Long non-coding RNAs transcribed from pericentromeric Satellite 3 repeats (HSAT3) are involved in stress-dependent splicing processes and maintenance of genome stability. The functions of the transcripts are largely unknown, which is mainly due to the predominant heterochromatic organization of satellite regions. HSAT3 expression is induced upon several cellular stressors and builds nuclear stress bodies (nSB), which recruit a large number of RNA-binding and regulatory proteins. Recently, we reported that HSAT3 transcripts induce resistance against the topoisomerase 2a (TOP2A) inhibitor etoposide in lung cancer. We show that HSAT3 transcripts and topoisomerase IIα (TOP2A) accumulate in nuclear stress bodies. Biochemical assays indicate that HSAT3 RNA can associate with TOP2A. Using single-molecule DNA fiber analysis, we demonstrate that depletion of HSAT3 delays replication re-initiation. Consistent with this, HSAT3 knockdown alters cell cycle distribution in the presence of the TOP2A inhibitor etoposide. In vivo, reduced HSAT3 levels lead to delayed tumor growth in xenograft models. In addition, we identify epigenetic compounds that suppress HSAT3 expression and enhance cellular sensitivity to etoposide, suggesting a potential strategy for combination treatments. Together, our findings support a functional link between stress-induced repetitive RNAs, nuclear compartmentalization, and replication-associated processes. These results provide a framework for understanding how pericentromeric lncRNAs contribute to genome stability and drug response in cancer cells.
DNA methylation is a key epigenetic mechanism of sex chromosome regulation. However, DNA methylation studies often exclude X chromosomes. Moreover, widely used DNA methylation assays interrogate biased subsets of CpGs, rather than assessing CpGs across the whole chromosome. Consequently, our understanding of X chromosome DNA methylation lags behind that of autosomes. Here we addressed this gap of knowledge by investigating X chromosome DNA methylation in 89 whole genome methylomes from neurons and oligodendrocytes. Using this unbiased and comprehensive data, we show that the combined methylation of the female X chromosomes is globally reduced (hypomethylated) across the chromosome compared with the male X and autosomes. DNA methylation patterns of promoters are more heterogeneous; while many promoters are more highly methylated (hypermethylated) in female X than in male X, a substantial number of promoters exhibit female hypomethylation. Notably, promoters of genes with higher expression in females—used here as a proxy for those that escape X chromosome inactivation—are significantly enriched among female hypomethylated promoters. Differentially methylated regions across the whole X chromosome corroborate the pervasive chromosome-wide reduction in the combined female X methylation and reveal many cell-type and sex-differentiated regions. Mechanistically, we show that female hyper- and hypo-methylation of promoters in large part associate with low and high density CpG promoters that tend to be highly and lowly methylated, respectively. These results provide chromosome-wide evidence that, in the examined human brain cell types, the combined female X chromosome methylation level is globally lower than that of the male X chromosome. More broadly, our findings support a model where relative hypomethylation is a general feature of transcriptionally inactive genomic regions compared to active genomic regions. Furthermore, we show that DNA methylation-mediated silencing of X chromosomes may preferentially occur in a subset of promoters with distinctive genomic and epigenetic features. This study provides a comprehensive and novel list of sex differentiated genomic regions on the X chromosome and furthers our understanding of epigenetic regulation of X chromosomes.
Lysine β-hydroxybutyrylation (Kbhb) is a histone posttranslational modification derived from the ketone body β-hydroxybutyrate (BHB), but its role in DNA replication has remained unexplored. N-acetyltransferase 10 (NAT10) is known for RNA acetylation; whether it functions as a β-hydroxybutyryltransferase to catalyze histone Kbhb and regulate replication dynamics is unknown. We demonstrate that NAT10 directly catalyzes histone Kbhb at multiple lysine residues via its β-hydroxybutyryltransferase activity, an activity abolished by the G641E and K989R mutants or by remodelin inhibition. NAT10-mediated Kbhb accelerates replication fork speed and shortens S-phase duration without affecting origin firing. BHB treatment via NAT10-mediated Kbhb reduces replication stress markers (γH2AX, ssDNA, RPA2, p-CHK1) and genomic instability (micronuclei, anaphase bridges, chromatid breaks)—effects abolished by NAT10 depletion or remodelin inhibition. Mechanistically, Kbhb reduces chromatin-bound NAT10 and enhances chromatin accessibility, as shown by ChIP-seq and ATAC-seq. These findings were validated in primary human fibroblasts (WI-38, 2BS). NAT10-mediated histone Kbhb constitutes a metabolic-epigenetic mechanism that promotes DNA replication fidelity while maintaining genome stability. This pathway offers a potential therapeutic target for replication stress-associated pathologies, including cancer and aging-related conditions.
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.
