A 5′- Regulatory Region and Two Coding Region Polymorphisms Modulate Promoter Activity and Gene Expression of the Growth Suppressor Gene ZBED6 in Cattle
Zinc finger, BED-type containing 6 (ZBED6) is an important transcription factor in placental mammals, affecting development, cell proliferation and growth. Polymorphisms in its promoter and coding regions are likely to impact ZBED6 transcription and growth traits. In this study, rapid amplification of 5’ cDNA ends (5'-RACE) analysis revealed two transcription start sites (TSS) for the bovine ZBED6 starting within exon 1 of the ZC3H11A gene (TSS-1) and upstream of the translation start codon of the ZBED6 gene (TSS-2). There was one SNP in the promoter and two missense mutations in the coding region of the bovine ZBED6 by sequencing of the pooled DNA samples (Pool-Seq, n = 100). The promoter and coding region are the key regions for gene function; polymorphisms in these regions can alter gene expression. Quantitative real-time PCR (qPCR) analysis showed that ZBED6 has a broad tissue distribution in cattle and is highly expressed in skeletal muscle. Eleven promoter-detection vectors were constructed, which enabled the cloning of putative promoter sequences and analysis of ZBED6 transcriptional activity by luciferase reporter gene assays. The core region of the basal promoter of bovine ZBED6 is located within region -866 to -556. The activity of WT-826G-pGL3 in driving reporter gene transcription is significantly higher than that of the M-826A-pGL3 construct (P < 0.01). Analysis of gene expression patterns in homozygous full-sibling Chinese Qinchuan cattle showed that the mutant-type Hap-AGG exhibited a lower mRNA level than the wild-type Hap-GCA (P < 0.05) in longissimus dorsi muscle (LDM). Moreover, ZBED6 mRNA expression was low in C2C12 cells overexpressing the mutant-type ZBED6 (pcDNA3.1+-Hap-GG) (P < 0.01). Our results suggest that the polymorphisms in the promoter and coding regions may modulate the promoter activity and gene expression of bovine ZBED6 in the skeletal muscles of these cattle breeds.
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