A significant decrease in MLL1 at the Cck promoter was observed in Clock∆19 mice (t 8=2.827, p = 0.0223) (n= 5 per group). d) Relative enrichment of MLL1 at the Cck promoter in Clock∆19 mice and WT littermates was assessed by performing ChIP assays with an MLL1-specific antibody. Induction of Cck-luc activity was not detected when the E-box element was mutated (n=5–8 per group). Co-transfection of 5μg of CLOCK and BMAL1 expression constructs resulted in a significant increase in Cck-luc activity (t 14=5.314, p =0.0001) when the E-box element was intact. ( c) Relative luciferase activity of PC12 cells transfected with a Cck-luc construct (318 bp) containing either an intact or mutated E-box element. Inset are representative agarose gels of q-PCR products from ChIP assays showing AcH3 positive control IPs, IgG negative control IPs, and CLOCK IPs (n=4–6 per genotype). One sample t-tests revealed that CLOCK is significantly enriched at the Cck promoter regions (~4–6 fold) above background in both WT (t 4=2.920, p = 0.0432) and Clock∆19 (Mut) (t 3=6.754. ( b) Fold enrichment at proximal promoter region following ChIP with a CLOCK specific antibody comparing Clock∆19 mutants and wild-type (WT) littermate controls. Additional important transcription factor binding sites and regulatory regions are highlighted.
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The region containing the proximal promoter, including the E-Box, was amplified by quantitative qPCR after ChIP assays were performed. These studies identify a key role for Cck in the development and treatment of mania, and describe some of the molecular mechanisms by which lithium may act as an effective antimanic agent. Human postmortem tissue from bipolar subjects reveals a similar increase in Cck expression in the VTA with mood stabilizer treatment.
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The decrease in Cck expression in the ClockΔ19 mice appears to be due to a lack of interaction with the histone methyltransferase, MLL1, resulting in decreased histone H3K4me3 and gene transcription, an effect reversed by lithium. Moreover, chronic treatment with lithium restores Cck expression to near wild-type and this increase is necessary for the therapeutic actions of lithium. Selective knockdown of Cck expression via RNA interference in the VTA of wild-type mice produces a manic-like phenotype. Here, we find that cholecystokinin (Cck) is a direct transcriptional target of CLOCK and levels of Cck are reduced in the ventral tegmental area (VTA) of ClockΔ19 mice. Mice with a mutation in the Clock gene (ClockΔ19) have been identified as a model of mania however, the mechanisms that underlie this phenotype, and the changes in the brain that are necessary for lithium's effectiveness on these mice remain unclear.