Is the Secret of VDAC Isoforms in Their Gene Regulation? Characterization of Human VDAC Genes Expression Profile, Promoter Activity, and Transcriptional Regulators

Abstract

VDACs (voltage-dependent anion-selective channels) are pore-forming proteins of the outer mitochondrial membrane, whose permeability is primarily due to VDACs’ presence. In higher eukaryotes, three isoforms are raised during the evolution: they have the same exon–intron organization, and the proteins show the same channel-forming activity. We provide a comprehensive analysis of the three human VDAC genes (VDAC1–3), their expression profiles, promoter activity, and potential transcriptional regulators. VDAC isoforms are broadly but also specifically expressed in various human tissues at different levels, with a predominance of VDAC1 and VDAC2 over VDAC3. However, an RNA-seq cap analysis gene expression (CAGE) approach revealed a higher level of transcription activation of VDAC3 gene. We experimentally confirmed this information by reporter assay of VDACs promoter activity. Transcription factor binding sites (TFBSs) distribution in the promoters were investigated. The main regulators common to the three VDAC genes were identified as E2F-myc activator/cell cycle (E2FF), Nuclear respiratory factor 1 (NRF1), Krueppel-like transcription factors (KLFS), E-box binding factors (EBOX) transcription factor family members. All of them are involved in cell cycle and growth, proliferation, differentiation, apoptosis, and metabolism. More transcription factors specific for each VDAC gene isoform were identified, supporting the results in the literature, indicating a general role of VDAC1, as an actor of apoptosis for VDAC2, and the involvement in sex determination and development of VDAC3. For the first time, we propose a comparative analysis of human VDAC promoters to investigate their specific biological functions. Bioinformatics and experimental results confirm the essential role of the VDAC protein family in mitochondrial functionality. Moreover, insights about a specialized function and different regulation mechanisms arise for the three isoform gene.

Renaissance of VDAC: New Insights on a Protein Family at the Interface between Mitochondria and Cytosol

“…The level of the VDAC1 and VDAC2 transcripts is comparable, while VDAC3 is lower than the other two isoforms, confirming previous experimental conclusions drawn by RT-PCR [ 59 ]. While confirming that VDAC isoforms are ubiquitously expressed, the comparison with the data present in a second repository (RNA-seq CAGE RIKEN FANTOM 5’ project) [ 60 ] of Expression Atlas repository of EMBL-EBI [ 61 ] revealed that the VDAC3 expression levels were higher than VDAC2 and VDAC1, whose transcripts are scarcely represented in all tissues [ 57 ]. The data emerging from this analysis highlight for the first time the prevalence of VDAC3 gene transcription as compared to other isoforms reflecting a higher promoter activity.…”

“…The studies of the structure, activity, and regulation of VDAC genes aim at obtaining a reliable picture of their differences in tissue expression or sensitivity to specific stimuli [ 57 ]. In humans, for each VDAC gene, several different transcript splice variants were identified: they did not vary in the coding region but mainly in the length of their 5′-UTR and 3′-UTR.…”

“…The organization of the core promoter is similar to that of most TATA-less human promoters of ubiquitously expressed genes, where the presence of abundant GC regions, alternative binding sites Inr, DPE, and BRE assure a basal levels of transcription. Interestingly, a non-canonical initiation site termed the TCT motif (polypyrimidine initiator), which is a target for translation regulation by the mTOR pathway, oxidative, and metabolic stress [ 62 ], was also identified in VDAC2 and VDAC3 promoters [ 57 ]. Gene reporter assays revealed that VDAC3 promoter had the highest transcriptional activity and VDAC1 promoter was, on the contrary, the least active [ 57 ].…”

“…Interestingly, a non-canonical initiation site termed the TCT motif (polypyrimidine initiator), which is a target for translation regulation by the mTOR pathway, oxidative, and metabolic stress [ 62 ], was also identified in VDAC2 and VDAC3 promoters [ 57 ]. Gene reporter assays revealed that VDAC3 promoter had the highest transcriptional activity and VDAC1 promoter was, on the contrary, the least active [ 57 ]. We proposed that a quantitative regulation of the transcript levels due to their different stability, or to maintain a high level of transcripts to promptly respond to a particular stimulation, is necessary for the cell.…”

“…VDAC2 promoter showed the presence of different factors specially active during the development of specialized tissues and organogenesis processes mainly related to nervous system genesis and growth [ 73 , 74 ]. VDAC3 promoter analyzed in [ 57 ] shows a particular abundance of transcription factors binding sites involved in the development of germinal tissues, organogenesis, and sex determination [ 39 , 75 ]. Converging evidence reported in the literature confirms the crucial role of VDAC isoforms in the specific context where the transcription factors that bind to their promoters exert a function.…”