DNA STRUCTURE AND ACCESSIBILITY IN CHROMATIN

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DESCRIPTION (Adapted from Applicant's Abstract): Each eukaryotic cell must execute a complex program of specific gene expression. The resolution of many genetically-based diseases will depend upon the elucidation of all factors contributing to this program. Recent work has demonstrate that, in some instances, chromatin structure plays an important role in bringing about this pattern of specific expression and it is likely that the structural elements of chromatin have been integrated into many transcriptional control mechanisms. This role may be active, actually facilitating the activity of the transcriptional machinery, or it may be more passive, whereby the precise alignment of protein-DNA contacts in chromatin with critical cis-acting DNA elements within promoters is such that pathways to transcription are not hindered. Most DNA-binding transcription factors will not be able to access these critical elements unless they are situated in the accessible regions of chromatin between nucleosome core structures. Further, even elements in these accessible regions may be rendered inaccessible by the binding of linker histones and the formation of higher order structures of chromatin. The specific aim of this proposal is to develop a detailed structural understanding of how nucleosome cores and linker histones organize DNA and modulate the accessibility of trans-acting factors to DNA in chromatin. This will be accomplished by exploiting the ability of a DNA fragment containing a Xenopus 5S RNA gene to position nucleosomes. A set of homogeneous and well- defined model chromatin complexes will be constructed which will be amenable to comprehensive structural analyses by the use of high resolution chemical probes of DNA structure and functional analysis by assessment of the binding of various transcription factors. This approach circumvents the problem of heterogeneity usually associated with preparation of native chromatin complexes and problems with structural analysis of these large complexes by traditional methods such as X-ray crystallography. The functional and structural extent of influence of the nucleosome core into the accessible regions of chromatin will be precisely determined as well as assessment of effects due to linker histones and folding of these accessible regions. This investigation will be relevant to proposed models for the role of chromatin in 5S gene regulation and to general understanding of the complex role of chromatin structure in promoter architecture. The proposed work may also bear on long-standing issues concerning the conformation and configuration of linker DNA in higher- order chromatin structures.