HIV-I- and HTLV-1-infected CD4+
T cells are activated and proliferate during the initial phase of infection that is accompanied by dysregulation of multiple genes (7
). The retroviral infection is limited by the quiescence of most circulating T cells that are therefore nonsupportive of viral replication. Formation of integrated provirus is obligatory for viral replication and subsequent development of AIDS (13
). The barrier for integration observed in host peripheral T cells can be overcome by T-cell activation (47
). Recent studies have shown that expression of the earliest HIV gene products, Nef and Tat, can increase T-cell activity (34
). Nef acts by lowering the activation threshold in T cells (41
), while Tat acts by deregulating the expression of several host genes, including proto-oncogenes and genes encoding metabolic enzymes, cytokines, and cytokine receptors (5
). Recent studies by Wu and Marsh solved the puzzle of how these proteins would function in the absence of viral replication. They reported that nef
genes are selectively transcribed in infected cells before integration (52
). Thus, the early events of HIV infection induce the IL-2 and IL-2R protein levels that are required for rapid proliferation of active T-cells which subsequently become the most preferable target cells for HIV infection. In the later episodes of HIV infection, infected T cells undergo apoptosis, and this may be responsible for the sudden decrease in T-cell numbers and the resulting drop in immunity of the infected patient. This is one of the host-pathogen interactions in which virus initially takes advantage of the host machineries for its efficient infection and rapid proliferation of host T cells. However, the molecular mechanisms of how this is accomplished, and especially the contribution of host factors, are not completely understood.
Tat activates host and viral transcription by recruiting host transcription factors and coactivators (7
). Contradictory observations describe the regulation of IL-2 and its receptor by Tat (19
). However, in studies where the expression of IL-2 and its receptor have been shown to be downregulated by Tat, stable expression of Tat was employed (36
). Lymphocytes from HIV-1 infected patients are known to express increased amounts of IL-2 and IL-2Rα (18
). We therefore argued that the molecular mechanism of this phenomenon could not be elucidated using only Tat-transfected T cells. Here, we have used HIV-1 infected T cells and evaluated the role of SATB1, a T-lineage-specific transcription factor (2
), in the regulation of IL-2 and IL-2Rα expression. Studies utilizing SATB1 null mice have established SATB1 as a new type of gene regulator responsible for a novel cagelike nuclear architecture, providing sites for tissue-specific organization of DNA sequences and regulating region-specific histone modification (6
). SATB1 is a predominant architectural protein in T cells (2
) that functions as a global suppressor by targeting chromatin remodeling to regulate genes over long distances (53
). We therefore reasoned that Tat would have to overcome the transcriptional suppression mediated by SATB1 in a manner that would allow activation of genes required for viral replication and T-cell activation.
The activation dependence and cell type specificity of IL-2 expression are mediated through a dynamic assembly of diverse transcription factors, including NF-AT, NF-κB, AP-1, and Oct-1 at its promoter (38
). Likewise, several studies have demonstrated the presence of multiple protein binding sites within the IL-2Rα upstream regulatory sequences in the region of base pairs −476 to −225 (28
). We have identified, for the first time, SATB1 binding sites in the promoters of IL-2 and its receptor. Furthermore, we narrowed down these binding sites to the IDR in the IL-2Rα promoter and a 37-mer ATC sequence in the IL-2 promoter. SATB1 may also bind to additional regions within these promoters; however, the presence of these two elements is essential since deletions of these elements abolish binding to longer overlapping regions. Studies using chromatin from thymocytes of wild-type and SATB1 null mice demonstrated the presence of a strong intronic SATB1 binding site within the IL-2Rα locus and a weak binding site within the promoter (53
). In the present study, we have narrowed down the binding site for SATB1 in the human IL-2Rα promoter and demonstrate that it consists of the IDRs flanking the κB-like motif. Whether a similar binding site preference is exhibited by SATB1 in the mouse IL-2Rα promoter remains to be investigated. In most of the earlier studies, contribution of specific transcription factors has been evaluated by using their minimal binding sites in the upstream regulatory regions of IL-2 and IL-2Rα (9
). We used regions of more than 500 bp to analyze the role of SATB1, ensuring that the promoters may be simultaneously occupied by other transcription factors whose contribution was considered as the baseline. Our data demonstrate that SATB1 binds to the upstream regulatory elements of IL-2 and IL-2Rα and negatively regulates their expression. We monitored IL-2 levels in infected PBMCs by RT-PCR analyses and observed that IL-2 expression peaks around day 4 postinfection and then gradually decreases with time (our unpublished observation). However, monitoring the expression of IL-2 in PBMCs infected in the presence of IL-2 is more complicated due to the fact that exogenously added IL-2 exerts diverse effects on virus production (3
) and the impaired IL-2 production during HIV disease may be attributed to a feedback mechanism wherein IL-2 induces CD8+
HIV suppressors (26
). However, Tat was previously shown to derepress the IL-2 promoter (12
), and the derepression is very clearly observed in the case of infected CEM cells.
SATB1 contains an MD and an HD towards the carboxy-terminal half, which confer the ability to recognize the core-unwinding element within a BUR element (10
). SATB1 harbors a novel dimerization domain in its N-terminal region, which is homologous to PDZ domains (16
). Recently SATB2, a homolog of SATB1, was identified; it also possesses the PDZ-like domain (our unpublished observations). However, SATB2 is specifically expressed in brain, testis, kidney, pre-B cells, and B cells but not in T cells (11
); therefore, it may not play a role in HIV pathogenesis at least during the early stages of infection. PDZ domains are protein interaction modules that are implicated in signal transduction (14
). Both HDAC1 and Tat bind SATB1 via this protein interaction domain. SATB1 interacts with the transactivation domain of Tat, a domain that is known to associate with many of Tat's interaction partners (reviewed in reference 24
). Thus, the competition within these factors would eventually determine the outcome of such interactions. Through direct physical interaction, Tat competitively displaces SATB1-bound HDAC1 in vivo. Strikingly, the transcriptional upregulation of IL-2 and IL-2Rα does not require the transactivation function of Tat. In the case of Tat-mediated regulation of LTR, the transcriptional activation is associated with remodeling of nucleosome 1 by acetylation. ChIP studies by He and Margolis suggested that histone acetyltransferases or other factors recruited to LTR by Tat may interact with HDAC1, YY1, or both and may regulate their functions or interaction (23
). Furthermore, treatment with trichostatin A or Tat transfection decreased HDAC1 occupancy at the LTR, presumably via displacement. However, further study is required to define the mechanisms by which trichostatin A and Tat decrease HDAC1 occupancy at the LTR.
Histone acetylation is typically associated with transcriptional activation (reviewed in reference 4
). Consequently, we reasoned that the loss of HDAC1 from the IL-2 and IL-2Rα promoters might likely lead to an increase in acetylation at the regulatory regions. We observed that the promoter chromatin was specifically acetylated in vivo in the infected cells, suggesting remodeling of the promoter to favor transcription. In the absence of Tat, SATB1 represses transcription of IL-2 and its receptor by recruiting the HDAC1 corepressor, which could be in the form of a larger repressor complex, and thereby promotes deacetylation of the promoter and represses transcription. When Tat is introduced either via HIV-1 infection or by transfection or transduction, it binds to the PDZ-like domain of SATB1 and competitively displaces HDAC1. Tat may further recruit the CBP/p300 histone acetyltransferase to promote acetylation of the promoter, thereby stimulating transcription (model depicted in Fig. ). Interestingly, transduction experiments using soluble Tat and its derivatives clearly demonstrated an inverse correlation between HDAC1 occupancy and acetylation of promoter chromatin and resulted in induction of IL-2 and its receptor, corroborating our hypothesis that competitive displacement of SATB1-bound HDAC1 by Tat is the cause for upregulation of IL-2 and its receptor. Our results further suggest that the induction of transcription may occur by abolition of the repressor function in addition to the induction of host factors upon viral infection. We propose that this mechanism may be employed by HIV-1 to overcome the SATB1-mediated repression of genes essential for its propagation in T cells.