Epigenetics & Chromatin最新文献

Background: The 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.

Results: While 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.

Conclusions: In 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.

Major pharmacologic advances the past few decades have transformed infection with Human Immunodeficiency Virus (HIV) from a fatal disease into a chronic, manageable condition for people with access to antiretroviral therapy (ART). However, a cure remains elusive because HIV persists in a latent state throughout the body, evading immune clearance after ART suppression. Our understanding of HIV latency has made tremendous strides the past few decades, but the specific epigenetic mechanisms underlying latency are still being elucidated. Insights might be gained from simpler retroviruses capable of endogenization, such as the gammaretrovirus Murine Leukemia Virus (MLV). Most vertebrates, including humans, exhibit evidence for ancient retroviral infections that have been epigenetically silenced during early embryogenesis, offering natural modes of viral repression. This review summarizes our current understanding of epigenetic and epitranscriptomic silencing of HIV-1, highlighting parallels and contrasts with MLV and other retroviruses throughout the animal kingdom. We also discuss epigenetic mechanisms of pre-integration latency and T cell-mediated control, made possible through comparative studies of retroviral infections in other species. Finally, we propose how insights from other retroviruses might inform strategies for durable HIV-1 suppression.

Background: Three-dimensional genome organization helps coordinate enhancer-promoter communication while insulating loci from inappropriate regulatory contacts. CTCF and cohesin contribute to this organization by forming topologically associating domains. However, how boundary elements at individual loci influence transcription remains context dependent.

Results: We investigated the conserved topological organization of the mammalian NOTCH1 locus. Across human cell types, NOTCH1 resides within a defined topologically associated domain with CTCF/cohesin occupancy at both 5' and 3' boundaries. In human K562 cells, CRISPR-Cas9 deletion of boundary CTCF sites increased transcription of NOTCH1 and the intradomain non-coding transcripts NALT1 and LINC01451. Boundary perturbations impaired proliferation and clonogenic growth. Chromatin conformation profiling revealed defects in domain insulation and a redistribution of regulatory contacts between NOTCH1 promoter and enhancers within the domain. Cross-species analyses showed that domain architecture is conserved in mouse, yet transcriptional and phenotypic effects associated to domain boundary disruption were cell-type specific and correlated with differential chromatin contexts.

Conclusions: CTCF-dependent boundary integrity at the NOTCH1 locus tunes transcriptional output and cellular phenotypes in a chromatin context-dependent manner, supporting a model in which conserved 3D architecture constrains regulatory communication but yields distinct outcomes across cellular states.

As a key component of freshwater ecosystem, Daphnia survives environmental challenges by entering a state of developmental arrest known as diapause. Diapause is not simply a sudden halt in growth and development, but a genetically regulated program involving a sequence of coordinated developmental processes. Histone modifications, as crucial epigenetic mechanisms that regulate gene expression by altering chromatin structure, are hypothesized to play a significant role in diapause regulation. This study focuses on the acetylation (H3K9ac) and tri-methylation (H3K9me3) at the H3K9 site. Given the established roles of H3K9ac in transcriptional activation and H3K9me3 in transcriptional silencing, we hypothesize that these antagonistic modifications play regulatory roles in the diapause developmental program of Daphnia. Via immunocytochemistry, we observed a significant overall decrease in H3K9ac and an increase in H3K9me3 levels in Daphnia magna diapausing cells, suggesting transcriptional silencing of H3K9-related genes. Notably, however, a subset of cells retained high H3K9ac and low H3K9me3 levels, indicating localized transcriptional activity of H3K9 related genes that may be essential for maintaining diapause and facilitating developmental resumption once conditions improve. We also examined the expression of the H3K9ac-related acetyltransferase gene kat2a and the H3K9me3-related methyltransferase gene suv39h1 via quantitative PCR. kat2a was highly expressed during active embryonic development (pre- and post-diapause) and maintained a basal level during diapause, potentially supporting the transcriptional activity of H3K9-related genes in selected cells. On the other hand, suv39h1 transcript levels remained low from ovulation through the entire diapause period, making its contribution to the elevated H3K9me3 levels during diapause unclear. However, the increase in suv39h1 expression toward the end of diapause suggests its potential role in diapause termination and subsequent post-diapause development. Our results illustrate dynamic changes in H3K9 modifications throughout the diapause developmental program in Daphnia, indicating their role in diapause regulation by modulating gene expression via alterations to the chromatin landscape.

Background: Obsessive Compulsive Disorder (OCD) affects 2 to 3% of the population and is marked by intrusive thoughts and repetitive behaviours. OCD is increasingly recognized as a polygenic disorder involving gene-environment interactions, with genetic risk largely driven by common variants and a smaller contribution from rare pathogenic variation. Despite these insights, the functional roles of implicated genes remain unclear. Recent genomic studies suggest that chromatin dysregulation contributes to the pathogenesis of OCD.

Results: We analysed the recently published large-scale genome-wide association study meta-analysis of OCD by Strom et al., which included 53,660 cases and over 2 million controls. This study identified 30 genome-wide significant loci and approximately 11,500 common variants, together explaining 90% of OCD heritability. We analysed the 251 prioritized genes mapped through six genomic methods identified in this study. Gene Ontology over representation analysis (ORA) showed enrichment in chromatin-related pathways, including nucleosome assembly and DNA packaging, mainly driven by histone H1 to H4 genes. We also analysed two additional studies, a whole-exome sequencing study involving 1313 cases by Halvorsen et al., and a whole-genome sequencing study involving 53 parent-offspring trios by Lin et al., both of which found an enrichment of chromatin regulation genes in OCD.

Conclusion: Chromatin remodeling regulates neuronal differentiation, synaptic gene expression, and immune signaling. Disruptions in these processes may alter neurotransmission and immune responses, contributing to OCD symptoms and their fluctuation with stress or infection. Our findings highlight chromatin dysregulation as a shared mechanism across common and rare OCD variants. This supports a gene environment model and suggests chromatin remodeling as a novel therapeutic target. Further epigenomic research is needed to investigate these potential mechanisms.

Background: Transcription factor (TF)-dependent DNA demethylation is associated with generation of specific DNA methylation profiles in normal cellular development and disease, although only a small fraction of TFs are known to promote DNA demethylation.

Results: Here, we systematically predicted which TFs have DNA-demethylation-promoting activity. Experiments with deletion mutants of the TFs RUNX1 and SPI1 revealed that this activity is associated with a relatively long intrinsically disordered region (IDR). Examination of the IDRs from eight TFs previously confirmed to have such activity revealed that at least one IDR was active in each TF. We constructed a Random Forest classifier based on 25 numeric physicochemical features extracted from length-controlled 26 positive (active) and 32 negative (inactive) IDRs. Four key features- aromaticity, aliphatic index, fractional charge ratio, and side chain hydrophobic density-were identified as the most informative contributors to prediction of positive IDRs. A model based on these features achieved an area under the receiver operating characteristic curve of 0.84, with an optimized decision threshold of 0.303. Applying this model to all TFs, we predicted 959 of 2364 IDRs to be positive, corresponding to 825 of 1308 TFs. The model correctly identified all of 14 previously validated positive TFs. The predicted positive TFs showed significant enrichment of Gene Ontology terms related to morphogenesis and development and may be clinically relevant to certain cancer types.

Conclusion: The developed model with high predictive performance and the predicted TFs with DNA-demethylation-promoting activity will be useful for further analysis of TFs involved in generation of DNA methylation profiles in normal cell development and disease.

Loss-of-function mutations in DNMT3A, a DNA methyltransferase, or NSD1, a histone methyltransferase, cause overgrowth syndromes. Conversely, disruption of the DNMT3A domain that binds NSD1-deposited H3K36 dimethylation (H3K36me2) results in growth restriction. To investigate the molecular basis of these opposing growth outcomes, we generated isogenic human embryonic stem cells carrying growth syndrome-associated mutations in DNMT3A and NSD1. Unexpectedly, both overgrowth- and growth restriction-associated DNMT3A mutations led to DNA hypomethylation in a shared subset of active enhancers, implicating H3K36me2 in directing enhancer methylation maintenance. In contrast, bivalent promoters-marked by both active and repressive histone modifications-showed divergent DNA methylation changes: hypermethylation in growth restriction-associated DNMT3A mutants and hypomethylation in overgrowth-associated DNMT3A or NSD1 loss-of-function mutants. These findings identify locus-specific DNA methylation defects as a common molecular feature and nominate dysregulated DNA methylation at bivalent promoters as a potential driver of abnormal growth phenotypes.

Background: CTCF (CCCTC-binding factor) is the best-studied architectural protein that is highly conserved among animals, including Drosophila and mammals. In Drosophila, CTCF is involved in the organization of functional promoters and insulators, in cooperation with many other architectural proteins, including Su(Hw) and Pita. These proteins, like many other architectural proteins, interact with CP190, which, along with its partners, recruits transcriptional complexes to target promoters. This study was conducted to investigate the cooperation between architectural proteins in the recruitment of CP190 to regulatory elements.

Results: We generated transgenic flies expressing double and triple mutants of dCTCF, Su(Hw), and Pita that cannot interact with CP190. The single mutants are fully viable; however, few triple mutants develop into adults. In the triple mutant, although the CP190 concentration is reduced, the level of gene transcription remains unaltered, suggesting that co-expression of the three mutant proteins is responsible for CP190 instability. ChIP-seq analysis showed that CP190 is required for dCTCF-chromatin binding. In the triple mutant, CP190 demonstrates an almost complete loss of association with the promoters and insulators to which the tested architectural proteins bind; however, these regulatory elements were found to retain their activity.

Conclusions: Architectural proteins cooperate to recruit CP190 to regulatory elements and determine its stability. Despite the important role of CP190 in transcriptional regulation, its functions may be partially performed by partner proteins.