Chromatin immunoprecipitation assays revealed CREB and serine 133 phospho-CREB binding to the CART gene proximal promoter

Abstract

Both over expression of cyclic AMP response element binding protein (CREB) in the nucleus accumbens (NAc), and intra-accumbal injection of cocaine- and amphetamine-regulated transcript (CART) peptides, have been shown to decrease cocaine reward. Also, over expression of CREB in the rat NAc increased CART mRNA and peptide levels, but it is not known if this was due to a direct action of P-CREB on the CART gene promoter. The goal of this study was to test if CREB and P-CREB bound directly to the CRE site in the CART promoter, using chromatin immunoprecipitation (ChIP) assays. ChIP assay with anti-CREB antibodies showed an enrichment of the CART promoter fragment containing the CRE region over IgG precipitated material, a non-specific control. Forskolin, which was known to increase CART mRNA levels in GH3 cells, was utilized to show that the drug increased levels of P-CREB protein and P-CREB binding to the CART promoter CRE-containing region. A region of the c-Fos promoter containing a CRE cis-regulatory element was previously shown to bind P-CREB, and it was used here as a positive control. These data suggest that the effects of CREB over expression on blunting cocaine reward could be, at least in part, attributed to the increased expression of the CART gene by direct interaction of P-CREB with the CART promoter CRE site, rather than by some indirect action.

Figure 1. Genomic DNA sequence of the rat CART proximal promoter region

Shown is the nucleotide sequence of the rat CART gene promoter (Genbank accession no. AF519794) originally identified and published by Barrett and colleagues [21] as well as the Genbank nucleotide numbering in the left-hand margin. The PCR primer forward and reverse sequences (corresponding to DNA sequences that are underlined, bolded and labeled as "5'-Start” and "3'-End", respectively) were recommended by Primer Express v3.0 software (Applied Biosystems, Foster City, CA). The CART gene promoter consensus CRE DNA cis-regulatory element is identified in bold and located between the flanking primers. That region of the promoter was amplified in PCR reactions. Both the TATA box necessary to initiate promoter-driven transcription and the +1 site of CART gene transcriptional initiation are also delineated in bold to orient the reader.

Figure 2. Composite figure of gels of DNA enriched in chromatin immunoprecipitation (ChIP) assays by anti-CREB and -P-CREB antibodies and amplified in PCR reactions

Four groups of lanes from agarose gels are shown and they correspond to four separate experiments. The experiments were used to verify that PCR amplicons produced by CART and c-Fos were their predicted sizes. Some non-specific amplification was present below 100 base pairs in lanes 3, 5, 6 and 8, which were likely primer dimers that arose because of low template DNA quantities. See figure and text for additional details.

Figure 3. Enrichment of the CART promoter CRE-containing region by ChIP assays after immunoprecipitation with a CREB-specific antibody

(A) Western blot analysis identified a CREB immunoreactive band from GH3 cell lysates (shown in triplicate), indicated by an arrow on the left of the gel. (B) Shown are representative PCR amplification plots of GH3 genomic DNA amplified by CART primers (a); anti-CREB IP DNA amplified by CART primers (b); IgG IP DNA amplified by CART primers (c); and H₂O-template negative control PCR reactions amplified by CART primers (d). The circles next to the letters in panel B identify the y-axis position of the cycle threshold (Ct) setting used in relative quantification calculations to determine fold-enrichment values. (C) Melting temperatures (Tm's) were identified at the peak of the curves. Lettered curves correspond to lettered samples in B. (D) CART promoter fragments isolated by anti-CREB IP were more abundant than those isolated with IgG IP (used as a non-specific IP control), as expected, indicating that CREB was bound to the CART promoter region containing the CRE cis-regulatory element. See text for additional details.

Figure 4. Forskolin stimulation enhanced P-CREB binding to the CART gene promoter region containing the CRE cis-regulatory element as determined by ChIP assays

(A) After 15 and 30 minutes (15m and 30m) of forskolin treatment, CART promoter fragments were enriched in GH3 cell lysates after anti-P-CREB immunoprecipitations (IP) compared to DNA from cell lysates immunoprecipitated with IgG. *p < 0.05, one-sample t-test; **p < 0.001, one-sample t-test; ^ap < 0.05, two-sample t-test. (B) Shown is a representative PCR amplification plot of CART promoter fragments enriched after: 30 min of forskolin and anti-P-CREB IP (a); DMSO and anti-P-CREB IP (b); DMSO and IgG IP (c); and H₂O-template negative control PCR reactions amplified with CART primers (d). A slight variation in one of the replicates of sample (a) makes it appear as two separate lines. The circles next to the letters in panel B indicate the y-axis position of the Ct setting used in relative quantitation calculations to determine fold-enrichment values. (C) The melt curves after 30 minutes of forskolin or DMSO treatment showed that melting temperature (Tm) values for amplicons amplified with CART primers were around 87.5°C, confirming that the PCR amplicons were the predicted size of 334 base pairs. Melt curves after 15 minutes of treatment (not shown) also revealed Tm values around 87.5°C for DNA enriched by anti-P-CREB IP. (D) In determining the fold-enrichment of CART DNA after anti-P-CREB IP compared to IgG IP, values were determined by normalizing the amount of CART promoter enriched by anti-P-CREB IP or IgG IP to the total amount of CART promoter present in those sample before antibody IP, defined here as the "starting material" (see statistical analysis for more detail). Shown are melt curves for starting material (without IP) amplified by CART primers which also had Tm's around 87.5°C, meaning the DNA fragments amplified in those PCR reactions were the predicted 334 base pairs long (a–c the same as in panel B).

Figure 5. Forskolin stimulation (15 min) enhanced P-CREB binding to the c-Fos gene promoter region containing the CRE cis-regulatory element

(A) After 15 minutes of forskolin treatment of GH3 cells and anti-P-CREB immunoprecipitations (IP), c-Fos promoter fragments were enriched compared to DNA from forskolin or vehicle (DMSO) treated cells immunoprecipitated with IgG. gDNA (a); forskolin treatment and anti-P-CREB IP (b); DMSO treatment and anti-P-CREB IP (c); forskolin treatment and IgG IP (d); and H₂O-template negative control PCR reactions with c-Fos primers (e). (B) Shown are representative PCR amplification plots. (C) The melt curves after 15 minutes of forskolin or DMSO treatment showed that melting temperature (Tm) values for DNA isolated by antibody IP's and gDNA amplicons were around 80.5°C, confirming the PCR amplicons were the predicted size of 104 base pairs. Letters correspond to samples in 5B. (D) Starting material amplified by c-Fos promoters, which was used to normalize the quantity of DNA enriched by antibody IP’s, also had melting temperatures around 80.5°C, meaning the DNA fragments amplified in PCR reactions were the predicted 104 base pairs long (b–d the same as in panel B). Starting material was the amount of c-Fos promoter present in the GH3 cell lysate of each ChIP sample before antibody IP. See text for additional details.

Figure 6. Forskolin time-dependently stimulated P-CREB levels in GH3 cells

A) Shown is a composite figure of representative P-CREB Western blots from the lysates of paired plates of GH3 cells incubated with either 20µM forskolin or DMSO (vehicle). Immunoreactive P-CREB bands are indicated by an arrow in the left-hand margin of the panel. Blots from the different time points had different raw optical densities because of differences in exposure times. Thus, the ratios of forskolin to DMSO treated pairs of dishes at the individual time points were compared. The mean ratios of P-CREB levels in forskolin treated cells versus DMSO treated cells at different time points are presented graphically in panel (B) (n of 3–5 groups/time point). The asterisk indicates a significantly greater ratio. See text for additional details.

Figure 7. CREB and P-CREB from the rat pituitary gland bound the rat CART CRE cis-regulatory element in EMSA/SS assays

Nuclear proteins were isolated from rat pituitaries, and binding to the CART CRE DNA cis-regulatory site was observed by electrophoretic mobility shift assays (EMSA) and antibody super shifts (SS) using an oligonucleotide identical in sequence to the rat CART promoter CRE site, and CREB and P-CREB antibodies. See figure and text for additional details.