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Epigenetic regulation of Hoxa1 and Hoxa2



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Hoxa1 and Hoxa2 are master regulators in the development of hindbrain, ear, palate, bone and cardiovascular development. There is little information on the epigenetic regulator(s) of Hoxa2 gene during development. In this thesis, I have determined whether regulation of Hoxa2 is occurring via a specific epigenetic pathway, and investigated the role of DNA methylation, noncoding RNAs (microRNAs and long non-coding RNAs) and histone protein modification. First, analysis of Hoxa2 promoter revealed the presence of three CpG islands near the Hoxa2 5′ regulatory region. Using methylation specific PCR (MSP) and the bisulfite specific PCR (BSP) primers followed by DNA sequencing, I found the methylation status of CpG island 1 remains unmethylated and that the DNA methylation status of the Hoxa2 promoter does not change with the spatio-temporal expression of Hoxa2 during palatogenesis. These findings indicate that DNA methylation does not appear to play a key role in the epigenetic regulation of Hoxa2 gene during palatogenesis. My second objective was to determine whether specific miRNAs impact Hoxa2 expression. I performed in-silico analysis and identified six miRNAs that have the potential to bind 3'UTR of the Hoxa2 gene. The miR-669b and miR-376c were capable of down regulating Hoxa2 expression at both transcriptional and translational level. Two direct miR-669b binding sites were identified on mouse Hoxa2 3'UTR. Luciferase assays showed that the two miR-669b binding sites appear to work independently of each other and that mutations within the seed sequences abrogated luciferase activity. I further analyzed the degree of sequence similarity of both miR-669b binding sites and found that binding site 1 is evolutionarily conserved between the five species (human, mouse, rat, chimpanzee and dog). In the developing mouse palate (from E13 to E15), miR-669b showed a complementary expression to that of Hoxa2. No direct interaction between miR-376c and Hoxa2 3'UTR was identified. Thus my results indicated that the miR-669b likely plays a role in regulating Hoxa2 expression during palate development My third objective was to characterize a new lncRNA (mHotairm1) that I identified between mouse Hoxa1 and Hoxa2 intergenic region. I demonstrated that mHotairm1 is involved in recruiting MLL1/WDR5 to Hoxa1 and Hoxa2 genes and regulating their expressions. In situ hybridization histochemistry of E14 developing palate showed expression of mHotairm1 in medial edge epithelia (MEE), indicating mHotairm1 may play a role in the palatal fusion. Downregulation of mHotairm1 in NIH 3T3 cells resulted in significantly decreased expression of Hoxa1 and Hoxa2 expression, whereas treatment with ATRA resulted in increased expression of mHotairm1, Hoxa1 and Hoxa2. Using capture hybridization analysis of RNA targets (CHART) and pull down assays, I found that the TrxG protein WDR5 is associated with mHotairm1, and knockdown of mHotairm1 resulted in reduced occupancy of gene activation mark H3K4me3 and increased occupancy of gene suppression mark H3K27me3, suggesting MLL1/WDR5 complex may be playing a role in the regulation of Hoxa1 and Hoxa2 gene expression through mHotairm1. Lastly, I found that WDR5 was sumoylated. This modification appears to be important for its interaction with mHotairm1 and MLL and for its cellular distribution, primarily to the nuclei. Following ATRA treatment, although the total WDR5 protein remained unchanged, an increase in sumoylated WDR5 was observed together with increased expression of mHotairm1, Hoxa1 and Hoxa2 gene. These findings reveal that sumoylated WDR5 with its interaction with mHotairm1 plays an important role in H3K4me3 and H3K27me3 occupancy and influencing the epigenetic regulation of Hoxa1 and Hoxa2 genes.



Epigenetics, Hoxa1, Hoxa2, miRNA, lncRNA



Doctor of Philosophy (Ph.D.)


Pharmacy and Nutrition




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