Date of Award

2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Klaus H. Kaestner

Abstract

Type 2 diabetes mellitus (T2DM) is a complex metabolic disease characterized by inadequate insulin secretion by the pancreatic β-cell in response to increased blood glucose levels. Despite compelling evidence that T2DM has a high rate of familial aggregation, known genetic risk variants account for less than 10% of the observed heritability. Consequently, post-transcriptional regulators of gene expression, including microRNAs and other noncoding RNAs, have been implicated in the etiology of T2DM, in part due to their ability to simultaneously regulate the expression of hundreds of targets.

To determine if microRNAs are involved in the pathogenesis of human T2DM, I sequenced the small RNAs of human islets from diabetic and non-diabetic organ donors. From this screen, I identified the maternally-expressed genes in the imprinted DLK1-MEG3 locus as highly- and specifically-expressed in human β-cells, but repressed in T2DM islets. Repression of this noncoding transcript was strongly correlated with hyper-methylation of the promoter that drives transcription of all the maternal noncoding RNAs including the long noncoding RNA MEG3, several microRNAs and snoRNAs. Additionally, I identified disease-relevant targets of DLK1-MEG3 microRNAs in vivo using HITS-CLIP, a technique to detect targets of RNA binding proteins. My results provide strong evidence for a role of microRNAs and epigenetic modifications, such as DNA methylation, in the pathogenesis of T2DM. In addition, my data set catalogs human islet microRNAs relevant to human T2DM pathogenesis and characterizes their target transcriptomes.

Despite being associated with T2DM and several other diseases, very little is known about the regulation of imprinting of the MEG3-DLK1 locus. Hence, I interrogated a newly described enhancer in this locus, as enhancers are known mediators of mono-allelic expression at other imprinted loci. I discovered allele-specific binding of this enhancer by critical islet transcription factors, including FOXA2 in human islets. In addition, I mapped long-range interactions of this enhancer in human islets using 4C-Seq. Overall, my findings provide novel insights into the regulation of an imprinted locus critical to β-cell health and function.

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