allocation requires further scrutiny. Array analyses deliver not only a detailed information about copy number changes, but also about allele distribution alterations, such as the presence of otherwise unidentifiable uniparental disomies. Bearing the ongoing trend to reduce treatment for very low-risk patients in mind, which especially also concerns HD leukemias, we advocate to implement this state-of-the-art technology together with mutation screening in treatment studies to comprehensively characterize the detailed genomic make-up of HD leukemias [HD leukemias have puzzled and confused researchers and clinicians for more than 50 years now. Novel findings that derive from array, mutation, and gene expression analyses offer now new opportunities to re-examine deeply engrained, yet largely unproven and unquestioned views about their origin, their mode of creation, and the interrelatedness of the diverse aneuploid sub-forms. Such fresh insights will eventually help to reconsider and refine the current classification system and thereby also influence the prognostic stratification and treatment of these subgroups. One example concerns the elucidation of the biological relatedness of classical, nonclassical (“duplicated hyperhaploids”), and pure hyperhaploid cases, which will help to better understand what drives their apparent different clinical behaviors. Another one concerns hyperdiploid forms with a high chromosome number. Near-triploid cases as well as mono- and bi-clonal hypodiploid cases can nowadays be easily ascertained, because over 90% of them are eukemias . Only su


Three-dimensional genome organization represents an additional layer in the epigenetic regulation of gene expression. Active transcription controlled by enhancers or super-enhancers has been extensively studied. Enhancers or super-enhancers can recruit activators or co-activators to activate target gene expression through long-range chromatin interactions. Chromatin interactions and phase separation play important roles in terms of enhancer or super-enhancer functioning. Silencers are another major type of cis-regulatory element that can mediate gene regulation by turning off or reducing gene expression. However, compared to active transcription, silencer studies are still in their infancy. This review covers the current knowledge of human silencers, especially the roles of chromatin interactions and phase separation in silencers. This review also proposes future directions for human silencer studies. Cis-regulatory elements (CREs) are non-coding sequences that play an important role in regulating these cell type-specific transcriptional programs that allow for heterogeneous gene expression from a common DNA template [Humans are complex multicellular organisms made up of 30–40 trillion cells, all containing the same DNA sequences . Yet, ditemplate ,3. CREs Extensive research has been done on two classes of activating CREs: promoters and enhancers. Promoters are located proximal to transcription start sites and contain binding sites for RNA polymerase II and other general transcription factors that initiate transcription ,6,7. EnhJust as activating CREs are required to activate selected genes for cell type-specific functions, so too are repressive CREs required to curb the expression of genes that need to be switched off. Although activating CREs are well characterized, repressive CREs remain poorly understood. Silencers are one type of repressive CRE, broadly defined as sequence-specific CREs that switch off or reduce the expression of their target genes. Unfortunately, very few human silencers have been identified and characterized. In Silencers can be classified into proximal and distal silencers depending on their position relative to the gene promoter. Promoter-proximal silencers are usually position dependent and contain binding sites for repressor proteins that can switch off gene expression by inhibiting transcriptional machinery binding or function . PromoteWith more and more individual silencer examples, systematic methods identifying human silencers have been proposed. Huang et al.  identifiDoni Jayavelu et al.  adopted HRH1, SYNE2, and CDH23 were validated via CRISPR/Cas9 deletion.In contrast to the above methods, Pang and Snyder  systematOur group followed a similar approach to Huang et al.  and idenAlthough various systematic methods have been proposed ,33,34,35IL4) gene expression in B cells by competing with signal transducer and activator of transcription 6 (STAT6) and CCAAT enhancer binding protein beta (CEBPB) for promoter binding [In general, silencers function in two ways: First, repressors may bind at silencers to block binding sites for activators and transcription machinery A. For ex binding .Second, silencers may prevent activators and/or GTFs from accessing promoters by establishing a repressive chromatin structure through the recruitment of histone modifiers or chromatin stabilizing factors B. In parHistone proteins are decorated by an array of post-translational modifications, including methylation and acetylation, that regulate chromatin accessibility and transcription factor binding . MethylaPolycomb group (PcG) proteins form Polycomb repressive complexes (PRC) that deposit H3K27me3 marks on facultative heterochromatin . Global STMN2) [SYN1) [Individual silencers have been shown to be enriched for repressors that recruit PcG proteins for their repressive function. The repressor element 1 (RE-1) is a silencer first identified in humans upstream of the stathmin 2 gene (STMN2) , and sub) [SYN1) . RE-1 wa) [SYN1) ,37. Usin) [SYN1) .The multifunctional transcriptional factor YY1 can repress gene expression in some specific conditions by recruiting PRC2 ,44,45, cBesides histone methylation, chromatin compaction is also correlated with histone deacetylation. Acetyl groups reduce the positive charges on histones, which decreases electrostatic interactions with chromatin, thereby relaxing and opening the chromatin structure for transcription factor binding . Hence, LHCGR) [TERT) [IL1A) [CDKN1A) [EPAS1) [CD4 gene [-/CD8- double-negative and CD4-/CD8+ single-positive T cells but not in CD4+/CD8- single-positive T cells [TSHB [CYP1A1 [POU1F1 [NOS2 [For example, SP1 and SP3 have been shown to function as repressors by recruiting SIN3A-HDAC complexes, inhibiting the expression of genes such as luteinizing hormone receptor (LHCGR) ,47, telo) [TERT) , interle) [IL1A) , cyclin [CDKN1A) , and end [EPAS1) . The RUNCD4 gene , recruitCD4 gene . NotablyCD4 gene ,54, and  T cells . POU2F1  T cells , inhibitls [TSHB , CYP1A1  [CYP1A1 , POU1F1  [POU1F1 , and NOSF1 [NOS2 .Just as enhancers or super-enhancers form chromatin interactions with distant gene promoters to activate transcription ,60,61, sLooping silencers have been well documented in Drosophila . The DroChromatin loops similarly connect H3K27me3-marked domains in mice. Using Chromatin Interactions Analysis with Paired-End Tag sequencing (ChIA-PET), Ngan et al.  enrichedABCC2 gene that connected to CPN1 through chromatin looping [ABCC2 and the distal CPN1, validating the long-range repressive function of this silencer. Apart from the validated ABCC2 looping silencer, the same study also intersected promoter capture Hi-C data from human primary blood cells and silencers identified from K562 cells, and identified around 4000 silencer–promoter chromatin interactions, suggesting a high prevalence of looping silencers [+ cells [In humans, few examples of looping silencers have been identified. Hi-C and ChIA-PET analysis in K562 cells identified a silencer proximal to the  looping . CRISPR/ilencers . Another[+ cells ; howeverIGF2 led to the upregulation of both proximal and distal genes, including genes associated with erythroid differentiation and cell adhesion, as well as growth inhibition in xenograft models, suggesting that looping silencers are important in establishing cell identity. Removing the silencer led to decreased H3K27me3 and increased H3K27ac histone modifications at distal loops, opening up the chromatin structure. This suggests that looping silencers function by maintaining a repressive chromatin structure. Taken together, these recent works demonstrate the importance of silencers in maintaining cell identity and show that silencer looping is likely to be a major mechanism of repression.In our recent work, we identified MRRs with exquisite enrichment of H3K27me3 as putative human silencers and found that MRRs were highly associated with chromatin interactions . MRRs shIGF2 looping silencer, there has been no detailed exploration on the mechanism and function of looping silencers. We speculate that repressors may facilitate the formation of chromatin interactions between repressed regions , concentrating transcription activators within biomolecular condensate compartments at promoter–enhancer interactions ,73,74,75The formation of constitutive heterochromatin domains is mediated by the chromobox 5 protein , which recognizes and binds to H3K9me2/3 ,79. CBX5Facultative heterochromatin compaction may also be mediated by LLPS of PcG proteins. Canonical Polycomb repressive complex 1 (cPRC1) is assembled from RING1A and RING1B histone ubiquitin ligase proteins, a Polycomb group ring-finger domain protein (PCGF2/4), a Polyhomeotic homologous protein (PHC1/2/3), and a chromobox protein (CBX2/4/6/7/8). CBX7/8 mediates the binding of cPRC1 to H3K27me3-marked regions , while CAlthough preliminary studies have provided strong evidence of heterochromatin condensates in vitro and in vivo, important questions about the formation of these condensates remain unanswered: How do these condensates assemble and disassemble dynamically, especially during cell cycles? Does the formation of these condensates require initial seeding of heterochromatin proteins at specific nucleation sites? The answers to these questions will greatly improve our understanding of chromatin organization and gene regulation.KCNQ1OT1 [Non-coding RNAs (ncRNA) are key regulators of gene expression. Recently, Long et al.  found thKCNQ1OT1 . These rKCNQ1OT1  found thXIST), which orchestrates X chromosome inactivation (XCI) in females [XIST ncRNA triggers a cascade of events, including loss of histone acetylation [XIST binds first to chromatin in close spatial proximity to its gene locus, before spreading further to binding sites across the entire chromosome [Although there are no examples of ncRNA-mediated silencers to date, various ncRNAs have been shown to play a direct role in silencing gene transcription. One of the most well-known examples is X inactive-specific transcript .Taken together, these pieces of evidence suggest that ncRNAs may play important roles in silencer function, potentially by recruiting repressors to proximal silencers, or mediating long-range chromatin interactions between distal silencers and target promoters. It would be interesting to dissect the exact role of ncRNAs regarding loop and TAD formation, and repressor recruiting. Specifically, revealing detailed looping silencer examples involving ncRNAs is necessary.CD4 gene was reported to repress CD4 gene expression during T cell lymphocyte development, which can play roles in cell date determination [L1CAM) and other target genes in non-neural tissues during early embryonic development [Human silencers have been shown to repress target genes during development and differentiation processes. For example, a lineage-specific silencer of mination . The RE1elopment . REST, telopment .CPED1 gene was identified in GM12878 cells, but the same genomic locus was identified as a super-enhancer in K562 cells, indicating the importance of silencers in the precise control of gene transcription in different cells [Genome-wide identification of human silencers has revealed that these CREs are cell line-specific and may function as enhancers or silencers depending on the cellular context ,32,33,34nt cells  and DoniABCC2 and ABCG2 increased chemo-resistance to doxorubicin, daunorubicin, and etoposide [Silencers have also been suggested to function in drug resistance. Deletion of silencer regions linked to the drug transporter genes toposide , suggestAltered expression of PcG proteins has been commonly observed in human cancers. EZH2, the enzymatic subunit of PRC2 , is recuSUZ12 inhibited cell apoptosis by inhibiting pro-apoptotic genes such as harakiri (HRK) [E2F1) gene expression [BMI1 occurs in both solid tumors and hematological cancers and is linked to proliferation, invasion, and poor patient survival [Besides EZH2, overexpression of other PcG proteins such as SUZ12 and BMI1 have also been shown to favor cancer progression. In epithelial ovarian cancer, high expression of ri (HRK) . In non-pression . Overexpsurvival . BMI1 issurvival ,114. TogApart from curbing the expression of neuronal-specific genes in non-neuronal tissues, REST also functions as a tumor suppressor gene . Loss ofTaken together, silencers play important roles such as cell development, differentiation, and perhaps even evolution. Several lines of evidence suggest that alteration of silencer sequence or the silencer-associated repressor can lead to various diseases, including cancers.Spatial and temporal control of gene expression is crucial for multicellular organism development, and dysregulation of these regulatory mechanisms can lead to various diseases such as cancer ,119. CREHuman silencers have been shown to exist to play gene repression roles, and multiple human silencer examples including proximal silencers and looping silencers have been elucidated in different cellular contexts. Although multiple experimental and bioinformatics methods have been proposed for systematic genome-wide identification of human silencers, ,32,33,34Different subclasses of silencers may be endowed with subclass-specific chromatin signatures or repressor binding profiles. In different cellular contexts and different developmental stages, the chromatin signatures and repressor binding profiles of these elements can switch between repressive and activating modes, functioning as both silencers and enhancers ,33,122, There are heated debates on whether super-enhancers are merely an assembly of nearby enhancers or whether the constituent enhancers work cooperatively to regulate the same gene ,125. EnhSuper-enhancers are enriched with various activators such as BRD4, and have been shown to control cell identity . In cancSimilar to super-enhancers, the importance of silencers towards cell identity has been demonstrated, and perturbation of silencers has been shown to lead to changes in cell identity . Genome-


Background: Doxorubicin resistance remains a major therapeutic challenge leading to poor survival prognosis and treatment failure in breast cancer. Although doxorubicin induces massive changes in the transcriptional landscape are well known, potential diagnostic or therapeutic targets associated with the reorganization of three-dimensional (3D) chromatin architecture have not yet been systematically investigated.Methods: Here we performed in situ high-throughput chromosome conformation capture (Hi-C) on parental and doxorubicin-resistant MCF7 (MCF7-DR) human breast cancer cells, followed by integrative analysis of HiC, ATAC-seq, RNA-seq and TCGA data.Results: It revealed that A/B compartment switching was positively correlated to genome-wide differential gene expression. The genome of MCF7-DR cells was spatially reorganized into smaller topologically associating domains (TADs) and chromatin loops. We also revealed the contribution of increased chromatin accessibility and potential transcription factor families, including CTCF, AP-1 and bHLH, to gained TADs or loops. Intriguingly, we observed two condensed genomic regions (∼20 kb) with decreased chromatin accessibility flanking TAD boundaries, which might play a critical role in the formation or maintenance of TADs. Finally, combining data from TCGA, we identified a number of gained and lost enhancer-promoter interactions and their corresponding differentially expressed genes involved in chromatin organization and breast cancer signaling pathways, including FA2H, FOXA1 and JRKL, which might serve as potential treatment targets for breast cancer.Conclusion: These data uncovered a close connection between 3D genome reorganization, chromatin accessibility as well as gene transcription and provide novel insights into the epigenomic mechanisms involving doxorubicin resistance in breast cancer. Breast cancer is one of the cancers with the highest morbidity and mortality rates, and its incidence has progressively increased over the last few decades in the female population . AlthougS. peucetius var. caesius  data, and mRNA sequencing data on the parental and doxorubicin-resistant MCF7 cell lines, and systematically investigated global alterations in the 3D chromatin architecture associated with differential gene transcription, including compartments, TADs, and enhancer-promoter interactions.In the present study, we applied integrated analyses combining 2 at 37°C in 10-cm dishes. Thereafter, the medium was renewed every 2–3 days until ready for sub-culturing. At about 80% confluence, cells were serially passaged by 1:4 dilution after trypsinization. The resistant-derived cells were maintained in 1 μM doxorubicin hydrochloride , and the parental MCF7 cells were grown in a drug-free medium for comparison. The MCF7-DR cells were maintained in the doxorubicin-free medium for about one week prior to further experiments.The MCF7 and doxorubicin-resistant MCF7 cell lines were originally obtained from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences  and cultured as previously described . Brieflyin situ Hi-C libraries was optimized according to previous protocols   was usedTADs and their corresponding boundaries were identified at different resolution with hicFindTADs command in HiCExplorer suite (v3.6)  with parAfter chromatin compartments analysis with HOMER suites (v4.10) , the anaChromatin loops were identified based on a strict candidate selection, negative binomial distributions and Wilcoxon rank-sum tests as described in the previous publication  using hiBy comparing the identified TAD boundaries and their separation scores, it was defined as a gained TAD boundary that was only identified as a boundary in MCF7-DR cells and possessed a lower TAD-separation score  compared to the parental cells; a lost TAD boundary was associated with a higher TAD-separation score  in MCF7-DR cells and was only detected in MCF7 cells; the boundaries detected in both MCF7 and MCF7-DR cells were defined as stable TAD boundaries.According to the genomic locations, the chromatin loops were classified into stable, gained or lost: stable, loops present in both MCF7 and MCF7-DR cells; gained, loops present in the MCF7-DR cells but not in the MCF7 cells; lost, loops present in the loops but not in the MCF7-DR cells.To measure the aggregate strength of stable, lost or gained chromatin loops, APA score was calculated as described previously  and visup value lower than 0.01 were kept for downstream analysis. Then the annotatePeaks.pl (https://ggplot2.tidyverse.org).To identify transcription factors potentially involved in the formation of TAD boundaries or loop anchors, motif enrichment analysis was performed using the findMotifsGenome.pl script from HOMER suite (v4.10)  with thePeaks.pl  was usedThe “bedtools intersect” command of BEDTools (v2.30.0)  was usedATAC-seq and RNA-seq data of MCF7 and MCF7-DR cells were obtained from our previous publication and are available in the NCBI Gene Expression Omnibus (GEO) repository under the accession number GSE174152 , includip value < 0.05.Functional annotation enrichment analysis with GO terms and KEGG pathways were performed using the clusterProfiler R package  for gene+; n = 363) and the other group . The mRNA expression levels (FPKM) of representative genes were compared between the Dox+ and Dox− subgroups using the Wilcoxon rank-sum test in R software (v 4.1.2). Pearson’s correlation coefficient was then calculated to assessing the associations between critical DEGs using the “corrplot” R package. The Kaplan-Meier survival curves and log-rank test were performed by utilizing the “survival” and “survminer” packages in R to analyze survival differences. p < 0.05 were regarded as statistically significant.Based on the clinical and genomic data from The Cancer Genome Atlas (TCGA) Breast Invasive Carcinoma (BRCA), the breast cancer samples were divided into two subgroups based on whether the doxorubicin (a.k.a. adriamycin) was used alone, namely the doxorubicin alone group  were calculated in the analysis. Significant differences for all quantitative data were considered when *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.If not specified, all bioinformatics analyses were performed with R software (v4.1.2) to compute statistics and generate plots throughout this manuscript. Wilcoxon rank-sum test or Student’s t-test was used to assess significance in paired comparisons. Pearson correlation coefficient was used to compute correlations between two conditions. Fold change, To probe the reorganization of 3D chromatin architecture in doxorubicin-resistant breast cancer, we generated Hi-C libraries from two independent biological replicates for doxorubicin-resistant MCF7 (MCF7-DR) cells and parental MCF7 cells , which aIncreasing evidence suggests that the mammalian genome is spatially organized into two major types of compartments, “A” and “B”: A compartments are mainly composed of euchromatin which is transcriptionally active whereas B compartments are characterized by inactive heterochromatin and exhibit high chromatin density . To addrTo further understand the link between compartment switching and differential gene expression associated with doxorubicin resistance, in combination with mRNA-seq data , we idenEach chromosome is composed of many distinct megabase-sized chromatin domains, referred to variably as topologically associating domains (TADs) that interact more frequently within themselves than with neighboring regions . DisruptChromatin loops are another level of hierarchical genome organization, ranging from Kbs to Mbs, and bring distal cis-regulatory elements such as enhancers into close physical proximity with promoters of target genes, which are vital for the regulation of gene transcription . Using tIn combination with ATAC-seq and RNA-seq data, we analyzed the distribution characteristics of chromatin accessibility, mRNA expression levels and TAD-separation scores around the TAD boundaries and loop anchors. Our data revealed that both TAD boundaries and loop anchors possessed lower TAD separation scores  and wereTranscription factors (TFs) regulate gene expression through binding directly to cis-regulatory elements of target genes in a sequence-specific manner dependent on the consensus motif . To examOur previous work demonstrated that differentially accessible regions (DARs), including 11686 hyper- and 6542 hypo-accessible regions, were positively correlated with nearest DEGs in MCF7-DR cells . To furtWhat is more, it is interesting that we have stumbled across two stably condensed chromatin regions (∼20 kb) with decreased chromatin accessibility  flanking (within ±30 kb) the stable, gained or lost TAD boundaries , 3C, whiIn order to identify TFs potentially involved in TAD reorganization associated with doxorubicin resistance, the sequences of changed TAD boundaries were scanned for significantly enriched TF motifs using HOMER software (v4.10) . As expeTo obtain potential TFs associated with chromatin loop changes, the identified loops were classified into three types based on the genomic location of loop anchors: 1283 stable loops, 6405 gained loops and 5483 lost loops . AggregaETS1, EGFR and WTAPP1  are increasingly being recognized as an important mechanism involved in epigenetic regulation of gene expression and chromatin organization . Using td WTAPP1 , whereasand UBR5 , indicatand UBR5 , which wand UBR5 , respectand UBR5 , suggestand UBR5 , such asost EPIs . The gai+; n = 363) and without  doxorubicin alone. Correlation analysis showed that the expression pattern of JRKL was positively correlated with CCDC82 expression (r = 0.85), both of which were negatively correlated with FOXA1; the expression pattern of E2F3 was positively correlated with EZH2 (r = 0.53) and RIOK1 (r = 0.53) expression (JRKL and RIOK1 was verified to be significantly (p < 0.05) associated with the poor prognosis of the Dox+ patients when compared to the Dox− patients, whereas lower expression of FA2H and GPRC5A was related with the poor prognosis of Dox+ patients (JRKL and RIOK1 were significantly (p < 0.05) higher in the Dox+ patients than Dox−, while FA2H and GPRC5A expression levels were lower in the Dox+ patients  BRCA (breast invasive carcinoma) and analyzed their expression levels in breast cancer patients treated with  TAD boundaries and loop anchors in MCF7-DR cells. Combining ATAC-seq and mRNA-seq data, we further found that TAD boundaries and loop anchors possessed higher chromatin accessibility/openness, which would be in favor of the recruitment of TFs and insulator element-binding proteins, including insulator proteins CTCF and BORIS, which have been proposed to be indispensable for the formation of TADs and loops . Indeed By comparing MCF7-DR cells with MCF7 cells, we observed that the gained TAD boundaries were enriched with higher ATAC-seq signals and vice visa, demonstrating a positive correlation between chromatin accessibility changes and TAD boundary strength. Besides CTCF and BORIS, members of AP-1 TF family are potentially involved in the formation of the gained TAD boundaries, while the lost TAD boundaries might be related to the weakened binding affinity of members of DLX and LHX TF families. Similarly, bHLH family members might contribute to gained chromatin loops associated with CTCF . In addiFA2H, FOXA1, JRKL and EZH2, many of which were involved in chromatin organization, Notch and breast cancer signaling pathways and have been known to be closely related to drug resistance or epigenetic regulation in human cancer  depletion could lead to decreased chemosensitivity to cisplatin via inhibition of AMPK and activation of mTOR/S6K1/Gli1 pathway in gastric cancer (JRKL and down-regulate FA2H may constitute potential diagnostic or therapeutic targets for reversing doxorubicin resistance in breast cancer.Alterations in the intrinsic chromatin interaction landscape are associated with differential gene expression , and we n cancer . Additioc cancer , and turc cancer . Here weOur findings provide genome-wide evidence for the reorganization of 3D genome structure and accompanying chromatin accessibility and gene expression changes occurring in doxorubicin-resistant breast cancer cells. However, additional validation and further investigations will be required to establish the direct link between 3D genome reorganization observed in doxorubicin-resistant cell lines and specific molecular changes that can be used as therapeutic targets in breast cancer patients. Collectively, these results extend our understanding and suggest a key epigenomic mechanism of doxorubicin resistance and offer a resource for further studies to explore future therapeutic applications.


Epigenetic memory, which refers to the ability of cells to retain and transmit epigenetic marks to their daughter cells, maintains unique gene expression patterns. Establishing programmed epigenetic memory at each stage of development is required for cell differentiation. Moreover, accumulating evidence shows that epigenetic memory acquired in response to environmental stimuli may be associated with diverse diseases. In the field of kidney diseases, the “memory” of acute kidney injury (AKI) leads to progression to chronic kidney disease (CKD); epidemiological studies show that patients who recover from AKI are at high risk of developing CKD. The underlying pathological processes include nephron loss, maladaptive epithelial repair, inflammation, and endothelial injury with vascular rarefaction. Further, epigenetic alterations may contribute as well to the pathophysiology of this AKI-to-CKD transition. Epigenetic changes induced by AKI, which can be recorded in cells, exert long-term effects as epigenetic memory. Considering the latest findings on the molecular basis of epigenetic memory and the pathophysiology of AKI-to-CKD transition, we propose here that epigenetic memory contributing to AKI-to-CKD transition can be classified according to the presence or absence of persistent changes in the associated regulation of gene expression, which we designate “driving” memory and “priming” memory, respectively. “Driving” memory, which persistently alters the regulation of gene expression, may contribute to disease progression by activating fibrogenic genes or inhibiting renoprotective genes. This process may be involved in generating the proinflammatory and profibrotic phenotypes of maladaptively repaired tubular cells after kidney injury. “Priming” memory is stored in seemingly successfully repaired tubular cells in the absence of detectable persistent phenotypic changes, which may enhance a subsequent transcriptional response to the second stimulus. This type of memory may contribute to AKI-to-CKD transition through the cumulative effects of enhanced expression of profibrotic genes required for wound repair after recurrent AKI. Further understanding of epigenetic memory will identify therapeutic targets of future epigenetic intervention to prevent AKI-to-CKD transition. Epigenetics refers to the study of modifications of gene expression patterns that are not caused by alterations in DNA nucleotide sequences . The abiIn the field of kidney diseases, the memory property of epigenetics is widely accepted as a contributor to “metabolic memory” in diabetic kidney disease (DKD) ; a serieWhile the prevalence of CKD and its subsequent progression to end-stage renal disease (ESRD), requiring renal replacement therapy, is increasing worldwide, there are no effective therapies for renal fibrosis . EvidencHere we focus on epigenetic memory in AKI-to-CKD transition: the epigenetic alterations induced by AKI that persist and contribute to the development of subsequent CKD. We review the most recent findings on the molecular basis of epigenetic memory and the pathophysiology of AKI-to-CKD transition. Accordingly, we present plausible hypothetical mechanisms of epigenetic memory that mediate this transition and the prospects for their potential therapeutic application.Renal homeostasis is maintained by multiple types of differentiated cells with specific functions that compose the kidneys . AKI-to-The kidney is physiologically prone to hypoxia because of its characteristic vascular structure and high metabolic demand . The perRenal epithelial cells possess remarkable regenerative capacity, which enables them to completely recover from mild injury with subsequent resolution of renal perfusion . HoweverThe limited regenerative capacity of the renal microvasculature, in contrast to renal tubules, explains the AKI-induced reduction in the number of capillaries, called “capillary rarefaction,” which results in chronic renal hypoxia, contributing to CKD progression . The undSubsequent renal hypoxia induces expression of proinflammatory and profibrotic gene of tubular epithelial cells through epigenetic alterations, which is called “hypoxic memory” reviewed  Nangaku. HypoxiaMyofibroblasts are activated forms of fibroblasts that produce large amounts of extracellular matrix (ECM) that contributes to tissue fibrosis formation . These cells are predominantly derived from resident fibroblasts and pericytes  other o and endoRenal hypoxia, as described above, contributes to the pathophysiology of AKI-to-CKD transition. Indeed, renal hypoxia occurs in various forms of CKD . For exaRecent studies suggest “metabolic reprogramming” of tubular epithelial cells is also an important factor that contributes to the AKI-to-CKD transition . As an aStudies suggest that successful switching metabolism from glycolysis to FAO after AKI is an important checkpoint for determining whether to progress to CKD. Identifying the mechanisms of maladaptive persistence of metabolic reprogramming following AKI even after tubular epithelial cells are successfully repaired, including the presence or absence of epigenetic memory, requires further investigation.The ischemia-reperfusion injury (IRI) model is a validated animal model of AKI-to-CKD transition that induces transient renal ischemia through clamping renal arteries, which generates tubulointerstitial fibrosis weeks later .Liu et al. conducted RNA sequencing (RNA-seq) of whole kidney using murine IRI models, from 2 h to 12 months following reperfusion, which reveal dynamic changes in gene expression, characterized by several functionally related temporal-specific patterns of gene expression, including tubular injury and repair, fibrosis, and the inflammatory response . These tProximal tubular cells are the most sensitive to AKI among renal cell populations. Their regenerative capacity is limited, and the success or failure of tubular repair may depend on the intensity of an injury . Acute iKim-1)  . In contr repair . Howeverr repair .Vcam1+/Ccl2+ was identified using time-course RNA-seq subsequent to IRI in mouse models, which is called the “failed repair proximal tubular cell” (FR-PTC)  . These “(FR-PTC) . Such ce(FR-PTC) . Further(FR-PTC) . This str” cells . This suEpigenetic memory affecting the acquisition of a pathogenic phenotype, such as proinflammatory and profibrotic phenotypes involved in the pathophysiology of AKI-to-CKD transition, if present as in DKD, may serve as promising therapeutic targets. Whereas renal fibrosis represents the hallmark of CKD, emerging evidence suggests a proregenerative effect of fibrosis on kidney repair following AKI, particularly during the early phase . Here weTubular remodeling following injury is mediated by a significant capacity to recover tubular cells  associated with kidney repair, providing insight into the appropriate timing of antifibrotic therapeutic intervention . Super-eIn this chapter, we emphasize the possible protective role of fibrosis during the early stage of kidney injury. In contrast, if these proregenerative responses maladaptively persist, those responses may switch upon subsequent progression of tubulointerstitial fibrosis. Further, epigenetic alterations are involved in gene expression dynamics after AKI through enhancer regulation, and intervention in the action of these factors may modulate postinjury responses. Epigenetic alterations trigger dynamic phenotypic changes by regulating enhancer and SE activity. If epigenetic memory induces maladaptive extension of the normal wound healing process, it may serve as a promising therapeutic target.Our group focuses on renal hypoxia as a common pathway driving the transition of AKI to CKD. We identified epigenetic alterations that may contribute to the progression of fibrosis induced by hypoxic stimuli.Hypoxia-inducible factor (HIF) is a master TF that mediates the adaptive response to hypoxia through the activation of target genes harboring hypoxia-responsive elements (HREs) within their regulatory domains . SignaliSLC2A3 transcription through its interaction with lysine-specific demethylase 3A (KDM3A), a HIF-1-induced protein that demethylates repressive dimethylated H3 lysine 9 (H3K9me2)  assay. Under normoxic conditions, the 35-kb upstream region of the SLC2A3 promoter (enhancer 1) is structurally close to the transcriptional start site (TSS) through the binding of HIF-1 located in both sites, forming a loop, which is lost when HIF-1 expression is inhibited by a specific small interfering RNA (siRNA). In contrast, under hypoxic conditions, HIF-1 additionally binds to the 24-kb upstream region of the SLC2A3 promoter (enhancer 2), and KDM3A is recruited via the interaction with HIF-1 located in the TSS, enhancer 1, and 2 with two loops, enabling robust expression of SLC2A3, which demonstrates that HIF-1-dependent epigenetic alterations in chromatin conformation regulate downstream gene expression , as revealed by analysis of a DNA microarray, detected early hypoxia-responsive genes, which are functionally associated with glycolysis, including the gene encoding solute carrier family 2A3: SLC2A3 (GLUT3) . We founH3K9me2) . Furtherpression . Moreove in vivo , suggestGenome-wide analysis of HIF-1 binding sites of cultured tubular cells exposed to a hypoxic environment using RNA-seq and ChIP-seq identified 44 long noncoding RNAs (lncRNAs) that are upregulated under hypoxia in multiple tubular cell lines . For exaAs described in Increasing evidence has demonstrated that epigenetic alterations are involved in the pathophysiology of AKI-to-CKD transition ; howeverThere are several studies focusing on the persistence of epigenetic alterations during AKI-to-CKD transition. Most of the animal models use IRI model because it is the most common cause of AKI.Zager et al. found that a single IRI in mice induces upregulation of histone-modifying enzymes (SET1 and BRG1), and elevation of gene-activating histone modifications (H3K4me3 and histone variant H2A.Z) at the promoters of the genes encoding the proinflammatory monocyte chemoattractant protein 1 (MCP1), the profibrotic TGFβ1, and collagen III . These eFurther, changes in DNA methylation patterns induced by IRI can be also epigenetic memory contributing to AKI-to-CKD transition. Several underlying mechanisms that IRI affects DNA methylation have been proposed: 1. IRI-induced oxidative stress causes DNA lesions, which affects the binding of DNA methyltransferases (DNMTs) and methyl-binding proteins; 2. Ten-eleven translocation (TET) enzymes (responsible for active DNA demethylation) is arrested during ischemia because of oxygen depletion; and 3. Hypoxia during ischemia directly modulates the expression of DNMTs and TET enzymes .Vegfα promoter throughout the observational periods  gene in rat kidney, thus implicating local C3 synthesis by tubular epithelial cells (C3 promoter in both ischemic control group (48 h of reperfusion) and chronic nephropathy group (6 months after transplantation), suggesting that aberrant DNA demethylation persists throughout disease progression to chronic allograft injury  is hypomethylated in cisplatin-induced AKI, accompanied by a marked induction of Irf8, which contributes to the pathogenesis of cisplatin-induced AKI , including TFIIB, TFIID, TFIIE, TFIIF, and TFIIH into a preinitiation complex (PIC) at the promoter domain . For PolThe mechanism underlying the ability of a TF to access closed chromatin is insufficiently understood despite evidence that suggest several models of chromatin accessibility remodeling for TF binding . First, As reviewed above, promoter accessibility must be established to initiate transcription, and highly dynamic chromatin accessibility is regulated by the interactions among TFs, active chromatin remodelers, and histone modifiers , which aReturning to the promoter recognition in PIC assembly, the TATA box-binding protein (TBP) subunit of TFIID binds to the TATA box in promoters in a sequence-specific manner  Sainsbu. AlthougThe promoter-TBP complex then binds to TFIIB, which is required for initiation through the recruitment of the Pol II-TFIIF complex to the promoter with its N-terminal domain (“B-ribbon”) bound to Pol II and its C-terminal domain (“B-core”) bound to DNA and TBP . TFIIB aThereafter, this core initiation complex assembles with TFIIE and TFIIH and forms a complete PIC  Sainsbu. The TFIThe multi-subunit coactivator complex Mediator regulates the initiation phase of transcription in various aspects . MediatoAfter promoter escape, Pol II forms the elongation complex that processively extends the nascent RNA chain . During In metazoan cells, Pol II typically pauses 20–80 nucleotides after the TSS, which is referred to as “promoter-proximal pausing” . Such paFollowing its escape from promoter-proximal pausing, Pol II moves downstream to the gene body and soon encounters nucleosomes as structural barriers against elongation  Core an. ElongatDrosophila cells, suggests that the proportion of genes displaying Pol II pausing is roughly conserved across species  polymerases (PARPs) contribute to this regulatory step of elongation . The rolThe persistent alterations of factors involved in these regulatory processes after initial stimulation may contribute to remodeling subsequent gene expression as epigenetic memory. For example, DNA methylation in the promoter region suppresses gene expression by either reducing chromatin accessibility for TFs or reader proteins that are involved in recruiting repressor complexes . HistoneTranscriptional memory is a highly conserved molecular mechanism that influences the rate or output of gene expression following a stimulus and is enhanced by previous exposure to the same stimulus to optimize gene expression patterns in a rapidly changing environment . This prTranscriptional memory requires changes in factors that control the degree of gene expression. Such factors are induced and conserved following the initial stimulus. In principle, the “memory” is retained in factors described above, which are involved in each step of transcriptional regulation. These factors, including TFs and those involved in chromatin opening, recruitment, initiation, elongation of Pol II, and enhancer-promoter interactions.The inducible gene encoding inositol-1-phosphate synthase (INO1) in budding yeast serve as models for transcriptional memory; moreover, emerging evidence suggests the contribution of transcriptional memory to immune responses of human cells . Here weINO1 upon exposure to the second stimulus  in the promoter, to which Sfl1 specifically binds, and the nuclear pore protein Nup100 , which may involve H3K4me2 inheritance during DNA replication , along with other mechanisms, epigenetically inheritable changes such as the incorporation of H2A.Z, H3K4me2, and poised Pol II at the promoter contribute to primed reactivation  collected from subjects with T2DM (T2D-PTECs) and without T2DM (N-PTECs) . They denditions . Notablytivation , possiblMoreover, transcriptional memory may be induced in tubular epithelial cells with “endotoxin preconditioning”. The mechanisms of endotoxin preconditioning have been extensively studied in immune cells, such as macrophages. Toll-like receptors (TLRs) exposed to bacterial endotoxin, such as LPS, induce the transient silencing of a subset of genes, including those encoding proinflammatory mediators, and exhibit a transient immunosuppressive state upon recurrent stimulation with LPS (“LPS tolerance”). This is one of the most important mechanisms that protects the host from dysregulated inflammation . Foster These studies suggest that renal tubular epithelial cells may also utilize the mechanism of transcriptional memory. It is quite possible that transcriptional memory is also involved in the expression of genes that promote inflammation and fibrosis in the pathogenesis of AKI-to-CKD transition as well.Numerous studies show that the molecules that confer epigenetic modifications involved in the pathophysiology of AKI-to-CKD transition show promise as future therapeutic targets . HoweverVCAM1+ proximal tubular cells” identified in healthy human kidneys , transcription initiation (regulated by factors mediating promoter escape), and elongation (regulated by factors mediating pause release). Alterations of these regulating factors caused by AKI that persist as “priming” memory may thereafter induce excessive responses to even a mild AKI, and the accumulation of such responses may lead to the progression of tubulointerstitial fibrosis.Therapies targeting epigenetic mechanisms that underlie AKI-to-CKD transition show promise because of their reversibility and accumulating favorable results in preclinical studies. Along with recent technical advances in epigenetic editing, identifying the detailed mechanisms of epigenetic memory mediating the transition from AKI to CKD will help resolve the low specificity of potential therapeutics and their wide range of off-target effects, which pose major clinical challenges. Importantly, temporal specificity should be considered along with locus specificity for optimizing the epigenetic intervention of AKI-to-CKD transition. Emerging evidence suggests that fibrosis plays a proregenerative role in the early phase of kidney repair, and antifibrotic therapy may exert opposing effects on disease progression, depending on the timing of treatment. Therefore, the most suitable timing to maximize therapeutic effects by targeting maladaptively maintained epigenetic memory must be determined.In conclusion, epigenetic memory that remains maladaptive after the completion of the tubular repair process shows great potential as a novel therapeutic target of AKI-to-CKD transition. Recent advances in structural and genome-wide analyses will illuminate in greater detail the epigenetic landscape during the transition from AKI to CKD. Timely, target-specific epigenetic intervention established according to a detailed understanding of epigenetic memory will resolve the challenges of low-specificity and broad off-target effects, and present the possibility of applying epigenetics to clinical practice.


The cellular mechanisms organizing chromosomal architecture in the eukaryotic genome are not yet well understood. Here, based on our demonstration that chemical disruption of nuclear condensate assembly weakens the insulation properties of a specific subset (∼20%) of topologically associated domain (TAD) boundaries, we were able to reveal that these boundary elements are characterized by high transcriptional activity, striking spatial clustering, and an augmented presence of long-distance/interchromosomal interactions between transcription units widely expressed in diverse cell types , thus providing insights into the physical basis for TAD-boundary establishment. Therefore, these results support a model whereby transcription contributes to chromatin compartmentalization and hierarchical genome organization, potentially through the association of transcriptional condensates with membraneless nuclear organelles. The eukaryotic genome is partitioned into distinct topological domains separated by boundary elements. Emerging data support the concept that several well-established nuclear compartments are ribonucleoprotein condensates assembled through the physical process of phase separation. Here, based on our demonstration that chemical disruption of nuclear condensate assembly weakens the insulation properties of a specific subset (∼20%) of topologically associated domain (TAD) boundaries, we report that the disrupted boundaries are characterized by a high level of transcription and striking spatial clustering. These topological boundary regions tend to be spatially associated, even interchromosomally, segregate with nuclear speckles, and harbor a specific subset of “housekeeping” genes widely expressed in diverse cell types. These observations reveal a previously unappreciated mode of genome organization mediated by conserved boundary elements harboring highly and widely expressed transcription units and associated transcriptional condensates. Microscopic studies and sequencing-based approaches have revealed that the eukaryotic genome is hierarchically organized and partitioned into distinct compartments , 2. For The emergence of the biophysical process of liquid–liquid phase separation (LLPS) as a potential driving force in forming several membraneless cellular organelles , 13 rais1319However, technically, it remains a challenge to determine whether the LLPS-based mechanisms contribute to the formation of the chromatin organizing units such as TADs and A/B compartments in the intact cells. Because of a clear overlap of transcriptionally less active heterochromatin with the B compartments  and tranTreatment of cells with the aliphatic alcohol 1,6-hexanediol  has been shown to disrupt many cellular membraneless organelles by weakening the hydrophobic interactions between molecules , 27. ThiThe aliphatic alcohol 1,6-HD has been used extensively to test the material property and the contribution of RNA–protein condensates in the assembly of membraneless compartments in vitro and in vivo –29. The SI Appendix, Dataset S1). We constructed contact count matrices using genomic bins of sizes 40 kb to identify TAD boundaries, 500 kb to generate A/B compartment profiles, and heat maps representing low-resolution intra and interchromosomal contacts. Correlation coefficient calculations exhibited high similarity between replicates . Therefore, the replicates were pooled for further analysis when needed. When we compared A/B compartment profiles in the different treatments, we found only rare changes in compartments, namely 0, 15, and 3 genomic bins for the 5 min, 30 min, and 3 h 1,6-HD–treated samples , respectively, out of 5,726 total bins when compared to the 2,5-HD control . This analysis revealed a significant change in the insulation score in ∼20% of boundaries in RUES1 cells  at the 5-min time point compared to 2,5-HD–treated samples . However, these boundaries gradually returned to normal insulation level by 3 h . This suggests that the recruitment of the architectural proteins CTCF and cohesin is equivalent on both boundary categories and is not the critical underlying feature of differential sensitivity to 1,6-HD. In contrast, cistrome analysis revealed that HDS boundaries were enriched in regulatory proteins such as RNA Polymerase II and other transcription regulators  database (cistrome.org/) of human transcription factors and regulators to search for known factors that are most enriched in each of the two classes of boundaries A. The mogulators A, suggesTo directly test the transcriptional activity of the TAD boundaries, we performed global run-on sequencing (GRO-seq)  or PRO-sSI Appendix, Fig. S2 C and D). Analysis targeting de novo transcripts revealed that 1,6-HD treatment led to 1,166 down-regulated genes and 520 up-regulated genes, suggesting dysregulation of transcription, with a preponderance of down-regulated genes . Next, we examined the PRO-seq profiles of all genes that overlap either with HDS or nonaffected boundaries . RUES1 cells were treated with flavopiridol and triptolide, and the samples were cross-linked and used for Hi-C library preparation . R. R35). Rparation B. This eparation C and D. paration E. These The eukaryotic genome is hierarchically organized into distinct compartments based on the transcriptional activity , 7. PhasFor further analysis, we filtered out boundaries that were 1 Mb proximal to centromere regions due to poor mappability. We generated contact matrices for all chromosomes to detect intrachromosomal and interchromosomal interactions using 4-Mb bins C. This rSI Appendix, Fig. S4A). Second, after 5 min of 1,6-HD treatment, interactions were reduced but subsequently recovered by 3 h after treatment . This suggests that highly expressed HDS boundaries utilize a mechanism of spatial chromatin organization using membraneless nuclear organelles as anchor points. Finally, to gain insight into the functional basis of this HDS-boundary organization, we examined 54 human tissue gene expression datasets from the Genotype-Tissue Expression (GTEx) database. We selected the top 1% of highly expressed genes common to all cell types . When taking into account that HDS boundary clusters account for ∼20% of the genome , we found that the top 1% of the highly expressed genes common to all cell types are fivefold more enriched on HDS-boundary clusters in comparison to the rest of the genome . These data suggest that highly transcribed HDS-boundary clusters are organized to ensure robust expression of a subset of essential genes, often referred to as “housekeeping genes,” required in most cell types.Actively transcribed genomic regions have been associated with nuclear speckles , 38. RecOur study provides insights into the physical mechanisms underlying the establishment of a subset of TAD-boundary regions. In this manuscript, we show that ∼20% of TAD-boundary elements, characterized by robust transcriptional activity and the recruitment of transcriptional apparatus, are sensitive to the disruption of hydrophobic interactions by the aliphatic alcohol 1,6-hexanediol. Because of the disruptive effects of 1,6-HD on a wide variety of biomolecular condensates , 27, theThe underlying mechanisms of chromosomal partitioning are not entirely understood. Studies have explored the enrichment of various chromatin-binding proteins and epigenomic markers to explain the organization of TADs. In mammalian cells, CTCF and cohesin subunits are highly enriched in a large proportion of boundary regions; however, CTCF and cohesin themselves are not sufficient to demarcate the boundary elements . OverrepMoreover, the fact that we could essentially reproduce the effect on boundary insulation with two drugs inhibiting transcription (flavopiridol and triptolide), which have very different mechanisms of action, suggests this finding is consistent and robust, despite the relatively small effect size. Our data, therefore, support the model that highly active transcription units maintain a subset of boundary regions. We propose that the transcriptional condensates assembled at high concentrations in these regions are required to maintain the boundary function. A study of early mouse development reported a lack of impact of transcription-inhibiting drugs on the chromatin architecture , perhapsAn equally intriguing observation is the resistance of the majority of TAD boundaries to 1,6-HD. It is possible that the condensates that organize these boundaries are not sensitive to 1,6-HD, as several types of molecular forces that drive the assembly of biomolecular condensates have differential sensitivities to various chemicals . It is a45Transcription has also been suggested to be responsible for short-range loop interactions, detected as “stripes,” that correlate to enhancer–promoter and promoter–promoter interactions even in the absence of CTCF or cohesin binding , 49. SucMany reported long-distance genomic interactions span multiple TADs and even different chromosomes –58. The In contrast to the mounting evidence for the spatial segregation of chromatin as a result of chromatin-activity states and phase separation –18, 64, SI Appendix, Fig. S4 B–D). We, therefore, hypothesize that these highly transcribed genes, critically required in most cell types, have become organized in HDS-sensitive, highly accessible transcriptional boundaries in association with membraneless nuclear compartments, ensuring the robust expression of critical genes required in most human cell types. Indeed, these findings are consistent with the emerging roles of LLPS and biomolecular condensates in cellular organization and genome compartmentalization (Our analysis reveals that HDS-boundary clusters are enriched for genes commonly transcribed at high levels in most cell types (lization , 67. ThrSI Appendix reports methods information on the antibodies used, cell culture treatments, RT-qPCR, DNA and RNA FISH, microscopy, image analysis, ChIP-seq, PRO-seq, in situ Hi-C, Hi-C data analysis and visualization, and Cistrome analysis.Supplementary FileSupplementary FileSupplementary File


Atherosclerosis is an important pathological basis of coronary heart disease, and the antisense non-coding RNA in the INK4 locus (ANRIL) is located in the genetically susceptible segment with the strongest correlation with it - the short arm 2 region 1 of chromosome 9 (Chr9p21). ANRIL can produce linear, circular and other transcripts through different transcriptional splicing methods, which can regulate the proliferation and apoptosis of related cells and closely related to the development of atherosclerotic plaques. Linear ANRIL can regulate proliferation of vascular smooth muscle cells (VSMCs) in plaques by chromatin modification, as well as affecting on proliferation and the apoptosis of macrophages at the transcriptional level; circular ANRIL can affect on proliferation and apoptosis of VSMCs by chromatin modification as well as interfering with rRNA maturation. In this review we describe the evolutionary characteristics of ANRIL, the formation and structure of transcripts, and the mechanism by which each transcript regulates the proliferation and apoptosis of vascular cells and then participates in atherosclerosis. In the early stage of atherosclerosis, long-term mechanical change in blood flow, high-fat and high-glucose environment, inflammatory infection damage, etc. The pathological factors of injury and pro-apoptotic factors increase, leading to the increase of endothelial cell injury and apoptosis, and the destruction of vascular endothelial integrity . SubsequNon-coding RNA (ncRNA) refers to RNAs that are not translated into proteins, mainly including microRNA (miRNAs), long non-coding RNA (lncRNA), and circular RNA (circRNA) ,6. They LncRNAs are a class of RNAs with transcripts longer than 200bp that do not encode proteins . LncRNAsThe antisense non-coding RNA  in the INK4 locus originated in placental mammals and acquired additional exons during evolution, and then part of the exons was gradually lost, and finally It is fully formed in apes . ANRIL iThis article reviews the research progress of ANRIL transcripts with different structures that affect the occurrence and development of atherosclerosis by regulating the proliferation and apoptosis of vascular cells.2Last few years the function of circular ANRIL has also been gradually explored, and with the progression of atherosclerosis, the expression level of circular ANRIL in plaque lesion tissue gradually decreases ,64. Prot3Cyclic ANRIL can also be used as a molecular scaffold for chromatin modification complexes, affecting target gene expression by regulating the covalent modification of histones, thereby affecting cell proliferation and apoptosis; or as a dynamic scaffold, binding, storing or transport transcription factors to specific subcellular locations such as mitochondria, affecting cell proliferation and metabolism ,70. Ther3.1Meseure et al. found by RNA co-immunoprecipitation method that linear ANRIL binds efficiently to polycomb family proteins such as CBX7 and SUZ12 . Polycom3.2trans-action, which can activate gene transcription by binding to target gene promoters such as cyclins, and affect the level of target gene transcription and expression. Cytologically, it is manifested as increased macrophage proliferation, enhanced metabolic activity, and decreased apoptosis [Linear ANRIL can affect the transcriptional expression levels of multiple genes and increase the risk of atherosclerosis by interfering with the transcription of target genes. Overexpression of linear ANRIL in macrophage cell lines can induce AEBP2, EZH2, Jumonji/Jmj Jarid2 C-domain family protein, MEL18 DNA-binding protein, YY1 transcriptional repressor proteins, Gli-Kruppel family proteins with transcription regulation functions, such as COREST/REST, and these proteins are enriched in Alu repeats with the most active transcription of genes in the chromosomal segment ,78. Alu poptosis . Holdt epoptosis . Also, lpoptosis .4More and more evidences show that ANRIL plays an important role in atherosclerotic cardiovascular disease, but what mechanism affects cell proliferation and apoptosis and participates in the occurrence and development of atherosclerosis still needs further research. With the continuous emergence of new technologies, studies have discovered the existence of more ANRIL transcripts, and there are more means to detect the transcriptional expression levels of target genes and protein molecules in different pathways, which provides an important guarantee for more effective mechanism exploration. However, due to the presence of many common exon fragments among different ANRIL transcripts, it is difficult to distinguish it, and the expression level of ANRIL in peripheral blood is low, and the degradation rate of linear ANRIL in vitro is high. These factors can become a serious obstacle to the process of studying the effect of ANRIL on the mechanism of the onset and development of atherosclerosis. Therefore, it is necessary to create a more stable system and a more accurate measurement method to further study the mechanism of action of ANRIL.Ilgiz Gareev, Valentin Kudriashov and Albert Sufianov conceptualized and designed the study. All authors participated in the acquisition, analysis and interpretation of the data. Sema Begliarzade, Tatiana Ilyasova and Yanchao Liang drafted the manuscript. Ozal Beylerli contributed to critical revisions of the manuscript. All authors agreed on the journal to which the article would be submitted, gave final approval for the version to be published, and agreed to be accountable for all aspects of the work.None.The authors declare that no conflicts of interest exist.


Herein, a proof of concept highlighting the possibility of the up- or downregulation of RNA transcription is presented. Hybrid DNA–RNA triplex nanostructures were rationally designed to produce bacterial transcription units with switchable promoters. The rate of RNA production was measured using the signal of a transcribed fluorescent RNA aptamer (i.e. Broccoli). Indeed, several designed bacterial promoters showed the ability of induced transcriptional inhibition, while other properly tailored sequences demonstrated switchable enhancement of transcriptional activity, representing an unprecedented feature to date. The use of RNA-regulated transcription units and fluorescent RNA aptamers as readouts will allow the realization of biocomputation circuits characterized by a strongly reduced set of components. Triplex forming RNA oligonucleotides are proposed as smart tools for transcriptional modulation and represent an alternative to current methods for producing logic gates using protein-based components.Triplex nanostructures can be formed   The tri control . Althoug core enzyme, one sigma factor, and their cognate DNA sequences ,25. It h in vivo ,12,26,27in vitro transcriptional circuits controlled by diffusing oligonucleotides were made previously, which were based on the completion or disruption of a nicked T7 promoter by toehold-mediated strand-displacement   . By contEscherichia coli promoter architecture, Figure In this report, an artificial transcriptional regulation system was developed, in which bacterial promoters were designed to contain one or two TTSs for ribonucleic TFOs. The sequences were developed starting from the general N,N,N’,N’-tetramethylethylenediamine (TEMED), and Tris were purchased from ROTH. Magnesium acetate was purchased from Fluka. SybrGold nucleic acid stain, Triton X-100 and ammonium persulfate were purchased from ThermoScientific. Acrylamide/bis-acrylamide solution was purchased from Sigma Aldrich/Merck. Broccoli fluorescent ligand (Z)-4--2-methyl-1--1H-imidazol-5(4H)-one (DFHBI-1T) was purchased from Lucerna. σ70-saturated E. coli RNA polymerase (holoenzyme) was purchased from New England Biolabs (NEB). Dithiothreitol was purchased from Biochemica.All DNA and RNA sequences were ordered from IDT  as freeze-dried material. Four synthetic ssRNA strands were used: TFO1 5’–UCCUCCUCUUCUCCU-3’, TFO2 5’-UGGUGGUGUUGUGGU-3’, TFO3 5’-UGGUGUUGUGGUGGU-3’ and TFO4 5’-AGGAGAAGAGGAGGA-3’. Each TFO was designed to form a triplex with a specific geometry with its respective target. Simply put, the target duplex should contain the exact same sequence as the TFO in the parallel or antiparallel orientation (except T is replacing U). All DNA sequences are detailed in 2, 1 mM DTT, 0.01% Triton X-100) supplemented with additional MgCl2 to a final concentration of 30 mM. The additional magnesium was needed to ensure the formation of Broccoli and the triplexes. A Clariostar plate reader from BMG labtech with samples loaded in 384-well plates from Corning was used to acquire the fluorescence data. A JascoV-750, equipped with a Peltier unit, was used to perform temperature-dependent denaturation experiments to determine melting temperatures of the triplex structures. Melting temperature experiments were carried out in the same Mg-supplemented NEB buffer used in all other experiments. Triplex structures for EMSA (electrophoretic migration shift assay) experiments were prepared in the following way: First, the duplex DNA TTS was prepared using a 4-h ramp between 90°C and 4°C, then the specific TFO was added using the same Mg-modified NEB buffer used in all other experiments. Electrophoretic runs in 12% polyacrylamide, supplemented with 10 mM Mg2+, were performed using standard electrophoresis equipment (50 V for 3 h at 10°C) and stained with SybrGold.Double-stranded DNA 10 μM stock solutions were prepared in 50 mM KCl using a Mastercycler Nexus-GX from Eppendorf. All RNA polymerization experiments were carried out at 30°C in the recommended NEB buffer , which was performed via polyacrylamide gel electrophoresis (PAGE) and temperature-dependent denaturation analysis (i.e. melting temperature determination). The full-length transcription units were re-designed and reduced to 15-base pair dsDNA constructs containing the TTS without adjacent sequences. The corresponding RNA TFOs 1, 2, 3, and 4 were designed to generate triplex structures at the TTS and form Hoogsteen interactions with the polypurine sequence in the parallel pyrimidine motif, the antiparallel purine motif, and the parallel/antiparallel mixed motif, respectively . Figure ometries ,21,30,31In comparison to TFO4, the higher stability of the complex generated by TTS and TFO1 suggests its higher potential as a tight-binding oligonucleotide to inhibit transcriptional activity. Therefore, a DNA construct was designed to contain the 15 base polypurine TTS (TTS-1) in the template strand of the promoter consensus sequence between positions –35 and –10 (termed P1). Similar as for protein-based transcription factors binding in the P1 region, the TFO is then expected to compete with the polymerase for binding to the duplex.E. coli RNAp saturated with σ70 factor (i.e. the protein interacting with the promoter conserved domains and the RNAp), for the rate of production of the Broccoli RNA aptamer (50) are reported in Table 50 can be interpreted as a measure of the effectiveness of the TFO in inhibiting the transcription. In this view, the calculated EC50 values for TFOs 1 and 4, 22 ± 1 and 190 ± 2 nM, respectively, indicate that TFO1 is a stronger inhibitor than TFO4. Noteworthy, the EC50 values are fully in accord with the dissociation constants. Namely, the stronger the binding to the P1 promoter, the stronger is the effect of the oligonucleotide in hindering transcriptional activity.The synthetic promoters were tested in the presence of the  aptamer  that forvide supra). Again, fully in harmony with the EMSA assay, results indicate that the effects on the Broccoli transcription rate depend on triplex formation and was found to be significant for TFOs 1 and 4, Figure Based upon a number of experimental and theoretical studies, the hypothesis that natural triplex structures can modulate transcription with both inhibitory and enhancing effects is not farfetched ,17–19,32The results for the promoters containing the polypurine sequences downstream of the –10 domain in the template (P3) or sense strand P4) are displayed in Figure  are dispSummarizing, when TTS-1 was located at the template strand, which represents the model target for inhibition, both TFO1 and 4 hindered transcription as expected. In addition, the inhibitory effect follows the stability of the binding. The situation was inverted when TTS-2 was located on the template strand, in fact both TFO1 and TFO4 enhanced the RNA production. Differently, when TTS-1 and 2 were located at the sense strand, TFO1 enhanced the transcriptional activity whereas TFO4 displayed inhibition. Thus, both TTS positions and TFO1 and 4 can be suitably designed to up- or downregulate transcription of the fluorescent aptamer.Kd values measured by EMSA and the EC50 values observed in the polymerization experiments seems to be relevant only when inhibition of transcription is involved. In fact, the phenomenon of inhibition was attributed to a competition mechanism of the RNAp and the TFO for the same binding site within the promoter region  were determined in the presence of TFOs 5–9.Firstly, TTS-1 and TTS-2 were modularly combined into engineered promoters that contained two triplex target sites (TTS-3) as depicted in Figure Ideally, for a single TFO (i.e. TFO1 or TFO4), eight regulatory regions of type TTS-3 can be constructed by simple combinations, where two strands comprising the same TFO sequence bind simultaneously within the promoter and in the downstream region . Furthermore, to maximize the observed effect, only configurations that form triplex structures with mutual enhancement or inhibitory effects were selected. Thus, four potential TTS-3 combinations were designed for hosting TFO1 or 4, respectively.50 values were calculated to be 12 ± 1nM and 63 ± 1nM, while the transcriptional activity was inhibited by 70% and 90%, respectively. Similarly, Figure 50 of 120 ± 1 nM. Noteworthy, the transcription was 2-fold enhanced for this combination (i.e. 210%) and it was the highest enhancement obtained in the present study, indicating that the enhancement effect is strengthened by the presence of both TTSs in the sense strand. Table The experimental results show that engineered TTS-3 promoters that exerted an effect on transcription were P2 + P4 for TFO1 while P1 + P4 and P2 + P4 showed regulation in the presence of TFO4. These promoters were subjected to increasing concentrations of the corresponding TFO as previously described for promoters with a single triplex target site. Figure 50 to higher values suggests a decrease in the affinity of the TFOs for their target site on the promoter. Notably, the effect on TTS-3 ranged from a doubling to almost silencing the transcriptional activity of the promoter, showing the tunability of the system which allows broad tuning of gene circuit parameters.In general, the shift of ECin situ by a system involving two transcription units. Transcription units were designed to contain the template for two purine-motif containing TFOs, separated by a spacer 4- to 8-nucleotides long. The kinetic analysis of Broccoli production from promoter P2 + P4 in the presence of increasing excesses of TFO transcription unit showed an inhibitory effect of 80% with an EC50 = 12 nM for two-tandem repeats (i.e. at a 0.4:1 ratio of TFO to Broccoli transcription unit). Figure 50 values and maximum effects, clearly, only a minute difference is detected among the different spacer length .In biological systems, transcription units belonging to different genomic regions can communicate and operate logic functions by their interaction with freely diffusing short nucleic acids and proteins acting as wiring molecules. Herein, to evaluate the effect of molecular wiring on the engineered P2 + P4 promoter, TFO4 or an extended TFO4 were directly produced in situ produced TFO to exert an effect on a secondary transcription unit containing the TTS (i.e. the Broccoli transcription unit).With respect to P2 + P4, the transcription units for the TFOs were used in the following concentration ratios: 0:1, 0.5:1, 0.75:1, 1:1, 1.25:1, 1.5:1, 1.75:1, 2:1, namely at the ratio 1:1 the TFO-transcription unit and P2 + P4 were added to the mixture at the same concentration. Moreover, to compensate for the increase of the total promoter concentration and account for resource sharing effects of the RNAp, a third transcription unit, producing a polyadenine (polyA) RNA strand, was introduced into the system. The results showed the feasibility of the It is known that the formation of a triplex structure at the promoter of a transcription unit inhibits RNA polymerization by RNAp. As a general concept, this effect can be ascribed to competitive binding of the TFO and the RNAp to the promoter, which reflects the quantitative relationship between the stability of the hybrid triplex in comparison to the transcription initiation complex.E. coli promoter  or purine (inhibition) triplexes, Figure Importantly, TFO-producing transcription units were able to affect the transcription from engineered downstream promoters. This showed the possibility to wire the different transcription units with inhibitory effect. Also, due to the geometry of the triplex, the spacer sequence separating the two TFO domains could, in principle, be further engineered to contain an additional regulatory sequence (e.g. an aptamer) to effectively turn the sequence into a riboswitch that would form the triplex only in the presence of the stimulus or ligand.in vivo as a new bio-orthogonal transcription modulation approach.In conclusion, we demonstrated the possibility to build engineered transcription units, which were properly regulated by hybrid RNA–DNA triplex structures formed at the promoter. While such a modulation mechanism is similar to protein-mediated transcription regulation, an RNA-only regulatory machinery based on triplex structure formation is unprecedented, especially for transcription rate enhancement . Most imThe data underlying this article are available in the article and in its online supplementary material.gkac1131_Supplemental_FileClick here for additional data file.


Male fertility throughout life hinges on the successful production of motile sperm, a developmental process that involves three coordinated transitions: mitosis, meiosis, and spermiogenesis. Germ cells undergo both mitosis and meiosis to generate haploid round spermatids, in which histones bound to the male genome are replaced with small nuclear proteins known as protamines. During this transformation, the chromatin undergoes extensive remodeling to become highly compacted in the sperm head. Despite its central role in spermiogenesis and fertility, we lack a comprehensive understanding of the molecular mechanisms underlying the remodeling process, including which remodelers/chaperones are involved, and whether intermediate chromatin proteins function as discrete steps, or unite simultaneously to drive successful exchange. Furthermore, it remains largely unknown whether more nuanced interactions instructed by protamine post-translational modifications affect chromatin dynamics or gene expression in the early embryo. Here, we bring together past and more recent work to explore these topics and suggest future studies that will elevate our understanding of the molecular basis of the histone-to-protamine exchange and the underlying etiology of idiopathic male infertility. Spermatogenesis ensures transmission of genetic information to the next generation by maintaining male fertility throughout life. Three biologically distinct processes safeguard the continuous generation of sperm: mitosis, meiosis, and spermiogenesis – the last of which involves extensive remodeling of both cytoskeleton and chromatin to achieve a significant compaction of the sperm head , 2. At bin vitro experimental systems to investigate mechanisms. In this review, we summarize the current understanding of chromatin dynamics during spermiogenesis and the advances made to understand sperm chromatin 3D organization. A greater understanding of sperm genome packaging and molecular organization will inform our understanding of how this process is dysregulated in infertility and will aid in the development of clinical assays and therapeutic approaches that may enhance clinical care and reproductive outcomes.Seminal work used biochemical and genetic approaches to identify intermediate proteins involved in the histone-to-protamine transition; however despite its biological importance, our insight into how chromatin-associated factors/remodelers are involved remains limited –9. We laThe histone-to-protamine transition is one of the most poorly understood aspects of spermiogenesis and the sequence of events is also known to vary across species. However, this remodeling process is believed to occur in a stepwise fashion, wherein canonical histones are sequentially replaced by testis-specific histone variants –13 folloin vitro studies have demonstrated that the incorporation of histone variants such as H3T, H2AL2, and TH2B induces nucleosome destabilization by altering histone-DNA binding and weakening the associations between H2A/H2B dimers and H3/H4 tetramers, to ultimately promote reorganization of the chromatin  leads to aberrant spermatid development and impaired fertility   . Howevers (ICSI) , underscPrevious dogma posited that TNPs are incorporated onto chromatin following histone eviction and occupy the majority of the genome in elongating spermatids, thereby acting as intermediates between histones and protamines . This inin vitro data shows that TNP1 has a >8-fold affinity for single-stranded DNA (ssDNA) over double-stranded DNA (dsDNA), and in contrast to H1 histone, TNP1 forms less stable structures with DNA even at higher ionic strength (50 mM NaCl), which is still below physiological salt concentrations  and undergoes selective proteolytic processing to produce its mature form (P2) once bound to DNA , 89. TruAcross species, the P1:P2 ratio is highly variable but maintenance of a species-specific P1:P2 ratio is critical for normal fertility –94. ConvGiven that protamines were assumed to bind uniformly in the genome and not believed to bear PTMs, their potential role as informational carriers has been largely overlooked. Recent biochemical and mass-spectrometry analysis by us and others led to the discovery that P1 and P2 proteins from mature sperm carry multiple PTMs, including phosphorylation, acetylation, and methylation , 99. Dynin vitro , with phenotypes ranging from subfertility to infertility, the infertility is not caused by changes in the hypothalamic pituitary axis or somatic cell numbers. Instead, the infertility appears to be germ cell-intrinsic; presenting as defects in spermatid elongation and condensation defects, consistent with CHD5 expression in steps 7-10 of spermatid maturation, immediately preceding and overlapping with the extensive chromatin remodeling  and ISWI (Imitation SWItch). A knockout of BRG1 (a SWI/SNF component and transcription activator) in germ cell progenitors resulted in a mid-pachytene arrest, preventing investigations in post-meiotic round spermatids . The zinin vitro biochemistry and biophysics experiments have provided fundamental insights into protamine-DNA interactions and the structure of sperm chromatin imposed by protamine binding. Early in vitro studies primarily relied on measuring the behavior and properties of either polyarginine/polylysine peptides or purified salmon or bull  sperm protamine , spermine, and spermidine, have also been shown to form DNA toroids  grants 1R21HD090371-01A1 (SSH), 1DP2HD091949-01 (SSH), R01HD104680 (SSH), training grants NSF 1256260 DGE (LM), Rackham Predoctoral Fellowship (LM), T32GM007315 (LM), and Open Philanthropy Grant 2019-199327 (5384) (SH).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.