DMMC Workshop: Unravelling Chromatin and the Role of Epigenetics in Disease

No abstract available at this time.     

综述: 表观遗传之染色质重塑

真核细胞中的染色质重塑因子种类繁多,多数以蛋白多聚体的形式存在于细胞中．不同的染色质重塑因子在特定时间定位于特定的核小体上,通过改变染色质结构,影响基因转录活性,进而确保细胞内各种生物学过程的正确运行．另外,染色质重塑因子根据所含功能结构域的不同,大致分为SWI/SNF、ISWI、CHD和INO80四大家族,不同的染色质重塑因子之间既有蛋白质结构和酶活性的相似性,各自又有其特异性．本综述的宗旨在于全面概括和总结染色质重塑因子的分类、结构特点以及其在细胞内的生物学功能,为深入研究染色质重塑因子的生物学功能,尤其是在发育和疾病发生中的作用机制提供理论基础．     

Remodeling of Human Sperm Chromatin Mediated by Nucleoplasmin from Amphibian Eggs

The molecular events associated with decondensation of human sperm nuclei were analyzed by incubating sperm with egg extracts from an amphibian, Bufo japonicus . Acid-urea-Triton polyacrylamide gel electrophoresis (AUT-PAGE) showed that the nuclear basic proteins of human sperm consist mainly of protamines (HPI, HPII) with minor amounts of nucleosomal histones. On incubation of lysolecithin (LC)- and dithiothreitol (DTT)-treated human sperm with the egg extract, the nuclei lost HPI and HPII within 15 min in association with extensive nuclear decondensation, and the acquirement of a whole set of nucleosomal histones. Incubation of LC-DTT-sperm with nucleoplasmin purified from Bufo eggs also induced nuclear decondensation and loss of protamines within 30 min. Native-PAGE and Western blot analyses of incubation medium indicated tight association of the released protamines to nucleoplasmin, strongly suggesting that protamines are removed from sperm nuclei not enzymatically but by their specific binding to nucleoplasmin. On incubation of LC-DTT-sperm with nucleoplasmin and exogenous nucleosomal core histones, micrococcal nuclease-protected DNA fragments were released, although their unit repeat length was slightly less than that of somatic nucleosomes. Thus remodeling of human sperm during fertilization can be mimicked under defined conditions with nucleoplasmin and exogenous histones.     

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex''s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.     

Meeting review: Epigenetics in Development and Disease

The 2002 Keystone symposia held in Taos, New Mexico (21-26 February) saw the convergence of two related fields; Epigenetics in Development and Disease, and RNA Interference, Cosuppression and Related Phenomena. The meeting highlights presented here concentrate upon the sessions within the Epigenetics in Development and Disease meeting, although there were joint sessions which will also be discussed. Of course epigenetic regulation is not restricted to the vertebrates but I have chosen, rightly or wrongly, to limit the highlights to those concerning vertebrates.     

Dynamics of Chromatin and Transcription during Transient Depletion of the RSC Chromatin Remodeling Complex

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染色质重塑和高等植物开花时间控制

染色质的结构和组成直接影响转录因子与基因启动子的结合,并最终导致基因的活化或沉默。多年来在酵母和动物等领域的研究已经证实,起关键调节作用的转录因子表达模式的建立和维持需要染色质重塑。外界和细胞内部信号介导的染色质重塑调控基因的表达,并最终调控细胞的分化和生物个体的发育。近几年人们发现高等植物也存在与动物和酵母同源的参与染色质重塑的蛋白质因子。最近的研究结果表明,决定高等植物开花时间关键基因的表达调控就是通过外界信号影响其染色质结构实现的。     

On Chromatin Remodeling in Mammary Gland Differentiation and Breast Tumorigenesis

Epigenetic programs have been extensively studied in embryonic stem cells. However, epigenetic controls in mammary gland development and in the differentiation of mammary epithelial stem cells have not been defined.The role of epigenetic programs, including DNA methylation, chromatin (histone) modification, and noncoding RNAs, in cellular differentiation and tumorigenesis is well established and, with recent technological improvements, increasingly well understood at the molecular level. Increasing evidence also implicates epigenetic alterations in mediating the long-term effects of environmental risk factors such as diet, exposure to allergens, and various chemicals in various human diseases, including cancer, asthma, and mental disorders (Heijmans et al. 2009; Feinberg 2010). DNA and histone modification patterns have been the most extensively studied in embryonic stem cells (ESCs) (Mikkelsen et al. 2007; Meissner et al. 2008; Lister et al. 2009), whereas the roles of epigenetic changes in mammary gland development and in the differentiation of mammary epithelial stem cells have not been analyzed in either humans or laboratory animals.Huang and Esteller (2011) provide an overview of epigenetic modifications and the technologies developed for their characterization and profiling studies performed in normal mammary epithelial cells and breast cancer. The role of epigenetic programs in regulating human mammary epithelial cell differentiation has not been defined, largely owing to difficulties and controversies associated with the purification and functional characterization of various progenitor and differentiated cells. As discussed by Borowsky (2011) and Visvader and Smith (2011), currently there is no consensus on the identity of bipotential human mammary epithelial stem cells and luminal and myoepithelial progenitors. Further hampering progress in this area are the lack of technologies suitable for the characterization of genome-wide DNA methylation and histone modification profiles of small numbers of cells that can be recovered from tissue samples. Advances in single-molecule sequencing platforms and their application to epigenetic studies will likely solve this problem as methods allowing genome-wide gene expression, DNA methylation, and histone methylation profiling of minute cell numbers have recently been described (Adli et al. 2010; Gu et al. 2010; Ozsolak et al. 2010). The lack of defined human mammary epithelial stem cell hierarchy also makes the interpretation of epigenetic alterations identified in breast cancer problematic, owing to uncertainties about what normal cell to use for comparison. This is especially problematic when using bulk tissue samples, which is the case in the majority of published studies. Numerous genes have been identified as being epigenetically altered in breast cancer and some of these are likely to reflect true malignancy-associated events, but many events may just reflect cell-type-specific differences between normal and cancer tissues. Although this issue does not influence the use of these markers for cancer diagnosis and prognostication, it complicates attempts to understand their potential role in tumorigenesis.One of the most exciting areas of investigation is the role of epigenetic alterations in the long-term effects of various life events on breast cancer risk. For example, in utero exposure to chemicals such as bisphenols (BPA) may increase breast cancer risk by inducing epigenetic alterations in mammary epithelial stem and progenitor cells. Similarly, the reduced risk of postmenopausal breast cancer associated with full-term pregnancy in young adulthood may also be explained by epigenetic alterations in stem cells. The development of new technologies and improved understanding of human mammary epithelial cell types will assure rapid progress in these areas.Finally, the most important question is how we can use the knowledge we have gained for the prevention and treatment of breast cancer. Drug discovery efforts aimed at the identification of inhibitors of specific DNA- (and histone) modifying enzymes will likely lead to the discovery of clinically useful agents. The number of studies published on these topics in the past few years and the number of pharmaceutical companies pursuing epigenetic targets guarantee that progress in these areas will be made soon.