Transcriptional control of CCAAT/enhancer binding protein zeta gene in chicken adipose tissue.

CCAAT/enhancer binding protein zeta (C/EBPZ) was differentially expressed in abdominal adipose tissues of fat and lean broilers and regulated adipogenesis in chicken. The objective of this study was to elucidate the transcriptional regulation of C/EBPZ gene in chicken adipose tissue. A 2,031-base pair (bp) chicken C/EBPZ sequence (2,025 nucleotides upstream to 6 nucleotides downstream from the initiator codon, -2,025/+6) was studied. The sequence exhibited a significant promoter activity (P < 0.05) and had some cis-acting elements, notably, a core promoter was identified in nucleotides -94 to +6. Additionally, DNA pull-down assay showed that proteins interacted with chicken C/EBPZ promoter (-173/+6) in preadipocytes were implicated in transcription, post-transcriptional regulation and translation. In addition, KLF2 facilitated the activities of chicken C/EBPZ promoter (-2,025/+6, -1,409/+6, -793/+6, -485/+6, -173/+6, and -94/+6) in preadipocytes (P < 0.05). The expression levels of KLF2 and C/EBPZ in chicken abdominal adipose tissue were substantially associated (r = 0.5978278, P < 0.0001), and KLF2 increased C/EBPZ expression in vitro (P < 0.05). Additionally, chromatin immunoprecipitation (ChIP)-PCR analysis revealed that KLF2 has the ability to interact with the chicken C/EBPZ promoter regions at least at the positions -1,245/-1,048 and -571/-397. Mutation analysis showed that the CGCAGCGCCCG motif located in the chicken C/EBPZ promoter at positions -45 to -35 is involved in regulating transcription and facilitates trans activation by KLF2. These results provided some information of transcription control of C/EBPZ in chicken adipose tissue.

Molecular function of Krüppel-like factor 7 in biology.

Krüppel-like factor 7 ( KLF7), also named ubiquitous KLF ( UKLF) based on its ubiquitous expression in adult human tissues, is a conserved gene in animals. There are few reports on KLF7 among KLFs; however, an increasing number of reports are demonstrating that KLF7 plays an important role in development and diseases. Genetic studies have shown that the DNA polymorphisms of KLF7 are associated with obesity, type 2 diabetes mellitus (T2DM), lachrymal/salivary gland lesions, and mental development in some populations of humans, and the DNA methylation of KLF7 is associated with the development of diffuse gastric cancer. In addition, biological function studies have shown that KLF7 regulates the development of the nervous system, adipose tissue, muscle tissue and corneal epithelium as well as the preservation of pluripotent stem cells. Additionally, disease-related studies have shown that KLF7 is involved in the development or progression of T2DM, hematologic diseases, lung cancer, gastric cancer, squamous cell carcinoma of the head and neck, pancreatic ductal adenocarcinoma, glioma, advanced high-grade serous ovarian cancer and osteosarcoma. This review provides research progress on the genetic association, molecular properties and biological function of KLF7, and it may shed light on the understanding of the molecular function of KLF7 in biology and the molecular mechanisms of some diseases.

C/EBPZ modulates the differentiation and proliferation of preadipocytes.

BACKGROUND/OBJECTIVES: This study investigated the functions of CCAAT/enhancer-binding protein zeta (C/EBPZ; Gene ID: 10153) in adipose tissue. SUBJECTS/METHODS: Bioinformatics analysis were used to study the expression pattern of C/EBPZ in human adipose tissue. The expression and function of C/EBPZ in adipose tissue were further studied using chicken as animal model in vivo and in vitro. RESULTS: The human C/EBPZ transcripts were greater and more stable in subcutaneous adipose tissue than in visceral adipose tissue (P < 0.01), and they were increased with age in adipose tissue (P < 0.05). In addition, the chicken C/EBPZ transcripts (C/EBPZ /ACTB) of visceral (abdominal) adipose tissue were significantly different between fat and lean broilers and decreased with age during development (P < 0.01). RNA-seq analysis showed that the C/EBPZ overexpression associated with adipose tissue development and DNA replication in chicken preadipocytes (P < 0.05). Additionally, overexpression of chicken C/EBPZ inhibited preadipocytes differentiation and promoted preadipoytes proliferation in vitro (P < 0.05). In addition, C/EBPZ overexpression suppressed the promoter activities of PPARγ, C/EBPα, FASN and LPL, and promoted the promoter activities of GATA2 and FABP4 in chicken preadipocytes (P < 0.05). CONCLUSIONS: C/EBPZ modulated the differentiation and proliferation of preadipocytes, and it might be a new negative regulator of adipogenesis.

Transcriptional control of chicken KLF7 promoter in preadipocytes.

Krüppel-like factor 7 (KLF7) has been reported to inhibit adipogenesis and regulate the development of the nervous system. However, transcription regulation of KLF7 remains poorly understood. In the current study, a 2196-bp-long 5'-flanking sequence of chicken KLF7 (-2286 bp to -91 bp, upstream of the translation start site) was studied for promoter activity, and there was a remarkable promoter activity in this sequence (P<0.05). The 5'-truncated mutation analysis showed that a minimal promoter was on the sequence from -241 bp to -91 bp. In addition, GATA2 overexpression facilitated the promoter activity of pGL3-KLF7(-2286/-91), pGL3-KLF7(-1215/-91), pGL3-KLF7(-521/-91), and pGL3-KLF7(-241/-91), and GATA3 overexpression inhibited the promoter activity of pGL3-KLF7(-1845/-91), pGL3-KLF7(-1215/-91), pGL3-KLF7(-521/-91), and pGL3-KLF7(-241/-91) in chicken preadipocytes (P<0.05). Knockdown of GATA2 expression inhibited the promoter activity of pGL3-KLF7(-1215/-91) and pGL3-KLF7(-241/-91), and knockdown of GATA3 expression facilitated the promoter activity of pGL3-KLF7(-521/-91) and pGL3-KLF7(-241/-91) (P<0.05). Additionally, overexpression and knockdown analyses showed that GATA3 inhibited KLF7 mRNA expression (P<0.05), and both overexpression and knockdown of GATA2 resulted in the downregulation of KLF7 mRNA expression in chicken preadipocytes (P<0.05). Western blot analysis in chicken preadipocytes showed that GATA2 facilitated KLF7 expression and GATA3 inhibited KLF7 expression. Mutation analysis showed that the motif of 'GGATCTATCA' (-107 bp/-98 bp) might be a cis-regulation element, which is involved in the KLF7 expression regulation by GATA3 in chicken preadipocytes. These results provided some details of KLF7 transcription regulation in chicken adipose tissue.

DNA methylation of CpG sites in the chicken KLF7 promoter and Exon 2 in association with mRNA expression in abdominal adipose tissue and blood metabolic indicators.

BACKGROUND: Our previous study found that chicken KLF7 was an important regulator in formation of adipose tissue. In the present study, we analyzed the association for DNA methylation in chicken KLF7 with its transcripts of abdominal adipose tissue and blood metabolic indicators. RESULTS: The KLF7 transcripts of the adipose tissue of Chinese yellow broilers were associated with age (F = 6.67, P = 0.0035). In addition, the KLF7 transcripts were negatively correlated with blood glucose levels (r = - 0.61841, P = 0.0140). The DNA methylation levels of 26 CpG loci in the chicken KLF7 promoter and Exon 2 were studied by Sequenom MassArray. A total of 22 valid datasets were obtained. None of them was significantly different in relation to age (P > 0.05). However, the DNA methylation levels in the promoter were lower than those in Exon 2 (T = 40.74, P < 0.01). Correlation analysis showed that the DNA methylation levels of PCpG6 and E2CpG9 were significantly correlated with KLF7 transcripts and blood high-density lipoprotein levels, respectively, and many CpG loci were correlated with each other (P < 0.05). The methylation data were subjected to principal component analysis and factor analysis. The six principal components (z1-z6) were extracted and named Factors 1-6, respectively. Factor analysis showed that Factor 1 had a higher load on the loci in the promoter, and Factors 2-6 loaded highly on quite different loci in Exon 2. Correlation analysis showed that only z1 was significantly correlated to KLF7 transcripts (P < 0.05). In addition, an established regression equation between z1 and KLF7 transcripts was built, and the contribution of z1 to the variation on KLF7 transcripts was 34.29%. CONCLUSIONS: In conclusion, the KLF7 transcripts of chicken abdominal adipose tissue might be inhibited by DNA methylation in the promoter, and it might be related to the DNA methylation level of PCpG6.

Loss of the third C2H2 zinc finger of chicken KLF7 affects its transcriptional regulation activities in adipose tissue.

KLF7, one of candidate genes in neurotherapy and metabolic syndrome, has been studied in adipogenesis of mammalian species and birds. However, the effect of the third C2H2 zinc finger of KLF7 for its transcriptional regulation in adipogenesis has not been well understood. Here, the wild-type chicken KLF7 (KLF7) overexpression plasmid, pCMV-myc-KLF7, and two plasmids of chicken KLF7 mutants, i.e. pCMV-myc-KLF7m1 with half of the third zinc finger (KLF7m1) and pCMV-myc-KLF7m2 without the third zinc finger (KLF7m2), were constructed. Luciferase reporter assay in DF1 cells showed that the effect of chicken KLF7 overexpression on the promoter activity of LPL was greater than those of KLF7m1 and KLF7m2 (P < 0.05). There was no significant difference among the overexpression of KLF7, KLF7m1 and KLF7m2 on the promoter activities of FASN, C/EBPα and FABP4 (P > 0.05). Additionally, the effects of KLF7, KLF7m1 and KLF7m2 overexpression on the promoter activity of PPARγ were different. KLF7 overexpression had no significant effect on the PPARγ promoter activity (P > 0.05), KLF7m1 overexpression suppressed PPARγ promoter activity (P < 0.05), while KLF7m2 overexpression facilitated the promoter activity of PPARγ (P < 0.05), consistent with the results of western blot analysis. Our results suggested that the third zinc finger of chicken KLF7 may play a role in its transcriptional regulation of LPL and PPARγ but has no effect on its regulation of C/EBPα, FASN and FABP4. The third zinc finger of KLF7 might be a target for the treatment of metabolic disorder in chicken.

[Effect of Kruppel-like factor 2 (KLF2) over-expression on activities of chicken PPARγ and C/EBPα promoters].

Objective To examine the effect of Kruppel-like factor 2 (KLF2) over-expression on the activities of chicken peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer binding protein alpha (C/EBPα) promoters. Methods Luciferase reporter assay was used to investigate the effect of KLF2 over-expression on reporter activities of 6 kinds of chicken PPARγ promoter construct and 5 kinds of chicken C/EBPα promoter construct in DF1 cells. Results KLF2 over-expression significantly inhibited the reporter activities of 4 kinds of chicken PPARγ promoter construct (-1978/-82, -1513/-82, -1254/-82 and -1019/-82), but had no significant effect on the reporter activities of 2 kinds of PPARγ promoter construct (-513/-82 and -320/-82). In addition, the effect of KLF2 over-expression on the reporter activities of 2 kinds of PPARγ promoter construct (-1513/-82 and -1254/-82) was significantly greater than that on the other 2 kinds of PPARγ promoter construct (-1978/-82 and -1019/-82). Additionally, KLF2 over-expression inhibited the reporter activity of one kind of C/EBPα promoter construct (-1863/+332), and enhanced the reporter activities of 4 kinds of C/EBPα promoter construct (-1318/+332, -891/+332, -538/+332 and -123/+332). There was no significant difference in the promoting effect of KLF2 over-expression among the 4 kinds of C/EBPα promoter construct (-1318/+332, -891/+332, -538/+332 and -123/+332). Conclusion The effect of KLF2 over-expression on the activities of chicken PPARγ and C/EBPα promoters are different among the constructs containing various lengths.

[Research progress of Krüppel-like factor 7].

Krüppel-like factor 7 (KLF7), a member of Krüppel-like transcription factors (KLFs), also known as ubiquitous Krüppel- like factor (UKLF), is ubiquitously expressed in various tissues of adult human beings. Genetics reports showed that the genetic polymorphism of KLF7 is associated with obesity, type 2 diabetes, mental development in human beings; and KLF7 methylation is associated with the development of diffuse gastric cancer (gastric adenocarcinoma). In addition, some genomics reports suggested that KLF7 is one of the key transcription factors in the regulatory networks of serum markers change during the cardiovascular disease. The function studies showed that KLF7 is involved in the regulation of the development and function of the nervous system and adipose tissue, type 2 diabetes, blood diseases, as well as pluripotent cells maintenance. This review summarizes the research progress of KLF7 in genetic characteristics, protein structure and gene function.

[Functional analysis of SNPs in porcine TLR4 gene].

Toll-like receptor 4 (TLR4) plays an important role in immune response and the polymorphism in it might affect protein signaling and host resistance/susceptibility to disease. This study was designed to characterize the functional relevance of 3 nonsynonymous single nucleotide polymorphisms (SNPs), c.611 T>A (p.Leu204His), c.1027 C>A (p.Gln343Lys), and c.1605 G>T (p.Leu535Phe), which were selected based on our previous studies. RT-PCR method was used to clone the complete coding sequence of porcine TLR4 gene and the PCR-based method was used to introduce the point mutation. The effects of 3 SNPs on the ligand recognition and signaling of porcine TLR4 were investigated in transiently transfected PK-15 cells using dual-luciferase reporter system and Western blotting method. At the same time, the distribution of c.1605 G>T among pig populations composed of Min pig, Yorkshire, Landrace, and Wild boar from northeastern China was studied by created restriction site PCR-RFLP method. The complete coding sequence of TLR4 gene in Min pig and 3 variants with single point mutations were obtained. Eukaryotic expression vectors containing different alleles of porcine TLR4 were constructed. SNP c.1605 G>T significantly decreased the TLR4 signaling (P<0.01) and the polymorphism only existed in Min pig and Wild boar from northeastern China with high frequencies. SNP c.1605 G>T in porcine TLR4 might affect the receptor function and host resistance/susceptibility to diseases.