Ancient mtDNA Genetic Variants Modulate mtDNA Transcription and Replication

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

Ancient mtDNA Genetic Variants Modulate mtDNA Transcription and Replication

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

Relative steady-state levels of mtDNA transcripts in cybrids harboring different mtDNA haplogroups.

<p>Each color represents the transcription level estimated by real time quantitative PCR (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000474#s4" target="_blank">Materials and Methods</a>) for mtDNA-encoded genes in the mentioned cybrids. Briefly, the measured transcripts levels were normalized to the geometric mean of the three housekeeping genes: GAPDH, β-Actin and β2 microglobulin. Cybrids haplogroup assignment was verified by PCR RFLP of selected polymorphic sites and sequencing and HVR1 and HVR2: cybrids 3861 and 1106 belong to haplogroup J1 and J1b2, respectively, while cybrids CB, LR, NN, SB and TL belong to haplogroup H. Notice that the transcript level of ND1 and ND4L genes were significantly lower in one of the haplogroup J1 cybrids (3861), and that the ND3 transcript level was lower in one of haplogroup H transcripts (NN).</p

Run-off <i>in vitro</i> transcription assays preformed using mtDNA templates with or without TFAM binding site variants.

<p>A. A schematic map of the mtDNA templates used in the run-off <i>in vitro</i> transcription assay, with the TFAM-binding sites (striped rectangles), light-strand promoter (LSP, bent arrow), and the location of the 242 and 295 variants (arrows) indicated. B. A representative in vitro transcription reaction using templates containing the indicated mtDNA variants. Equal amounts of the linear mtDNA templates (ethidium bromide-stained at the bottom) were used in the <i>in vitro</i> transcription assay with a partially purified POLRMT (i.e. mtRNA polymerase) fraction from HeLa cells. The 223-nt full-length run-off transcript and a second major, shorter transcript of ∼160 nt (see Discussion) are indicated. Quantification of the 223-nt, full-length transcript and the ∼160-nt truncated product from multiple independent experiments is shown in C. and D., respectively. Both mutant templates showed a trend toward increased LSP transcription activity when either the full-length or shorter product was analyzed, but only in the case of shorter, ∼160 transcript from the haplogroup J template (C295T) was this difference statistically significant (p-value 0.034).</p

Primers used for the real time PCR-based estimation of transcript levels and mtDNA copy numbers.

<p>Lines 1–30 (excluding column titles): primers used for Real Time PCR-based analysis of mtDNA transcripts levels in cybrids. Lines 31–34: primers used for Real Time PCR-based analysis of mtDNA copy number in cybrids.</p

Increased TFAM binding to oligonucleotide probes harboring the C295T mtDNA variant.

<p>A. EMSA analysis of TFAM binding to double-stranded oligonucleotide probes containing the C295T mtDNA variant compared to its “wild-type” control (wt-295). Shown are autoradiograms of representative EMSA gels performed at a 10∶1 (TFAM 10×) and 25∶1 (TFAM 25×) TFAM∶probe molar ratio. The locations of the free and TFAM-bound (shifted) probe are indicated. B. Graphical representation and statistical analysis of the gels in A. The data of binding in the C295T variant is normalized to that of the wt-295, which was given a value of 1.0. The mean+/−one standard deviation (error bars) is shown as is the corresponding p-value.</p

Natural genetic variation in selected mtDNA regulatory elements.

<p>The columns represent the number of times that mutation events (change) occurred in a given nucleotide position.</p>*<p>represents fixed changes - mutations that were represented in more than 5 different mtDNA sequences belonging to the same phylogenetic branch (lineage, haplogroup). Letters within the ‘Fixation events’ column correspond to distinct human mtDNA haplogroups.</p

Increased mtDNA copy number in haplogroup J cybrids compared to haplogroup H cybrids.

<p>The copy number of mtDNA was measured using an mtDNA marker (ND2) and a nuclear DNA marker (18S rRNA gene). Data were obtained from three independent measurements for each of the tested cybrids, 2 of haplogroup J and 5 from haplogroup H. Standard deviation is shown for the mtDNA/nDNA ratios obtained from haplogroup J and haplogroup H cybrids.</p

List of double-stranded oligonucleotides containing the mtDNA TFAM-binding sites.

<p>Variants within two of the TFAM-binding sites (position 233–260 and 276–303) are underlined in bold. Type of mtDNA genetic background is shown in parentheses in the first column.</p

Schematic representation of the D-loop regulatory region.

<p>The names of regulatory motifs are indicated: the three conserved sequence blocks (CSB1, CSB2, and CSB3), light-strand promoter (LSP), heavy-strand promoter (HSP), the conserved termination-associated sequence (TAS), and origin of H-strand DNA replication (O_H). Blue rectangles represent TFAM-binding sites and the red rectangle represents the mTERF binding site. The arrows pointing left and right show transcription orientation of H (H1 and H2) and L strands, respectively. The location of ribosomal RNA genes (12S and 16S) and the genes encoding tRNAs for phenylalanine, valine and leucine (F, V, and L, respectively) is indicated.</p