Elucidation of mechanisms generating 5-hydroxymethylcytosine (5hmC) in mammalian mitochondria.

摘要

DNA methylation plays a pivotal role in governing cellular processes including genomic imprinting, gene expression, and development. Recently, the Tet family of methylcytosine dioxygenases(Tet1, Tet2 and Tet3) was found to catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), an intermediate in the pathway of DNA demethylation. Tet enzymes catalyze this hydroxylation in a 2-oxoglutarate and Fe2+ dependent manner.;We have recently reported significant levels of 5mC and 5hmC modification in immunoprecipitates of mammalian mitochondrial DNA(mtDNA). We provide the first evidence that a DNA Methyltransferase-1 isoform (mtDNMT1) translocates to the mitochondria using an N-terminal mitochondrial targeting sequence. mtDNMT1 expression is upregulated by NRF1 and PGC1α, master regulators of mitochondrial biogenesis and function, as well as by loss of p53. Altered mtDNMT1 expression asymmetrically affects mtDNA transcription. We are now pursuing the role of Tet proteins in generating 5hmC in mtDNA. Using an in vitro enzyme assay, we have successfully detected Tet activity in crude and percoll purified mitochondrial fractions of HCT116 cells. Mitoprot analysis on Tet family predicts that Tet1 may be translocated to the mitochondria. Immunoblot analysis indicates that a band of expected size(235kDa) is present on immunoblots of mitochondrial fraction from mouse embryonic stem cells with an antibody directed against Tet1. This band, however, is not protected from trypsin treatment of mitochondria indicating that Tet1 may not be transported to the mitochondrial matrix. The putative Tet1 mitochondrial targeting sequence (MTS) fails to carry heterologous protein to the mitochondria. Knock out of Tet1 in mouse ES cells also does not alter 5hmC signal in hydroxyMeDIP assay. We now seek to determine if Tet2/Tet3 may be involved in 5hmC generation. In the nucleus, 5hmC serves as an intermediate in the process of DNA demethylation through the combined action of cytidine deaminases and the base excision repair pathway. We plan to investigate if 5hmC holds the same functional significance in the mitochondria as it does in the nucleus. Our overall goal is to understand epigenetic regulation of normal mitochondrial function and changes that occur in diseases involving mitochondrial dysfunction such as ischemic heart disease, neurodegenerative diseases like Parkinsons disease, and cancer.