Transcriptional regulation of the human secretin gene
Abstract of thesis entitled
HUMAN SECRETIN GENE
Lee Tsz On
for the degree of Doctor of Philosophy
at The University of Hong Kong
in April 2004
Secretin, the first hormone ever to be identified, is a member of the glucagon/secretin/vasoactive intestinal peptide (VIP) family. Secretin has recently aroused considerable research interest, primarily because of its potential therapeutic effect on autistic patients. This thesis aims to unravel the mechanism that regulates the human secretin gene expression in various cell lineages. Five kilobases of the 5?flanking region of the human secretin gene were investigated. Since secretin is expressed in the brain and gut, a human duodenal adenocarcinoma cell (Hutu-80), and a human neuroblastoma cell (SH-SY5Y) were employed to study the regulation of secretin in different cell types.
In duodenal cells, an E-box and two GC-boxes located at the 5?flanking region were found to be essential for the human secretin promoter activity. In vivo and in vitro binding studies (gel mobility shift and chromatin immunoprecipitation, ChIP) showed that these motifs were binding sites for transcription factors, namely, Sp1,
Sp3, NeuroD and E2A. Expression of various ratios of Sp1 and Sp3 in Drosophila SL2 cells (a cell line that does not express Sp transcription factors) and Hutu-80 cells reflected the importance of the stoichiometric ratio of Sp1 and Sp3 on the control of secretin gene expression. The activation effect of Sp1 and NeuroD was additive and such effect was further confirmed when Sp1 and NeuroD were co-expressed in Hutu-80 cells. The GC-boxes are located within a putative CpG island suggesting a role of CpG methylation in human secretin gene regulation. This hypothesis was confirmed later by: (1) cell-specific methylation patterns of the promoter, (2) augmentation of the secretin transcript levels in 5?Aza-2?eoxycytidine (5?Aza-dC) treated non-secretin expressing cells (PANC-1 and HepG2); and (3) the changes of Sp1, Sp3 and NeuroD levels after 5?Aza-dC treatment.
In the second part of this study, a well established neuronal differentiation cell line, SH-SY5Y, was used. The secretin expression level was increased and the secretin promoter activity was activated upon the treatment of all-trans retinoic acid (RA), a drug routinely used for inducing neuronal differentiation in SH-SY5Y cells. As in the case of duodenal cells, the Sp-protein binding motifs were found to be functional. Sp4 together with Sp1 and Sp3 was able to bind to the GC-Box within the core promoter region. In addition, a DNA motif close to the GC-box was found to be partially responsible for the RA induction. Gel mobility shift assay and southwestern blot analysis revealed that the transcriptional factor, Nuclear Factor I (NFI), was responsible for interacting with this motif. Among the NFI proteins (NFI-A, NFI-B, NFI-C, and NFI-X), only the over-expression of NFI-C was able to increase the promoter activity upon RA treatment. These data suggest that this DNA motif is at least partially responsible for the RA-responsiveness of the human secretin gene promoter. In summary, this study suggests that co-operations of Sp-proteins and
NFI-C are involved in the transcriptional regulation of the human secretin gene in the neuronal cells.
School:The University of Hong Kong
School Location:China - Hong Kong SAR
Source Type:Master's Thesis
Keywords:secretin genetic transcription regulation
Date of Publication:01/01/2004