Gene expression array of HTLV type 1-infected T cells: Up-regulation of transcription factors and cell cycle genes

By utilizing a human cDNA expression array blot (588 genes), we have observ ed overexpression of various transcription factors, cell cycle regulated ki nases, and DNA repair genes in HTLV-1-infected T cells. One of the genes of interest, and focus in this study, is the cyclin-dependent kinase inhibito r, p21/waf1, The p21/wafl1 transcription and protein is overexpressed in al l HTLV-1-infected cell lines tested as well as ATL and HAM/TSP patient samp les. While p21/waf1 has been shown to display a selectivity for G(1)/S cycl in/cdk complexes, we have observed p21/waf1 to be complexed with cyclin A/c dk2, Functionally, the association of p21/cyclin A/cdk2 decreased the histo ne H1 phosphorylation in vitro, as observed in immunoprecipitations followe d by kinase assays, as well as affecting other substrates such as the C-ter minus of Rb protein involved in c-Abl and HDAC1 regulation. Wild-type, but not a mutant form (M47) of Tax, was found to be able to transactivate the p 21/waf1 promoter in a p53-independent manner. We found that the minimal pa1 /waf1 promoter (-49 to +49 sequence) was activated by Tax and the minimal p romoter contained two E2A transcription factor binding sites located betwee n the TATA box and the initiation site. E2A proteins, E12 and E47, as well as a related helix-loop-helix protein, HEB, are all up-regulated in HTLV-1- infected T cells. When using band shift analysis, we found that only the E1 site (overlapping the transcription start site) was a functional DNA bindi ng site. By using a chromatin immunoprecipitation (ChIP) assay, we observed that histone H4, and not histone H3, was acetylated from the endogenous p2 1/waf1 promoter in vivo, implying that CBP/p300, and not the SAGA complex, was critical in complexing with E2A in up-regulation of p21/waf1 in HTLV-1- infected cells.