利用拟南芥杂交组合研究siRNA与等位基因DNA甲基化调控中的联系

近年来,越来越多的实验结果表明,表观遗传因子,如DNA甲基化、小RNA、组蛋白修饰等在杂种优势形成中起到重要作用,然而对于这些表观遗传因子在F。中遗传调控方式的认识仍很有限．本实验室先前工作曾以拟南芥C24和Ler两种生态型及其正反交子一代为材料,运用新一代测序方法获得该杂交组合中DNA甲基化及小RNA单碱基分辨率的全基因组图谱．本文进一步对这批数据中的等位基因DNA甲基化水平进行分析,区分DNA甲基化遗传过程中的顺式与反式调控方式,并发现这两种调控方式均有重要的贡献．研究发现,siRNA与DNA甲基化的两种调控方式有密切联系,尤其在DNA甲基化的反式调控中,Fl中DNA甲基化变化程度越大,该区域内siRNA富集程度越强,二者可能存在某种调控机制．通过等位基因表观遗传组的分析研究杂交过程中DNA甲基化和小RNA遗传调控的规律,为更好地理解杂种优势机制提供了帮助．     

Aberrant epigenetic landscape in cancer: how cellular identity goes awry

Appropriate patterns of DNA methylation and histone modifications are required to assure cell identity, and their deregulation can contribute to human diseases, such as cancer. Our aim here is to provide an overview of how epigenetic factors, including genomic DNA methylation, histone modifications, and microRNA regulation, contribute to normal development, paying special attention to their role in regulating tissue-specific genes. In addition, we summarize how these epigenetic patterns go awry during human cancer development. The possibility of "resetting" the abnormal cancer epigenome by applying pharmacological or genetic strategies is also discussed.     

An atypical epigenetic mechanism affects uniparental expression of Pol IV-dependent siRNAs

Background

Small RNAs generated by RNA polymerase IV (Pol IV) are the most abundant class of small RNAs in flowering plants. In Arabidopsis thaliana Pol IV-dependent short interfering (p4-si)RNAs are imprinted and accumulate specifically from maternal chromosomes in the developing seeds. Imprinted expression of protein-coding genes is controlled by differential DNA or histone methylation placed in gametes. To identify epigenetic factors required for maternal-specific expression of p4-siRNAs we analyzed the effect of a series of candidate mutations, including those required for genomic imprinting of protein-coding genes, on uniparental expression of a representative p4-siRNA locus.

Results

Paternal alleles of imprinted genes are marked by DNA or histone methylation placed by DNA METHYLTRANSFERASE 1 or the Polycomb Repressive Complex 2. Here we demonstrate that repression of paternal p4-siRNA expression at locus 08002 is not controlled by either of these mechanisms. Similarly, loss of several chromatin modification enzymes, including a histone acetyltransferase, a histone methyltransferase, and two nucleosome remodeling proteins, does not affect maternal expression of locus 08002. Maternal alleles of imprinted genes are hypomethylated by DEMETER DNA glycosylase, yet expression of p4-siRNAs occurs irrespective of demethylation by DEMETER or related glycosylases.

Conclusions

Differential DNA methylation and other chromatin modifications associated with epigenetic silencing are not required for maternal-specific expression of p4-siRNAs at locus 08002. These data indicate that there is an as yet unknown epigenetic mechanism causing maternal-specific p4-siRNA expression that is distinct from the well-characterized mechanisms associated with DNA methylation or the Polycomb Repressive Complex 2.     

Glutathione-S-transferase pi 1(GSTP1) gene silencing in prostate cancer cells is reversed by the histone deacetylase inhibitor depsipeptide

Gene silencing by epigenetic mechanisms is frequent in prostate cancer (PCA). The link between DNA hypermethylation and histone modifications is not completely understood. We chose the GSTP1 gene which is silenced by hypermethylation to analyze the effect of the histone deacetylase inhibitor depsipeptide on DNA methylation and histone modifications at the GSTP1 promoter site. Prostate cell lines (PC-3, LNCaP, and BPH-1) were treated with depsipeptide; apoptosis (FACS analysis), GSTP1 mRNA levels (quantitative real-time PCR), DNA hypermethylation (methylation-specific PCR), and histone modifications (chromatin immunoprecipitation) were studied. Depsipeptide induced apoptosis in PCA cells, but not a cell cycle arrest. Depispeptide reversed DNA hypermethylation and repressive histone modifications (reduction of H3K9me2/3 and H3K27me2/3; increase of H3K18Ac), thereby inducing GSTP1 mRNA re-expression. Successful therapy requires both, DNA demethylation and activating histone modifications, to induce complete gene expression of epigenetically silenced genes and depsipeptide fulfils both criteria.     

Epigenetic "bivalently marked" process of cancer stem cell-driven tumorigenesis

Silencing of tumor suppressor genes (TSGs), by DNA methylation, is well known in adult cancers. However, based on the "stem cell" theory of tumorigenesis, the early epigenetic events arising in malignant precursors remain unknown. A recent report demonstrates that, while pluripotent embryonic stem cells lack DNA methylation and possess a "bivalent" pattern of activating and repressive histone marks in numerous TSGs, analogous multipotent malignant cells derived from germ cell tumors (embryonic carcinoma cells) gain additional silencing modifications to those same genes. These results suggest a possible mechanism by which aberrant differentiation, mediated by histone and DNA methylation, instigates tumor progression.