ABSTRACT
Rebound of HIV viremia after interruption of anti-retroviral therapy is due to the small population of CD4+ T cells that remain latently infected. HIV-1 transcription is the main process controlling post-integration latency. Regulation of HIV-1 transcription takes place at both initiation and elongation levels. Pausing of RNA polymerase II at the 5' end of HIV-1 transcribed region (5'HIV-TR), which is immediately downstream of the transcription start site, plays an important role in the regulation of viral expression. The activation of HIV-1 transcription correlates with the rearrangement of a positioned nucleosome located at this region. These two facts suggest that the 5'HIV-TR contributes to inhibit basal transcription of those HIV-1 proviruses that remain latently inactive. However, little is known about the cell elements mediating the repressive role of the 5'HIV-TR. We performed a genetic analysis of this phenomenon in Saccharomyces cerevisiae after reconstructing a minimal HIV-1 transcriptional system in this yeast. Unexpectedly, we found that the critical role played by the 5'HIV-TR in maintaining low levels of basal transcription in yeast is mediated by FACT, Spt6, and Chd1, proteins so far associated with chromatin assembly and disassembly during ongoing transcription. We confirmed that this group of factors plays a role in HIV-1 postintegration latency in human cells by depleting the corresponding human orthologs with shRNAs, both in HIV latently infected cell populations and in particular single-integration clones, including a latent clone with a provirus integrated in a highly transcribed gene. Our results indicate that chromatin reassembly factors participate in the establishment of the equilibrium between activation and repression of HIV-1 when it integrates into the human genome, and they open the possibility of considering these factors as therapeutic targets of HIV-1 latency.
Subject(s)
Chromatin/metabolism , HIV-1/genetics , Saccharomyces cerevisiae/genetics , Transcription, Genetic , DNA Helicases/genetics , DNA Helicases/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , HIV-1/metabolism , Humans , Jurkat Cells , Models, Biological , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , Saccharomyces cerevisiae/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
We have developed a simple reporter assay useful for detection and analysis of mutations and agents influencing mRNA biogenesis in a gene length-dependent manner. We have shown that two transcription units sharing the same promoter, terminator and open reading frame, but differing in the length of their 3'-untranslated regions, are differentially influenced by mutations affecting factors that play a role in transcription elongation or RNA processing all along the transcription units. In contrast, those mutations impairing the initial steps of transcription, but not affecting later steps of mRNA biogenesis, influence equally the expression of the reporters, independently of the length of their 3'-untranslated regions. The ratio between the product levels of the two transcription units is an optimal parameter with which to estimate the efficiency of gene length-dependent processes in mRNA biogenesis. The presence of a phosphatase-encoding open reading frame in the two transcription units makes it very easy to calculate this ratio in any mutant or physiological condition. Interestingly, using this assay, we have shown that mutations in components of the SAGA complex affect the level of mRNA in a transcript length-dependent fashion, suggesting a role for SAGA in transcription elongation. The use of this assay allows the identification and/or characterization of new mutants and drugs affecting transcription elongation and other related processes.