Identification of a novel ovine LH-beta promoter region, which dramatically enhances its promoter activity

Abstract

The luteinizing hormone beta subunit (LH-beta) gene plays a critical role in reproduction. In order to characterize and analyze the promoter region of LH-beta in sheep, a genomic library was constructed in phage lambda gt 10 and screened. A novel region of 1,224 bp upstream from the targeted LH-beta gene was identified. Blasting this sequence showed a perfect homology for the first 721 bp sequence with an upstream ovine LH-beta sequence in the database. However, the remaining 5'-503 bp showed no sequence matching. DNA from Moroccan breeds was isolated and the whole region was amplified and confirmed by sequencing. To further confirm the promoter activity of this region, an in vitro analysis using a luciferase assay was carried out. An increase in the promoter activity of the whole region was demonstrated compared to the empty vector. More interestingly, the unpublished region significantly enhanced the promoter activity compared to the known region alone. To predict putative transcription factor binding-sites (TFBSs), an in silico analysis was performed using the TFSEARCH program. The region features many TFBSs and contains two palindrome sequences of 17- and 18-bp. Taken together, a novel region was identified and confirmed in sheep which contained a promoter activity rich with binding sites for a putative regulatory element as shown in silico.

Keywords: LH-beta; Luciferase assay; Promoter; Reproduction.

Fig. 1

Schematic representation of the promoter region of the LH beta gene from a library screening of the Moroccan sheep database sequence: Homology sequence analysis revealed that the two sequences shared more than 96% homology with the reference sequence available in the database after alignment (Brown et al. 1993).

Fig. 2

Investigation of LH-beta promoter region activity in Moroccan sheep. The promoter region including the published, unpublished and entire regions were amplified and analyzed for promoter activity using the luciferase assay. The luciferase gene alone was used as a negative control. The position of the two palindromic sequences is also shown. P-values of <0.05 were considered as statistically significant (*p < 0.05, **p < 0.01, and ***p < 0.001).

Fig. 3

In-silico transcription factor binding sites (TFBS) analysis of the whole promoter region of the LH beta gene. The in silico analysis of the promoter region was carried out using the TFSEARCH software program. The localization of each TF is shown in the figure according to its position from the ATG start codon. The first (unpublished) part of the promoter is shown.

Fig. 4

In-silico transcription factor binding sites (TFBS) analysis of the whole promoter region of the LH beta gene. The in silico analysis of the promoter region was carried out using the TFSEARCH software program. The localization of each TF is shown in the figure according to its position from the ATG start codon. The second (published) part of the promoter is shown.