The epigenetic promotion of osteogenic differentiation of human adipose-derived stem cells by the genetic and chemical blockade of histone demethylase LSD1

Human adipose-derived stem cells (hASCs) are a highly attractive source in bone tissue engineering. It has become increasingly clear that chromatin regulators play an important role in cell fate determination. However, how osteogenic differentiation of hASCs is controlled by epigenetic mechanisms is not fully understood. Here we use genetic tools and chemical inhibitors to modify the epigenetic program of hASCs and identify lysine-specific demethylase 1 (LSD1), a histone demethylase that specifically catalyzes demethylation of di- and mono- methyl histone H3 lysine 4 (H3K4me2/1), as a key regulator in osteogenic differentiation of hASCs. Specifically, we demonstrated that genetic depletion of LSD1 with lentiviral strategy for gene knockdown promoted osteogenic differentiation of hASCs by cell studies and xenograft assays. At the molecular level, we found that LSD1 regulates osteogenesis-associated genes expression through its histone demethylase activity. Significantly, we demonstrated LSD1 demethylase inhibitors could efficiently block its catalytic activity and epigenetically boost osteogenic differentiation of hASCs. Altogether, our study defined the functional and biological roles of LSD1 and extensively explored the effects of its enzymatic activity in osteogenic differentiation of hASCs. A better understanding of how LSD1 influences on osteogenesis associated epigenetic events will provide new insights into the modulation of hASCs based cell therapy and improve the development of bone tissue engineering with epigenetic intervention.     

piRNA pathway targets active LINE1 elements to establish the repressive H3K9me3 mark in germ cells

Transposable elements (TEs) occupy a large fraction of metazoan genomes and pose a constant threat to genomic integrity. This threat is particularly critical in germ cells, as changes in the genome that are induced by TEs will be transmitted to the next generation. Small noncoding piwi-interacting RNAs (piRNAs) recognize and silence a diverse set of TEs in germ cells. In mice, piRNA-guided transposon repression correlates with establishment of CpG DNA methylation on their sequences, yet the mechanism and the spectrum of genomic targets of piRNA silencing are unknown. Here we show that in addition to DNA methylation, the piRNA pathway is required to maintain a high level of the repressive H3K9me3 histone modification on long interspersed nuclear elements (LINEs) in germ cells. piRNA-dependent chromatin repression targets exclusively full-length elements of actively transposing LINE families, demonstrating the remarkable ability of the piRNA pathway to recognize active elements among the large number of genomic transposon fragments.     

组蛋白H3K9甲基修饰酶在2周、4周和6周龄小鼠肝脏中的表达

目的 探讨不同周龄小鼠肝脏组蛋白H3K9甲基转移酶(KMT)和去甲基化酶(KDM)的表达.方法 取2周、4周和6周龄BALB/c雄性小鼠肝脏组织,分别提取总mRNA和蛋白质,应用实时定量反转录-聚合酶链式反应(RT-qPCR)确定几种H3K9甲基转移酶和去甲基化酶的表达差异,使用Western blotting 检测两...     

Recognition of H3K9 methylation by GLP is required for efficient establishment of H3K9 methylation,rapid target gene repression,and mouse viability

GLP and G9a are major H3K9 dimethylases and are essential for mouse early embryonic development. GLP and G9a both harbor ankyrin repeat domains that are capable of binding H3K9 methylation. However, the functional significance of their recognition of H3K9 methylation is unknown. Here, we report that the histone methyltransferase activities of GLP and G9a are stimulated by neighboring nucleosomes that are premethylated at H3K9. These stimulation events function in cis and are dependent on the H3K9 methylation binding activities of ankyrin repeat domains of GLP and G9a. Disruption of the H3K9 methylation-binding activity of GLP in mice causes growth retardation of embryos, ossification defects of calvaria, and postnatal lethality due to starvation of the pups. In mouse embryonic stem cells (ESCs) harboring a mutant GLP that lacks H3K9me1-binding activity, critical pluripotent genes, including Oct4 and Nanog, display inefficient establishment of H3K9me2 and delayed gene silencing during differentiation. Collectively, our study reveals a new activation mechanism for GLP and G9a that plays an important role in ESC differentiation and mouse viability.     

Inhibited expression of hematopoietic progenitor kinase 1 associated with loss of jumonji domain containing 3 promoter binding contributes to autoimmunity in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by T cell overactivation and B cell hyper-stimulation. Hematopoietic progenitor kinase 1 (HPK1, also called MAP4K1) negatively regulates T cell-mediated immune responses. However, the role of HPK1 and the mechanisms that regulate HPK1 expression in SLE remain poorly understood. Using chromatin immunoprecipitation (ChIP) microarray data, we identified markedly increased histone H3 lysine 27 trimethylation (H3K27me3) enrichment at the HPK1 promoter of SLE CD4+ T cells relative to controls, and confirmed this observation using ChIP and real-time PCR experiments. We further found that HPK1 mRNA and protein levels were significantly decreased in CD4+ T cells of patients with SLE, and that this decrease was not caused by exposure to standard SLE medications. Down-regulating HPK1 in healthy CD4+ T cells significantly accelerated T cell proliferation and production of IFNγ and IgG. Consistent with these findings, overexpressing HPK1 in SLE CD4+ T cells caused a significant decrease in T cell reactivity. In addition, we observed a striking decrease in jumonji domain containing 3 (JMJD3) binding, but no marked change in enhancer of zeste homolog 2 (EZH2) binding, at the HPK1 promoter region in SLE CD4+ T cells compared to healthy controls. SiRNA knock down of JMJD3 in healthy CD4+ T cells led to decreased JMJD3 binding and increased H3K27me3 enrichment at the HPK1 promoter region, thus inhibiting the expression of HPK1. Concordantly, plasmid-induced overexpression of JMJD3 in SLE CD4+ T cells led to increased JMJD3 binding, decreased H3K27me3 enrichment, and up-regulated HPK1 expression. Our results show for the first time that inhibited HPK1 expression in SLE CD4+ T cells is associated with loss of JMJD3 binding and increased H3K27me3 enrichment at the HPK1 promoter, contributing to T cell overactivation and B cell overstimulation in SLE. These findings suggest that HPK1 may serve as a novel target for effective SLE therapy.     

A dual flip-out mechanism for 5mC recognition by the Arabidopsis SUVH5 SRA domain and its impact on DNA methylation and H3K9 dimethylation in vivo

Cytosine DNA methylation is evolutionarily ancient, and in eukaryotes this epigenetic modification is associated with gene silencing. Proteins with SRA (SET- or RING-associated) methyl-binding domains are required for the establishment and/or maintenance of DNA methylation in both plants and mammals. The 5-methyl-cytosine (5mC)-binding specificity of several SRA domains have been characterized, and each one has a preference for DNA methylation in different sequence contexts. Here we demonstrate through mobility shift assays and calorimetric measurements that the SU(VAR)3-9 HOMOLOG 5 (SUVH5) SRA domain differs from other SRA domains in that it can bind methylated DNA in all contexts to similar extents. Crystal structures of the SUVH5 SRA domain bound to 5mC-containing DNA in either the fully or hemimethylated CG context or the methylated CHH context revealed a dual flip-out mechanism where both the 5mC and a base (5mC, C, or G, respectively) from the partner strand are simultaneously extruded from the DNA duplex and positioned within binding pockets of individual SRA domains. Our structure-based in vivo studies suggest that a functional SUVH5 SRA domain is required for both DNA methylation and accumulation of the H3K9 dimethyl modification in vivo, suggesting a role for the SRA domain in recruitment of SUVH5 to genomic loci.     

UHRF2在羟基脲所致的DNA损伤应答中促进H3K9乙酰化抑制细胞增殖

目的探讨UHRF2在羟基脲所致的DNA损伤应答中对H3K9乙酰化及细胞增殖的影响。方法体外培养HEK293细胞,构建羟基脲所致的DNA损伤细胞模型;转染p CMV-3×Flag-UHRF2质粒,用Western blot评估p CMV-3×Flag-UHRF2转染效率及H3K9乙酰化水平;CCK8法检测HEK293细胞增殖。结果 HEK293细胞经2.5 mmol/L羟基脲处理12 h后,DNA出现了明显的损伤;转染p CMV-3×Flag-UHRF2质粒后,UHRF2蛋白表达显著升高(P0.01);DNA损伤应答中H3K9乙酰化显著增加(P0.05);细胞增殖能力明显下降(P0.05)。结论 UHRF2在羟基脲所致的DNA损伤应答中促进组蛋白H3K9乙酰化抑制细胞增殖。     

Ing1 functions in DNA demethylation by directing Gadd45a to H3K4me3

Active DNA demethylation regulates epigenetic gene activation in numerous processes, but how the target site specificity of DNA demethylation is determined and what factors are involved are still poorly understood. Here we show that the tumor suppressor inhibitor of growth protein 1 (Ing1) is required for targeting active DNA demethylation. Ing1 functions by recruiting the regulator of DNA demethylation growth arrest and DNA damage protein 45a (Gadd45a) to histone H3 trimethylated at Lys 4 (H3K4me3). We show that reduced H3K4 methylation impairs recruitment of Gadd45a/Ing1 and gene-specific DNA demethylation. Our results indicate that histone methylation directs DNA demethylation.     

Protective antigen and extractable antigen 1 based chimeric protein confers protection against Bacillus anthracis in mouse model

Recombinant bivalent chimeric protein was generated comprising of domain 4 of protective antigen (PA4) and carboxy terminal region of extractable antigen 1 (EA1C) by overlap extension PCR. The immunogenicity and protective efficacy of recombinant chimeric protein (PE) and protein mixture (PAEA) along with the individual components, PA4 and EA1C were evaluated in this study. We found that PE and PAEA exhibited higher endpoint titer and elevated IgG1 response. Compared to PA4 and EA1C, the chimeric protein PE and protein mixture PAEA exhibited 1.52 and 1.39 times more proliferative effect on lymphocytes in vitro. The spore uptake by anti-PE and anti-PAEA antibodies was significantly more than the individual components. We further evaluated the effects of antisera on the toxins in vitro and in vivo. Anti-PE and anti-PAEA antibodies displayed nearly 80% protection against crude toxin activity on RAW 264.7 cell lines. We further demonstrated that the anti-PE and anti-PAEA antibodies displayed better protection in controlling the edema induced by crude toxin. Passive immunization with anti-PE and anti-PAEA provided protection against toxin challenge in mice. The present study reveals that the chimeric protein consisting of heterologous regions of PA and EA1 can render better protection than PA4 or EA1C alone against toxins and bacilli.     

The localization of proteins encoded by CRYM, KIAA1199, UBA52, COL9A3, and COL9A1, genes highly expressed in the cochlea

Genes that are highly expressed in the inner ear, as revealed by cDNA microarray analysis, may have a crucial functional role there. Those that are expressed specifically in auditory tissues are likely to be good candidates to screen for genetic alterations in patients with deafness, and several genes have been successfully identified as responsible for hereditary hearing loss. To understand the detailed mechanisms of the hearing loss caused by the mutations in these genes, the present study examined the immunocytochemical localization of the proteins encoded by Crym, KIAA1199 homolog, Uba52, Col9a3, and Col9a1 in the cochlea of rats and mice. Confocal microscopic immunocytochemistry was performed on cryostat sections. Ultrastructurally, postembedding immunogold cytochemistry was applied using Lowicryl sections. Crym protein was predominantly distributed in the fibrocytes in the spiral ligament, as well as the stria vascularis in rats. KIAA1199 protein homolog was localized in various supporting cells, including inner phalangeal, border, inner and outer pillar, and Deiters' cells. Uba52 protein was restrictedly localized within the surface of the marginal cells of the stria vascularis. Collagen type IX was found within the tectorial membrane as well as fibrocytes in the spiral ligament. The present results showed cell-specific localization of the encoded proteins of these highly expressed genes, indicating that the coordinated actions of various molecules distributed in different parts of the cochlea are essential for maintenance of auditory processing in the cochlea.