In vivo evidence of enhanced di-methylation of histone H3 K4 on upregulated genes in adipose tissue of diabetic db/db mice

Di-methylation of histone H3 lysine (K) 4, a component of the epigenetic memory, is associated with gene transactivation. In this study, we examined whether the development of diabetes induces di-methylation of histone H3 K4 on the upregulated genes. We searched for upregulated genes in mesenteric adipose tissue of insulin-resistant/diabetic db/db mice compared with non-diabetic db/m mice using microarray analysis. We also performed chromatin immunoprecipitation assays for di-methylation of histone H3 K4 in the upregulated genes in mesenteric adipose tissue of db/m and db/db mice. Di-methylation of histone H3 K4 was enhanced at the upstream and/or transcribed regions of upregulated genes including Atp6v0d2, Mmp12, Trem2 and Clec4d genes in mesenteric adipose tissue of db/db mice, as compared with db/m mice. These results suggest that di-methylation of histone H3 K4 is involved in the induction of Atp6v0d2, Mmp12, Trem2 and Clec4d in mesenteric adipose tissue in db/db mice.     

沙棘H3K9乙酰化修饰全基因组分析

为了绘制沙棘H3K9乙酰化修饰图谱,确定H3K9乙酰化修饰所调控的基因,该实验通过Western blot验证抗体与组蛋白的结合能力和ChIP-seq验证抗体富集效率,获得全基因组范围内沙棘H3K9乙酰化修饰图谱和调控基因。实验结果表明,H3K9ac抗体与复合物具有较强的结合能力。对富集到的DNA片段进行高通量测序,分别获得2.2×10~7和3.6×10~7条原始序列;唯一比对序列广泛分布于沙棘基因组中,并且在结构基因中的两端具有明显的富集。对富集区进行峰的预测结果显示,共预测出1 011个峰;对峰所处部位基因进行功能预测结果发现,H3K9ac对于沙棘细胞代谢和信号转导基因的表达具有重要调控作用。沙棘片段化DNA的富集以及高通量测序结果证明,抗体能够用于研究沙棘的组蛋白修饰类型,并且绘制了沙棘第一张H3K9乙酰化修饰遗传图谱草图,鉴定出沙棘H3K9乙酰化修饰所调控的基因,为今后研究组蛋白修饰对沙棘基因表达的调控方式奠定了基础。     

G9α‐dependent histone H3K9me3 hypomethylation promotes overexpression of cardiomyogenesis‐related genes in foetal mice

Alcohol consumption during pregnancy can cause foetal alcohol syndrome and congenital heart disease. Nonetheless, the underlying mechanism of alcohol‐induced cardiac dysplasia remains unknown. We previously reported that alcohol exposure during pregnancy can cause abnormal expression of cardiomyogenesis‐related genes, and histone H3K9me3 hypomethylation was observed in alcohol‐treated foetal mouse heart. Hence, an imbalance in histone methylation may be involved in alcohol‐induced cardiac dysplasia. In this study, we investigated the involvement of G9α histone methyltransferase in alcohol‐induced cardiac dysplasia in vivo and in vitro using heart tissues of foetal mice and primary cardiomyocytes of neonatal mice. Western blotting revealed that alcohol caused histone H3K9me3 hypomethylation by altering G9α histone methyltransferase expression in cardiomyocytes. Moreover, overexpression of cardiomyogenesis‐related genes (MEF2C, Cx43, ANP and β‐MHC) was observed in alcohol‐exposed foetal mouse heart. Additionally, we demonstrated that G9α histone methyltransferase directly interacted with histone H3K9me3 and altered its methylation. Notably, alcohol did not down‐regulate H3K9me3 methylation after G9α suppression by short hairpin RNA in primary mouse cardiomyocytes, preventing MEF2C, Cx43, ANP and β‐MHC overexpression. These findings suggest that G9α histone methyltransferase‐mediated imbalance in histone H3K9me3 methylation plays a critical role in alcohol‐induced abnormal expression cardiomyogenesis‐related genes during pregnancy. Therefore, G9α histone methyltransferase may be an intervention target for congenital heart disease.     

Pygo2 expands mammary progenitor cells by facilitating histone H3 K4 methylation

Recent studies have unequivocally identified multipotent stem/progenitor cells in mammary glands, offering a tractable model system to unravel genetic and epigenetic regulation of epithelial stem/progenitor cell development and homeostasis. In this study, we show that Pygo2, a member of an evolutionarily conserved family of plant homeo domain–containing proteins, is expressed in embryonic and postnatal mammary progenitor cells. Pygo2 deficiency, which is achieved by complete or epithelia-specific gene ablation in mice, results in defective mammary morphogenesis and regeneration accompanied by severely compromised expansive self-renewal of epithelial progenitor cells. Pygo2 converges with Wnt/β-catenin signaling on progenitor cell regulation and cell cycle gene expression, and loss of epithelial Pygo2 completely rescues β-catenin–induced mammary outgrowth. We further describe a novel molecular function of Pygo2 that is required for mammary progenitor cell expansion, which is to facilitate K4 trimethylation of histone H3, both globally and at Wnt/β-catenin target loci, via direct binding to K4-methyl histone H3 and recruiting histone H3 K4 methyltransferase complexes.     

Narrow H3K4me2 is required for chicken PGC formation

The development of primordial germ cells (PGCs) undergoes epigenetic modifications. The study of histone methylation in regulating PGCs is beneficial to understand the development and differentiation mechanism of germ stem cells. Notably, it provides a theoretical basis for directed induction and mass acquisition in vitro. However, little is known about the regulation of PGC formation by histone methylation. Here, we found the high enrichment of H3K4me2 in the blastoderm, genital ridges, and testis. Chromatin immunoprecipitation sequencing was performed and the results revealed that genomic H3K4me2 is dynamic in embryonic stem cells, PGCs, and spermatogonial stem cells. This trend was consistent with the H3K4me2 enrichment in the gene promoter region. Additionally, narrow region triggered PGC‐related genes (Bmp4, Wnt5a, and Tcf7l2) and signaling pathways (Wnt and transforming growth factor‐β). After knocking down histone methylase Mll2 in vitro and vivo, the level of H3K4me2 decreased, inhibiting Cvh and Blimp1 expression, then repressing the formation of PGCs. Taken together, our study revealed the whole genome map of H3K4me2 in the formation of PGCs, contributing to improve the epigenetic study in PGC formation and providing materials for bird gene editing and rescue of endangered birds.     

Histone deacetylase OsHDA706 increases salt tolerance via H4K5/K8 deacetylation of OsPP2C49 in rice

High salt is a major environmental factor that threatens plant growth and development. Increasing evidence indicates that histone acetylation is involved in plant responses to various abiotic stress; however, the underlying epigenetic regulatory mechanisms remain poorly understood. In this study, we revealed that the histone deacetylase OsHDA706 epigenetically regulates the expression of salt stress response genes in rice (Oryza sativa L.). OsHDA706 localizes to the nucleus and cytoplasm and OsHDA706 expression is significantly induced under salt stress. Moreover, oshda706 mutants showed a higher sensitivity to salt stress than the wild-type. In vivo and in vitro enzymatic activity assays demonstrated that OsHDA706 specifically regulates the deacetylation of lysines 5 and 8 on histone H4 (H4K5 and H4K8). By combining chromatin immunoprecipitation and mRNA sequencing, we identified the clade A protein phosphatase 2 C gene, OsPP2C49, which is involved in the salt response as a direct target of H4K5 and H4K8 acetylation. We found that the expression of OsPP2C49 is induced in the oshda706 mutant under salt stress. Furthermore, the knockout of OsPP2C49 enhances plant tolerance to salt stress, while its overexpression has the opposite effect. Taken together, our results indicate that OsHDA706, a histone H4 deacetylase, participates in the salt stress response by regulating the expression of OsPP2C49 via H4K5 and H4K8 deacetylation.     

Induction by Fructose Force-Feeding of Histone H3 and H4 Acetylation at Their Lysine Residues around the Slc2a5 Gene and Its Expression in Mice

It has been reported that fructose force-feeding rapidly induced jejunal Slc2a5 gene expression in rodents. We demonstrate in this study that acetylation at lysine (K) 9 of histone H3 and acetylation at K5 and K16 of histone H4 were more enhanced in the promoter/enhancer to transcribed regions of the Slc2a5 gene in fructose force-fed mice than in glucose force-fed mice. However, fructose force-feeding did not induce acetylation at K14 of histone H3, or at K8 and K12 of histone H4 around the Slc2a5 gene. These results suggest that fructose force-feeding induced selective histone acetylation, particularly of H3 and H4, around the jejunal Slc2a5 gene in mice.     

Reduced Histone H3K9 Acetylation of Clock Genes and Abnormal Glucose Metabolism in ob/ob Mice

Recent chronobiological studies found significant correlation between lack of clock function and metabolic abnormalities. We previously showed that clock gene expressions were dampened in the peripheral tissues of obese and diabetic ob/ob mice. However, the molecular mechanism of the disturbance remained to be determined. In this study, we demonstrated for the first time that acetylation levels of histone H3 lysine 9 (H3K9) at the promoter regions of clock genes, such as Dbp, Per2, and Bmal1, in the adipose tissue of ob/ob mice were significantly reduced compared with those of its control C57BL/6J mice. Treatment with histone deacetylase (HDAC) inhibitors increased Dbp, but not Per2 or Bmal1, mRNA expression in adipose tissue, and it decreased blood glucose in these animals. In addition, 2-deoxyglucose uptake activity was significantly suppressed by silencing Dbp expression in cultured adipocytes. These results suggest that reduced H3K9 acetylation and subsequent decreased mRNA expression of the Dbp gene in adipose tissue are involved in the mechanism of development of abnormal glucose metabolism in ob/ob mice. (Author correspondence: akiofuji@jichi.ac.jp)     

β-Carotene enhances the expression of inflammation-related genes and histone H3 K9 acetylation,K4 dimethylation,and K36 trimethylation around these genes in juvenile macrophage-like THP-1 cells

β-Carotene is converted into vitamin A in the body and can remove reactive oxygen species. However, it is still unclear whether β-carotene alters the expression levels of inflammation-related genes in macrophages and how this is regulated. In the present study, we investigated whether the administration of β-carotene under hyperglycemic conditions altered the expression level of inflammation-related genes and whether any observed differences were associated with changes in histone modifications in juvenile macrophage-like THP-1 cells. THP-1 cells (from a human monocytic leukemia cell line) were cultured in low glucose (5 mM), high glucose (25 mM), or high glucose (25 mM) + β-carotene (5 μM) media for 1 day, and mRNA expression levels of genes related to oxidative stress and inflammation, and histone modifications were determined by mRNA microarray and qRT-PCR analyses, and chromatin immunoprecipitation assays, respectively. The expression of inflammation-related genes, such as IL31RA, CD38, and NCF1B, and inflammation-associated signaling pathway genes, such as ITGAL, PRAM1, and CSF3R, were upregulated by β-carotene under high-glucose conditions. Under these conditions, histone H3 lysine 4 (K4) demethylation, H3K36 trimethylation, and H3K9 acetylation around the CD38, NCF1B, and ITGAL genes were higher in β-carotene-treated cells than in untreated cells. Treatment of juvenile macrophage-like THP-1 cells with β-carotene under these high glucose conditions induced the expression of inflammation-related genes, K9 acetylation, and K4 di- and K36 trimethylation of histone H3 around these genes.     

Biofilm Formation on Biotic and Abiotic Surfaces in the Presence of Antimicrobials by Escherichia coli Isolates from Cases of Bovine Mastitis

Escherichia coli is a highly adaptive microorganism, and its ability to form biofilms under certain conditions can be critical for antimicrobial resistance. The adhesion of four E. coli isolates from bovine mastitis to bovine mammary alveolar (MAC-T) cells, biofilm production on a polystyrene surface, and the expression profiles of the genes fliC, csgA, fimA, and luxS in the presence of enrofloxacin, gentamicin, co-trimoxazole, and ampicillin at half of the MIC were investigated. Increased adhesion of E. coli isolates in the presence of antimicrobials was not observed; however, increased internalization of some isolates was observed by confocal microscopy. All of the antimicrobials induced the formation of biofilms by at least one isolate, whereas enrofloxacin and co-trimoxazole decreased biofilm formation by at least one isolate. Quantitative PCR analysis revealed that all four genes were differentially expressed when bacteria were exposed to subinhibitory concentrations of antimicrobials, with expression altered on the order of 1.5- to 22-fold. However, it was not possible to associate gene expression with induction or reduction of biofilm formation in the presence of the antimicrobials. Taken together, the results demonstrate that antimicrobials could induce biofilm formation by some isolates, in addition to inducing MAC-T cell invasion, a situation that might occur in vivo, potentially resulting in a bacterial reservoir in the udder, which might explain some cases of persistent mastitis in herds.