ChIP analysis trial of human TFB2M and POLRMT interaction with mtDNA

In many mammalian tissues and cells, two classes of mitochondrial DNA (mtDNA) replication intermediates have been observed. One involves leading-strand synthesis in the absence of synchronous lagging-strand synthesis (strand-asynchronous replication), and the other has properties of coupled leading- and lagging-strand synthesis (strand-coupled replication). While strand-asynchronous replication is primed by long RNA synthesized from a defined transcription initiation site, little is known about the commencement of strand-coupled replication. To investigate it, we attempted to abolish strand-asynchronous replication in cultured human cybrid cells by knocking out the components of the transcription initiation complexes, mitochondrial transcription factor B2 (TFB2M/mtTFB2) and mitochondrial RNA polymerase (POLRMT/mtRNAP). Surprisingly, removal of either protein resulted in the complete mtDNA loss, demonstrating for the first time that TFB2M and POLRMT are indispensable for human mtDNA maintenance. Moreover, the lack of TFB2M could not be compensated for by mitochondrial transcription factor B1 (TFB1M/mtTFB1). These findings indicate that TFB2M and POLRMT are crucial for the priming of not only strand-asynchronous but also strand-coupled replication, providing deeper insights into the molecular basis of strand-coupled replication initiation. Overall design: After 1% formaldehyde crosslinking of cells (containing mitochondria), immunoprecipitation was carried out. DNA fragments obtained were subjected to deep sequencing to examine the binding sites for TFB2M (a mitochondorial transcription initiation factor) and POLRMT (the mitochondorial RNA polymerase). Although the obtained data showed strong enrichment of ChIP reads in the NCR for TFB2M, POLRMT, and HA (for HA-tagged TFB2M), such enrichment was also seen in the respective negative cntorol samples (control IgG). Therefore, we note that the ChIP-seq data may contain artificial enrichments with unknwon reasons.

在众多哺乳动物组织与细胞中，已观测到两类线粒体DNA（mitochondrial DNA, mtDNA）复制中间体。其一为前导链合成与滞后链合成不同步的复制模式（链异步复制，strand-asynchronous replication），其二则具备前导链与滞后链偶联合成的特征（链偶联复制，strand-coupled replication）。目前，链异步复制的起始由特定转录起始位点合成的长RNA引物所介导，而人们对链偶联复制的起始机制却知之甚少。为探究该机制，我们尝试在培养的人类胞质杂种细胞（cybrid cells）中敲除转录起始复合物组分：线粒体转录因子B2（mitochondrial transcription factor B2, TFB2M/mtTFB2）与线粒体RNA聚合酶（mitochondrial RNA polymerase, POLRMT/mtRNAP），以阻断链异步复制。令人意外的是，单独移除任一蛋白均会导致线粒体DNA完全丢失，这首次证实了TFB2M与POLRMT对人类线粒体DNA维持不可或缺。此外，线粒体转录因子B1（mitochondrial transcription factor B1, TFB1M/mtTFB1）无法代偿TFB2M的功能。上述发现表明，TFB2M与POLRMT不仅对链异步复制的引物合成至关重要，亦参与链偶联复制的引物起始过程，为解析链偶联复制起始的分子机制提供了更深入的认知。实验整体设计：对含线粒体的细胞进行1%甲醛交联后，开展免疫沉淀实验。将获取的DNA片段进行深度测序，以检测TFB2M（一种线粒体转录起始因子）与POLRMT（线粒体RNA聚合酶）的结合位点。尽管所得数据显示，TFB2M、POLRMT及HA标签的TFB2M的染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq）读段在非编码区（non-coding region, NCR）呈现显著富集，但此类富集现象在对应的阴性对照样本（对照IgG）中亦有出现。因此，本研究提示，本次ChIP-seq数据可能存在未知原因导致的人工富集。