Abstract
Pituitary adenylate cyclase-activating polypeptide (PACAP) is an important hypophysiotrophic factor as well as a regulator for immune, reproductive, and neural tissues. We recently found that TTF-1, a homeodomain-containing transcription factor essential for the development of the fetal diencephalon, is postnatally expressed in the hypothalamic area and plays a transcription regulatory role for certain neurohormones. Based on the similarity of synthesis sites between PACAP and TTF-1 and, moreover, on the presence of conserved core TTF-1 binding motifs in the 5'-flanking region of the PACAP gene, we sought to uncover a regulatory role of TTF-1 in PACAP gene transcription. The TTF-1 homeodomain binds to six of the seven putative binding domains observed in the 5'-flanking region of the PACAP gene. In the C6 glioma cell-line, TTF-1 activates the PACAP promoter in a dose-dependent manner. This transactivation of PACAP by TTF-1 was totally removed when the core TTF-1 binding motif at -369 was deleted. RNase protection assays showed that TTF-1 and PACAP mRNAs have daily fluctuations in the rat hypothalamus. They both were at low levels during the day and high levels during the night. Intracerebroventricular administration of an antisense TTF-1 oligodeoxynucleotide significantly decreased the PACAP mRNA level as well as TTF-1 protein content in the rat hypothalamus, suggesting that TTF-1 also regulates PACAP transcription in vivo. Moreover, the TTF-1 promoter was inhibited by molecular oscillators of CLOCK and BMAL-1. Taken together, these data suggest that TTF-1 plays an important regulatory role in the gene transcription for PACAP, which may be important for the generation of a daily rhythm of hypothalamic PACAP gene expression.
Highlights
Pituitary adenylate cyclase-activating polypeptide (PACAP)1 was originally isolated from the ovine hypothalamus [1, 2] and was classified as a member of the secretin-glucagon-vasoactive intestinal peptide family [1]
Identification of Consensus TTF-1 Binding Motifs in the Rat PACAP Promoter—To find possible TTF-1 binding motifs in the 5Ј-flanking region of the rat PACAP gene, the sequence deposited in the NCBI GenBankTM data base by us (Ref. 7; accession number AF163322) was analyzed again with the aid of a search program
The 5Ј-flanking sequence of the rat PACAP gene contains several consensus motifs involved in transcriptional control (Fig. 2): two cAMP response element (CRE), a TPA response element (TRE), three growth hormone factor-1 (GHF-1), and one GATA-1 binding sites
Summary
Primer Extension for PACAP Transcription Initiation Site Determination—To ascertain the transcription initiation site of the PACAP gene, primer extension of mRNA samples from the rat brain was employed. Serial Deletion of the PACAP Promoter and Site-directed Deletion of TTF-1 Binding Core Motifs—The rat PACAP promoter, luciferase reporter (pGL3-basic) vector was previously described [7]. Serial deletion from this promoter was produced with restriction enzymes that cut uniquely in the promoter at the 5Ј-end (PstI for Ϫ1007, BxtI for Ϫ708, XbaI for Ϫ481, and AatII for Ϫ199) and with SpeI of the vector’s multicloning site at the 3Ј-end. The second round of PCR was performed with a specific nested primer set inside the span between RV3 and GL2 to link the upstream and downstream fragments These deletion mutants were cloned into the pGL-3-basic vector. Changes observed between two groups were analyzed by Student’s t test
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