amanitins and dimethyl-sulfate

Chromatin organization of eukaryotic promoters is increasingly recognized as an important factor in the regulation of transcription in vivo. To determine the role of chromatin in HIV-1 expression, we have examined the nucleosome organization of the promoter of HIV-1 under low and high transcription rates. Independently of the cell line examined, nucleosomes are precisely positioned in the viral 5' long terminal repeat (5' LTR) and define two large nucleosome-free regions encompassing nt 200-450 and 610-720. A nucleosome positioned between these two regions, immediately after the transcription initiation site (nuc-1), is disrupted following TPA or TNF-alpha treatment. The disruption of nuc-1 from DNA is independent of DNA replication since it is completed in 20 min and independent of transcription as it is alpha-amanitin insensitive. A model is proposed in which nuc-1 plays an organizing role in the HIV-1 promoter to bring in close proximity factors bound to DNA in the two nucleosome-free regions, upstream and downstream of the site of transcription initiation. These results define chromatin as an integral component of the HIV-1 transcriptional regulatory machinery and identify a chromatin transition associated with activation of viral gene expression.

Topics: Alkylating Agents; Amanitins; Base Sequence; Cell Line; Chromatin; Deoxyribonuclease I; DNA Replication; DNA, Viral; HIV Long Terminal Repeat; HIV-1; Micrococcal Nuclease; Molecular Sequence Data; Nucleosomes; Promoter Regions, Genetic; Restriction Mapping; Sulfuric Acid Esters; Tetradecanoylphorbol Acetate; Transcriptional Activation