A transcriptionally permissive epigenetic landscape at the vasoactive intestinal peptide receptor-1 promoter suggests a euchromatin nuclear position in murine CD4 T cells

Abstract

T cells express receptors for neuropeptides that mediate immunological activities. Vasoactive intestinal peptide receptor-1 (VPAC1), the prototypical group II G protein coupled receptor, binds two neuropeptides with high-affinity, called vasoactive intestinal peptide and pituitary adenylate cyclase activating polypeptide. During T cell signaling, VPAC1 mRNA expression levels are significantly downregulated through a Src kinase dependent mechanism, thus altering the sensitivity for these neuropeptides during an immune reaction. Presently, it is unknown whether the mechanism that regulates VPAC1 during T cell signaling involves epigenetic changes. Therefore, we hypothesized that the epigenetic landscape consisting of diacetylation at H3K9/14 and trimethylation at H3K4, two transcriptionally permissive histone modifications, would parallel VPAC1 expression showing high enrichment in untreated T cells, but lower enrichment in alpha-CD3 treated T cells. To this end, quantitative chromatin immunoprecipitation (ChIP) analysis of H3K9/14ac and H3K4me3 was conducted using purified CD4(+) T cells, with CD45R(+) B cells as a negative control. Our data revealed that these histone modifications at the VPAC1 promoter did indeed parallel its mRNA levels between T and B lymphocytes, but did not decrease during T cell signaling. Collectively, these data strongly imply a euchromatin nuclear position for the VPAC1 locus irrespective of the activation status of T cells.

Figure 1. The mouse VPAC1 promoter in CD4 T cells is enriched for H3K9/14ac and H3K4me3

A. Schematic representation of the VPAC1 5’ flanking region (-1000 to +1 bp; horizontal bar) with an arrow indicating the transcriptional start site. This region was segmented into five ≈ 200 bp regions named 1-5 and demarcated by lines underneath the promoter. The dotted line represents the minimal VPAC1 promoter. B. Splenic, naïve CD4⁺ T cells were analyzed by ChIP analysis for enrichment of H3K9/K14ac (mono- and di-acetylated), and H3K4me3 histone signals as indicated. Numbers to the left indicate the primer set used for semi-quantitative PCR amplification. Gels were separated on an ethidium bromide stained 3% agarose gel and visualized by a digital CCD camera. Data is representative of three independent experiments. C. Densitometry from diacetylated H3K9/14 and H3K4me3 enrichment at the VPAC1 promoter obtained in C. Square, IgG; Triangle, diacetylated H3K9/K14; circle, H3K4me3.

Figure 1. The mouse VPAC1 promoter in CD4 T cells is enriched for H3K9/14ac and H3K4me3

A. Schematic representation of the VPAC1 5’ flanking region (-1000 to +1 bp; horizontal bar) with an arrow indicating the transcriptional start site. This region was segmented into five ≈ 200 bp regions named 1-5 and demarcated by lines underneath the promoter. The dotted line represents the minimal VPAC1 promoter. B. Splenic, naïve CD4⁺ T cells were analyzed by ChIP analysis for enrichment of H3K9/K14ac (mono- and di-acetylated), and H3K4me3 histone signals as indicated. Numbers to the left indicate the primer set used for semi-quantitative PCR amplification. Gels were separated on an ethidium bromide stained 3% agarose gel and visualized by a digital CCD camera. Data is representative of three independent experiments. C. Densitometry from diacetylated H3K9/14 and H3K4me3 enrichment at the VPAC1 promoter obtained in C. Square, IgG; Triangle, diacetylated H3K9/K14; circle, H3K4me3.

Figure 2. Differential enrichment of H3K9/14ac and H3K4me3 histone modifications at the VPAC1 promoter

A. Flow cytometric analysis of mouse CD4⁺ T cells and CD45R⁺ B cells. Left Panel: Non-adherent splenocytes were stained with anti-CD4-FITC pre- (red line) and post- (blue line) CD4 positive magnetic bead separation (Materials and Methods) with typical purities ≥93% (n=3). Right Panel: CD4 depleted non-adherent splenocytes were subsequently stained pre- (red line) and post- (blue line) CD45R-FITC positive magnetic bead separation (Materials and Methods) with typical purities ≥97% (n=3). B. Bar graph for Taqman qPCR measurements for VPAC1 from indicated cell type. A hash mark indicates a shift in the y-axis. Data is represented as means +/- SEM for relative VPAC1 levels normalized to HPRT and calculated by 2^(-ΔCt) formula from three independent experiments. The asterisk (*) represents a p≤ 0.05 as compared to CD4⁺ T cells. C-D. ChIP analysis was performed using purified CD4⁺ T and CD45R⁺ B cells followed by quantitative SYBR green PCR using VPAC1 primer set 2 or genomic negative control. C. Amplification reactions from SYBR green qPCR using primer set 2 were separated on a 3% agarose gel, stained with ethidium bromide and visualized by a CCD camera (Syngene). This experiment was repeated twice with similar results. D. Bar graph showing fold-enrichment over IgG control of H3K9/14ac and H3K4me3 from the indicated cell types. Cycle thresholds (Ct) were subtracted from input Ct values to obtain relative ΔCt values. Fold increases were calculated by 2^-(ΔCt) formula with non specific IgG levels normalized to input arbitrarily set to 1 (not shown). Data is presented as means +/- SEM from three biologically independent experiments (*, p≤0.05).

Figure 2. Differential enrichment of H3K9/14ac and H3K4me3 histone modifications at the VPAC1 promoter

A. Flow cytometric analysis of mouse CD4⁺ T cells and CD45R⁺ B cells. Left Panel: Non-adherent splenocytes were stained with anti-CD4-FITC pre- (red line) and post- (blue line) CD4 positive magnetic bead separation (Materials and Methods) with typical purities ≥93% (n=3). Right Panel: CD4 depleted non-adherent splenocytes were subsequently stained pre- (red line) and post- (blue line) CD45R-FITC positive magnetic bead separation (Materials and Methods) with typical purities ≥97% (n=3). B. Bar graph for Taqman qPCR measurements for VPAC1 from indicated cell type. A hash mark indicates a shift in the y-axis. Data is represented as means +/- SEM for relative VPAC1 levels normalized to HPRT and calculated by 2^(-ΔCt) formula from three independent experiments. The asterisk (*) represents a p≤ 0.05 as compared to CD4⁺ T cells. C-D. ChIP analysis was performed using purified CD4⁺ T and CD45R⁺ B cells followed by quantitative SYBR green PCR using VPAC1 primer set 2 or genomic negative control. C. Amplification reactions from SYBR green qPCR using primer set 2 were separated on a 3% agarose gel, stained with ethidium bromide and visualized by a CCD camera (Syngene). This experiment was repeated twice with similar results. D. Bar graph showing fold-enrichment over IgG control of H3K9/14ac and H3K4me3 from the indicated cell types. Cycle thresholds (Ct) were subtracted from input Ct values to obtain relative ΔCt values. Fold increases were calculated by 2^-(ΔCt) formula with non specific IgG levels normalized to input arbitrarily set to 1 (not shown). Data is presented as means +/- SEM from three biologically independent experiments (*, p≤0.05).

Figure 3. Transcriptionally permissive modifications, H3K9/K14ac and H3K4me3, are not removed during T cell signaling

ChIP analysis was performed using purified CD4 T cells incubated +/- plate bound α-CD3 (Materials and Methods) followed by quantitative SYBR green PCR using VPAC1 primer set 2. All data is from three independent biological experiments. A. Flow cytometric analysis of early activation markers, CD69 (left two panels, increase expected) and CD127 (right far panel, decrease expected) using CD4⁺ T cells treated +/- anti-CD3. B. Bar graph representing relative VPAC1 expression levels in CD4⁺ T cells treated +/- anti-CD3 as indicated. Data is presented as means +/- SEM and an * represents a p≤0.05 as compared to cells without anti-CD3. C. Semi-quantitative amplification reactions using immunoprecipitated chromatin and primer set 2 were separated on a 3% agarose gel, stained with ethidium bromide, and visualized by a CCD camera (Syngene). D. Bar graph of fold enrichment over IgG control of H3K9/14ac and H3K4me3 from the indicated cell types. Cycle thresholds (Cts) were subtracted from input Ct values to normalize to input and fold increases were calculated by 2^-(ΔCt) formula. Non-specific IgG levels were also normalized to input and arbitrarily set to 1 (not shown). Data is presented as means +/-SEM.

Figure 3. Transcriptionally permissive modifications, H3K9/K14ac and H3K4me3, are not removed during T cell signaling

ChIP analysis was performed using purified CD4 T cells incubated +/- plate bound α-CD3 (Materials and Methods) followed by quantitative SYBR green PCR using VPAC1 primer set 2. All data is from three independent biological experiments. A. Flow cytometric analysis of early activation markers, CD69 (left two panels, increase expected) and CD127 (right far panel, decrease expected) using CD4⁺ T cells treated +/- anti-CD3. B. Bar graph representing relative VPAC1 expression levels in CD4⁺ T cells treated +/- anti-CD3 as indicated. Data is presented as means +/- SEM and an * represents a p≤0.05 as compared to cells without anti-CD3. C. Semi-quantitative amplification reactions using immunoprecipitated chromatin and primer set 2 were separated on a 3% agarose gel, stained with ethidium bromide, and visualized by a CCD camera (Syngene). D. Bar graph of fold enrichment over IgG control of H3K9/14ac and H3K4me3 from the indicated cell types. Cycle thresholds (Cts) were subtracted from input Ct values to normalize to input and fold increases were calculated by 2^-(ΔCt) formula. Non-specific IgG levels were also normalized to input and arbitrarily set to 1 (not shown). Data is presented as means +/-SEM.

Figure 4. VPAC1 is highly expressed in naïve T cells but not naïve B cells

A. Mouse non-adherent splenocytes were stained with anti-CD4-PE/Cy5, anti-CD25-PE and anti-CD44-FITC. CD4⁺/CD25^-/CD44^- T cells were sorted by flow cytometry. B. CD4⁺/CD25^-/CD44^- T cells were used for total RNA isolation, cDNA synthesis and used for qPCR (Materials and Methods). Bar graph representing VPAC1 mRNA expression levels measured by qPCR from the indicated T cell populations. Data is graphed as means +/- SEM from three independent biological experiments. C. Mouse non-adherent splenocytes were stained with anti-CD45R-FITC and anti-CD43-PE. CD45R⁺/CD43^- B cells were sorted by flow cytometry. D. CD45R⁺/CD43^- B cells were used for total RNA isolation, cDNA synthesis and used for qPCR (Materials and Methods). Scatter plot representing VPAC1 mRNA expression levels measured by qPCR from the indicated T cell populations is shown. Means from duplicate replicates (circles) from two or three independent biological experiments (means are represented as horizontal lines) is presented. Samples that showed no VPAC1 detection is labeled N.D. standing for not detected.

Figure 4. VPAC1 is highly expressed in naïve T cells but not naïve B cells

A. Mouse non-adherent splenocytes were stained with anti-CD4-PE/Cy5, anti-CD25-PE and anti-CD44-FITC. CD4⁺/CD25^-/CD44^- T cells were sorted by flow cytometry. B. CD4⁺/CD25^-/CD44^- T cells were used for total RNA isolation, cDNA synthesis and used for qPCR (Materials and Methods). Bar graph representing VPAC1 mRNA expression levels measured by qPCR from the indicated T cell populations. Data is graphed as means +/- SEM from three independent biological experiments. C. Mouse non-adherent splenocytes were stained with anti-CD45R-FITC and anti-CD43-PE. CD45R⁺/CD43^- B cells were sorted by flow cytometry. D. CD45R⁺/CD43^- B cells were used for total RNA isolation, cDNA synthesis and used for qPCR (Materials and Methods). Scatter plot representing VPAC1 mRNA expression levels measured by qPCR from the indicated T cell populations is shown. Means from duplicate replicates (circles) from two or three independent biological experiments (means are represented as horizontal lines) is presented. Samples that showed no VPAC1 detection is labeled N.D. standing for not detected.

Figure 5. Working model of VPAC1 regulatory mechanism

A. Schematic representation of the epigenetic state at the VPAC1 promoter between T and B cells generated from a common haematopoietic stem cell (HSC). The font size of ac or me3 indicates the extent of enrichment of these modifications as determined from this study. The black arrow size at the transcriptional start site (TSS) indicates the transcriptional rate. B. TCR signaling in CD4 T cells decreases VPAC1 steady-state mRNA expression levels through a Src→JNK kinase dependant mechanism [8, 11]. Possible mechanisms by which TCR signaling downregulates VPAC1 steady-state mRNA levels are shown. Change in chromatin state is crossed out as H3K9/14ac and H3K4me3 levels remain unchanged, suggesting that the VPAC1 promoter remains in euchromatin, and cannot explain such regulation. Question marks indicate those mechanisms that cannot be ruled out and are major future goals of our laboratory.