Ancient mtDNA genetic variants modulate mtDNA transcription and replication.
Ancient mtDNA genetic variants modulate mtDNA transcription and replication.
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DOI：
10.1371/journal.pgen.1000474
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发表时间：
2009-05
期刊：
影响因子：
4.5
通讯作者：
Mishmar D
中科院分区：
文献类型：
--
作者：
Suissa S;Wang Z;Poole J;Wittkopp S;Feder J;Shutt TE;Wallace DC;Shadel GS;Mishmar D
尽管线粒体DNA （mtDNA）遗传背景（单倍型、单倍群）的功能后果已被疾病关联研究和细胞培养实验证明，但尚不清楚单倍群中哪些突变具有功能意义，哪些突变是“进化沉默的搭便车者”。我们开始通过体外转录研究mtDNA转录/复制调控区内单倍群定义突变的功能，假设mtDNA调控基序内发生的单倍群定义突变可能影响这些过程。因此，我们在实验建立的蛋白质结合位点和调控区域（每个mtDNA共241bp）中筛选了代表全世界所有主要人群的bbbb2500个完整的人类mtDNA，以寻找自然变异。我们的筛选显示，241个位点中有77个显示点突变，这些突变可以分为非固定突变（57/ 77,74%）和单倍群/亚单倍群定义突变（即群体固定突变，20/ 77,26%）。定义高加索单倍群J （C295T）的变异增加了TFAM（电迁移转移试验）的结合和体外l链转录的能力，特别是位于保守序列块1 （CSB1）上游的较短转录物，这是一个与RNA启动mtDNA复制相关的区域。与这一发现一致的是，含有单倍群J mtDNA的细胞系（即具有相同核遗传背景但mtDNA背景不同的细胞）与含有单倍群H的细胞系相比，mtDNA拷贝数增加了50倍，而mtDNA编码转录物的稳态水平没有明显差异。因此，单倍群J调控区突变影响mtDNA复制或稳定性，这可能部分解释了该单倍群的表型影响。因此，我们的分析首次证明了特定mtDNA单倍群定义控制区突变的功能影响，为评估线粒体基因组中固定和非固定遗传变异的功能铺平了道路。线粒体是细胞的“发电厂”，它有自己独特的基因组（mtDNA），其序列因全球个体而异。这种变异是由进化过程中突变（变异）的积累形成的，似乎改变了对常见复杂疾病（如帕金森病和糖尿病）的易感性。然而，由于随着时间的推移mtDNA突变的积累会导致新的组合（遗传背景）的形成，因此尚不清楚哪些突变是功能性的，哪些是“进化沉默的搭便车者”。因此，我们旨在评估mtDNA遗传变异的功能，重点关注mtDNA调控区域内的变异，并假设它们可能影响mtDNA的活性和维持。我们发现，与其他遗传背景相比，定义mtDNA遗传背景“J”的变异显著提高了细胞中的转录效率和mtDNA拷贝数。因此，mtDNA调控区变异可以影响mtDNA的维持，这可能部分解释了这种遗传背景与疾病易感性的关系。我们的分析首次证明了在进化过程中固定的特定mtDNA变体的功能影响。此外，我们的研究结果强调了mtDNA变体在进化可变调控区域的功能。
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. Mitochondria, the ‘power plant’ of the cell, have their own distinct genome (mtDNA), whose sequence varies among individuals around the globe. This variation, which was formed by the accumulation of mutations (variants) during the course of evolution, appears to alter the susceptibility to common complex diseases (such as Parkinson's disease and diabetes). However, since the accumulation of mtDNA mutations over time results in the formation of new combinations (genetic backgrounds), it is not clear which of the mutations are functional and which are “evolutionary silent hitchhikers”. Thus we aimed at assessing the functionality of mtDNA genetic variants, focusing on variants within the mtDNA regulatory region, hypothesizing that they could affect mtDNA activity and maintenance. We found that a variant defining mtDNA genetic background ‘J’ significantly increased the transcriptional efficiency and elevated mtDNA copy numbers in cells, as compared to other genetic backgrounds. Hence, mtDNA regulatory region variants can affect mtDNA maintenance, which may partially account for the involvement of this genetic background in disease susceptibility. Our analysis demonstrates, for the first time, the functional impact of a particular mtDNA variant that was fixed during evolution. Moreover, our findings underline the functionality of mtDNA variants in the evolutionary variable regulatory region.
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