DeltaFosB indirectly regulates Cck promoter activity

Abstract

Some of the important biochemical, structural, and behavioral changes induced by chronic exposure to drugs of abuse appear to be mediated by the highly stable transcription factor DeltaFosB. Previous work has shown that DeltaFosB overexpression in mice for 2weeks leads to an increase in the expression of numerous genes in striatum, most of which are later downregulated following 8weeks of FosB expression. Interestingly, a large number of these genes were also upregulated in mice overexpressing the transcription factor CREB. It was unclear from this study, however, whether short-term DeltaFosB regulates these genes via CREB. Here, we find that 2weeks of DeltaFosB overexpression increases CREB expression in striatum, an effect that dissipates by 8weeks. The early induction is associated with increased CREB binding to certain target gene promoters in this brain region. Surprisingly, one gene that was a suspected CREB target based on previous reports, cholecystokinin (Cck), was not controlled by CREB in striatum. To further investigate the regulation of Cck following DeltaFosB overexpression, we confirmed that short-term DeltaFosB overexpression increases both Cck promoter activity and gene expression. It also increases binding activity at a putative CREB binding site (CRE) in the Cck promoter. However, while the CRE site is necessary for normal basal expression of Cck, it is not required for DeltaFosB induction of Cck. Taken together, these results suggest that while short-term DeltaFosB induction increases CREB expression and activity at certain gene promoters, this is not the only mechanism by which genes are upregulated under these conditions.

Figure 1

CREB levels increase with ΔFosB overexpression. Western blot analysis of lysates from (A) mouse striata from 11A mice off dox for 2 or 8 weeks or (B) PC12 cells overexpressing ΔFosB. Data in A are shown as percent change in off dox compared to on dox animals. Data in B are normalized to GAPDH levels. * p<0.05 vs on dox animals (A) or pCDNA transfected cells (B).

Figure 2

Short-term overexpression of ΔFosB increases Cck gene expression. (A) Cck mRNA expression in striatum following ΔFosB overexpression in 11A mice for 1, 2, 4, or 8 weeks. Microarray analysis was performed on striatal samples and Cck levels were compared between off dox and on dox animals (data from large array set published in McClung and Nestler, 2003). Levels at 2 and 8 weeks off of dox were replicated by qPCR in a separate group of animals (data not shown). (B) A Cck-luciferase reporter plasmid was co-transfected into PC12 cells along with a ΔFosB expression plasmid or pCDNA, and luciferase activity was assayed 2 or 3 days after transfection. Data were normalized to cells cotransfected with the Cck reporter and pCDNA. *** p<0.05. * significant difference when compared to no ΔFosB control, ** significant difference between 2 and 3 days ΔFosB.

Figure 3

Protein binding at the Cck promoter. (A, B) Electrophoretic mobility shift assay using the Cck CRE-like site with striatal tissue from animals overexpressing ΔFosB for 2 weeks (A) or 8 weeks (B). In (A), competition with excess unlabeled competitor DNA was performed to demonstrate probe specificity. (C) Binding shown in A and B was quantified using densitometry. (D) Supershift assay using the Cck-like CRE site and a CREB antibody (lanes 2, 4) on lysates from ΔFosB overexpressing mice on (lanes 1-2) or off (lanes 3-4) dox for 2 weeks. (E) Supershift assay using a consensus CRE site and a CREB antibody (lanes 2, 4) on lysates from ΔFosB overexpressing mice on (lanes 1-2) or off (lanes 3-4) dox for 2 weeks. (F) Electrophoretic mobility shift assay using a consensus CRE site with striatal tissue from animals overexpressing ΔFosB for two weeks. Excess unlabeled competitor DNA was used to demonstrate probe specificity. For A, D, E, and F representative experiments are shown. * p<0.05 vs on dox animals.

Figure 4

The Cck-like CRE site is not necessary for Cck induction by ΔFosB. (A) Cck-luciferase activity was measured 2 days after transfection with either normal Cck-luciferase or one in which the CRE-like site was mutated. * p<0.05 (B) Cck-luciferase reporter activity (either normal promoter or one containing a mutation in the CRE-like site) was measured in cells co-transfected with a ΔFosB expression plasmid or pCDNA. Luciferase activity was assayed 2 or 3 days after transfection. Data are presented as fold change compared to the reporter cotransfected with pCDNA.

Figure 5

cFos binds to the Cck promoter. Chromatin immunoprecipitation assays were performed with a specific antibody for cFos using striatal tissue from ΔFosB overexpressing mice either on dox, or after 2 weeks of dox removal. Real time PCR analysis was performed on immunoprecipitated DNA using primers for the Cck promoter. Representative products from the inputs (inp), on dox controls (On), off dox animals (Off), or control IgG pulldowns (IgG) are shown in (A). The fold change of off dox animals relative to on dox animals is plotted in (B).