The objectives of this project are to study the function of the evolutionarily diverged but highly conserved basal H2A isoprotein, H2A.Z, in chromatin and to study the structure and function of the gene for H2A.Z. The human H2A.Z gene has been isolated and completely sequenced. The gene contains four introns and has two Alu sequences in the 5' upstream region. There is single functional copy of the gene and one or more pseudogene copies. The expression of the gene does not vary through the cell cycle but is downregulated as cells differentiate and enter a quiescent state. Hybridization probes specific for the functional copy of the gene are being used to determine if in different states of cell proliferation there is differential methylation of cytosine residues in the highly GC-rich promoter region and first intron. These probes are also being used to locate DNase I hypersensitive sites and thusly determine the regions of the H2A.Z gene that may be bound by transcription factors. CAT constructs have been assembled from various fragments of the H2A.Z gene promoter region. The core promoter is located in the first 234 base pairs of sequence upstream from the start of transcription. A short stretch of sequence just upstream from the core promoter up regulates the promoter in embryonic (proliferating) cells and down-regulates the promoter in differentiated or quiescent cells. Cotransfection experiments have shown that the H2A.Z promoter is down-regulated by the HIV-TAT gene product. Experiments are in progress to determine the effect of HIV infection on histone biosynthesis.
