Regulation of HIV-1 gene expression by histone acetylation and factor recruitment at the LTR promoter

Abstract

In HIV-1 infected cells, the LTR promoter, once organized into chromatin, is transcriptionally inactive in the absence of stimulation. To examine the chromosomal events involved in transcriptional activation, we analyzed histone acetylation and factor recruitment at contiguous LTR regions by a quantitative chromatin immunoprecipitation assay. In chronically infected cells treated with a phorbol ester, we found that acetylation of both histones H3 and H4 occurs at discrete nucleosomal regions before the onset of viral mRNA transcription. Concomitantly, we observed the recruitment of known cellular acetyl-transferases to the promoter, including CBP, P/CAF and GCN5, as well as that of the p65 subunit of NF-kappa B. The specific contribution of the viral Tat transactivator was assayed in cells harboring the sole LTR. We again observed nucleosomal acetylation and the recruitment of specific co-factors to the viral LTR upon activation by either recombinant Tat or a phorbol ester. Strikingly, P/CAF was found associated with the promoter only in response to Tat. Taken together, these results contribute to the elucidation of the molecular events underlying HIV-1 transcriptional activation.

Fig. 1. Competitive PCR for quantitative ChIP. (A) Schematic structure of the integrated HIV-1 LTR promoter (left side) and of the control genomic region on human chromosome 19 (right side). On the LTR sequence, the positions of the nucleosomes are indicated, alongside those of some of the relevant transcriptional control elements; the transcription start site is indicated by an arrow pointing rightward. On both regions, the position of the primer pairs used for PCR amplification is indicated by arrows. (B) Schematic representation of the multicompetitor DNA used for DNA quantification by competitive PCR. This DNA fragment contains all the primer recognition sites, as indicated, arranged in order to generate PCR amplification products of different but comparable size to those obtained by the amplification of genomic DNA. The gray area indicates the original DNA fragment used as a core for competitor construction, flanked by the β-globin primers PCO3 and PCO4. (C) Quantification by competitive PCR of crosslinked, non-immunoprecipitated (input) DNA from U1 cells without, or following, stimulation with TPA. After crosslinking, a fixed amount of total sonicated DNA was mixed with decreasing concentrations of the multicompetitor segment, as indicated at the top of each lane, and amplified with the primer pairs for nuc-0, PPR, nuc-1, nuc-2 and B13 (Table I). After amplification, the PCR products were resolved by polyacrylamide gel electrophoresis and stained with ethidium bromide; the specific bands were quantified by densitometric analysis. The number of DNA molecules corresponding to each genomic region was calculated according to the principles of competititve PCR by evaluating the slope of the curve fitting the values corresponding to the competitor:genomic ratios (Diviacco et al., 1992). The calculated number of DNA molecules in the analyzed sample dilution is reported in the table below the gels. The results of a representative experiment are shown. All experiments were independently repeated at least three times; the detected variation was <25%. M, molecular weight markers; C, PCR band corresponding to competitor DNA; G, PCR band corresponding to genomic or HIV DNA.

Fig. 2. Localized histone acetylation at the integrated LTR in U1 cells following TPA stimulation. (A) Schematic representation of the multicompetitor DNA used for quantification of RNA by competitive RT–PCR. (B) Induction of HIV-1 transcription in U1 cells by TPA measured using quantitative RT–PCR. Cells were either mock- or TPA- (10^–7 M final concentration) treated for 1, 3 or 5 h. Decreasing amounts of competitor DNA were mixed with fixed amounts of cDNA, and PCR was performed either with β-actin or HIV-specific primers. (C) Histone H3 acetylation. Formaldehyde-crosslinked chromatin from U1 cells induced with TPA (t = 0, 1, 3 and 5 h) was immunoprecipitated with anti-acetylated histone H3 (AcH3) antibody, and the relative acetylation of histone H3 was quantitated by competitive PCR as described above. The results of fold enrichment for regions nuc-0, PPR, nuc-1 and nuc-2 over control B13, calculated on the basis of the estimated number of DNA molecules, are shown graphically in the histograms below the gels. (D) Histone H4 acetylation, as for (C), with the immunoprecipitation performed with the anti-acetylated histone H4 antibody.

Fig. 3. Nucleosomes at the LTR show distinct acetylation patterns at specific lysines of H3 and H4. Chromatin immunoprecipitation, performed as in Figure 2, was performed using antibodies against acetylated Lys9 (A) and Lys14 (B) of histone H3, and against acetylated Lys5 (C), Lys8 (D), Lys12 (E) and Lys16 (F) of histone H4.

Fig. 4. Factor recruitment to the integrated viral promoter in U1 cells following TPA treatment. The binding of transcription factors USF (A), p50 (B) and p65 (C), and the time-dependent recruitment of histone acetyltransferases CBP (D) GCN5 (E) and P/CAF (F) (1, 3 and 5 h after TPA induction) to the viral regions nuc-0, PPR and nuc-1, and to the control B13 region, were analyzed by quantitative ChIP, as described in the legend of Figure 2. The results of the estimated enrichments for each transcription factor in the regions nuc-0, PPR and nuc-1 over the control B13 region are presented. Calculations were made as in Figure 2.

Fig. 5. Induction of HIV-1 transcription in HL3T1 cells. (A) Schematic structure of the integrated LTR-CAT cassette. Converging arrows indicate the position of the specific primers used for amplification. (B) Multicompetitor used for the PCR quantification of DNA from HL3T1 cells. (C) Transcriptional activity of the LTR-CAT promoter upon stimulation with GST, GST–Tat or TPA as measured by CAT assays. GST or GST–Tat were delivered to cells at a final concentration of 5 µg/ml, while final TPA was 1 × 10^–7 M. Five hours after treatment, cells were extensively washed with PBS and new medium was added, and the CAT assay was performed 20 h later. (D) Kinetic analysis of LTR activation in Tat-treated HL3T1 cells. One, 3 and 5 h after stimulation, total RNA was extracted and reverse-transcribed with random primers. A fixed amount of cDNA from each time point was mixed with increasing amounts of competitor molecules and used for competitive PCR amplification with CAT- or β-actin primers. The quantification was performed as in Figure 2. (E) Kinetic analysis of LTR activation in control cells and in cells treated for 1, 3 and 5 h with TPA.

Fig. 6. Localized histone acetylation at the integrated LTR in HL3T1 cells following TPA treatment; acetylation precedes the onset of transcription. (A and B) Cells were treated for 1, 3 or 5 h with TPA, and the immunoprecipitated chromatin was analyzed by PCR. The competitive PCR analysis of two viral and two genomic regions immunoprecipitated with anti-acetylated H3 and H4 antibodies was performed as described above. (C) Cells were treated for 1.5 h with α-amanitin prior to TPA treatment (for 3.5 h); chromatin was then immunoprecipitated with anti-acetylated H3 and H4 antibodies as described above and analyzed for enrichment in the nuc-0 region. (D) Graphs show the kinetics of histone acetylation at nuc-0 and the levels of transcription at different time points after TPA addition.

Fig. 7. Localized histone acetylation at the integrated LTR in HL3T1 cells following treatment with recombinant Tat. (A and B) Acetylation of histones H3 and H4 upon Tat stimulation, shown as in Figure 6. (C) Graphic comparison of the kinetics of histone acetylation and CAT mRNA synthesis upon Tat activation of the integrated LTR-CAT cassette.

Fig. 8. Factor binding to the LTR promoter in HL3T1 cells induced with either TPA or Tat. Immunoprecipitation of crosslinked chromatin with antibodies against transcription factors USF (A), p50 (B) and p65 (C) after TPA or Tat treatment in HL3T1 cells for 5 h. The time course of the binding of histone acetyltransferases GCN5, CBP and P/CAF to the viral regions nuc-0, PPR and nuc-1 was analyzed by quantitative ChIP after TPA (D) or Tat (E) treatment. Calculations were made as in Figure 2.