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. 2005 Feb;3(2):e44.
doi: 10.1371/journal.pbio.0030044. Epub 2005 Feb 8.

HIV-1 Tat stimulates transcription complex assembly through recruitment of TBP in the absence of TAFs

Tamal Raha  1 , S W Grace Cheng, Michael R Green
Affiliations

HIV-1 Tat stimulates transcription complex assembly through recruitment of TBP in the absence of TAFs

Tamal Raha et al. PLoS Biol. 2005 Feb.

Abstract

The human immunodeficiency virus type I (HIV-1) transactivator protein Tat is an unusual transcriptional activator that is thought to act solely by promoting RNA polymerase II processivity. Here we study the mechanism of Tat action by analyzing transcription complex (TC) assembly in vivo using chromatin immunoprecipitation assays. We find, unexpectedly, that like typical activators Tat dramatically stimulates TC assembly. Surprisingly, however, the TC formed on the HIV-1 long terminal repeat is atypical and contains TATA-box-binding protein (TBP) but not TBP-associated factors (TAFs). Tat function involves direct interaction with the cellular cofactor positive transcription elongation factor b (P-TEFb). Artificial tethering of P-TEFb subunits to HIV-1 promoter DNA or nascent RNA indicates that P-TEFb is responsible for directing assembly of a TC containing TBP but not TAFs. On the basis of this finding, we identify P-TEFb-dependent cellular promoters that also recruit TBP in the absence of TAFs. Thus, in mammalian cells transcription of protein-coding genes involves alternative TCs that differ by the presence or absence of TAFs.

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Figures

Figure 1
Figure 1. Diverse Activators Function by Increasing TC Assembly
(A) Gal4-VP16. Left: a CAT reporter construct containing the E1b core promoter and four Gal4-binding sites (G4E1bCAT) was transiently trans-fected into 293T cells, together with a plasmid expressing Gal4-VP16. Middle: transcription levels were determined by quantitating CAT reporter activity. Right: TC assembly was analyzed by a ChIP assay using the indicated antibodies. The percentage of DNA immunoprecipitated relative to input is indicated. The location of the primers used in the ChIP assay to analyze the promoter (black) or ORF (gray) is schematically shown on the left. (B) SV40 enhancer. Shown on the left is a schematic diagram of a construct containing a minimal rabbit β-globin promoter and harboring or lacking the SV40 enhancer. Also shown is transcription analysis by CAT assay (middle) and TC assembly (right) in transiently transfected HeLa cells. (C) Ad E1a. Left: a CAT reporter construct containing the Ad E4 promoter and upstream ATF-binding sites (E4CAT) was transiently transfected into HeLa cells, together with a plasmid expressing Ad E1a. Middle: transcription analysis by CAT assay is shown. Right: TC assembly is shown.
Figure 2
Figure 2. The HIV-1 Tat Protein Stimulates TC Assembly through Recruitment of TBP in the Absence of TAFs
(A) A CAT reporter construct containing the TAR element and Sp1-binding sites ([−83]HIV LTRCAT) was transiently transfected into 293T cells, together with a plasmid expressing Tat. Left: transcription analysis by CAT assay is shown. Middle and right: TC assembly is shown. Bottom: a schematic diagram of the CAT reporter construct is shown. (B) TC assembly was monitored in a chronically HIV-1-infected cell line, 8E5/LAV, that harbors an integrated provirus that is constitutively transcribed. Virus production was confirmed by analysis of p24 levels and reverse transcriptase activity in the culture medium (data not shown). (C) TC assembly was monitored in the chronically infected cell line U1 on the integrated HIV-1 LTR in the presence or absence of PMA.
Figure 3
Figure 3. TAFs Are Not Recruited to the HIV 1-LTR and Not Required by Tat for Transcription Activation
(A) TC assembly was analyzed by a ChIP assay using antibodies directed against nine additional human TAFs, four subunits of the S TAGA complex, and Mot1. TAFs that are also present in S TAGA are indicated by asterisks. Bottom: schematic diagrams of the promoter constructs are shown. (B) Transcription analysis in ts13 cells, which harbor a temperature-sensitive mutation in TAF1. Ts13 cells grown at the permissive or non-permissive temperature were transiently transfected with a luciferase reporter plasmid and a plasmid expressing a transcriptional activator. Transcription was monitored by luciferase activity, and normalized relative to activity at the permissive temperature. (C) Left: immunoblot analysis is shown. 293A cells were transfected with a TAF5 or TAF12 shRNA expression vector or an empty vector (control) and analyzed by immunoblotting using the indicated antibodies. Right: transcription levels were monitored by luciferase reporter activity in shRNA-treated cells, and normalized relative to luciferase activity in non-shRNA-treated cells.
Figure 4
Figure 4. Tat and P-TEFb Direct Recruitment of TBP in the Absence of TAFs When Tethered to DNA
Transcription (left) and TC assembly (right) were examined using an HIV-1 LTR derivative that lacks the TAR element and contains upstream Gal4-binding sites (G6[−83]HIV LTRΔTARCAT; bottom) and a series of Gal4 fusion proteins, as indicated.
Figure 5
Figure 5. Tat and P-TEFb Direct Recruitment of TBP in the Absence of TAFs When Tethered to RNA
Transcription (left) and TC assembly (right) were examined using a Rev-Tat or Rev-CycT1 fusion protein and an HIV-1 LTR derivative in which the TAR element was replaced by stem loop IIB (SLIIB), a Rev-protein-binding site (HIV SLIIBCAT; bottom).
Figure 6
Figure 6. The Cellular Activator CIITA Directs Recruitment of TBP in the Absence of TAFs
TC assembly was monitored on the promoters of HLA-DM and HLA-DR, two MHC class II genes known to use CIITA, and, as a negative control, GAPDH.
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References

    1. Orphanides G, Lagrange T, Reinberg D. The general transcription factors of RNA polymerase II. Genes Dev. 1996;10:2657–2683. - PubMed
    1. Burley SK, Roeder RG. Biochemistry and structural biology of transcription factor IID (TFIID) Annu Rev Biochem. 1996;65:769–799. - PubMed
    1. Albright SR, Tjian R. TAFs revisited: More data reveal new twists and confirm old ideas. Gene. 2000;242:1–13. - PubMed
    1. Green MR. TBP-associated factors (TAFIIs): Multiple, selective transcriptional mediators in common complexes. Trends Biochem Sci. 2000;25:59–63. - PubMed
    1. Ptashne M. How eukaryotic transcriptional activators work. Nature. 1988;335:683–689. - PubMed

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