A Novel Single-Nucleotide Polymorphism in WNT4 Promoter Affects Its Transcription and Response to FSH in Chicken Follicles

The signaling pathway of the wingless-type mouse mammary tumor virus integration site (Wnt) plays an important role in ovarian and follicular development. In our previous study, WNT4 was shown to be involved in the selection and development of chicken follicles by upregulating the expression of follicle-stimulating hormone receptors (FSHR), stimulating the proliferation of follicular granulosa cells, and increasing the secretion of steroidal hormones. FSH also stimulates the expression of WNT4. To further explore the molecular mechanism by which FSH upregulates WNT4 and characterize the cis-elements regulating WNT4 transcription, in this study, we determined the critical regulatory regions affecting chicken WNT4 transcription. We then identified a single-nucleotide polymorphism (SNP) in this region, and finally analyzed the associations of the SNP with chicken production traits. The results showed that the 5′ regulatory region from −3354 to −2689 of WNT4 had the strongest activity and greatest response to FSH stimulation, and we identified one SNP site in this segment, −3015 (G > C), as affecting the binding of NFAT5 (nuclear factor of activated T cells 5) and respones to FSH stimulation. When G was replaced with C at this site, it eliminated the NFAT5 binding. The mRNA level of WNT4 in small yellow follicles of chickens with genotype GG was significantly higher than that of the other two genotypes. Moreover, this locus was found to be significantly associated with comb length in hens. Individuals with the genotype CC had longer combs. Collectively, these data suggested that SNP−3015 (G > C) is involved in the regulation of WNT4 gene expression by responding FSH and affecting the binding of NFAT5 and that it is associated with chicken comb length. The current results provide a reference for further revealing the response mechanism between WNT and FSH

The stability of DTT and DTNF proteins.

<p>(A) Thermal stability of DTT and DTNF proteins measured by DSC. Left panel, temperature dependent heat capacity of DTT and DTNF proteins. Right panel: melting temperatures of DTT and DTNF proteins. (B) SDS-PAGE analysis of the soluble fraction of DTT and DTNF proteins stored at 4°C for 2 days (Top) and 10 days (Bottom).</p

The molecular dynamics of DTNF proteins.

<p>(A) Left panel: Molecular dynamic simulation of DTNF proteins. The secondary structure conformation was shown for residues belong to TNF-α epitope and DTT at positions 97–111. Right panel: Pie charts showing fraction of time the amino acid residue adopting indicated conformation during 300 ns simulation. (B)(C)(D) The RMSF trajectories of DTT and DTNF proteins. (E) Molecular dynamic simulation of the tertiary structure of DTNF7. The dashed circle highlights the positions of TNF-α epitope peptide at indicated time.</p

The cytokine levels in mice sera immunized with DTT or DTNF7.

<p>Serum samples were collected from CIA mice on day 28 and 56 after the first injection with chicken type II collagen. The cytokine concentrations in sera were determined by Bio-Plex assay. The average of four mice with standard variation was plotted for each cytokine. Data are presented as the mean ± SD.</p

An atlas of regulatory elements in chicken: A resource for chicken genetics and genomics.

A comprehensive characterization of regulatory elements in the chicken genome across tissues will have substantial impacts on both fundamental and applied research. Here, we systematically identified and characterized regulatory elements in the chicken genome by integrating 377 genome-wide sequencing datasets from 23 adult tissues. In total, we annotated 1.57 million regulatory elements, representing 15 distinct chromatin states, and predicted about 1.2 million enhancer-gene pairs and 7662 super-enhancers. This functional annotation of the chicken genome should have wide utility on identifying regulatory elements accounting for gene regulation underlying domestication, selection, and complex trait regulation, which we explored. In short, this comprehensive atlas of regulatory elements provides the scientific community with a valuable resource for chicken genetics and genomics