TRIM24 controls induction of latent HIV-1 by stimulating transcriptional elongation.

Binding of USF1/2 and TFII-I (RBF-2) at conserved sites flanking the HIV-1 LTR enhancer is essential for reactivation from latency in T cells, with TFII-I knockdown rendering the provirus insensitive to T cell signaling. We identified an interaction of TFII-I with the tripartite motif protein TRIM24, and these factors were found to be constitutively associated with the HIV-1 LTR. Similar to the effect of TFII-I depletion, loss of TRIM24 impaired reactivation of HIV-1 in response to T cell signaling. TRIM24 deficiency did not affect recruitment of RNA Pol II to the LTR promoter, but inhibited transcriptional elongation, an effect that was associated with decreased RNA Pol II CTD S2 phosphorylation and impaired recruitment of CDK9. A considerable number of genomic loci are co-occupied by TRIM24/TFII-I, and we found that TRIM24 deletion caused altered T cell immune response, an effect that is facilitated by TFII-I. These results demonstrate a role of TRIM24 for regulation of transcriptional elongation from the HIV-1 promoter, through its interaction with TFII-I, and by recruitment of P-TEFb. Furthermore, these factors co-regulate a significant proportion of genes involved in T cell immune response, consistent with tight coupling of HIV-1 transcriptional activation and T cell signaling.

Identification and functional analysis of a second RBF-2 binding site within the HIV-1 promoter.

Transcription from the HIV-1 long terminal repeat (LTR) is mediated by numerous host transcription factors. In this study we characterized an E-box motif (RBE1) within the core promoter that was previously implicated in both transcriptional activation and repression. We show that RBE1 is a binding site for the RBF-2 transcription factor complex (USF1, USF2, and TFII-I), previously shown to bind an upstream viral element, RBE3. The RBE1 and RBE3 elements formed complexes of identical mobility and protein constituents in gel shift assays, both with Jurkat T-cell nuclear extracts and recombinant USF/TFII-I. Furthermore, both elements are regulators of HIV-1 expression; mutations in LTR-luciferase reporters and in HIV-1 molecular clones resulted in decreased transcription, virion production, and proviral expression in infected cells. Collectively, our data indicate that RBE1 is a bona fide RBF-2 binding site and that the RBE1 and RBE3 elements are necessary for mediating proper transcription from the HIV-1 LTR.

Specific interaction of TFII-I with an upstream element on the HIV-1 LTR regulates induction of latent provirus.

RBF-2 is a factor comprised of a USF1/2 heterodimer, whose association with a highly conserved upstream element (RBEIII) on the HIV-1 LTR requires a co-factor TFII-I. We have identified specific nucleotides, immediately 3' of RBEIII that are required for stable association of TFII-I with this region of the LTR. Mutations that inhibit interaction of TFII-I with DNA also prevent stimulation of USF binding to RBEIII, and render the integrated LTR unresponsive to T cell signaling. These results demonstrate an essential role of TFII-I bound at an upstream LTR element for viral replication.

Factors controlling chromatin organization and nucleosome positioning for establishment and maintenance of HIV latency.

Transcription of the integrated HIV provirus is subject to regulation by chromatin organization and must employ host cell transcription factors and chromatin modifying complexes to promote the formation of latency, and then reverse this process to replicate in response to T cell activation. The repressed latent HIV-1 proviral 5' LTR is organized into a defined structure where two de-acetylated and positioned nucleosomes flank the enhancer region, presumably imposing a block to transcriptional initiation and elongation. LTR-associated nucleosomes undergo further histone H3 K9 trimethylation, to cause silencing by recruitment of HP1. In this article, we review current understanding of how the transcriptionally silenced provirus might be established through the function of transcription factors that bind conserved cis-elements, including SP1, YY1, NF-kappaB, CBF-1 and RBF-2 (USF/TFII-I), and propose mechanisms by which factors bound to the repressed LTR can enable reactivation in response to cell signaling.

Induction of chromosomally integrated HIV-1 LTR requires RBF-2 (USF/TFII-I) and Ras/MAPK signaling.

The HIV-1 LTR is regulated by multiple signaling pathways responsive to T cell activation. In this study, we have examined the contribution of the MAPK, calcineurin-NFAT and TNFalpha-NF-kappaB pathways on induction of chromosomally integrated HIV-1 LTR reporter genes. We find that induction by T-cell receptor (CD3) cross-linking and PMA is completely dependent upon a binding site for RBF-2 (USF1/2-TFII-I), known as RBEIII at -120. The MAPK pathway is essential for induction of the wild type LTR by these treatments, as the MEK inhibitors PD98059 and U0126 block induction by both PMA treatment and CD3 cross-linking. Stimulation of cells with ionomycin on its own has no effect on the integrated LTR, indicating that calcineurin-NFAT is incapable of causing induction in the absence of additional signals, but stimulation with both PMA and ionomycin produces a synergistic response. In contrast, stimulation of NF-kappaB by treatment with TNFalpha causes induction of both the wild type and RBEIII mutant LTRs, an effect that is independent of MAPK signaling. USF1, USF2 and TFII-I from unstimulated cells are capable of binding RBEIII in vitro, and furthermore can be observed on the LTR in vivo by chromatin imunoprecipitation from untreated cells. DNA binding activity of USF1/2 is marginally stimulated by PMA/ ionomycin treatment, and all three factors appear to remain associated with the LTR throughout the course of induction. These results implicate major roles for the MAPK pathway and RBF-2 (USF1/2-TFII-I) in coordinating events necessary for transition of latent integrated HIV-1 to active transcription in response to T cell signaling.

Identification of protein interaction antagonists using the repressed transactivator two-hybrid system.

The repressed transactivator (RTA) yeast two-hybrid system was developed to enable genetic identification of interactions with transcriptional activator proteins. We have devised modifications of this system that enable its use in screening for inhibitors of protein interactions from small molecule compound libraries. We show that inhibition of protein interactions can be measured by monitoring growth in selective medium containing 3-aminotriazole (3-AT) and using this assay have identified inhibitors of four independent protein interactions in screens with a 23,000 small molecule compound library. Compounds found to inhibit one of the tested interactions between FKBP12 and the transforming growth factor beta receptor (TGFbeta-R) were validated in vivo and found to inhibit calcineurin-dependent signaling in T cells. One of these compounds was also found to cause elevated basal expression of a TGFbeta-R/SMAD-dependent reporter gene. These results demonstrate the capability of the RTA small molecule screening assay for discovery of potentially novel therapeutic compounds.

TFII-I regulates induction of chromosomally integrated human immunodeficiency virus type 1 long terminal repeat in cooperation with USF.

Human immunodeficiency virus type 1 (HIV-1) replication is coupled to T-cell activation through its dependence on host cell transcription factors. Despite the enormous sequence variability of these factors, several cis elements for host factors are highly conserved within the 5' long terminal repeats (LTRs) of viruses from AIDS patients; among these is the RBEIII upstream element for the Ras response element binding factor 2 (RBF-2). Here we show that RBF-2 is comprised of a USF1/USF2 heterodimer and TFII-I, which bind cooperatively to RBEIII. Recombinant USF1/USF2 binds to the RBEIII core sequence 160-fold less efficiently than it binds to an E box element, but the interaction with RBEIII is stimulated by TFII-I. Chromosomally integrated HIV-1 LTRs bearing an RBEIII mutation have slightly elevated basal transcription in unstimulated Jurkat cells but are unresponsive to cross-linking of the T-cell receptor or stimulation with phorbol myristate acetate (PMA) and ionomycin. Induction is inhibited by dominant interfering USF and TFII-I but not by the dominant negative I-kappaB protein. USF1, USF2, and TFII-I bind to the integrated wild-type LTR in unstimulated cells and become phosphorylated during the induction of transcription upon stimulation with PMA. These results demonstrate that USF1/USF2 and TFII-I interact cooperatively at the upstream RBEIII element and are necessary for the induction of latent HIV-1 in response to T-cell activation signals.

Mitogen-responsive expression of RhoB is regulated by RNA stability.

The small GTPase-encoding gene RhoB is strongly induced as part of the immediate early response of serum-stimulated fibroblasts. In this report, we have characterized the mechanism for growth factor responsiveness of RhoB in Rat-2 fibroblasts. By Northern blotting and ribonuclease protection, we observed low or barely detectable levels of RhoB mRNA in quiescent cells, but expression was transiently induced in response to serum stimulation, such that the mRNA peaked within 30 min and then declined over the next hour. Analysis of the rat promoter revealed cis-elements conserved with the mouse and human genes, including a pair of CEBP sites near the transcriptional start site. However, in contrast to the analysis of RNA, RhoB promoter fusions were constitutively expressed in quiescent cells in transient transfections, and were unaffected by serum. Similarly, stable RhoB promoter integrants were highly expressed in quiescent cells, and growth factor caused a slight decrease in activity. This indicates that growth factor-inducible RhoB expression cannot be mediated by transcriptional activation. We then examined decay of the RhoB mRNA and found that serum caused significant stabilization. Additionally, fusion of the 3' RhoB untranslated region (UTR) to a constitutively expressed reporter gene caused serum and growth factor as well as DNA damage-inducible expression. These observations are consistent with the view that RhoB mRNA is produced constitutively but its abundance is controlled in response to growth factors, and other signals including DNA damage, by stabilization through elements within the 3' UTR.

Organization and expression of the Cyr61 gene in normal human fibroblasts.

We have examined the human Cyr61 gene and its expression in normal fibroblasts. The core promoter, second intron, and 3' untranslated region (UTR) are highly conserved between the human and mouse genes. Cyr61 expression was induced slightly slower but more transiently in human fibroblasts compared to Rat-2 fibroblasts. These differences may relate to the absence of a serum response element in the human Cyr61 promoter, and the presence of additional AU-rich elements within the 3' UTR. Cycloheximide causes accumulation of human Cyr61 RNA in the absence of growth factors, and EGF prevents decay of transcripts in actinomycin-D-treated cells, which suggests that induction by growth factors may partially involve mRNA stabilization. We detect an alternative RNA in serum-stimulated fibroblasts containing an in-frame deletion within exon 4 which disrupts the thrombospondin type 1 repeat. Constitutive expression of the full hCyr61 genomic DNA in rodent fibroblasts causes production of multiple protein species, whereas expression of hCyrDelta4 produces a single stable protein of the expected size. We also observed multiple hCyr61 protein species in normal fibroblasts following serum stimulation, indicating that Cyr61 may normally be produced as alternative isoforms.