Grant: Discovery Grants Program - Individual

Genomic DNA controls cellular morphology and function via epigenetic mechanisms that instruct the on and off state of genes. An important type of epigenetic modifications is DNA methylation (DNAm), which controls gene transcription, cellular identity/fate decision, and function. DNAm machinery includes 3 types of factors, the readers, writers, and erasers (or modifiers). The readers bind DNAm and interpret them. DNA methyl transferases (DNMT) are the writers that catalyze DNAm in the form of 5-methylcytosine (5mC). Ten eleven translocation (TET) enzymes are the erasers that oxidize 5mC to another form of DNAm; called 5-hydroxymethylcytosine (5hmC). The harmonic action of these factors is critical for chromatin stability (chromatin is the combination of DNA and DNA-bound proteins) and epigenome integrity, maintaining proper cellular phenotype and function. Multiple DNAm readers exist, one of them (MeCP2) binds to both unmethylated and methylated DNA (5mC and 5hmC). MeCP2 binding to unmethylated DNA blocks DNMT (5mC formation), and its binding to 5mC inhibits TET activity (5hmC formation). Thus, DNAm integrity can be impaired by altered level of MeCP2. Change in cellular DNAm affects the chromatin structure that is important for organ development, specially in the brain where DNAm level is the highest. MeCP2 is the most prominent DNAm reader in the brain, acting as the protector of chromatin structure. Thus, the precisely controlled MeCP2 level is essential to maintain the structural organization and function of brain cells. The work from my past NSERC-DG programs reported the developmental time-point, sex-specific, and brain region-dependent regulatory role of DNAm. The long-term objective of my NSERC-DG supported research program is to uncover the epigenetic mechanisms that control the establishment and maintenance of the chromatin structure in the developing brain. In short-term, we will explore the interplay between DNMT and TET, with MeCP2. Our model will be specific brain cell types isolated from MeCP2-deficient (knockout) and duplication (overexpressed) mice. In objective 1, we will map the genomic localization of DNMT/TET and position relative to 5mC/5hmC. In Objective 2, we will apply transcript sequencing to study the expressed genes, and associated impact on gene expression, DNAm levels (5mC/5hmC), and DNAm machinery (expression/enzymatic activities). State-of-the art sequencing, cellular and molecular biology experiments, as well as enzymatic assays, and microscopic imaging will be used to study how DNAm maintains the structural properties and composition of specific brain cell types. Our studies will help to better understand how DNAm controls chromatin stability and epigenome composition in different brain cell types  determine their cell fate commitments. My research program has and will continue to involve and train highly qualified personnel in cell and molecular biology, chromatin biology, and neuroscience techniques.