Date on Master's Thesis/Doctoral Dissertation

8-2013

Document Type

Master's Thesis

Degree Name

M.S.

Department

Oral Biology

Committee Chair

Darling, Douglas S.

Author's Keywords

Amylase; MIST1; PSP; Parotid gland; NUPR1

Subject

Parotid glands; Salivary glands

Abstract

BACKGROUND: Salivary secretion aids in digestion, host defense, and lubrication. Parotid salivary gland defects from Sjogren’s Syndrome affect more than one million Americans and cause dry mouth leading to oral disease. The Amylase 1 gene and the PSP gene are markers of parotid gland differentiation and indicate normal parotid gland function. Transcriptional activators of genes involved in parotid differentiation have not been characterized. HYPOTHESIS: Characterizing transcriptional activating sites in gene promoter regions will confirm upstream activating factors in bioinformatic network pathways. METHODS: Two promoter regions of mouse amylase 1 were identified using the ECR Browser, PCR amplified and ligated into a pGL4.10 luciferase vector. Transcriptional repressor binding sites were identified and removed from the 1 kbp region, followed by PCR amplification to produce fragments that were ligated into the minimal promoter pGL4.23 luciferase vector. These promoter clones were then transfected into three cell lines and tested for promoter activity. Bioinformatic analysis of microarray data determined correlations between increased transcription factor expression and markers of parotid differentiation. A hypothetical transcription factor network was created. Two interactions from the network predicted to activate the NUPR1 gene were Cited2/p300 and IRF2/PCAF. Mist1 was suggested to activate the PSP gene based on the hypothetical network. The promoter regions of NUPR1 and PSP were transfected into a parotid acinar cell line, and promoter activity was determined using dual luciferase assays with an internal Renilla control. RESULTS: The four amylase promoter clones, R1, R2-R1, 900bp, and 700bp were repressed as compared to a promoterless negative control, pGL4.10. The NUPR1 promoter was not activated by the co-transfected combination of Cited2/p300 or IRF2/PCAF. The PSP promoter alone was not activated by Mist1 transcription factor. However, PSP was activated when Mist1 was co-transfected with Tcf3 transcription factor and when intron regions containing E-box binding sites were added to the PSP promoter clone. CONCLUSION: Amylase 1 was not activated in the regions tested; therefore the active regions of the amylase 1 gene must be outside the 3 kbp region tested. NUPR1 is not activated by the predicted interactions of Cited2/p300 or IRF2/PCAF, so the results do not support the hypothetical transcription factor network. The PSP proximal promoter is not activated by Mist1, but a PSP promoter + intron construct is activated when the transcription factor Tcf3 + Mist1 are co-transfected.

Download

Share

COinS