2016染色质研究技术与原理.ppt

5. 体外利用Western Blot和特异组蛋白修饰抗体检测组蛋白修饰 6. 利用质谱方法精确分析组蛋白修饰 Genome-wide structural organization of PICs ChIP-exo approach to examine the precise location of 6045 PICs in yeast genome. PICs were positioned within promoters and excluded from coding regions. Rhee Pugh Nature 2013 四、体外染色质重组 The majority of chromatin assembly is coupled to DNA replication in the cell Incorporation of the H3-H4 tetramer by histone chaperones Addition of two H2A-H2B dimers to form a core particle by chaperones ATP dependent remodeling factors to establishe a regular spacing and histones are de-acetylated Incorporation of linker histones Further folding 体外重组染色质（核小体）方法 1. ATP-independent method 2. ATP-dependent method The minimal nucleosome reconstitution process involves the DNA template, core histones and a chaperone that functions both to allow the proper deposition of positively charged histones onto the negatively charged DNA and to prevent undesired interactions of the histones with other molecules. This chaperone function can be mediated by histone-binding proteins — such as NAP-1 or nucleoplasmin— by negatively charged polymers (such as polyglutamate or RNA) or by relatively high concentrations of a salt, such as NaCl. In the absence of a chaperone, core histones and DNA will form an insoluble, non-nucleosomal aggregate at low ionic strengths (such as in a buffer containing 0.1 M NaCl). ATP-independent random deposition of histones onto DNA and the ATP-dependent assembly of periodic nucleosome arrays. The ATP-independent reconstitution process involves core histones, DNA and a histone chaperone (such as NaCl or NAP-1), and typically results in the random deposition of histones onto DNA. The assembly of periodic nucleosome arrays, similar to those seen in bulk native chromatin, is an ATP-dependent reaction and requires ATP-utilizing chromatin assembly factors, which include ACF (ATP-utilizing chromatin assembly and remodeling factor) or RSF (remodeling and spacing factor), or relat