Integrative Analysis of Methylation and Transcriptional Profiles to Reveal the Genetic Stability of Cashmere Traits in the Tβ4 Overexpression of Cashmere Goats

Abstract

DNA methylation alteration is frequently observed in exogenous gene silencing and may play important roles in the genetic stability of traits. Cashmere is derived from the secondary hair follicles (SHFs) of cashmere goats, which are morphogenetically distinct from primary hair follicles (PHFs). Here, in light of having initially produced 15 Tβ4 overexpression (Tβ4-OE) cashmere goats which had more SHFs than the wild type (WT) goats, and produced more cashmere, we produced Tβ4-OE offsprings both via somatic cell nuclear transfer (SCNT) and via natural mating (NM). However, the desired trait exhibited lower fixation in the line-bred offspring compared to the SCNT offspring. Integrative analysis of methylation and transcriptional profiles showed that this might be due to the influence of methylation on the expression of differentially expressed genes (DEGs) between generations, which was mutually consistent with the results of the functional and pathway enrichment analysis of differentially methylated regions (DMRs) and DEGs. Overall, our study systematically describes the DNA methylation characteristics between generations of cashmere goats and provides a basis for improving genetic stability.

Keywords: Tβ4; genetic stability; methylation; somatic cell nuclear transfer; transcriptome.

Figure 1

Establishment of the thymosin β4 overexpression (Tβ4-OE) germline. (A) A schematic showing germline transmission. (B) Tβ4-OE first filial generation of 2014 (F1-1, a total of 11; via NM, F1-1-NM, a total of 9; via SCNT, F1-1-SCNT, a total of 2), and 2015 (F1-2, a total of 12; via NM, F1-2-NM, a total of 8; via SCNT, F1-2-SCNT, a total of 8). (C) PCR genotyping, showing the insertion of donor DNA in the Tβ4-OE goats. SCNT: offspring produced by somatic nuclear transfer: NM: offsprings produced by natural mating. (D) Body weights of the F1-1 and F1-2 from birth to four years old. CON: control. (E) Physiological indexes of the F1-1 and F1-2. Analytical measurements included routine blood count (white blood cell (WBC), red blood cells (RBC), hemoglobin (HGB) and, platelet (PLT)), liver function (gamma-glutamyltransferase (GGT), aspartate aminotransferase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), total protein (TPROT), albumin (ALB), globulin (GLOB), and total bilirubin (TBIL)) and renal function tests (blood urea nitrogen (BUN), serum creatinine (CRE), and blood uric acid (URIC).

Figure 2

Comparison of Tβ4 expression in the hair follicles of Tβ4-OE first filial generation. (A) Immunohistochemical (IHC) staining of Tβ4 expression in representative samples of hair follicles; hair follicles of F1-1 and F1-2 cashmere goats are shown; F1-1, Tβ4-OE first filial generation of 2014; F1-2, Tβ4-OE first filial generation of 2015 (B) qPCR and (C) western blot showing Tβ4 expression in the skin of F1-1 and F1-2 cashmere goats. Asterisks indicate that the mean expression was significantly different from the control (*, p < 0.05). Bars indicate the means of three replicates; error bars indicate the standard error of the mean.

Figure 3

Comparison of the number of hair follicles and cashmere indicators of Tβ4-OE first filial generation. (A) Comparison of the cashmere growth in different offspring produced by SCNT and NM; SCNT, somatic cell nuclear transfer; NM, natural mating; WT, wild type. (B) Hematoxylin-eosin (H&E) staining of the backside skin from F1-1 and F1-2 cashmere goats; F1-1, Tβ4-OE first filial generation of 2014; F1-2, Tβ4-OE first filial generation of 2015; PHF, primary hair follicle; SHF, secondary hair follicles (**, p < 0.01). (C) Cashmere weight, cashmere thickness, and fiber length for the F1-1 and F1-2 goats; CON, control (*, p < 0.05).

Figure 4

Methylation level profiles for the skin samples of cashmere goats. (A) Circus plot showing the genome-wide relationship between the DNA methylation and gene expression of the skin samples. The outermost ring displays the chromosome numbers and bands. The second ring (red) shows the methylation of CHG (H = A, C, or T). The third ring (green) shows the methylation of CG. The fourth ring (blue) shows the methylation of CHH (H = A, C, or T). The inner most circle (black) represents gene expression. 5mC, 5-methylcytosine. P0-GM-1, a male from P0 for an generational methylation study; P0-GM-2, a female from P0 for an generational methylation study; F1-GM-1, the one of twin offspring from F1-2-NM for an generational methylation study; F1-GM-2, the another of twin offspring from F1-2-NM for an generational methylation study. (B) a violin plot showing the methylation levels of the skin samples with 2kb sliding windows. (C) Distribution of differentially methylated CpGs and genes of differentially methylated regions (DMRs); CGI, CpG iland. (D) Comparison of DMR length distribution between hyper and hypo. Hyper, hypermethylation; Hypo, hypomethylation.

Figure 5

Transcriptional level profiles for the skin samples of cashmere goats and an integrative analysis of the methylation and transcriptional profiles. (A) A volcano plot of all genes in the skin samples from the P0-GM and F1-GM cashmere goats; showing genes with >2-fold difference and an adjusted p < 0.01 among groups; P0-GM, the two parents (a female and a male from P0 for an generational methylation study); F1-GM, their twin offspring (from F1-2-NM for an generational methylation study). (B) The heat map of gene expression profiles in the skin samples from the P0-GM and F1-GM cashmere goats. The colored bars illustrate relative expression. In the map, each sample group is clustered. Skin samples were taken from the back and belly of cashmere goats. (C) A Venn diagram illustrating a comparison of differentially methylated genes and differentially expressed genes. The red circles represent the genes selected. The box shows information about the genes selected. DE-high, the differentially expressed genes (DEGs) of high expressed; DE-low, the DEGs of low expressed; DM-high, differentially methylated regions (DMRs) of hypermethylation; DM-low, DMRs of hypomethylation.

Figure 6

Comparison of the GO analysis of DMRs and DEGs. (A) GO analysis of DMRs. (B) GO analysis of DEGs. GO, Gene ontology enrichment analysis; DMRs, differentially methylated regions; DEGs, differentially expressed genes.

Figure 7

Comparison of KEGG analysis of DMRs and DEGs. (A) KEGG analysis of DMRs. (B) KEGG analysis of DEGs. KEGG, Encyclopedia of Genes and Genomes enrichment analysis; DMRs, differentially methylated regions; DEGs, differentially expressed genes.