Date on Master's Thesis/Doctoral Dissertation

8-2016

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Biochemistry and Molecular Biology

Degree Program

Biochemistry and Molecular Biology, PhD

Committee Chair

Darling, Douglas

Committee Co-Chair (if applicable)

Clark, Barbara

Committee Member

Clark, Barbara

Committee Member

Kalbfleishch, Ted

Committee Member

Li, Jiaxu

Committee Member

Samuelson, David

Author's Keywords

microRNA; Salivary gland; Systems Biology; Oral Biology; acinar cell; differentiation

Abstract

Objective: This project sought to understand the gene regulatory networks that drive parotid salivary gland acinar cells to terminally differentiate, and drive expression of terminal differentiation genes in dedifferentiated ParC5 cells. Methodology: Laser capture microdissection was used to isolate acinar cells at multiple time points during differentiation. This important step allowed us to measure gene expression in a single and important cell type. A systems biology approach was taken to measure global mRNA and microRNA expression across acinar cell terminal differentiation in the rat parotid salivary gland. In ParC5 cells, the ER stress activator tunicamycin was used to stimulate Xbp1 activity. Results: Profiles of statistically significant changes of mRNA expression, combined with reciprocal correlations of microRNAs and their target mRNAs, suggest a putative network involving Xbp1 and Mist1 (BHLHA15). The network suggests that a molecular switch involving Prdm1, Sox11, and Pax5 progressively decreases repression of Xbp1 transcription, in concert with decreased translational repression by miR-214. Transfection studies validate each of the tested network interactions. Treatment of ParC5 cells with tunicamycin increases expression of Mist1 downstream of Xbp1. However, further downstream effectors of Xbp1 and Mist1 (i.e. PSP, Connexin32) remain unchanged. The Mist1 target gene Rab3D is repressed. However, transfection of Mist1 cDNA, increases Rab3d expression. Conclusion: This study identified numerous novel transcription factor expressionpatterns during parotid acinar differentiation, including Pparg, Klf4, and Sox11. Many differentially expressed microRNAs were also measured which have not previously been described in salivary development. Network analysis identified a gene regulatory network driving expression of terminal differentiation genes. Stimulating Xbp1 activity in ParC5 cells increases Mist1 expression as predicted in the network, but other factors or epigenetic changes may be required for full expression of the network.

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