The long-term objective of this project is to better understand the molecular mechanism governing albumin gene expression as a model system for a highly transcribed polymerase II gene in mammalian liver. Although constitutively expressed, albumin can fluctuate with age of the individual and with dietary regimes reflecting a deviation from the normal status of the hepatocyte. The observation that histone H1 can interact at specific regions within the 5'-end of the albumin gene suggest this gene may have a unique higher order chromatin structure which may, at some level, regulate gene expression.
The specific aim of this proposal is to confirm, characterize, and demonstrate the role histone H1 has in the higher order chromatin organization of the rat serum albumin gene. To confirm the specific DNA binding activity associated with histone H1, DNA binding sites within three large restriction fragments of the albumin gene will be isolated by preferential binding of histone H1. These purified sites will be sequenced by chemical cleavage technology and histone H1 protection from DNase I digestion will identify the specific site of interaction. The role the specific H1 interaction has on higher order gene structure will be determined by mapping nucleosomes across the albumin gene with emphasis on that region neighboring the H1-DNA site of interaction. Nucleosomes will be mapped by measuring restriction enzyme accessibility on soluble rat liver chromatin by Southern blot hybridization with DNA fragments that contain the H1 binding site. Correlation of structural elements with the H1 binding sites will determine the role H1 binding has on structural components of the albumin gene. Delineation of the individual H1-binding sites by DNase fragments assays and DNase and dimethylsulfate footprinting technology will lead to a consensus sequence for H1 binding. The information obtained will provide a model of higher order chromatin structure that will settle the controversial question of nucleosome phasing across the 5'-end of the albumin gene. The model can subsequently be tested by in vitro reconstitution studies and its role in gene regulation tested by in vitro transcriptional assays.
| Sevall, J S (1988) High-resolution analysis of a histone H1 binding site in a rat albumin gene. Biochemistry 27:5038-44 |
