Diagnosis and molecular bases of mitocondrial diseases due to dysfunctions in the protein transport to mitochondria's

Abstract

Objective: Identification of the key transcription factors that regulate the expression of human TIMM8A gene. Background: TIMM8A belongs to a family of evolutionary conserved proteins located in the mitochondrial space involved in mediating import and insertion of hydrophobic membrane proteins into the mitochondrial inner membrane. The purpose of this study was to look at the promoter sequence of the TIMM8A gene and determine which transcription factors play a vital role in the successful transcription of the TIMM8A gene. Methods: Using the promoter region of the TIMM8A gene, gene reporter constructs with serial deletions and site-directed mutagenesis of the promoter region were developed and amplified via PCR. The PCR constructs were then ligated to the pGL3-basic vector and transformated to competent cells for growth. Plasmids were isolated via minipreps and proper insertion verified by restriction digestion and gel electrophoresis. The plasmids were then transfected to HeLa S3 and A204 cells and luciferase activity was recorded. Results: Luciferase showed a major decrease in activity between constructs 7 (-101) and 8 (-29) indicating that construct 7 contains the binding site(s) for the transcription factor(s) regulating the expression of TIMM8A but construct 8 lacks this(these) factor(s). In fact, binding sites for NRF-1 and Sp1 are in the promoter region encompassed by construct 7. Conclusions: We obtained preliminary results indicating that NRF-1 and Sp1 are the major transcription factors involved in the regulation of human TIMM8A expression. New constructs and other experiments (EMSAs, ChIP…) are necessary to precisely identify the role of these factors. Additional constructs were developed around the area of interest.