Chromatin assembly on cloned DNA in vitro: Histone H5 mediated nucleosome alignment
Abstract
Eukaryotic chromatin consists of extensive, ordered nucleosome arrays along the DNA chain. The distance between nucleosome centers, known as the nucleosome spacing periodicity or repeat length, varies in a tissue- and species-specific manner. In nature repeat lengths ranging from 160 to 250 base pairs have been observed; however, the mechanisms by which nucleosome arrays are generated, the precision of the spacing, and the factors which might influence nucleosome alignment are not understood. We have achieved physiological nucleosome spacing on cloned DNA using a fully defined in vitro chromatin assembly system. This is the first time anyone has achieved physiological nucleosome spacing using highly purified DNA and histones, and clearly demonstrates that all the information for nucleosome alignment is contained in the histones and the DNA. Histone H1- or H5-mediated nucleosome alignment has been found to nucleate from certain regions of DNA, which we call "chromatin organizing regions" or "CORs." A COR was initially identified in pBR327 by Shin Wu Jeong; sequences with COR-like activity have now been identified in the chicken ovalbumin gene. Nucleosome alignment in our chromatin assembly system appears to be essentially an all-or-none mechanism. A necessary requirement for histone H5-mediated nucleosome alignment is that the total DNA length must be close to an integral multiple of the repeat length generated for small $(<$4 kb) plasmids, a type of boundary effect. Only plasmids with DNA lengths close to integral multiples of 210 $\pm$ 3 bp permitted nucleosome alignment. This suggests that nucleosome arrays can be "quasi-crystalline," and are capable of transmitting information over a distance of more than 2 kb. Taken together, these data suggest a possible mechanism through which chromatin can regulate eukaryotic gene expression. It is possible that nucleosome spacing periodicities might in some way be specified by different CORs. Different cell types might use different CORs to alter chromatin structure and function in a manner analogous to the way different cell types might use different replication origins and chromosomal scaffold attachment regions. Changes in the boundary conditions, by binding of a regulatory protein for example, might be sufficient to affect the chromatin structure within that particular domain thereby altering the expression of genes within the affected region.
Degree
Ph.D.
Advisors
Stein, Purdue University.
Subject Area
Molecular biology|Genetics
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.