Analysis of the Promoter Regions of gga-miR-31 and Its Regulation by RA and C-jun in Chicken

Abstract

The role of gga-miR-31 in chicken germ cell differentiation and spermatogenesis is of significant importance. The transcriptional properties of gga-miR-31 are crucial in establishing the foundation for the formation of chicken spermatogonia stem cells and spermatogenesis. In this study, a series of recombinant vectors including varying lengths of the gga-miR-31 promoter were predicted and constructed. Through the utilization of the dual luciferase reporting system, the upstream -2180~0 bp region of gga-miR-31 was identified as its promoter region. Furthermore, it was predicted and confirmed that the activity of the gga-miR-31 promoter is increased by retinoic acid (RA). The binding of RA to the gga-miR-31 and Stra8 promoter regions was found to be competitive. Through the deletion of C-jun binding sites and the manipulation of C-jun expression levels, it was determined that C-jun inhibits the activity of the gga-miR-31 promoter. Furthermore, the combined treatment of C-jun and RA demonstrated that the positive regulatory effect of RA on the gga-miR-31 promoter is attenuated in the presence of high levels of C-jun. Overall, this study establishes a foundation for further investigation into the regulatory mechanisms of gga-miR-31 action, and provides a new avenue for inducing chicken embryonic stem cells (ESC) to differentiate into spermatogonial stem cells (SSC), and sperm formation.

Keywords: C-jun; RA; chicken; gga-miR-31; promoter.

Figure 1

gga-miR-31 promoter core region analysis. (A) Online prediction of gga-miR-31 promoter region. Quadrilaterals filled with red, yellow, and light blue in Figure 1 mean we used online prediction software to predict the promoter sites contained in 2180 bp upstream of gga-miR-31; BDGP, promoter 2.0, and TSSW, respectively. (B) Dual luciferase activity assay with different truncated carriers for relative luciferase activity. Note: For PGL3-2180, the target fragment (−2180~+1 bp, chrZ:83217373-83215193) was ligated to the pGL3-Basic vector. For PGL3-1344, the target fragment (−1344~+1 bp, chrZ:83216537-83215193) was ligated to the pGL3-Basic vector. For PGL3-584, the target fragment (−584~+1 bp, chrZ:83215777-83215193) was ligated to the pGL3-Basic vector. The marker “*” represents a significant difference (p < 0.05), while “**” represents a highly significant difference (p < 0.01).

Figure 2

Conservation of the promoter region and the prediction of binding transcription factors. (A) gga-miR-31 promoter sequence (−2180~+1 bp) phylogenetic tree of sixteen species; (B) Predicted transcription factors that may bind to the promoter region of gga-miR-31.

Figure 3

Activation of gga-miR-31 transcription by RA. (A) The DF-1, ESC, and SSC cell types were transfected with pGL3-miR-31 and pGL3-Stra8. Twelve hours after transfection, the fluid was changed and one group was cultured with 10⁻⁵ mol/L RA, 10% FBS DMEM, and the other group was cultured with 10% FBS DMEM. After 36 h, the cells were collected for dual luciferase activity assays and calibrated with the control group. Relative activity was calculated as RA group/Control group. (B) Using pEGFP-Bmp4 as a negative control for the RAR binding site, different groups were set up to cotransfect DF-1 cells with the gga-miR-31 promoter and the Stra8 promoter for 12 h. The dual luciferase activity was detected after treatment with 10⁻⁵ mol/L RA, 10% FBS DMEM. (C) Determination of RA concentration in different cell types. The marker “*” represents a significant difference (p < 0.05), while “**” represents a highly significant difference (p < 0.01).

Figure 4

Effect of deletion of the C-jun binding site on the activity of the gga-miR-31 promoter. (A) Venn analysis of the transcription factor binding sites of the gga-miR-31 and the Stra8 promoter regions. The transcription factors marked in red are the transcription factors differentially expressed between ESC and SSC. (B) RNA-seq data of C-jun in ESC and SSC. (C) Relative fluorescence activity of various recombinant plasmids after deletion of C-jun binding site in the promoter region of gga-miR-31. The marker “*” represents a significant difference (p < 0.05), while “**” represents a highly significant difference (p < 0.01).

Figure 5

The transcription factor C-jun inhibited the transcription of gga-miR-31, and combined with RA, affected the promoter activity of gga-miR-31. (A) Effect of overexpression/knockdown of C-jun on pGL3-2180 vector activity. (B) Effect of overexpression of C-jun on gga-miR-31 promoter activity after deletion of the C-jun binding sites. (C) Effect of C-jun in combination with RA on gga-miR-31 promoter activity. OE-C-jun means the overexpression vector of C-jun. OENC means negative control of the C-jun overexpression vector. KD-C-jun means the knockdown vector of C-jun. KDNC means negative control of the C-jun knockdown vector. The marker “*” represents a significant difference (p < 0.05), while “**” represents a highly significant difference (p < 0.01).

Figure 6

Schematic drawing of how RA and C-jun regulate the transcription of gga-miR-31 in chicken cells.