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. 2020 Oct 7;21(19):7388.
doi: 10.3390/ijms21197388.

Is the secret of VDAC Isoforms in their gene regulation? Characterization of human VDAC genes expression profile, promoter activity, and transcriptional regulators

Federica Zinghirino  1 , Xena Giada Pappalardo  1 , Angela Messina  2   3   4 , Francesca Guarino  1   3   4 , Vito De Pinto  1   3   4
Affiliations

Is the secret of VDAC Isoforms in their gene regulation? Characterization of human VDAC genes expression profile, promoter activity, and transcriptional regulators

Federica Zinghirino et al. Int J Mol Sci. .

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.

Keywords: VDAC isoforms; core promoter; expression profile; gene structure; mitochondrial function; transcription factor binding sites.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Human VDAC (voltage-dependent anion-selective channels) isoforms gene structures and functions. Overview of the gene structure of human VDAC isoforms and their most relevant functional and structural sites. (a) hVDAC1 gene location on Chr5:133,340,230-133,340,830 (GRCh37/hg19) from UCSC Genome Browser; (b) hVDAC2 gene location on Chr10:76,970,184-76,970,784; hg19; (c) hVDAC3 gene location on Chr8:42,248,998-42,249,598; hg19. In each panel, a black box encloses the 600 bp promoter sequence indicated as PVDAC1, PVDAC2, and PVDAC3, respectively, and aligned with the annotated sequence from EPDnew (v.006). This allows the profile of transcriptional activity of the gene promoter region to be highlighted by CpG island identification, levels of enrichment of the H3K4me1 and H3K4me3 histone marks, and RNA Pol2 and Chromatin State Segmentation ChIP-seq data. Functional elements of Chromatin state segmentation by HMM of nine different cell lines are identified using different colors as follows: bright red: active promoter; light red: week promoter; orange: strong enhancer; yellow: weak/poised enhancer; blue: insulator; dark green: transcriptional transition/elongation; light green: transcriptional transcribed.
Figure 2
Figure 2
Human VDAC genes expression by Genotype-Tissues-Expression (GTEx) database. RNA-seq data from Genotype-Tissues-Expression (GTEx) project were collected from the Expression Atlas repository, where all data are manually curated and subject to standardized analysis pipelines. The data are displayed in a heatmap (a) with different colors representing a range of transcripts per kilobase million (TPM) mRNA expression levels. The range of expression levels reported for VDAC genes is between 0 and 1280 TPM, as indicated by the bar. In panel (b), the specific expression values of each human VDAC isoforms, for representative tissues, are reported.
Figure 3
Figure 3
Human VDAC genes expression by RIKEN functional annotation of the mammalian genome 5 (RIKEN FANTOM 5) project. RNA-seq cap analysis gene expression (CAGE) data from the RIKEN FANTOM 5 project was collected from the Expression Atlas repository, where all data are manually curated and subject to standardized analysis pipelines. The data are displayed in a heatmap (a) with different colors representing a range of TPM mRNA expression levels. In Panel (b), the specific expression values of each human VDAC isoform, for representative tissues, are reported.
Figure 4
Figure 4
Experimental analysis of human VDAC gene expression and their promoter activity in HeLa cells. (a) Human VDAC genes expression. VDAC genes expression was detected by Real-Time PCR as described in methods. After normalization with the housekeeping gene β-actin, the variation of VDAC2 and VDAC3 transcripts was expressed using human VDAC1 as reference. The ΔΔCt method was applied. (b) VDAC promoter activity detection. To study the promoter activity, 600 bp sequence encompassing the transcription start site (TSS) (from −400 to +200 in the gene sequence) was placed upstream of the luciferase gene in pGL3 plasmid. The assay was performed in HeLa cells transfected with PVDAC1-pGL3, PVDAC2-pGL3, PVDAC3-pGL3 constructs after 48 h of transfection. Luciferase activity of cell lysates was calculated by referring to empty-pGL3 transfected cells and following normalization with Renilla activity. Three independent experiments were performed and results statistically analyzed by one-way ANOVA. A value of p < 0.05 was taken as significant. Significance was determined as reported and indicated as p < 0.05, * p < 0.01, and *** p < 0.001.
Figure 5
Figure 5
Canonical core promoter elements of human VDAC gene isoforms. The results of core promoter elements identified for hVDAC1, hVDAC2, and hVDAC3 genes by predictive tools are the sequence stretches with a high scoring consensus based on position weight matrix (PWM). (a) PVDAC1 (chr5:133,340,230-133,340,830; hg19) encompassed an Inr element (at −261 bp), two GC-boxes (at −85 bp; −49 bp), five B recognition element (BRE) motifs (at −255 bp; −217 bp; −78 bp; −42 bp; −27 bp), and two downstream promoter element (DPE) motifs (at −298 bp; −266). (b) PVDAC2 (chr10:76,970,184-76,970,784; hg19) a Polypyrimidine initiator (TCT) motif, as an alternative Inr (at +2 bp), Inr element (at +8 bp), two BRE motifs (at + 93 bp; +119 bp), a DPE motif (at −173 bp). (c) PVDAC3 (chr8:42,248,998-42,249,598; hg19) encompasses three Initiator element (Inr) elements (at −205 bp; −8 bp; +77 bp), a TCT (at −329 bp), a DPE (at −1 bp) and a BRE (at +170 bp). TSS site is indicated by a red arrow. Nucleotide sequence before TSS is shown in lowercase.
Figure 6
Figure 6
Identification of ChIP-seq peak regions in the human VDAC promoters. (a) The histogram shows the number of transcription factor binding sites (TFBS) experimentally validated by ChIP-Seq data (ENCODE project v3) among those predicted by the software Genomatix (MatInspector) in PVDAC1, PVDAC2, and PVDAC3 sequences. (b) Venn diagram showing the number of common and unique predicted binding sites that overlap with a ChIP-Seq region in PVDAC1, PVDAC2, and PVDAC3, based on Genomatix analysis.
Figure 7
Figure 7
Identification of common and unique transcription factor binding site clusters of VDACs promoter sequences. Distribution of a common set (enclosed in a blue box) and specific sets (enclosed in a red box) of Transcription Factor Binding Sites (TFBSs) in VDAC isoforms promoters as reported in different cell lines by ChIP-Seq analysis in the ENCODE Project (shown as vertical color bars in the gray segments). (a) hVDAC1, (b) hVDAC2, and (c) hVDAC3.
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