Upregulation of TFAM and mitochondria copy number in human lymphoblastoid cells

Highlights

• Hyper diploid copies TFAM in LCLs when compared to non-transformed cells.

• Increased mtDNA copy number in LCL

• Increased mitochondrial mass, intracellular ROS and membrane potential in the LCL

• Elevated mitochondrial biogenesis during EBV mediated cell transformation

Abstract

Mitochondria are central to several physiological and pathological conditions in humans. In the present study, we performed copy number analysis of nuclear encoded mitochondrial genes, in peripheral blood mononuclear cells (PBMCs) and its representative lymphoblastoid cells (LCLs). We have observed hyper diploid copies of mitochondrial transcription factor A (TFAM) gene in the LCLs along with increased mtDNA copy number, mitochondrial mass, intracellular ROS and mitochondrial membrane potential, suggesting elevated mitochondrial biogenesis in LCLs. Gene expression analysis confirmed TFAM over-expression in LCLs when compared to PBMC. Based on our observation, we suggest that increased copy number of TFAM gene upregulates its expression, increases mtDNA copy numbers and protects it from oxidative stress induced damage in the transformed LCLs.

Introduction

Mitochondria, an essential component of the eukaryotic cell, involved in a number of cellular events such as oxidative phosphorylation, calcium signaling, and apoptosis (Legros et al., 2004). The composition, architecture and the significance of human mitochondrial DNA (mtDNA) has been reviewed at length (Iborra et al., 2004). Mitochondrial genome has several unique features that include high copy number, maternal inheritance and higher mutation rate as compared to nuclear DNA. The first mitochondrial genome sequence was published in the year 1981 (Anderson et al., 1981). Since then, several investigators have published the whole mitochondria genome sequences for identification of novel mutations in age related degenerative diseases, cancers and for evolutionary studies (Filosto et al., 2011, Roshan et al., 2012, Thangaraj et al., 2006, Zheng et al., 2012). EBV (Epstein-Barr virus) transformed lymphoblastoid cell lines (LCLs) provides a convenient alternative for genome wide association studies due to its replicability and ease of performing functional genomic studies (Redon et al., 2006, Sie et al., 2009, Simon-Sanchez et al., 2007). However, there is a growing concern about potential genomic changes that can be introduced as a result of cell transformation and their possible impact on the genetic studies (Jeon et al., 2007, Redon et al., 2006). Recently, studies have shown that creation of lymphoblastoid cell lines through EBV induced transformation induces changes in DNA methylation, gene expression patterns and copy number variations (CNVs) (Aberg et al., 2012, Caliskan et al., 2011, Jeon et al., 2007, Londin et al., 2011, Shirley et al., 2012). Previously, we have shown that LCLs do not show gross aneuploidies and accumulate up to 56.6 Mb (1.82%) of changes in nuclear DNA represented through copy number variations (CNVs) (Chakrabarty et al., 2012). Simultaneously, we have observed a significant increase in copy number in the nuclear-encoded mitochondrial genes exclusively in the LCL genome when compared with CNVs identified in peripheral blood mononuclear cells (PBMC) DNA from the same individual (Chakrabarty et al., 2012). TFAM, a nuclear encoded mitochondria specific transcription factor, reported to activate transcription of genes within mitochondria (Campbell et al., 2012, Hallberg and Larsson, 2011, Kelly and Scarpulla, 2004). TFAM binds with higher affinity to sequences around both mitochondrial promoter (P_L and P_H) in the D-loop of mtDNA and appears to be ubiquitously expressed (Garrido et al., 2003). TFAM has been implicated in the maintenance of mitochondrial structure and function in number of studies (Ekstrand et al., 2004, Takamatsu et al., 2002). In cancer cells, TFAM truncating mutation and altered protein levels has been correlated with mtDNA depletion (Guo et al., 2011). Earlier, studies have shown TFAM gene over expression in LCLs when compared with PBMCs (Jeon et al., 2007). In our study, we have investigated the role of TFAM and regulation of mitochondrial copy number in PBMCs and LCLs.