We initially identified four putative Egr binding
We initially identified four putative Egr1 Minocycline HCl (−39/−36, −105/−102, −107/−104, and −227/−224) on the DBH proximal promoter. The deletion and mutagenesis experiments indicate that the motif at −227/−224 is required for Egr1-elicited reduction in DBH promoter activity. It remains to be determined whether the other motifs also contribute to the regulation. Since pCMV ETTL had no effect on DBH promoter-driven transcription or on endogenous mRNA levels, the Egr1 binding domain must be required for the suppression of DBH transcription. Both ChIP and EMSA experiments revealed that Egr1 could bind to a region of the DBH promoter within −247 to −204. An oligonucleotide containing this sequence was able to compete for binding of a consensus Egr1 oligonucleotide. How might Egr1 suppress DBH transcription? There are several mechanisms whereby Egr1 may be mediating the decreased DBH promoter activity. It could be due to Egr1 directly binding to DNA or Egr1 indirectly interacting with other transcriptional coactivators, such as the cAMP-response-element-binding protein (CREB)-binding protein (CBP). Egr1 has been shown to interact with CBP/p300 physically and functionally (Silverman et al., 1998). CBP/p300 are large multifunctional nuclear proteins and their levels are limited (reviewed by (Goldman et al., 1997)). Egr1, when overexpressed, might compete with other transcriptional factors for a limited amount of CBP/p300. However, this does not appear to be the case for TH, which is elevated by overexpression of Egr1 under the same conditions. It is also possible that Egr1 might be inhibiting the activation of DBH promoter-driven transcription by decreasing the availability of other transcription factors, such as Sp1. Egr1 has been implicated in inhibition of c-met expression by sequestering Sp1 as a transcriptional activator of c-met. The Sp1/Egr1 complex that was formed presumably resulted in a decreased availability of unbound Sp1 as a transcriptional activator (Zhang and Liu, 2003). Therefore, overexpressed Egr1 could repress DBH promoter-driven transcription by decreasing free Sp1. DBH gene transcription is also regulated by AP1 proteins, especially c-Jun and JunD (Swanson et al., 1998). Overexpressed Egr1 has been shown to suppress the c-Jun mRNA expression induced by interleukin-17 (Faour et al., 2005). Levkovitz and Baraban (Levkovitz and Baraban, 2002) showed that the zinc finger domain of Egr1 interacts with c-Jun, and then reduces transcriptional activation by c-Jun in response to nerve growth factor. Therefore, sequestration of c-Jun protein may mediate the Egr1-elicited reduction of the basal level of DBH gene expression. The newly identified Egr1 binding site at −227/−224 is very close to the DBH silencer site (Shaskus et al., 1995). Even though the DBH silencer site is only functional in noncatecholaminergic cells, it remains to be determined if an interaction between Egr1 and the DBH silencer site plays a role in the repression of DBH promoter activity observed here. In this regard, the DNA sequences near the Egr1 binding site were also shown to be important in determining whether Egr1 regulates gene transcription positively or negatively (Gashler et al., 1993). What is the impact of the ability of Egr1 to reduce DBH gene expression? It was surprising that Egr1 attenuates DBH gene transcription, while it was previously found to elevate transcription of TH and PNMT genes. A reduction of DBH gene expression might increase the dopamine levels in noradrenergic cells. In this regard, an elevated DA/NE ratio resulting from decreased DBH expression or activity is associated with increased vulnerability to psychotic depression (Meyers et al., 1999, Schatzberg and Rothschild, 1992). Interestingly, with a single exposure of rats to immobilization stress, TH and PNMT mRNAs in the adrenal medulla are elevated (transiently) as high as with repeated exposures to this stress (Nankova et al., 1994, Viskupic et al., 1994, Wong et al., 2002), while induction of DBH mRNA is not as high as with repeated stress (Kvetnansky and Sabban, 1998, McMahon et al., 1992). Both single and repeated exposures to stress induce Egr1 expression in the adrenal medulla (Papanikolaou and Sabban, 1999), but there are additional transcriptional changes with repeated stress. These include prolonged phosphorylation of CREB and induction of Fra-2 (Nankova et al., 2000, Sabban et al., 2006), which might overcome any inhibition of DBH by Egr1. Moreover, in the locus coeruleus, where Egr1 is not induced by immobilization stress (Hebert et al., 2005), DBH gene transcription is raised to a similar extent with both single and repeated exposures (Serova et al., 1999).