cMyc and YY1 bind to the HIV-1 LTR and regulate transcription through recruitment of HDACs.8,11
We sought to determine whether selective antilatency therapy could be specifically targeted to transcription factors that recruit HDACs to the HIV-1 LTR. Depletion of cMyc, YY1, or cMyc and YY1 did not significantly affect transcription or protein levels of the HDAC proteins, which indicates that the effects on HIV-1 transcription are not a secondary effect of changes in HDAC expression. Depletion of YY1 resulted in a significant increase in mRNA expression and the percentage of cells expressing GFP protein from the HIV-1 LTR in the 2D10 cells but not in J89 cells (). However, in contrast to studies in HeLa cells, single knockdown of cMyc did not have a significant effect on transcription of mRNA from the HIV-1 LTR or on the induction of GFP protein expression from the HIV-1 LTR (). Furthermore, depletion of cMyc and YY1 together did not induce HIV-1 transcription in either cell line. As cMyc has been show to be required for Tat-mediated elongation of the HIV-1 promoter,14
it is possible that cMyc's role in elongation may account for the lack of HIV-1 transcription that was observed after depletion of YY1and cMyc and may explain why the significant increase in histone 3 acetylation did not correlate with an increase in expression. Depletion of cMyc, YY1, or cMyc and YY1, followed by activation with PMA, induced a significant amount of expression from the HIV-1 LTR in 2D10 and J89 cells (Supplementary Fig. S1
). Therefore, we can conclude that cMyc is not absolutely required for activation of the HIV-1 promoter by PMA. However, because depletion of cMyc and YY1 resulted in less activation of the HIV-1 promoter than depletion of YY1 alone, the mechanism of repression that is mediated by YY1 may act through a pathway that requires cMyc. These results are particularly surprising in light of previous studies that found disruption of cMyc or YY1 was sufficient to disrupt quiescent HIV-1 proviruses. Because YY1 depletion did not activate HIV-1 transcription in both cell lines, targeting YY1 as part of a future antilatency therapy may not broadly disrupt latency in all proviral integrants.
Knockdown of cMyc, YY1, or cMyc and YY1 did not significantly affect histone 3 occupancy of the HIV-1 LTR. Although cMyc and YY1 are known to recruit HDACs to the HIV-1 LTR in HeLa cells, the results from this study indicate that they are not absolutely required to recruit HDAC1, HDAC2, or HDAC3 to the HIV-1 LTR in Jurkat cells. Contrary to previous findings, these results indicate that in T cells other transcription factors that bind to the HIV-1 LTR may be able to compensate for the loss of cMyc, YY1, or both and maintain HDAC occupancy. Specifically, NF-κB and CBF1 have been implicated in recruitment of HDAC1 to the HIV-1 LTR and may be able to compensate for the loss of cMyc and YY1.7,9
Furthermore, these or other proteins may contribute to the repression of HIV-1 transcription in T cells. Additionally, these finding highlight an important difference between HeLa cell line models of HIV-1 latency and T cell line models of HIV-1 latency and indicate that the epigenetic environment surrounding the HIV-1 LTR in HeLa cells may be less stable than in T cells. Although Jurkat cells more closely resemble primary CD4+
T cells, it is still important to develop more advanced tools and model systems for the future study of the epigenetics of quiescent HIV-1 proviruses.
Interestingly, targeting HDAC recruitment through depletion of YY1 in conjunction with HDAC inhibition resulted in a significant increase in GFP protein expression from the HIV-1 promoter. This finding indicates that targeting multiple restrictive mechanisms at the HIV-1 LTR may be an innovative method for disrupting HIV-1 latency. YY1 is involved in several malignancies and some new classes of chemotherapeutics have been demonstrated to decrease expression of YY1.26
Such approaches might be used for the treatment of latent HIV-1 infection, and may be able to augment the effects of SAHA (N. Archin, unpublished results). However, additional development of drugs that directly target the protein interaction domains of YY1 could be pursued.
Previous studies have found that the chromatin environment surrounding the HIV-1 insertion site may affect transcription. Furthermore, in vivo infected CD4+ T cells have a range of insertion sites. To account for the effect of insertion site differences, we performed our studies in two Jurkat cells lines with distinct proviral insertion sites. The differences seen between the two cells lines may be attributable to the difference in proviral locations. However, apart from the effects of YY1 depletion, most of the results were seen in both cell lines, indicating that the proviral location may have had only modest effects. The differences in results found in this study as compared to previous studies in HeLa cells highlight the importance of conducting studies in multiple lineages of cell lines. Although Jurkat cells are derived from CD4+ T cells, there are still some important differences between these cells and in vivo HIV-1-infected cells. Therefore, our laboratory and others are working to develop models of HIV-1 quiescence that more accurately mimic the in vivo environment of HIV-1 quiescence.
In conclusion, we find that depletion of the transcription factor YY1 using transduction of shRNAs in Jurkat cells is sufficient to disrupt the repression of the HIV-1 promoter in select cellular contexts. However, depletion of the transcription factor cMyc does not induce HIV-1 expression in Jurkat cells. Importantly, we found that depletion of cMyc, YY1, or cMyc and YY1 is not sufficient to disrupt the binding of HDAC complexes to the HIV-1 LTR in Jurkat cells. Therefore, we can conclude that the mechanism of maintenance of HIV-1 transcriptional repression is complex and may require a combination of therapies that target multiple levels or several factors to reverse transcriptional repression of the HIV-1 LTR.