Transcriptional regulatory mechanism of NR2F2 and ZNF423 in avian preadipocyte differentiation.

In the poultry industry, excessive abdominal fat deposition is not conducive to meat quality. Therefore, selection for optimal fat content levels in poultry has become a major breeding goal. We previously constructed NR2F2 overexpression (NR2F2OE) and knockout (NR2F2Δ/Δ/83-125aa) cell lines using Piggybac and CRISPR/Cas9 techniques, and confirmed that the transcription factor NR2F2 can significantly inhibit the differentiation of avian preadipocytes. In this study, we identified a downstream gene ZNF423 regulated by NR2F2, which is also involved in regulating avian fat deposition. First, we performed transcriptome analysis of the NR2F2-edited lines, which has been proven to be an inhibitor of avian fat deposition in our previous studies. Our findings revealed that NR2F2 affects a series of candidate regulators related to adipogenesis. Among these, we focused on ZNF423, which was significantly down-regulated in the NR2F2OE cell line and up-regulated in the NR2F2Δ/Δ/83-125aa cell line. Next, dual luciferase reporter assay results showed that the DNA-binding domain (DBDΔ72-143aa) of transcription factor NR2F2 may negatively affect the expression of downstream target gene ZNF423 by binding to its distal promoter region (-2356 to -2346). Moreover, we constructed a function analytical model and found that overexpression of ZNF423 significantly facilitated the differentiation of adipocytes in immortalized chicken preadipocytes (ICP1). Consistent with these findings, global transcriptome analysis of the ZNF423-overexpressed cell line (ZNF423OE) further demonstrated that the process of adipogenesis was significantly enriched. These results indicate that ZNF423 is a positive regulator of avian adipocyte differentiation. Overexpression of ZNF423 in the NR2F2OE cell line compensated for the inhibition of fat deposition phenotype, further suggesting that ZNF423 is a downstream target gene of NR2F2. These findings uncover a novel function of ZNF423 in avian adipocyte differentiation and analyzed the transcriptional regulation by its upstream transcription factor NR2F2. Additionally, we identified a list of functional candidate genes, providing important insights for further research on the mechanism of avian fat deposition.

Research Note: Identification of core promoter region of the polyunsaturated fatty acid synthesis-related gene family in chicken.

Chicken is considered an ideal model species to study the synthesis of polyunsaturated fatty acids (PUFAs) due to its appropriate proportions of fatty acids and abundant content of PUFAs, suitable for human consumption. However, the molecular mechanisms regulating poultry PUFA synthesis remain unclear. Here, we systematically explored the transcriptional regulation activity of the gene family related to PUFA synthesis in chicken by carrying out the Dual-Luciferase Reporter Assay. We identified the core promoter regions of members of the chicken PUFA synthesis-related gene family, including ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, FADS1, FADS2, FADS6, SCD, and SCD5. Additionally, changes in relative fluorescence values of different truncated segments in the upstream regulatory region of these genes indicate the existence of regulatory regions. Furthermore, we predicted the transcription factors that bind to the identified core promoter regions of multiple genes, including Sp1, NF-1, C/EBPalpha, etc. These findings provide a basis for the molecular mechanisms regulating poultry PUFA synthesis and offer new scientific insight into the potential improvement of poultry meat quality in the future.

A novel candidate gene CLN8 regulates fat deposition in avian.

BACKGROUND: The fat deposition has a crucial role in animal meat flavor, and fat deposition-related traits are vital for breeding in the commercial duck industry. Avian fat-related traits are typical complex phenotypes, which need a large amount of data to analyze the genetic loci. RESULTS: In this study, we performed a new phenotypic analysis of fat traits and genotyped whole-genome variations for 1,246 ducks, and combed with previous GWAS data to reach 1,880 ducks for following analysis. The carcass composition traits, subcutaneous fat weight (SFW), subcutaneous fat percentage (SFP), abdominal fat weight (AFW), abdominal fat percentage (AFP) and the body weight of day 42 (BW42) for each duck were collected. We identified a set of new loci that affect the traits related to fat deposition in avian. Among these loci, ceroid-lipofuscinosis, neuronal 8 (CLN8) is a novel candidate gene controlling fat deposition. We investigated its novel function and regulation in avian adipogenesis. Five significant SNPs (the most significant SNP, P-value = 21.37E-12) and a single haplotype were detected in the upstream of CLN8 for subcutaneous fat percentage. Subsequently, luciferase assay demonstrated that 5 linked SNPs in the upstream of the CLN8 gene significantly decreased the transcriptional activity of CLN8. Further, ATAC-seq analysis showed that transcription factor binding sites were identified in a region close to the haplotype. A set of luciferase reporter gene vectors that contained different deletion fragments of the CLN8 promoter were constructed, and the core promoter area of CLN8 was finally identified in the -1,884/-1,207 bp region of the 5' flanking sequences, which contains adipogenesis-related transcription factors binding sites. Moreover, the over-expression of CLN8 can remarkably facilitate adipocyte differentiation in ICPs. Consistent with these, the global transcriptome profiling and functional analysis of the over-expressed CLN8 in the cell line further revealed that the lipid biosynthetic process during the adipogenesis was significantly enriched. CONCLUSIONS: Our results demonstrated that CLN8 is a positive regulator of avian adipocyte differentiation. These findings identify a novel function of CLN8 in adipocyte differentiation, which provides important clues for the further study of the mechanism of avian fat deposition.