Access Type

Open Access Dissertation

Date of Award

January 2016

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Pharmacology

First Advisor

Douglas Ruden

Abstract

Prenatal and postnatal exposure to pervasive neuro-toxicants such as Lead (Pb) has been reported to causes extensive and diverse changes in the epigenetic profile. Among epigenetic modification, DNA methylation (5mC) is perhaps the most widely studied and has been proposed to be potential early biomarkers for Pb toxicity. Several studies have demonstrated the association between Pb-exposure and 5mC. However most of these studies are restricted to looking at a specific set of target genes or repetitive elements. Therefore, one of the main objectives of our study was to use an unbiased genome-wide approach to look at Pb-exposure associated changes in 5mC. To this end, we used the Human methylation 450K (HM450K) high density array to quantitatively measure the Pb-associated 5mC changes. The sample for this study consisted of DNA extracted from neonatal and current blood spots from a mother-infant cohort in Detroit, USA and Umbilical cord blood DNA from a mother-infant cohort from Mexico City, Mexico. We observed that Pb-exposure associated 5mC changes in whole blood and UCB are sex-specific. Furthermore, some of these 5mC changes are heritable and can be transmitted from the grandmother to the grandchildren. To further our understanding of the relationship between Pb-exposure and 5mC, we wanted to look at the impact of Pb-exposure on DNA demethylation, specifically the dynamic changes in 5-hydroxymethylcytosine (5hmC) profile. To study these changes in the 5hmC profile, we used a novel modification of the HM450K, which we named HMeDIP-450K array. Using the HMeDIP-450K array we demonstrated that 5hmC showed a much larger number of sex-independent changes. Interestingly, a vast majority Pb-dependent 5mC and 5hmC clusters mapped to either gene implicated in neurodegeneration and regulation of mitochondrial processes such as NINJ2, VAMP5, GSTM1, GSTM5 etc. 5mC and 5hmC are potent regulators of gene expression and their dysregulation can cause widespread changes in the transcriptome and may contribute to neurodegenerative phenotype. Besides 5mC and 5hmC, transcriptomic changes can also be regulated by dynamic changes in histone methylation profile and alternative splicing. To study these changes, especially in context of neurodegeneration we used a Drosophila model of traumatic brain injury (TBI). Using a modified version of this model, we subjected w1118 fruit flies to mild closed head trauma. To determine the transcriptomic changes which contribute to survival post TBI, we collected fly heads from the survivors at 2 time points; 4 hours and 24 hours’ post-trauma. Mild TBI using our modified TBI protocol had limited impact on the expression profile of genes but showed large perturbations in alternative splicing (AS) regulation 24 hours’ post-trauma. Classification of these AS changes showed selective retention of long introns (>81bps). Some of these genes also showed a significant reduction in transcript abundance and were specifically involved in mitochondrial metabolism. The retained introns were enriched for CA-rich motifs known to bind to Smooth (SM), an hnRNPL class of splicing factor. Mutating SM (sm1/DF) resulted in reversal of intron retention observed 24 hours’ post-trauma. This observation suggested that SM is critical regulator of Intron retention in fly heads. Interestingly, ChIP-sequencing for H3K36me3 revealed increased levels in retained introns post-trauma. Additionally, higher H3k36me3 was also observed around intronic SM-binding motifs post-trauma which suggested that increased level of H3k36me3 might be recruiting SM to their Intronic Splicing Suppressor sites and cause RI in the Drosophila model of TBI. Together our studies in human cohort and Drosophila sheds some light on the complex multi-layered mechanism regulating gene-expression especially under neurotoxic and neurodegenerative conditions.

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