A Novel 6-bp Repeat Unit (6-bp RU) of the 13th Intron Within the Conserved EPAS1 Gene in Plateau Pika Is Capable of Altering Enhancer Activity

Abstract

The plateau pika (pl-pika), a resilient mammal of the Qinghai-Tibet Plateau, exhibits remarkable adaptations to extreme conditions. This study delves into mutations within the Endothelial PAS Domain Protein 1 (EPAS1) gene, crucial for high-altitude survival. Surprisingly, a novel 6-bp insertion/deletion (indel) mutation in EPAS1's Intron 13, along with an additional repeat unit downstream, was discovered during PCR amplification. Genetic analysis across altitude gradients revealed a correlation between this indel's frequency and altitude, hinting at its role in altitude adaptation. Fluorescence enzyme assays unveiled enhancer activity within Intron 13, where the deletion of repeat units led to increased activity, indicating potential transcription factor binding. Notably, GCM1 emerged as a candidate transcription factor binding to the indel site, suggesting its involvement in EPAS1 regulation. These findings enrich our comprehension of high-altitude adaptation in plateau pikas, shedding light on the intricate interplay between genetic mutations, transcriptional regulation, and environmental pressures in evolutionary biology.

Keywords: EPAS1 gene; enhancer; indel; plateau pika; transcription factor.

Figure 1

Genotype identification of mutation sites on the pl-pikas EPAS1 gene: (A) The reported G/A mutation at the 5′ splice site of Intron 14 is of the AA genotype in all tested pl-pikas, suggestive of the absence of circadian rhythm. (B) Sequencing profile showing a 6-bp deletion mutation in Intron 13 of the pl-pikas EPAS1 gene. (C) Agarose gel electrophoresis reveals the presence of three genotypes (II, ID, and DD) for a 6-bp indel site identified in pl-pikas. Lanes 1, 3, and 5 represent the II genotype, lane 2 represents the DD genotype, lanes 4 and 6 represent the ID genotype, and lane 7 represents the Marker 2000.

Figure 2

The EPAS1 6-bp indel mutation of pl-pikas are differentially distributed between the low-altitude group (3–4 km) and the high-altitude group (4–5 km): (A) Distribution of I and D alleles at the 6-bp indel mutation site in high-altitude and low-altitude groups. (B) Independent chi-square test showing the differential distribution of genotypes at the 6-bp indel mutation site in high-altitude and low-altitude groups. (C) The relative expression levels of the EPAS1 gene between individuals with the II genotype in the low-altitude group and individuals with the DD genotype in the high-altitude group.

Figure 3

The enhancer activity of the 13th intron of the EPAS1 gene and the transcription factor binding analysis: (A) Dual-luciferase enhancer reporter gene assays are performed on the Wild type and different deletion sequences of the EPAS1 gene’s 13th intron. # and ## represent p < 0.05 and p < 0.01, respectively, compared to the Wild group. (B) Transcription factors binding to the 6-bp indel site are predicted using Animal TFDB and JASPAR databases. (C) The targeted relationship evaluation between GCM1 transcription factor and different deletion types of the EPAS1 sequence. # represents p < 0.05 compared to the pcDNA3.1 + Wild group; *** and **** represent p < 0.001 and p < 0.0001, respectively, compared to the pcDNA3.1-GCM1 + Wild group; ns indicates no significant difference (p > 0.05).