Analyzing the effects of ethanol in early stage of embryo development using transcriptomic and epigenomic approaches

Abstract

Fetal alcohol spectrum disorders (FASD) is an umbrella term that describes a diverse set of ethanol-induced defects. The phenotypic variation is generated by numerous factors, including timing and dosage of ethanol exposure as well as genetic background. We are beginning to learn about how the concentration, duration, and timing of ethanol exposure mediate variability within ethanol teratogenesis. However, little is known about the genetic susceptibilities in FASD. Studies of FASD animal models are beginning to implicate a number of susceptibility genes that are involved in various pathways. In this dissertation, I studied the overall effect of ethanol on genome especially gene expression. Beside this, I applied DNA methylome analysis to correlate ethanol mediated epigenetic control of differentially expressed genes.

Firstly, a cDNA microarray analysis was used to profile mRNA expression in embryoid bodies (EB) at day 7 with or without ethanol treatment. The aim was to provide a profile of important regulator that exerts ethanol related genomic alteration. A total of 493 differentially expressed genes were identified in response to 50 mM ethanol exposure, in which 111 of them were up-regulated and 382 were down-regulated. Gene ontology term enrichment analysis revealed that the genes are involved in the important biological processes like neurological system process, cognition, behavior, sensory perception of smell, taste and chemical stimuli and synaptic transmission. In consistent, disease enrichment chart showed relevant categories like neurological diseases, developmental disorders, skeletal and muscular disorders, connective tissue disorders. Furthermore, I have shorted out a group of 26 genes that encode transcription factors. I validated relative gene expression of several transcription factors from the list by quantitative real time PCR.

Secondly, I employed RNA sequencing to profile differentially expressed genes in EBs at day 1 in presence and absence of 50 mM ethanol. A total of 739 differentially expressed genes were identified in response to ethanol exposure, in which 436 of them were up-regulated and 303 were down-regulated. Functional annotation using bioinformatics tools reveal significant enrichment of different cellular development and developmental disorders. Furthermore, a group of 41 transcription factor encoding genes are screened out from the all differentially expressed genes. I validated relative gene expression of several transcription factors from the list by quantitative real time PCR.

Thirdly, to better understand the alcohol mediated epigenetic alterations, I performed a genome-wide DNA-methylome analysis of ethanol exposed EBs at day 1. The Methylated DNA Binding Domain sequencing (MBD-seq) analysis identified 15,210 differentially methylated regions (DMRs) between control and ethanol treated EBs. Among these DMRs, 2,412 regions (15.85%) were hypermethylated and 12,798 regions (84.14%) were hypomethylated. I examined associations with gene expression by intersecting RNA-seq and MBDseq data sets. In total, 282 DMRs were associated with significant changes in RNA expression levels between control and ethanol treated EBs. To profile a set of ethanol responsive DNMT3A/3B-target genes that are associated with DMRs, I compared ethanol responsive DMR associated 282 genes and ethanol responsive DNMT3A/3B-target genes. I found 101 genes common between these two set of common genes. Thus, it suggests that these 101 genes are epigenetically regulated by DNA methylation exerted by ethanol trough modulation DNMT3A/3B expression.

Fourthly, I examined the effects of chronic ethanol on gene expression during early stages of neuronal differentiation. NCCIT cells were differentiated into neuronal precursors/lineages in the presence or absence of ethanol. I performed RNA sequencing at day 1 and cDNA microarray analysis at day 21 to profile gene expression in different stages of differentiation. Pathway analysis demonstrated that ethanol deregulates many genes and pathways that are involved in neuronal differentiation and development, auxonogenesis, neurite growth and other functions that are involved in early brain development.

Finally, I studied the effect of ethanol on a rodent model. I reported that maternal alcohol consumption alters several important genes that are involved in nervous system development in the mouse hippocampus at embryonic day 18 (ED18). A cDNA microarray analysis revealed that Nova1, Ntng1, Gal, Neurog2, Neurod2 and Fezf2 gene expressions are altered in the fetal hippocampus. Pathway analysis also revealed the association of the calcium signaling pathway in addition to other pathways with the differentially expressed genes during early brain development. I also observed the reduced expression of glial transcription factor Nfia and altered expression of its target genes in fetal hippocampus. Additionally, I did another microarray analysis for ethanol exposed mouse embryonic neural stem cell (mNSC) differentiation at day 2. I found that ethanol suppress mNSC differentiation through Wnt-signaling pathway. Collectively, alteration of such important genes and dynamics of the signaling pathways may cause neurodevelopmental disorders.

In summary, this works represents a broad range of information that includes ethanol mediated alteration of gene expression and their regulation through DNA methylation. I hope that this study substantially contributes to the understanding of the molecular mechanism underlying the pathology of alcohol mediated anomalies and ease further research.