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. 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.