The long-term objectives of these proposals are directed to an understanding of the structure and function of active chromatin. Evidence has accumulated to support the view that the functional states of chromatin domains are associated with variables such as: 1) acetylation of core histones; 2) probable depletion or loss of histone Hl; 3) binding of HMG proteins 14 and 17; 4) ubiquitin modifications of H2A and H2B; 5) DNA methylation; 6) DNA supercoiling and polymorphism. Presumably such changes allow access for specific proteins to identify and bind to DNA regulatory sites and also provide the correctly structured template and DNA topology for transcription. Our cell cycle studies associate only the highest states of acetylation of H3 and H4 with transcription and show also that uH2A and uH2B disappear in prophase immediately before metaphase and reappear rapidly in anaphase. These specific regions of chromatin labeled with uH2A and uH2B appear to be packaged last into metaphase chromosomes. It has been suggested that uH2A is located in potentially transcribable chromatin. Studies are in progress of the effects of histone acetylation, ubiquitination and the binding of HMG proteins 14 and 17 on the structures and structural transitions of oligonucleosomes and on the structures, DNA topologies and transcriptional efficiencies of closed circular pBR322 minichromosomes containing gene inserts. These studies will allow a test of our earlier proposal that histone acetylation destabilizes the 34 nm solenoid. To avoid the previous ambiguities resulting from the use of mixed states of histone acetylation, we are fractionating the different states of acetylated histones for clean studies of structure/function relationships. We have evidence for an unusual type of interaction of the H4 peptides (1-23) and (1-37) with DNA which is suppressed by acetylation. Detailed studies of these peptides with the DNA dodecamer of known structure should provide an understanding of this interaction and how it might affect chromatin structure. We are growing mouse mammary ts85 cells which at the non-permissive temperature arrest in early G2 and have no uH2A; at the permissive temperature H2A is ubiquitinated, although it is subsequently deubiquitinated just prior to metaphase. We propose to isolate and characterize uH2A containing nucleosomes and oligonucleosomes to ask whether they are associated with the potentially active genes of that cell type. To relate in vitro studies of chromatin, we propose to isolate and characterize transcribed and non-transcribed pieces of ribosomal DNA chromatin from nucleoli at different states of the life cycle of Physarum polycephalum.
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