A 5-Methylcytosine Site of Growth Differentiation Factor 9 (GDF9) Gene Affects Its Tissue-Specific Expression in Sheep

Growth differentiation factor 9 (GDF9) plays an important role in the early folliculogenesis of sheep. This study investigated the mRNA expression of ovine GDF9 in different tissues by real-time PCR. GDF9 exhibits significantly higher levels of expression (p < 0.01) in the ovary, relative to other tissues, indicating that its expression is tissue specific. To explore the regulatory mechanism of this tissue-specific expression, the methylation level of one CpG island (−1453 to −1854) of GDF9 promoter in ovary and heart was determined. In this region (−1987 to −1750), only the mC-4 site was present in the Sp4 binding site showed differential methylation between the heart and ovary; with increased (p < 0.01) methylation being observed in the heart. Additionally, the methylation level was negatively correlated with GDF9 mRNA expression (R = −0.75, p = 0.012), indicating that the methylation of this site plays an important role in transcriptional regulation of GDF9. The methylation effect of the mC-4 site was confirmed by using dual-luciferase. Site-directed mutation (methylation) of mC-4 site significantly reduced (p < 0.05) basal transcriptional activity of GDF9 promoter in oocytes. These results imply that methylation of GDF9 promoter CpG island mC-4 site may affect the binding of the Sp4 transcription factor to the GDF9 promoter region in sheep, thereby regulating GDF9 expression and resulting in a tissue-specific expression.

Isolation and characterisation of peroxiredoxin 6 promoter from sheep (Ovis aries)

Introduction: Peroxiredoxin 6 (Prdx6) is a bifunctional protein and a unique 1-Cys Prdx of the peroxiredoxin family. The expression and regulation of Prdx6 are implicated in numerous physiological and pathological processes.Material and Methods: Eight stepwise truncated DNA fragments obtained from the 5′-flank region of the Prdx6 gene were prepared and subcloned into the pSEAP2-Enhancer vectors. To investigate the transcriptional activity of the truncated DNA fragments, the recombinant plasmids were transfected into the COS-1 cells and the transcriptional activity was measured via assaying the expression of the reporter gene of the secreted alkaline phosphatase.Results: A 3.4 kb 5′-upstream flank region of the Prdx6 gene was cloned and sequenced. The region from −108 nt to −36 nt of the 5′-flanking region of the Prdx6 gene contained basal transcriptional activity.Conclusion: This result provides the basis for further studies on the gene regulation of the Prdx6-mediated biological processes and on screening for the transacting factors that interact with cis-acting elements of the Prdx6 gene promoter.

Regulation of the Mechanism ofTWIST1Transcription by BHLHE40 and BHLHE41 in Cancer Cells

BHLHE40 and BHLHE41 (BHLHE40/41) are basic helix-loop-helix type transcription factors that play key roles in multiple cell behaviors. BHLHE40/41 were recently shown to be involved in an epithelial-to-mesenchymal transition (EMT). However, the precise mechanism of EMT control by BHLHE40/41 remains unclear. In the present study, we demonstrated that BHLHE40/41 expression was controlled in a pathological stage-dependent manner in human endometrial cancer (HEC). Ourin vitroassays showed that BHLHE40/41 suppressed tumor cell invasion. BHLHE40/41 also suppressed the transcription of the EMT effectorsSNAI1,SNAI2, andTWIST1. We identified the critical promoter regions ofTWIST1for its basal transcriptional activity. We elucidated that the transcription factor SP1 was involved in the basal transcriptional activity ofTWIST1and that BHLHE40/41 competed with SP1 for DNA binding to regulate gene transcription. This study is the first to report the detailed functions of BHLHE40 and BHLHE41 in the suppression of EMT effectorsin vitro. Our results suggest that BHLHE40/41 suppress tumor cell invasion by inhibiting EMT in tumor cells. We propose that BHLHE40/41 are promising markers to predict the aggressiveness of each HEC case and that molecular targeting strategies involving BHLHE40/41 and SP1 may effectively regulate HEC progression.

Zinc finger transcription factor Egr-1 is involved in stimulation of NHE2 gene expression by phorbol 12-myristate 13-acetate

The apical membrane Na+/H+exchanger isoforms NHE2 and NHE3 are involved in transepithelial Na+absorption in the intestine. However, they exhibit differences in their pattern of tissue expression and regulation of their activity by various molecular signals. To study the mechanisms involved in the transcriptional regulation of these genes, we characterized cis-acting elements within the human NHE2 promoter that regulate NHE2 promoter expression in C2BBe1 cells. A small DNA region (−85/+249) was involved in the regulation of basal transcriptional activity of the NHE2 promoter as determined by transient transfection assays. RT-PCR analysis showed that NHE2 mRNA was upregulated in response to phorbol 12-myristate 13-acetate (PMA). Results from actinomycin D-treated cells indicated that the regulation of the NHE2 gene by PMA occurs in part at the transcriptional level. Furthermore, PMA treatment led to a 100% increase in promoter activity through elements located on the −415/+249 DNA fragment. A PMA-induced nuclear factor that bound to the NHE2 promoter was identified as the transcription factor Egr-1. We identified two PMA response elements in the −415/+1 promoter region that bind to Sp1 and Sp3 in untreated nuclear extracts and to Egr-1 in PMA-treated nuclear extracts. In cotransfection experiments, Egr-1 was able to transactivate the NHE2 promoter. Our data indicate that Egr-1 may play a key role in regulated expression of the human NHE2 gene.