HIV-1 Chromatin, Transcription, and the Regulatory Protein Tat

Upon integration into the host cell genome, the nucleosomal organization and epigenetic control of the HIV-1 provirus play an active role in its transcriptional regulation. Therefore, characterization of the chromatin changes that occur in the viral promoter region in response to different cellular stimuli or drug treatments represents an important aspect of our understanding of HIV-1 transcription. Moreover, the viral transactivator Tat protein potently activates HIV-1 transcription by recruiting the cellular positive transcription elongation factor p-TEFb to the TAR element located at the 5′ end of all nascent viral transcripts, thereby promoting efficient elongation. This chapter describes two complementary techniques for analyzing chromatin structure. The first technique is called indirect end-labeling and uses DNase I, micrococcal nuclease (MNase) or specific restriction enzymes to provide a view of nucleosome positions and of nucleosome-free regions within genes that are usually associated with transcriptional regulatory elements. The second technique, called chromatin immunoprecipitation (ChIP), provides a detailed analysis of chromatin structure by determining the pattern of histone modification marks in the DNA region of interest and by identifying the transcription factors as well as the components of the transcriptional initiation and elongation machineries that are recruited in vivo to this chromosomal region.