Mutation of Signal Transducer and Activator of Transcription 5 (STAT5) Binding Sites Decreases Milk Allergen αS1-Casein Content in Goat Mammary Epithelial Cells

Abstract

α_S1-Casein (encoded by the CSN1S1 gene) is associated with food allergy more than other milk protein components. Milk allergy caused by α_S1-casein is derived from cow milk, goat milk and other ruminant milk. However, little is known about the transcription regulation of α_S1-casein synthesis in dairy goats. This study aimed to investigate the regulatory roles of signal transducer and activator of transcription 5 (STAT5) on α_S1-casein in goat mammary epithelial cells (GMEC). Deletion analysis showed that the core promoter region of CSN1S1 was located at -110 to -18 bp upstream of transcription start site, which contained two putative STAT5 binding sites (gamma-interferon activation site, GAS). Overexpression of STAT5a gene upregulated the mRNA level and the promoter activity of the CSN1S1 gene, and STAT5 inhibitor decreased phosphorylated STAT5 in the nucleus and CSN1S1 transcription activity. Further, GAS site-directed mutagenesis and chromatin immunoprecipitation (ChIP) assays revealed that GAS1 and GAS2 sites in the CSN1S1 promoter core region were binding sites of STAT5. Taken together, STAT5 directly regulates CSN1S1 transcription by GAS1 and GAS2 sites in GMEC, and the mutation of STAT5 binding sites could downregulate CSN1S1 expression and decrease α_S1-casein synthesis, which provide the novel strategy for reducing the allergic potential of goat milk and improving milk quality in ruminants.

Keywords: CSN1S1 promoter; STAT5; goat mammary epithelial cells; milk allergy; αS1-casein.

Figure 1

Schematic representation of the goat α_S1-casein gene (CSN1S1) promoter region. The underlined sequence of bases indicates the putative binding sites, and names of transcription factors appear below the underline. +1 represents transcriptional start site and gamma-interferon activation site (GAS) represents the binding site of signal transducer and activator of transcription 5 (STAT5).

Figure 2

Deletion analysis of the goat CSN1S1 promoter. (A) Relative luciferase activity of full-length CSN1S1 promoter. (B) Relative luciferase activity of the CSN1S1 promoter in different lengths. Serial CSN1S1 promoter deletions (−2018/+183, −1715/+183, −1354/+183, −1049/+183, −598/+183, −346/+183, −110/+183, −18/+183 bp) were transfected into GMEC and incubated at 48 h for luciferase assays. Data are shown as means ± SEM for 3 biological replicates. ** p < 0.01. Differences between 2 groups are considered significant at lowercase letters, p < 0.05.

Figure 3

Effects of STAT5 on the expression and the promoter activity of the CSN1S1 gene in goat mammary epithelial cells (GMEC). (A) The mRNA expression levels of STAT5a and CSN1S1 and (B) the promoter activity of CSN1S1 in GMEC transfected with pcDNA3.1-STAT5a or pcDNA3.1-NC for 48 h. (C) The mRNA expression level and (D) the promoter activity of CSN1S1 in GMEC treated with STAT5 inhibitor STAT5-IN-1 (200 μM) or DMSO for 48 h. (E) GMEC were treated with STAT5-IN-1 (200 μM) or DMSO. After 48 h incubation, p-STAT5a and α_S1-casein expression were examined. Relative expression of α_S1-casein was normalized by comparison to β-actin. Relative expression of nuclear p-STAT5a was normalized by comparison to Histone H3. Data are shown as means ± SEM for 3 biological replicates. * p < 0.05, ** p < 0.01.

Figure 4

Relative luciferase activities of mutagenesis of STAT5 binding sites (GAS) in the CSN1S1 promoter region. Data are shown as means ± SEM for 3 biological replicates. Differences between 2 groups are considered significant at lowercase letters, p < 0.05.

Figure 5

STAT5 regulates the CSN1S1 promoter activity by directly binding to the GAS sites in the CSN1S1 promoter region. (A) Effects of STAT5a overexpression on site-mutations of CSN1S1 promoter activity. GMEC were transfected with the wild-type pGL3-CSN1S1 (or GAS site-mutated constructs) followed by pcDNA3.1-STAT5a (or pcDNA3.1-NC) transfection, respectively. At 48 h treatment, cells were measured for luciferase activity. (B) Effects of STAT5 inhibition on site-mutations of CSN1S1 promoter activity. GMEC were treated with STAT5-IN-1 (200 μM, or DMSO) before wild-type pGL3-CSN1S1 (or GAS site-mutated constructs) transfection, respectively. At 48 h treatment, cells were measured for luciferase activity. Data are shown as means ± SEM for 3 biological replicates. * p < 0.05, ** p < 0.01. ns, no significance.

Figure 6

Effects of prolactin on the expression and promoter activity of the CSN1S1 gene. (A) The mRNA expression level and (B) promoter activity of CSN1S1 in GMEC incubated with prolactin (2 mg/L) for 48 h in culture medium. (C) GMEC seeded in culture medium were treated with STAT5-IN-1 (200 μM, or DMSO) before prolactin (2 mg/L) treatment for 48 h, then total protein was extracted. Relative expression of α_S1-casein was normalized by comparison to β-actin. Relative expression of p-STAT5a was normalized by comparison to STAT5a. (D) Relative luciferase activity of CSN1S1 promoter activity in GMEC co-treated with prolactin (2 mg/L) and the wild-type pGL3-CSN1S1 (or GAS site-mutated constructs) for 48 h in culture medium. Data are shown as means ± SEM for 3 biological replicates. ** p < 0.01. Differences between 2 groups are considered significant at lowercase letters, p < 0.05. ns, no significance.

Figure 7

Transcription of the CSN1S1 gene is regulated by direct STAT5 binding to GAS sites in GMEC. (A) GAS1 and (C) GAS2 binding sites in CSN1S1 promoter region were recognized by STAT5 directly using chromatin immunoprecipitation (ChIP)-PCR analysis. (B) GAS1 and (D) GAS2 binding sites in the CSN1S1 promoter region were recognized by STAT5 directly using ChIP-qPCR analysis. Data are shown as means ± SEM for 3 biological replicates. ** p < 0.01.