Neural tube defects (NTDs) are severe congenital malformations resulting from incomplete neural tube closure during embryogenesis, with maternal diabetes mellitus being a well-established risk factor. However, the underlying epigenetic mechanisms remain poorly understood. In this study, we established a maternal diabetes mouse model by intraperitoneal injection of streptozotocin at embryonic day (E) 5.5 and examined embryos during the critical neural tube closure period (E8.5–E10.5). Embryos from diabetic dams developed both cranial and spinal NTDs, with a marked predominance of cranial defects; we therefore focused our analyses on cranial neural tissues from affected embryos. In the cranial neural tissue of NTD-affected embryos, we observed significant accumulation of crotonyl-coenzyme A (Cro-CoA), accompanied by elevated levels of lysine crotonylation (Kcr) and histone H3 lysine 18 crotonylation (H3K18cr). Notably, maternal hyperglycemia-induced downregulation of HDAC1 and HDAC2, together with crotonyl-CoA accumulation, promoted a global increase in H3K18cr in cranial neural tissues. However, despite this global elevation, H3K18cr enrichment was paradoxically reduced at the promoters of AKT and PIKFYVE in cranial NTD embryos, correlating with their transcriptional silencing and contributing to cranial NTD pathogenesis. In HT-22 hippocampal neurons, exogenous glucose and crotonate recapitulated the metabolic and epigenetic alterations, leading to Cro-CoA accumulation and a global increase in H3K18cr. However, at the promoters of AKT and PIKFYVE, H3K18cr levels were decreased and consequently suppressed cell proliferation. Our findings demonstrate that maternal diabetes induces NTDs, at least in part, through aberrant elevation of H3K18cr, which suppresses the expression of AKT and PIKFYVE and impairs neural cell proliferation. Our findings reveal a potential regulatory mechanism by which abnormal H3K18cr elevation contributes to the occurrence of cranial NTDs induced by maternal diabetes.
Abstract Background Three-dimensional genome organization is maintained in part by chromatin insulators, DNA–protein complexes that create boundaries between active and inactive chromatin and regulate enhancer-promoter interactions. In Drosophila, the Homing insulator at eve (Homie) sequence prevents repressive Polycomb (PcG) chromatin at even-skipped ( eve ) from spreading into the neighboring essential gene, TER94 . The exact mechanisms controlling Homie barrier activity are understudied. Results Here, use of an in vivo reporter assay identified the requirement of the hnRNP M homolog, Rumpelstiltskin (Rump), for Homie barrier activity. Interestingly, Rump depletion resulted in no changes to chromatin insulator protein binding at Homie but extensive chromatin association changes to the Polycomb response element (PRE)-associated protein Crooked legs (Crol) at Crol-bound PREs. Furthermore, global chromatin association changes were detected for a subset of Polycomb repressive complex 1 and 2 (PRC1 and PRC2) components. Finally, we observed increased cis- compaction at Polycomb domains genome-wide including eve and increased distances between eve and other Polycomb domains in trans after Rump depletion. Conclusions These data identify Rump as a novel regulator of Homie barrier activity and suggest a regulatory role of Rump for both cis and trans interactions of PcG proteins. Importantly, this regulatory role likely helps to maintain Homie barrier activity independently of chromatin insulator proteins.
DNA methylation is a critical epigenetic modification that occurs on cytosine residues and plays essential roles in regulating gene expression, cellular differentiation, and genomic imprinting. However, the existence of cytosine methylation in mitochondrial DNA (mtDNA) remains highly controversial. Whole-genome bisulfite sequencing (WGBS) has been widely used to assess mtDNA methylation, but signals detected in mitochondrial DNA are frequently influenced by technical artifacts. We systematically evaluated technical factors that influence mtDNA methylation measurements using zebrafish WGBS datasets derived from a wide range of tissues and experimental conditions, complemented by analyses of human and mouse datasets. mtDNA methylation measurements varied widely among samples within CpG, CHG, and CHH contexts, and mtDNA CpG methylation estimates above 2
Protein arginine methyltransferases (PRMTs) are abnormally expressed in various tumors and participate in multiple tumorigenic processes, including proliferation, metastasis, drug resistance, and metabolic reprogramming. However, in recent years, with the in-depth exploration of the functions of PRMTs and their associated tumorigenic mechanisms, the complex crosstalk relationships among different PRMTs have been increasingly highlighted. Different PRMTs may not only exhibit synergistic tumor-promoting effects but also demonstrate antagonistic effects in certain tumor biological functions. This makes the scientific and rational use of PRMT inhibitors a key factor for effective tumor suppression. The review aims to provide a comprehensive overview of the functional roles of different PRMTs in tumors, with a particular focus on their cross-functional regulatory mechanisms within the complex regulatory networks of tumors. Meanwhile, it summarizes the current research progress on PRMT inhibitors and elaborates on some limitations associated with PRMT-targeted therapy. Through the integration and analysis of existing research findings, this review offers a novel perspective on the complex regulatory roles of different PRMTs jointly participating in biological functions. Accumulating evidence suggests that implementing a multi-targeted intervention strategy for PRMTs and conducting stage-specific inhibition of PRMTs according to the progression stage of tumor development hold the potential to exert more significant therapeutic effects in the field of tumor treatment.
Orofacial clefts, particularly cleft palate, are among the most common congenital anomalies, imposing substantial functional impairment and psychosocial burden on affected individuals. Emerging evidence implicates epigenetic modifications, especially histone lysine methylation, as critical regulators of embryonic development, yet their precise roles in palatogenesis remain poorly defined. The H3K4 methyltransferase complexes, which require the core subunit ASH2L for full enzymatic activity, govern transcriptional activation during development; however, whether ASH2L-mediated H3K4me3 deposition contributes to palatal morphogenesis is unknown. Here, we show that conditional deletion of Ash2l in Six2-positive palatal mesenchymal cells results in complete cleft palate in mice. Multi-omics analyses (RNA-seq and CUT Tag-seq) reveal that Ash2l loss causes genome-wide reduction of H3K4me3 enrichment at key developmental genes, most notably Wnt5b, leading to their transcriptional downregulation. Functionally, Ash2l deficiency impairs proliferation and osteogenic differentiation of palatal mesenchymal cells, resulting in stunted palatal shelf growth and disrupted osteogenesis. Pharmacological activation of WNT signaling downstream of Wnt5b partially rescues these cellular defects, confirming the functional relevance of this axis. These findings establish ASH2L-mediated H3K4me3 as a critical epigenetic regulator of palatal development through WNT5B modulation, and identify potential therapeutic targets for cleft palate.
Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) are abundant viral noncoding RNAs that associate with host chromatin, but the promoter-scale consequences of those contacts remain unclear. We re-analyzed GSE281522 in EBV-positive GM12878 cells integrating RNA–DNA contacts, RNAPII ChIA-PET, RNA-seq and internal ATAC-seq after EBER depletion. Promoters were classified according to increased RNAPII-associated promoter-looping signal after EBER depletion relative to the matched control conditions, defined as LoopRecovery; increased transcriptional output, defined as ExprUp; or the coordinated occurrence of both responses, defined as Recovery + Up. EBER-contacted promoters were markedly depleted for Recovery + Up relative to non-contacted promoters (0.42
Cold stress is a significant challenge to cotton (Gossypium hirsutum L.) production during seed emergence and early seedling establishment, as cotton is native to tropical and subtropical environments. Low temperatures during these sensitive stages impair photosynthetic efficiency, damage cellular structures, and reduce yield. Although cotton responses to cold stress have been extensively investigated at physiological, molecular, and transcriptional levels, increasing evidence suggests that transient gene expression changes alone are insufficient to explain sustained stress performance. This review synthesizes current knowledge on cotton cold-stress responses, emphasizing the regulatory roles of key histone modifications histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3) in transcriptional control and within-generation (somatic) epigenetic priming. Although cotton cold epigenome profiling is beginning to emerge, cotton-specific, time-resolved chromatin datasets that span chilling, recovery, and recurrent chilling (stress-recovery-re-stress) remain limited; therefore, several mechanistic inferences necessarily rely on indirect evidence from cotton studies under other conditions and on well-characterized model plant systems. H3K27me3 is implicated in Polycomb-mediated gene silencing and may regulate gene reactivation during cold stress and recovery, whereas H3K4me3 is proposed to support rapid induction of cold-responsive genes. Bivalent chromatin domains containing H3K4me3 and H3K27me3 may maintain stress-related genes in a poised transcriptional state, enabling swift activation while preserving developmental regulation. We highlight key knowledge gaps and experimental priorities for establishing cotton-specific chromatin mechanisms of cold memory and for translating these insights into epigenome-assisted breeding and biotechnological strategies to develop cotton varieties with improved and stable cold resilience.
Molecular subtyping is essential for precision oncology, enabling the classification of tumors into biologically and clinically relevant categories. DNA methylation has emerged as a promising biomarker for cancer subtyping, yet its application remains limited by high dimensionality, batch effects, and the lack of automated, user-friendly analytical tools. Here, we present CancerSubtyper, an end-to-end computational framework for deep learning–based cancer subtyping using DNA methylation data, which is accessible through an intuitive web interface designed to support interactive exploration and downstream analysis. CancerSubtyper integrates two complementary models: a semi-supervised classifier for cancers with well-established subtypes, and a hybrid framework that integrates supervised and unsupervised learning to identify novel subtypes. The framework automatically performs preprocessing, feature selection, batch correction, and cancer subtyping while offering interactive visualization for subtype exploration and validation. By providing an automated, end-to-end workflow accessible through a user-friendly web interface, CancerSubtyper lowers the barrier to large-scale methylation analysis and provides a powerful tool for molecular subtyping and precision oncology research. The framework is freely accessible at https://github.com/ycheung5/cancersubtyper/.
Asthma is a clinically heterogeneous airway disorder characterized by complex interactions between environmental exposures, immune activation, and molecular regulatory programs, whose underlying mechanisms are not fully elucidated by known genetic loci. DNA methylation serves as a mechanistic interface bridging genetic predisposition and environmental influences; however, most epigenetic studies remain confined to isolated CpG sites, lacking robust biological interpretability. We developed a cross-tissue, multi-cohort, and mechanistically interpretable epigenetic framework to delineate pathway-level methylation mechanisms underlying asthma. Leveraging data from 908 participants across one combined training cohort and three independent validation cohorts, we constructed a linear support vector classifier based on pathway-derived methylation scores. Additionally, SHapley Additive exPlanations (SHAP) were applied to quantify the contributions of individual pathways. To assess the statistical significance of pathway contributions, one-sample t-tests were performed for each pathway’s SHAP values against zero, followed by Benjamini–Hochberg false discovery rate (FDR) correction to obtain adjusted p values. The model exhibited reproducible and cross-tissue performance, achieving area under the curve (AUC) values of 0.792 (95
This study aimed to elucidate the mechanism of Lactate Dehydrogenase A (LDHA) in senescent fibroblast-derived exosomes during skin photoaging, focusing on the molecular pathway by which it regulates Acyl-CoA Synthetase Long-Chain Family Member 4 (ACSL4) expression through histone lactylation, thereby inducing ferroptosis and accelerating skin photoaging. An ultraviolet B (UVB)-induced senescence model was established using human foreskin fibroblasts. Exosomes were isolated from senescent fibroblasts and characterized. Their features and uptake were assessed using Western blot, transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and immunofluorescence. Small interfering RNA was employed to knock down LDHA and ACSL4 gene. Pharmacological inhibitors (FX11, Ferrostatin-1) and sodium lactate rescue experiments were utilized. Lactate levels, histone H3K18 lactylation modification, ACSL4 transcriptional activity, and ferroptosis markers were detected to assess the effects of ACSL4 gene lactylation on ferroptosis. The biological effects of exosomal LDHA in photoaged tissue were validated using an in vivo UVB-irradiated mouse model. UVB irradiation induced fibroblast senescence and significantly upregulated LDHA expression. Exosomes from senescent fibroblasts were effectively taken up by HaCaT cells, leading to increased lactate levels and enhanced histone H3K18 lactylation in recipient cells. LDHA knockdown or inhibition downregulated ACSL4 expression and suppressed ferroptosis, whereas exogenous lactate partially restored these effects. RNA sequencing and ChIP-qPCR results indicated that LDHA-mediated lactylation modification was enriched at the ACSL4 promoter region, enhancing its transcriptional activity. In vivo experiments further confirmed that senescent exosomes accelerated UVB-induced skin collagen degradation and ferroptosis, while LDHA intervention significantly alleviated photoaging damage. Senescent fibroblast-derived exosomes deliver LDHA, promoting histone lactylation modification, which upregulates ACSL4 expression and activates the ferroptosis pathway, ultimately accelerating skin photoaging. This study reveals the coupling mechanism between metabolic signaling and epigenetic regulation in skin aging, providing new molecular targets and a theoretical basis for anti-photoaging therapy.
Cardiomyopathies constitute a heterogeneous group of myocardial disorders representing leading causes of heart failure and cardiovascular mortality worldwide. While genetic mutations have been extensively characterized across different cardiomyopathy subtypes, the mechanistic links between genotype and phenotype remain incompletely understood. This review synthesizes current knowledge regarding chromatin remodeling complexes and their roles in cardiac gene regulation under physiological and pathological conditions. Moreover, disease-specific chromatin remodeling patterns were examined across dilated, hypertrophic, arrhythmogenic, and restrictive cardiomyopathies, highlighting both conserved mechanisms and subtype-specific alterations. Chromatin remodeling alterations contribute significantly to cardiomyopathy pathogenesis across multiple subtypes. The reversibility of epigenetic modifications presents therapeutic opportunities not available with genetic interventions. Selective HDAC inhibitors and EZH2 antagonists show promise in preclinical models, though clinical translation requires development of cardiac-specific delivery systems. CRISPR-based epigenetic editing technologies offer future potential for precise genomic locus-specific interventions to reverse pathological transcriptional programs. Chromatin remodeling complexes including SWI/SNF (BAF), NuRD, Polycomb, ISWI, CHD, and INO80 families- modulate disease expression, progression, and phenotypic variability through epigenetic modifications and ATP-dependent chromatin remodeling. Emerging evidence demonstrates that chromatin remodelers interact dynamically with DNA methylation machinery, histone-modifying enzymes, and cardiac transcription factors to orchestrate pathological gene expression programs. Understanding these epigenetic mechanisms offers unprecedented opportunities for developing novel therapeutic strategies targeting the chromatin regulatory apparatus, potentially reversing maladaptive transcriptional programs that drive disease progression.
Nucleosome positioning critically regulates chromatin functions, yet species-specific mechanisms remain incompletely understood. This study revisits nucleosome organization in Schizosaccharomyces pombe (S. pombe) using a DNA deformation energy model and a high-resolution nucleosome map. We demonstrate that DNA bending energy—not shearing energy—accurately predicts rotational positioning (72.2–77.4
Streptococcal septicemia is associated with high mortality and morbidity, yet the underlying epigenetic mechanisms, particularly those involving mitochondrial genes, remain poorly understood. This study employed a multi-omics Mendelian randomization (MR) framework to investigate the causal role of mitochondrial gene regulation in streptococcal septicemia, with an emphasis on epigenetic influences. We utilized genetic instruments—including cis-methylation quantitative trait loci (mQTLs), cis-expression QTLs (eQTLs), and cis-protein QTLs (pQTLs)—for 1,136 mitochondrial-related genes from MitoCarta3.0. These were analyzed for causal associations with streptococcal septicaemia (FinnGen R12: 3,239 cases, 439,048 controls). Epigenetic and transcriptomic data were integrated with MR analyses of 731 immune cell traits. Single-cell RNA sequencing (GSE175453: 4 sepsis patients, 5 controls) and bulk transcriptomic data (GSE57065: 28 septic shock patients, 25 controls) were used for validation. Additionally, single-cell eQTLs for PARK7 across 14 immune cell types were applied in MR to evaluate cell-type-specific causality. MR analyses identified 280 mQTLs, 79 eQTLs, and 29 pQTLs with causal links to streptococcal septicemia. Integrative multi-omics analysis revealed PARK7 as the only gene consistently implicated across epigenetic, transcriptomic, and proteomic levels. Epigenetic regulation via mQTLs was notably prominent. Single-cell RNA sequencing demonstrated significant PARK7 upregulation in T and NK cells from sepsis patients, corroborated by elevated PARK7 expression in septic shock patients across multiple timepoints. Temporal correlation analyses indicated that PARK7 levels inversely correlated with depleted CD8 + T cells, naive CD4 + T cells, and resting NK cells. Critically, sc-eQTL MR confirmed that genetically predicted PARK7 upregulation in two immune subsets—CD4 + KLRB1+ T cells and CD4 + KLRB1- T cells—causally increased septicemia risk. Our multi-omics genetic approach establishes PARK7 as a causal gene in streptococcal septicemia, mediated in part by epigenetic mechanisms. PARK7 upregulation in specific T and NK cell subsets contributes to susceptibility, linking mitochondrial epigenetic regulation to immune dysfunction and underscoring its potential as a therapeutic target.
BACKGROUND: Synovium derived mesenchymal stem cells (SMSCs) are considered promising for orthopedic application due to easy accessibility and strong differentiation potential. However, the transcription factors (TFs) that orchestrate the SMSCs osteogenic commitment, as well as the dynamic landscape of associated cis-regulatory elements, remain largely unclear. In addition, donor-specific epigenetic memory may lead to heterogeneous gene-regulatory profiles. RESULTS: In this study, we isolated porcine SMSCs from two pig breeds (German Saddleback, GS; German Landrace, GL) with distinct intrinsic metabolic characteristics and profiled their dynamic chromatin accessibility and transcriptomes during osteogenic induction. GO terms related to ossification and mesenchymal cell differentiation emerged earlier in the chromatin landscape (ATAC-seq, day 7) than at the transcriptional level (RNA-seq, day 21), indicating that chromatin accessibility captures lineage-specific programs prior to overt gene expression changes. Donor-specific differences in chromatin accessibility were minimal at baseline (day 0), became evident early after induction, and diminished over time. Footprinting analysis showed stronger binding affinity of C/EBP family members in osteogenic-induced SMSCs, whereas the FOS::JUN heterodimer exhibited greater occupancy in control cells. Interestingly, RUNX2 footprints displayed a slight decrease from day 0 to day 21 despite its established role in osteogenesis. De novo motif analysis further revealed TF-binding motif in differentially accessible regions, with RUNX2/RUNX motifs enriched in regions of reduced accessibility and CEBPs enriched in regions of increased accessibility. CONCLUSIONS: This study characterizes chromatin accessibility dynamics in SMSCs during osteogenic differentiation, driven mainly by differentiation state and time rather than donor metabolic differences. Integrated ATAC-seq/RNA-seq highlights key transcription factors and networks guiding osteogenic commitment, supporting porcine SMSCs as a translational model for bone regeneration.
DEK is a chromatin-associated, DNA-binding protein with unique properties that place it in its own protein class. First discovered in 1992 as a fusion protein in acute myeloid leukemia, DEK gained further attention in the mid-2000s as a growing number of studies identified its connections to chromatin architecture and subsequent impact on pathologies such as cancer and autoimmune diseases. Current evidence indicates that DEK can alter the topology of nucleic acids in a variety of biological processes, including DNA replication, DNA repair, chromatin organization, epigenetic modification, transcription, and mRNA splicing. Interestingly, DEK is highly evolutionarily conserved among higher eukaryotes, and this, combined with its involvement in such a diverse array of processes, highlights its biological significance. Interest in DEK is further driven by its status as a potent oncogene, as it is transcriptionally upregulated in most solid tumors tested to date, with high DEK expression correlating with poor survival and more aggressive tumors. Recently, advances in cryogenic electron microscopy have made it possible to visualize the structural basis of DEK-nucleosome interactions, providing concrete mechanistic insights into how DEK influences gene regulation and transcription. These findings clarify prior observations and open new avenues for exploring the biological and clinical relevance of DEK.
The ASH2L–DPY30 interaction is a structurally conserved and functionally essential component of the COMPASS family of histone methyltransferases responsible for H3K4 trimethylation. This minimalist helix–groove interface plays a critical allosteric role in stabilizing ASH2L, aligning the catalytic SET domain on nucleosomes, and enabling efficient methylation of chromatin targets. Recent structural, biochemical, and genetic studies have demonstrated that disrupting this contact—whether by point mutation, domain deletion, or competitive peptides—leads to widespread collapse of H3K4me3, transcriptional silencing of oncogenic programs, and suppression of cell proliferation, particularly in MLL-rearranged and MYC-driven cancers. In parallel, chemical-biology tools and fragment-based screening efforts have begun to yield the first ligandable scaffolds, setting the stage for drug discovery targeting this axis. This review synthesizes the current knowledge surrounding the ASH2L–DPY30 interface, covering its molecular architecture, catalytic importance, disease relevance, and therapeutic tractability. We also discuss resistance mechanisms, assay platforms, and the challenges and opportunities for translating this target into a first-in-class epigenetic therapy.
Epigenetic dysregulations are linked to several diseases, including cancer. Among them, breast cancer is the second leading cause of cancer-related deaths in women, with 50
Background Comprehensive profiling of epigenetic states is essential for understanding gene regulation and disease mechanisms. Sequencing-based methods such as ChIP-seq, Hi-C, and RNA-seq provide genome-wide views of histone modifications and 3D genome organization, but lack spatial resolution within single nuclei. Results Here we present an image-based epigenetic profiling framework that combines high-speed super-resolution microscopy with deep learning. Using models of (i) histone deacetylase inhibition in HEK293T cells and (ii) Rett syndrome iPS cells carrying MECP2 mutations, our approach accurately discriminated their epigenetic states (99.6% and 96.1% accuracy, respectively) and identified the nuclear periphery as a hotspot of H3K27ac and CTCF redistribution. Sequencing-based analyses showed compartment switching and lamina-associated domain alterations consistent with the image-based features. These results demonstrate that high-speed super-resolution imaging, when combined with deep learning, provides a powerful tool for epigenetic profiling. Conclusions Our framework offers a generalizable strategy for image-based epigenetic profiling to uncover chromatin alterations in development, disease, and therapeutic response.
BackgroundEmerging evidence has shown the common occupancy of dozens to hundreds of transcription factors (TFs) on cis-regulatory elements (CREs), yet the underlying details are largely unknown.ResultsIn this study, leveraging extensive collections of TF ChIP-seq data of more than 1000 TFs in human HepG2 and K562 cells, we located highly focused TF binding sites (FBSs) within CREs as single-nucleosome depleted regions, which accommodate the majority of the total TF binding events. Approximately 25,000 strong FBSs were identified in each cell type. For more than 90% of TFs, including some pioneer factors such as GATA1 and JUN, their binding sites out of FBSs barely show nucleosome depletion. Essential cellular function related motifs and phenotypically causal variants are strongly enriched in the FBSs, but not in their immediate flanking regions within CREs. Most TFs bind to FBSs not containing their canonical motifs.ConclusionOur study revealed the critical connection between highly focused TF binding and the nucleosome depleted status of DNA in vivo. Meanwhile, we constructed high-resolution maps of chromatin accessibility at distal CREs in the two human cells. We propose a model of TF co-binding in vivo and suggest that a short DNA residence time of most TFs underlies the requirement of a large number of TFs for sustained nucleosome depletion at CREs.
BackgroundThe use of programmable nucleases has transformed genome editing and functional genomics. Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) was developed such that targeted genomic lesions [usually DNA double-stranded breaks (DSBs)] could be introduced in vivo with ease and precision. In the presence of homology donors, these lesions facilitate high-efficiency precise genome editing (PGE) via homology-directed repair (HDR) pathways. Because DSBs can lead to genomic instability, however, a large amount of effort has been invested in methodologies (e.g., base editors) that only require nicking the chromosomal DNA on one strand. Indeed, we have demonstrated in human cells that oligodeoxynucleotide (ODN)-mediated PGE using nickase variants of Cas9 can proceed by at least two HDR subpathways termed synthesis-dependent strand annealing (SDSA) and single-stranded DNA incorporation (ssDI). Which pathway is utilized is determined by which chromosomal strand (sense or anti-sense/Watson or Crick) is nicked and by the strandedness (sense or anti-sense/Watson or Crick) of the donor ODN.ResultsWhile the mechanism of mammalian SDSA is moderately well understood, that of ssDI is not. To gain genetic insight into ssDI, we carried out a genome-wide CRISPR knockout screen to identify those genes which, when absent, enable increased ssDI. This screen identified the protein lysine methyl transferase (PKMT) Su(var)3-9, enhancer-of-zeste and trithorax (SET) domain bifurcated histone lysine methyltransferase 1 (SETDB1):activating transcription factor 7-interacting protein (ATF7IP) heterodimer and the downstream human silencing hub (HUSH) complex as strong negative regulators of ssDI. Consistent with their well-known biological effects, the negative regulation of ssDI by SETDB1/ATF7IP and HUSH was specific for transgenic reporters and for a HUSH-regulated single-copy gene, but was not observed at other (non-HUSH regulated) single-copy endogenous loci.ConclusionsIn toto, these experiments underscore the profound impact that chromatin modifiers - and by extension, chromatin structure - have on PGE outcomes. Specifically, we have identified SETDB1/ATF7IP and the HUSH complex as major negative regulators of the HDR subpathway, ssDI, when the target is a transgene. These experiments are a proof-of-principle that chromatin can act as a potent barrier to genetic recombination and they strongly support the feasibility of extending similar chromatin modulating strategies to enhance PGE efficiency at endogenous single-copy loci.
