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. 2012 Jun;4(6):661-71.
doi: 10.1039/c2ib20009k. Epub 2012 May 3.

Chromatin Accessibility at the HIV LTR Promoter Sets a Threshold for NF-κB Mediated Viral Gene Expression

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Free PMC article

Chromatin Accessibility at the HIV LTR Promoter Sets a Threshold for NF-κB Mediated Viral Gene Expression

Kathryn Miller-Jensen et al. Integr Biol (Camb). .
Free PMC article

Abstract

Higher order chromatin structure in eukaryotes can lead to differential gene expression in response to the same transcription factor; however, how transcription factor inputs integrate with quantitative features of the chromatin environment to regulate gene expression is not clear. In vitro models of HIV gene regulation, in which repressive mechanisms acting locally at an integration site keep proviruses transcriptionally silent until appropriately stimulated, provide a powerful system to study gene expression regulation in different chromatin environments. Here we quantified HIV expression as a function of activating transcription factor nuclear factor-κB RelA/p65 (RelA) levels and chromatin features at a panel of viral integration sites. Variable RelA overexpression demonstrated that the viral genomic location sets a threshold RelA level necessary to induce gene expression. However, once the induction threshold is reached, gene expression increases similarly for all integration sites. Furthermore, we found that higher induction thresholds are associated with repressive histone marks and a decreased sensitivity to nuclease digestion at the LTR promoter. Increasing chromatin accessibility via inhibition of histone deacetylation or DNA methylation lowered the induction threshold, demonstrating that chromatin accessibility sets the level of RelA required to activate gene expression. Finally, a functional relationship between gene expression, RelA level, and chromatin accessibility accurately predicted synergistic HIV activation in response to combinatorial pharmacological perturbations. Different genomic environments thus set a threshold for transcription factor activation of a key viral promoter, which may point toward biological principles that underlie selective gene expression and inform strategies for combinatorial therapies to combat latent HIV.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1
In vitro models of HIV gene expression provide an experimental system to study RelA-mediated gene expression in a range of chromatin environments. (A) There is general interest in how gene expression probability varies as a function of transcription factor availability and quantitative features of the local chromatin environment. (B) Schematic describing RelA-mediated gene expression in the HIV vectors before and after the Tat-mediated positive feedback loop is activated. (C) Representative flow cytometry histograms of GFP expression for the panel of clones each infected with a single integration of an inactive HIV provirus under basal conditions (left) and after stimulation with TNFα (20 ng/ml) for 48 hours (right). Percentage of TNFα-activated cells is indicated in parentheses. Clones are ordered according to increasing basal gene expression. (D) Infected clonal populations were stimulated with 400 nM TSA for 24 hours (light gray bars) or 5 μM 5-aza-dC for 48 hours (dark gray bars). Experiments were performed in biological triplicate. Data are presented as the mean ± standard deviation.
Fig. 2
Fig. 2
Inducing HIV gene expression by overexpression of RelA reveals an induction threshold of gene activation. (A) Schematic of the inducible RelA (iRelA) vector. (B) Immunoblot of total RelA-Cherry fusion protein and endogenous protein levels in clone 6.3 infected with iRelA 4 days after DOX induction. (C) Microscopy picture of clone 6.3 infected with iRelA 4 days after induction with 30 ng/ml DOX. (Left) DAPI and mCherry overlay. (Right) GFP and mCherry overlay. (D) Combined flow cytometry data for HIV-infected clones expressing iRelA in response to a range of DOX concentrations. More than 50,000 single cell events were divided into 256 bins of mCherry fluorescence, and the fraction of GFP+ cells was calculated and plotted for each bin. (Inset) Least squares fit line for clone 15.4 and E3. (E) Induction threshold (defined as the mCherry- RelA level at which 5% of the population expressed GFP) and (F) activation coefficient (defined as the Hill coefficient calculated from fitting Hill functions to the curves in (D)) for each clone. Error bars in (D–F) represent standard deviations and were calculated by bootstrapping.
Fig. 3
Fig. 3
Chromatin accessibility is correlated with RelA induction threshold. (A) Heterochromatin fraction was quantified with a DNAse I sensitivity assay. Quantitative PCR was performed in triplicate and normalized to a hemoglobin-β (HBB) reference gene. (B–C) Correlation of heterochromatin fraction with (B) induction threshold and (C) activation coefficient extracted from the fits in Fig. 2D–F. (D–F) Chromatin immunoprecipitation for (D) total H3, (E) H3K9me3 and (F) acetylated H3 bound to the HIV promoter in unstimulated clones was correlated to the induction threshold. Quantitative PCR was performed in triplicate and normalized to an input control. Data are presented as the mean ± standard deviation. Differences are labeled as significant (*) if p < 0.05. Pearson correlation coefficient R is indicated on plot.
Fig. 4
Fig. 4
Induction of gene expression is associated with a decrease in heterochromatin fraction. (A) Selected clones were treated with 20 ng/ml DOX to hold the clonal populations at the point at which gene expression in the population is just induced (arrow). (Inset) Flow histograms showing a low fraction of cells expressing GFP for each clone at the point of induction. (B) Heterochromatin fraction as quantified by nuclease sensitivity for clones at basal (white) and induction (gray) level of RelA. Quantitative PCR was performed in triplicate and normalized to a HBB reference gene. (C–D) Chromatin immunoprecipitation comparing (C) RNA polymerase II and (D) phospho- Ser5 RNAPII bound to the LTR promoter at basal (white) and induction (gray) level of RelA. Quantitative PCR was performed in triplicate and normalized to a GAPDH control gene. Data are presented as the mean ± standard deviation. Changes are labeled as significant (*) if p < 0.05.
Fig. 5
Fig. 5
Increasing chromatin accessibility via drug treatment lowers the RelA induction threshold. (A) Heterochromatin fraction for clone 15.4 was quantified with a DNAse I sensitivity assay following stimulation with 400 nM TSA for 4 hours or with 5 μM 5-aza- dC for 48 hours. Quantitative PCR was performed in triplicate and normalized to the hemoglobin reference gene. Relative heterochromatin fraction was calculated by normalizing clone 15.4 with drugs, B5 and D3 to the unstimulated 15.4 control. (B) Combined flow cytometry data for 15.4 expressing iRelA in response to a range of DOX concentrations and simultaneous stimulation with 400 nM TSA for 24 hours (dark blue), 5 μM 5-aza-dC for 48 hours (red), and no drug treatment controls at 24 and 48 hours (black and light gray, respectively). iRelA dose response curves for clone B5 (green) and D3 (dark gray) without TSA or 5-aza-dC are included for comparison. (C) Relative change in induction threshold versus relative change in heterochromatin fraction for clones 15.4 (circles), 8.4 (diamonds) and E3 (triangles). Data for 15.4 are calculated from results presented in (A) and (B), and data for 8.4 and E3 are calculated from experiments presented in Fig. S10. All points are calculated by normalizing the value of heterochromatin fraction or threshold for the clone in the presence of drugs to the corresponding value for the unstimulated control clone. Data are presented as the mean ± standard deviation. Changes are labeled as significant (*) if p < 0.05. Pearson correlation coefficient R is indicated on plot.
Fig. 6
Fig. 6
Gene activation function accurately predicts synergistic activation of HIV gene expression by simultaneous treatment with TNFα and HDAC or DMT inhibitors. (A) The empirically-derived gene activation function for clone 15.4+TSA was used to predict its response to combinatorial perturbation with TSA and TNFα. Approximate mCherry- RelA increases associated with TNFα treatment alone were estimated by locating the point on the gene activation curve for basal clone 15.4 that corresponded to the percentage of GFP+ cells that responded to TNFα treatment (~12%) (black line). This estimated TNFα-induced value of mCherry-RelA was used to predict the fraction of GFP+ cells expected for a combination of TNFα and TSA by solving the gene activation function for 15.4 treated with TSA (blue line). (B–C) Predicted (bars) and observed (dots) percentage of GFP+ cells following stimulation with TSA+TNFα or 5-aza-dC+TNFα based on (B) gene activation functions or (C) a Bliss independence model of drug response. Experiments were performed in biological triplicate and are presented as the mean ± standard deviation. Error bars for prediction were calculated as described in Materials and Methods.
Fig. 7
Fig. 7
3-D surface plot demonstrates gene activation as a function of RelA for different genomic locations. The plot was empirically derived by combining the gene activation functions for a subset of clones ranging from high to low repression. Surface plot provides a quantitative depiction of the function hypothesized in Fig. 1A. Yellow and red points and arrows describe behavior in different regimes of promoter repression. See text for discussion.

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