Transcriptional regulation of TFAP2A on glomerulosclerosis-related gene ADCK4

doi:10.11958/20231028

Abstract

Abstract:

Objective To exploring the mechanism of transcriptional regulation of TFAP2A on glomerulosclerosis-related gene aarF structural domain-containing kinase 4 (ADCK4) in cells and the specific binding region between TFAP2A and ADCK4. Methods Bioinformatics was used to analyze volcano map of glomerulosclerosis genes, and the relationship between ADCK4 and TFAP2A expression levels. JASPAR database was used to predict that ADCK4 gene transcription start site -464 bp/+206 bp region containing TFAP2A transcription factor binding site. TFAP2A siRNA concentrations were 5, 10 and 15 μmol/L, and the mass concentrations of TFAP2A overexpression plasmid were 50, 100 and 300 μg/L, respectively. The regulatory effect of TFAP2A on the promoter level of the ADCK4 gene was verified by dual-luciferase reporter gene assay. TFAP2A siRNA and TFAP2A overexpression plasmid were used to transfect cells. Real-time fluorescence quantitative PCR was used to detect TFAP2A and ADCK4 mRNA expression. Protein immunoblotting assay was used to detect TFAP2A and ADCK4 protein expression. Chromatin immunoprecipitation assay was used to confirm TFAP2A binding to a specific region of the ADCK4 promoter. Results Bioinformatics analysis showed that 273 genes were up-regulated and 219 genes were down-regulated. The expression levels of ADCK4 and TFAP2A were positively correlated (P<0.01). Dual luciferase reporter gene assay demonstrated that the relative luciferase activity of ADCK4 promoter was enhanced with TFAP2A siRNA concentrations of 10 and 15 μmol/L, and that the luciferase activity of ADCK4 promoter was reduced when TFAP2A overexpression plasmid mass concentrations of 100 and 300 μg/L (P < 0.05). ADCK4 mRNA and protein expression levels were increased in the TFAP2A siRNA group compared with the control siRNA group. ADCK4 mRNA and protein expression levels were decreased in the TFAP2A overexpression plasmid group compared with the pENTER plasmid group (P<0.05). Chromatin immunoprecipitation assay revealed that TFAP2A can bind to specific regions of ADCK4 promoter. Conclusion Negative regulation of ADCK4 gene expression by the transcription factor TFAP2A increases the number of transcription factor members that regulate genes important for podocytes.

Key words: glomerulosclerosis, focal segmental, transcription factor AP-2, promoter region, genetic, transcriptional regulation, aarF structural domain-containing kinase 4

CLC Number:

R726.92

Figures/Tables 11

Tab.1 Small interference RNA sequences

基因名称	序列（5'→3'）
TFAP2A siRNA	上游：CCUGCUCACAUCACUAGUATT
TFAP2A siRNA	下游：UACUAGUGAUGUGAGCAGGTT
control siRNA	上游：UUCUCCGAACGUGUCACGU
control siRNA	下游：ACGUGACACGUUCGGAGAATT

Tab.2 qRT-PCR primers and ChIP primers

基因名称	引物序列（5'→3'）	产物大小/bp
GAPDH	上游：ATGACATCAAGAAGGTGGTG	177
GAPDH	下游：CATACCAGGAAATGAGCTTG	177
ADCK4	上游：TTCCGATTCATGCAGACTGA	182
ADCK4	下游：CTGGACTTCTGCAGGACACA	182
TFAP2A	上游：ATTGACCTACAGTGCCCAGC	118
TFAP2A	下游：TCCATGAAAATGCTTTGGAA	118
ChIP-ADCK4	上游：GTGACGGTGGACAAAGTAAACA	303
ChIP-ADCK4	下游：AATCTGAAGAACACTGGGATAGG	303
ChIP-GAPDH	上游：TACTAGCGGTTTTACGGGCG	166
ChIP-GAPDH	下游：TCGAACAGGAGGAGCAGAGAGCGA	166

Fig.1 Bioinformatics analysis of glomerulosclerosis gene expression volcano map

Fig.2 JASPAR database predicting two TFAP2A transcription factor binding sites

Fig.3 Comparison of luciferase activity of TFAP2A siRNA with different concentrations

Fig.4 Comparison of luciferase activity of TFAP2A overexpression plasmids with different concentrations

Fig.5 Effects of TFAP2A siRNA on the expression of TFAP2A and ADCK4 mRNA

Fig.6 Effects of TFAP2A overexpression on TFAP2A and ADCK4 mRNA expression

Fig.7 Expression levels of ADCK4 and TFAP2A protein detected by Western blot assay

Tab.3 Changed expression levels of TFAP2A protein and ADCK4 protein after small interference and overexpression

组别	TFAP2A	t	ADCK4	t
对照组	1.00±0.21	2.936^＊	1.00±0.07	3.040^＊
实验组	0.37±0.03	2.936^＊	1.37±0.01	3.040^＊
过表达对照组	1.00±0.06	2.917^＊	1.00±0.08	9.409^＊
过表达实验组	1.55±0.18	2.917^＊	0.25±0.02	9.409^＊

Fig.8 TFAP2A binded directly to the ADCK4 promoter region

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