Research Article: Ancient mtDNA Genetic Variants Modulate mtDNA Transcription and Replication

Date Published: May 8, 2009

Publisher: Public Library of Science

Author(s): Sarit Suissa, Zhibo Wang, Jason Poole, Sharine Wittkopp, Jeanette Feder, Timothy E. Shutt, Douglas C. Wallace, Gerald S. Shadel, Dan Mishmar, Rob DeSalle

Abstract: Although the functional consequences of mitochondrial DNA (mtDNA) genetic backgrounds (haplotypes, haplogroups) have been demonstrated by both disease association studies and cell culture experiments, it is not clear which of the mutations within the haplogroup carry functional implications and which are “evolutionary silent hitchhikers”. We set forth to study the functionality of haplogroup-defining mutations within the mtDNA transcription/replication regulatory region by in vitro transcription, hypothesizing that haplogroup-defining mutations occurring within regulatory motifs of mtDNA could affect these processes. We thus screened >2500 complete human mtDNAs representing all major populations worldwide for natural variation in experimentally established protein binding sites and regulatory regions comprising a total of 241 bp in each mtDNA. Our screen revealed 77/241 sites showing point mutations that could be divided into non-fixed (57/77, 74%) and haplogroup/sub-haplogroup-defining changes (i.e., population fixed changes, 20/77, 26%). The variant defining Caucasian haplogroup J (C295T) increased the binding of TFAM (Electro Mobility Shift Assay) and the capacity of in vitro L-strand transcription, especially of a shorter transcript that maps immediately upstream of conserved sequence block 1 (CSB1), a region associated with RNA priming of mtDNA replication. Consistent with this finding, cybrids (i.e., cells sharing the same nuclear genetic background but differing in their mtDNA backgrounds) harboring haplogroup J mtDNA had a >2 fold increase in mtDNA copy number, as compared to cybrids containing haplogroup H, with no apparent differences in steady state levels of mtDNA-encoded transcripts. Hence, a haplogroup J regulatory region mutation affects mtDNA replication or stability, which may partially account for the phenotypic impact of this haplogroup. Our analysis thus demonstrates, for the first time, the functional impact of particular mtDNA haplogroup-defining control region mutations, paving the path towards assessing the functionality of both fixed and un-fixed genetic variants in the mitochondrial genome.

Partial Text: Mitochondria are the major sources for cellular energy, through the process of oxidative phosphorylation (OXPHOS), and thus play a central role in cell life and death. Since mitochondrial DNA (mtDNA) encodes 13 essential proteins of the energy production apparatus and 24 key factors of their translation machinery (i.e. 22 tRNAs and 2 rRNAs) it is not surprising that numerous association studies have demonstrated the involvement of mtDNA genetic backgrounds (haplotypes, haplogroups) in complex human disorders as well as in selective events that transpired during human evolution [1]. Such implied functional potential of natural mtDNA variants has gained recent experimental support in human and murine cytoplasmic hybrids (cybrids) [2],[3]. Further support was provided by back-cross experiments in Drosophila and rat as well as by inter-populations crosses in Tigriopus Americana revealing that the interaction of mtDNA with the nuclear genetic background is under selective constraint [4],[5],[6]. This implies that mtDNA variants underlying population divergence have phenotypic consequences. Nevertheless, all the above mentioned studies analyzed complete haplotypes, thus masking the effects of single nucleotide changes within haplogroups.

We have screened more than 2500 whole mtDNA sequences from various human populations worldwide ( for natural variants in experimentally established protein binding sites and regulatory regions in the mtDNA control region (Table 1). These include recognition sites for mitochondrial transcription factor A (TFAM) and transcription termination factor (mTERF), the conserved sequence blocks (CSBs), and mitochondrial recognition sites for proteins of the replication machinery (TAS and origin of replication of the light strand – OL), comprising a total of 241 bp in each mtDNA. This screen identified that 77/241 of the total screened sites harbored polymorphic point mutations which can be divided into non-fixed changes (57/77, 74%) and haplogroups/sub-haplogroup-defining changes (i.e. population-fixed changes), comprising 20/77 (26%) of the variable nucleotide positions.

Our results reveal that some genetic variants that became fixed during human evolution affect protein binding to mtDNA. Moreover, a point variant in a TFAM binding-site (C295T) increased in vitro transcription ∼2.5 fold. These findings support the view that some evolutionarily fixed mtDNA variants lead to functional consequences. The C295T TFAM binding-site variant did not clearly align with other mammalian sequences, other than those of the great apes (humans, chimpanzees, gorillas and orangutans). Nevertheless, the C295T nucleotide position underwent only a single fixation event during human phylogeny in the branch leading to haplogroup J, suggesting that, although poorly conserved, the low human variability at this nucleotide position is in line with its functional potential. We thus suggest that the degree of variability of a nucleotide position within humans may serve as a clue for functionality. To experimentally test for the generality of this prediction the functionality of additional variable nucleotide positions should be tested.