The primary goal of this proposal (""""""""Rapid and inexpensive epi/genetic profiling of the human mitochondrial genome"""""""") is to apply our new method of isolating and sequencing the mitochondrial genome (mtDNA) and epigenome (mtDNA-methylation) to large sets of samples in order to derive biological and clinical insights. We will characterize the variability in the mitochondrial genome and epigenome. The inexpensive and rapid method developed here will enable large-scale epidemiological studies of the role of mtDNA in aging and in the initiation and progression of a variety of human disorders.
In specific aim 1 we will develop the techniques to efficiently sequence the mtDNA. We will sequence the mtDNA isolated from commonly used human cell-lines and human blood samples in order to establish a baseline for comparison with future studies. This will also help us understand the nature of variations supported by mtDNA. We will also develop analytical techniques to determine the quality of mtDNA isolation, assemble the reads into a complete genome and identify variations in the genome.
In specific aim 2, we will modify and apply the technique of specific aim 1 to bisulfite treated mtDNA. This will allow us to profile DNA-methylation across the mtDNA in an unbiased manner. We will use the same samples as in specific aim 1 to quantify the levels of mtDNA methylation and its variability between samples, both within cell-lines and between individual blood samples.
In specific aim 3 will apply the techniques of specific aims 1 and 2 to placental samples from a newborn cohort established at Mount Sinai School of Medicine. Since placentas are rich in mitochondria and hold the key to early development, we believe this will give us insights into developmental differences between newborns. We will correlate the phenotypes (birth weight, length and head circumference) to mtDNA profiles and generate hypotheses on the role of variants in determining the phenotypes. This will be the first epidemiological use of mtDNA profiling. The results of this proposal will have a broad impact on a range of fields, from bacteril genomics and genetics of chloroplasts in plants to disorders such as cancer and diabetes in humans.
The role of the mitochondrial genome (mtDNA) and epigenome (mtDNA-methylation) in various human disorders is not well understood due to the difficulty of measuring changes in both. The novel techniques being developed for this proposal will enable accurate and inexpensive profiling of mtDNA and its methylation states, and make feasible its use on a large-scale in clinical and research settings, such as monitoring mtDNA in placentas. This proposal will have a major impact on the study of mitochondrial and chloroplast genomes in a variety of organisms.
|Jayaprakash, Anitha D; Benson, Erica K; Gone, Swapna et al. (2015) Stable heteroplasmy at the single-cell level is facilitated by intercellular exchange of mtDNA. Nucleic Acids Res 43:2177-87|
|Mudbhary, Raksha; Hoshida, Yujin; Chernyavskaya, Yelena et al. (2014) UHRF1 overexpression drives DNA hypomethylation and hepatocellular carcinoma. Cancer Cell 25:196-209|
|Cecere, Germano; Hoersch, Sebastian; O'Keeffe, Sean et al. (2014) Global effects of the CSR-1 RNA interference pathway on the transcriptional landscape. Nat Struct Mol Biol 21:358-65|
|Cora, Elisa; Pandey, Radha R; Xiol, Jordi et al. (2014) The MID-PIWI module of Piwi proteins specifies nucleotide- and strand-biases of piRNAs. RNA 20:773-81|
|Malone, Colin D; Mestdagh, Claire; Akhtar, Junaid et al. (2014) The exon junction complex controls transposable element activity by ensuring faithful splicing of the piwi transcript. Genes Dev 28:1786-99|
|Howarth, Deanna L; Lindtner, Claudia; Vacaru, Ana M et al. (2014) Activating transcription factor 6 is necessary and sufficient for alcoholic fatty liver disease in zebrafish. PLoS Genet 10:e1004335|
|Pandey, Radha Raman; Tokuzawa, Yoshimi; Yang, Zhaolin et al. (2013) Tudor domain containing 12 (TDRD12) is essential for secondary PIWI interacting RNA biogenesis in mice. Proc Natl Acad Sci U S A 110:16492-7|