KLF4, a Key Regulator of a Transitive Triplet, Acts on the TGF-β Signaling Pathway and Contributes to High-Altitude Adaptation of Tibetan Pigs

Abstract

Tibetan pigs are native mammalian species on the Tibetan Plateau that have evolved distinct physiological traits that allow them to tolerate high-altitude hypoxic environments. However, the genetic mechanism underlying this adaptation remains elusive. Here, based on multitissue transcriptional data from high-altitude Tibetan pigs and low-altitude Rongchang pigs, we performed a weighted correlation network analysis (WGCNA) and identified key modules related to these tissues. Complex network analysis and bioinformatics analysis were integrated to identify key genes and three-node network motifs. We found that among the six tissues (muscle, liver, heart, spleen, kidneys, and lungs), lung tissue may be the key organs for Tibetan pigs to adapt to hypoxic environment. In the lung tissue of Tibetan pigs, we identified KLF4, BCL6B, EGR1, EPAS1, SMAD6, SMAD7, KDR, ATOH8, and CCN1 genes as potential regulators of hypoxia adaption. We found that KLF4 and EGR1 genes might simultaneously regulate the BCL6B gene, forming a KLF4-EGR1-BCL6B complex. This complex, dominated by KLF4, may enhance the hypoxia tolerance of Tibetan pigs by mediating the TGF-β signaling pathway. The complex may also affect the PI3K-Akt signaling pathway, which plays an important role in angiogenesis caused by hypoxia. Therefore, we postulate that the KLF4-EGR1-BCL6B complex may be beneficial for Tibetan pigs to survive better in the hypoxia environments. Although further molecular experiments and independent large-scale studies are needed to verify our findings, these findings may provide new details of the regulatory architecture of hypoxia-adaptive genes and are valuable for understanding the genetic mechanism of hypoxic adaptation in mammals.

Keywords: Tibetan pig; gene network; hypoxia adaptation; multitissue; transcriptome.

FIGURE 1

Schematic diagram of feedforward loop.

FIGURE 2

Clustering dendrogram of 36 tissue samples of Tibetan pigs and Rongchang pigs. The figure shows the clustering of a total of 36 tissue samples of Tibetan pigs and Rongchang pigs, where “T” represents Tibetan pigs, and “R” represents Rongchang pigs. For example, “T_muscle1” represents the muscle sample of the first individual Tibetan pig.

FIGURE 3

Weighted gene co-expression network analysis of Tibetan pigs. (A) Network topology of different soft-thresholding power of Tibetan pig co-expression network. The left panel displays the influence of soft-thresholding power (x-axis) on scale-free fit index (y-axis). The right panel shows the influence of soft-thresholding power (x-axis) on the mean connectivity (degree, y-axis). (B) Gene clustering module of Tibetan pig co-expression network. The dissimilarity was based on topological overlap. The “Merged dynamic” is the result of merging modules with a correlation higher than 0.9. The y-axis is the distance determined by the extent of topological overlap. (C) Heatmap of the correlation between module eigengenes and the six tissues of Tibetan pigs. The x-axis is the six tissues of Tibetan pigs, and the y-axis is the module eigengene (ME). In the heatmap, red represents high adjacency (positive correlation), and blue represents low adjacency (negative correlation). In brackets is the p-value of the correlation test.

FIGURE 4

Multitissue expression patterns of genes in key modules of lung tissue of two pig breeds. (A) Multitissue expression patterns of key module genes in Tibetan pig lung tissues. (B) Multitissue expression patterns of key module genes in Rongchang pig lung tissues. The 1, 2, 3, 4, 5, and 6 in the figure represent the muscle, liver, heart, spleen, kidneys, and lung tissues, respectively. The yellow- or green-colored lines correspond to genes with low membership value; the red- and purple-colored lines correspond to genes with high membership value.

FIGURE 5

Gene regulatory network of six tissues of Tibetan pigs. In each network in the figure, the yellow dots represent TFs, the green dots represent miRNAs, and the hub genes are represented by triangles. The red edges with arrows represent the regulatory relationship between TFs and miRNAs and target genes. The gray edge indicates that there is only a co-expression relationship between the two genes.

FIGURE 6

The triad significance profile (TSP) of Tibetan pig lung gene regulatory network. The ordinate in the figure is the normalized Z value, and the abscissa is the 13 motif types. The point marked with “*” is that the frequency of the corresponding motif in the lung tissue gene regulatory network is significantly different from that of random networks (p < 1E-04). The motifs are FFL (7), Regulated mutual (9), Regulating mutual (10), and Clique (13) in that order.

FIGURE 7

The “KLF4–EGR1–BCL6B” complex and their regulated genes in Tibetan pig lung tissues. The complex formed by KLF4–EGR1–BCL6B regulates the EPAS1, SMAD6, SMAD7, KDR, ATOH8, and CCN1 genes and mediates the TGF-β and PI3K-Akt signaling pathways by regulating SMAD6, SMAD7, and KDR genes, respectively. The green edge in the figure represents regulation, and the red edge represents inhibition.

FIGURE 8

Three other groups (Diqing Tibetan pig, Tibetan sheep, and yak) verification network. (A–C) The verification results of the Diqing Tibetan pig, Tibetan sheep, and yak, respectively. The peach-colored rectangle in the middle part of each subgraph represents the KLF4–EGR1–BCL6B complex, the sky blue circle represents the target gene, and the white circle represents that this gene is not included in the data set. The solid green line in the figure represents the edge we verified, and the dotted line represents the unverified edge.