Regulation of mitochondrial transcription and mtDNA copy number in mammals

Abstract: Functional mitochondria are essential for wellbeing of the cell and the whole organism. Gene expression from the mitochondrial genome (mtDNA) is indispensable for oxidative phosphorylation, but also for the replication of mtDNA, as the replication primers are processed from mtDNA transcripts. Mitochondrial transcription factor A (TFAM) is a key transcriptional activator that is also necessary for the maintenance of mtDNA. In this thesis we have focused on characterizing the relationship between TFAM protein levels and mtDNA copy number and expression in mammals. mtDNA copy number seems to correlate with TFAM protein levels, which is strikingly apparent in human and mouse testis. We characterized the Tfam gene and its expression pattern in rat and could identify a set of conserved features concerning mtDNA and TFAM expression during mammalian spermatogenesis. These features include expression of alternate TFAM/Tfam transcripts and downregulation of TFAM protein levels and mtDNA copy number. These features most likely constitute one of the mechanisms involved in preventing paternal transmission of mtDNA. We studied the gene-dosage effects of TFAM in vivo by creating transgenic mice carrying the whole human TFAM gene and various amounts of flanking DNA. Enhanced TFAM protein levels led to increased mtDNA copy numbers. However, only L-strand transcription was affected with 5o-6o % higher transcript levels, while there was no effect on H-strand transcript levels. Also respiratory chain enzyme activities were unaffected. Comp lementation analyses demonstrated that human TFAM protein cannot functionally replace the endogenous mouse TFAM protein. Nevertheless, the Tfam null embryos carrying the human TFAM gene contain mtDNA, whereas the homozygous Tfam knockout embryos do not. These results suggest that human TFAM protein is able to activate L-strand transcription and support mtDNA maintenance in mouse. In this thesis we have also sought to identify "missing" factors required for mitochondrial transcription. With the help of profile-based BLAST searches and Mtf1p peptide sequence, we were able to identify two novel mammalian mitochondrial transcription factors called TFB1M and TFB2M. Using pure recombinant mitochondrial RNA polymerase, TFAM and TFB1M or TFB2M, and a template containing the LSP and HSP, we could for the first time reconstitute faithful mitochondrial transcription in vitro. In yeast, there is only one mitochondrial RNA polymerase specificity factor, Mtf1p. The existence of two such factors in human, mouse and Drosophila prompted us to study their origin and evolutionary conservation. Cluster analyses indicate that the TFBMs most likely originate from a common ancestral gene that duplicated early in metazoan evolution