Dataset Information

Tissue Expression Analysis and Characterization of Smad3 Promoter in Bovine Myoblasts and Preadipocytes.

ABSTRACT: The transforming growth factor-? (TGF?) pathway plays many key roles in regulating numerous biological processes. In addition, the effects of TGF? are mediated by the transcription factor Smad3. However, the regulation of Smad3 activity is not well understood. In the present study, quantitative real-time PCR revealed that the Smad3 gene was expressed ubiquitously in 11 bovine tissues and displayed different expression patterns between muscle and adipose tissue. We further explored the expression and regulation of Smad3 gene by cloning the bovine Smad3 gene promoter; a dual-luciferase reporter assay identified that the core promoter region -337 to -41?bp was located in a CpG island. In addition, mutational analyses and electrophoretic mobility shift assays provided evidence that the KLF6, KLF15, MZF1, and KLF7 binding sites within the Smad3 promoter were responsible for the regulation of Smad3 transcription. These findings were confirmed by executing further RNA interference assays in bovine myoblasts and preadipocytes, which indicated that KLF6, KLF15, MZF1, and KLF7 are important transcriptional activators of Smad3 in both adipose and muscle tissue. These results will provide an important basis for an improved understanding of the TGF? pathway and new insights in cattle breeding.

SUBMITTER: Zhang L

PROVIDER: S-EPMC5985903 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Tissue Expression Analysis and Characterization of Smad3 Promoter in Bovine Myoblasts and Preadipocytes.

Zhang Le L Ning Yue Y Li Peiwei P Guo Hongfang H Zan Linsen L

DNA and cell biology 20180419 6

The transforming growth factor-β (TGFβ) pathway plays many key roles in regulating numerous biological processes. In addition, the effects of TGFβ are mediated by the transcription factor Smad3. However, the regulation of Smad3 activity is not well understood. In the present study, quantitative real-time PCR revealed that the Smad3 gene was expressed ubiquitously in 11 bovine tissues and displayed different expression patterns between muscle and adipose tissue. We further explored the expression ...[more]

PMID: 29672161

Similar Datasets

Project description:This study aimed to explore the regulatory role of FOXO1 promoter methylation on its transcriptional level and unravel the effect of FOXO1 on the proliferation and differentiation of bovine myoblasts. Bisulfite sequencing polymerase chain reaction (BSP) and real-time quantitative PCR were performed to determine the methylation status and transcript levels of the FOXO1 promoter region at different growth stages. BSP results showed that the methylation level in the calf bovine (CB) group was significantly higher than that in the adult bovine (AB) group (p < 0.05). On the other hand, qRT-PCR results indicated that the mRNA expression level in the AB group was significantly higher than that in the CB group (p < 0.05), suggesting a significant decrease in gene expression at high levels of DNA methylation. CCK-8 and flow cytometry were applied to determine the effect of silencing the FOXO1 gene on the proliferation of bovine myoblasts. Furthermore, qRT-PCR and Western blot were conducted to analyze the expression of genes associated with the proliferation and differentiation of bovine myoblasts. Results from CCK-8 revealed that the short hairpin FOXO1 (shFOXO1) group significantly promoted the proliferation of myoblasts compared to the short-hairpin negative control (shNC) group (p < 0.05). Flow cytometry results showed a significant decrease in the number of the G1 phase cells (p < 0.05) and a significant increase in the number of the S phase cells (p < 0.05) in the shFOXO1 group compared to the shNC group. In addition, the expression of key genes for myoblast proliferation (CDK2, PCNA, and CCND1) and differentiation (MYOG, MYOD, and MYHC) was significantly increased at both mRNA and protein levels (p < 0.05). In summary, this study has demonstrated that FOXO1 transcription is regulated by methylation in the promoter region and that silencing FOXO1 promotes the proliferation and differentiation of bovine myoblasts. Overall, our findings lay the foundation for further studies on the regulatory role of epigenetics in the development of bovine myoblasts.

Project description:BACKGROUND:The Pregnancy-associated glycoproteins (PAGs) belong to a large family of aspartic peptidases expressed exclusively in the placenta of species in the Artiodactyla order. In cattle, the PAG gene family is comprised of at least 22 transcribed genes, as well as some variants. Phylogenetic analyses have shown that the PAG family segregates into 'ancient' and 'modern' groupings. Along with sequence differences between family members, there are clear distinctions in their spatio-temporal distribution and in their relative level of expression. In this report, 1) we performed an in silico analysis of the bovine genome to further characterize the PAG gene family, 2) we scrutinized proximal promoter sequences of the PAG genes to evaluate the evolution pressures operating on them and to identify putative regulatory regions, 3) we determined relative transcript abundance of selected PAGs during pregnancy and, 4) we performed preliminary characterization of the putative regulatory elements for one of the candidate PAGs, bovine (bo) PAG-2. RESULTS:From our analysis of the bovine genome, we identified 18 distinct PAG genes and 14 pseudogenes. We observed that the first 500 base pairs upstream of the translational start site contained multiple regions that are conserved among all boPAGs. However, a preponderance of conserved regions, that harbor recognition sites for putative transcriptional factors (TFs), were found to be unique to the modern boPAG grouping, but not the ancient boPAGs. We gathered evidence by means of Q-PCR and screening of EST databases to show that boPAG-2 is the most abundant of all boPAG transcripts. Finally, we provided preliminary evidence for the role of ETS- and DDVL-related TFs in the regulation of the boPAG-2 gene. CONCLUSION:PAGs represent a relatively large gene family in the bovine genome. The proximal promoter regions of these genes display differences in putative TF binding sites, likely contributing to observed differences in spatial and temporal expression. We also discovered that boPAG-2 is the most abundant of all boPAG transcripts and provided evidence for the role of ETS and DDVL TFs in its regulation. These experiments mark the crucial first step in discerning the complex transcriptional regulation operating within the boPAG gene family.

Dataset Information

Tissue Expression Analysis and Characterization of Smad3 Promoter in Bovine Myoblasts and Preadipocytes.

Publications

Tissue Expression Analysis and Characterization of Smad3 Promoter in Bovine Myoblasts and Preadipocytes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets