9th Annual Workshop, October 28-31, 1999
Co-sponsored by the U.S. Department of Energy
Stepwise chromatin restructuring and unexpected rules for interaction of distributed cis-elements that form a locus control region in vivo
Catherine Ley-Ebert, Carolyn Florio, John Maier, Chris Cost and Bruce Aronow
Children's Hospital Research Foundation and the University of Cincinnati, Cincinnati, Ohio, USA
We have performed a mutational analysis of the human adenosine deaminase (ADA) gene thymic locus control region in transgenic mice using CAT reporter genes. We examined in detail gene expression, chromatin structure based on low and high resolution MNase and DNAse hypersensitivity, restriction enzyme accessibility, and histone acetylation state analyses using chromatin immune precipitations. Mutations and perturbations of LCR cis-elements led to variably compromised LCR function and the formation of several common distinguishable chromatin structures.
Our results suggest that the ADA thymic LCR is formed by the interaction of distributed cis-elements that consist of a 200 97bp enhancer region which is bilaterally flanked by two 1 kb regions that contain specific cis-elements distinct from those of the enhancer. The flanking sequences facilitate the stepwise formation of an intricate chromatin architecture at the enhancer core. The central enhancer elements are partially redundant with each other and, at a stage prior to hypersensitive site formation, are required for the nucleosomal array dephasing and histone acetylation of an extended nucleosomal array associated with the inactive LCR. Unexpected rules govern the interactions of the flanking facilitative cis-elements with the enhancer core and suggest that a supranucleosomal organization, beyond the phased nucleosomes, is critical for proper LCR function in developmental gene activation.