PU.1 interacts with KLF7 to suppress differentiation and promote proliferation in chicken preadipocytes.

Krüppel-like factor 7 (KLF7) is a negative regulator of preadipocyte differentiation. Our previous KLF7 ChIP-seq analysis showed that the binding motif of PU.1 was found among the KLF7 binding peaks, indicating that an interaction between KLF7 and PU.1 at preadipocyte gene promoters and other regulatory elements might be common. Here, Co-IP and FRET assays are used to confirm that PU.1 can directly bind to KLF7 and enhance the transcription activity of cyclin-dependent kinase inhibitor 3 ( CDKN3), which is a downstream target gene of KLF7. We show that the PU.1 expression level is decreased during preadipocyte differentiation. Furthermore, PU.1 overexpression and knockdown experiments reveal that PU.1 negatively regulates chicken preadipocyte differentiation, as evidenced by appropriate changes in lipid droplet accumulation and altered expressions of PPARγ, FAS, and PLIN. In addition, PU.1 overexpression promotes preadipocyte proliferation, while knockdown of PU. 1 inhibits preadipocyte proliferation. We further demonstrate that PU.1 inhibits differentiation and promotes proliferation in preadipocytes, in part by directly interacting with KLF7.

KLF7 promotes preadipocyte proliferation via activation of the Akt signaling pathway by Cis-regulating CDKN3.

Krüppel-like transcription factor 7 (KLF7) promotes preadipocyte proliferation; however, its target gene in this process has not yet been identified. Using KLF7 ChIP-seq analysis, we previously showed that a KLF7-binding peak is present upstream of the cyclin-dependent kinase inhibitor 3 gene ( CDKN3) in chicken preadipocytes. In the present study, we identify CDKN3 as a target gene of KLF7 that mediates the effects of KLF7 on preadipocyte proliferation. Furthermore, 5'-truncating mutation analysis shows that the minimal promoter is located between nt -160 and nt -7 (relative to the translation initiation codon ATG) of CDKN3. KLF7 overexpression increases CDKN3 promoter activity in the DF-1 and immortalized chicken preadipocyte (ICP1) cell lines. Deletion of the putative binding site of KLF7 abolishes the promotive effect of KLF7 overexpression on CDKN3 promoter activity. Moreover, CDKN3 knockdown and overexpression assays reveal that CDKN3 enhances ICP1 cell proliferation. Flow cytometry analysis shows that CDKN3 accelerates the G1/S transition. Furthermore, we find that KLF7 promotes ICP1 cell proliferation via Akt phosphorylation by regulating CDKN3. Taken together, our results suggest that KLF7 promotes preadipocyte proliferation by activating the Akt signaling pathway by cis-regulating CDKN3, thus driving the G1/S transition.

Anti-obesity effects of corn peptide on 3T3-L1 preadipocytes and C57BL/6J obese mice.

Corn peptide (CP) is a small, natural, biologically active peptide obtained by protease-catalysed hydrolysis of corn. CP exerts antihypertensive, hypoglycaemic, antihyperlipidemic, antioxidant, and antitumor effects, as well as prevents cardiovascular and cerebrovascular diseases. Although CP plays a role in preventing obesity-related diseases, its role in reducing obesity has not yet been determined. In this study, we analysed the inhibitory effects of CP on lipid droplet accumulation in 3T3-L1 preadipocytes and high-fat diet (HFD)-induced C57BL/6J Obese Mice. The results show that CP could inhibit preadipocyte differentiation and oil accumulation in 3T3-L1 preadipocytes. Oral CP administration reduced serum triglyceride (TG) content, epididymal fat weight, abnormal liver fat droplet accumulation, and C/EBPα expression. Furthermore, combination of CP administration and exercise reduced body, liver, and adipose tissue weights; decreased serum total cholesterol (TC), triglyceride and low-density lipoprotein (LDL) levels; and inhibited hepatic lipid droplet accumulations and epididymal fat cell hypertrophy. Additionally, this combination inhibited the expression of transcription factors, C/EBPα, C/EBPß, and PPARγ, and adipogenic factors, FABP4 in mice. In conclusion, oral administration of CP inhibited lipid droplet accumulation and counteracted HFD-induced obesity in mice.

CpG site DNA methylation of the CCAAT/enhancer-binding protein, alpha promoter in chicken lines divergently selected for fatness.

CCAAT/enhancer-binding protein (C/EBP), alpha (CEBPA) is a master regulator of adipogenesis and, together with peroxisome proliferator-activated receptor gamma (PPARG), plays a critical role in adipocyte differentiation. Previous studies have demonstrated that CEBPA is regulated by DNA methylation and involved in the osteogenesis and adipogenesis of mouse C3H10T1/2 and bone marrow stromal cells. However, it is unclear whether CEBPA is regulated by DNA methylation in adipose tissues. Thus, the objectives of the present study were to investigate CpG site methylation in a 357-bp CEBPA promoter region and to assess the correlation between promoter CpG site methylation and CEBPA gene expression in the abdominal adipose tissues of Northeast Agricultural University broiler lines divergently selected for abdominal fat content. The results showed that the methylation percentage of the analyzed CEBPA promoter region was significantly higher in lean broilers than in fat broilers at 2 weeks (80.3% vs. 43.4%, P < 0.0001), 3 weeks (95.4% vs. 74.0%, P < 0.0001) and 7 weeks of age (82.6% vs. 57.2%, P < 0.0001). Real-time quantitative RT-PCR analysis showed that CEBPA expression was significantly higher in the fat vs. the lean line at 2 weeks of age (P = 0.0013) but not at 3 or 7 weeks of age. The correlation analysis showed that only at 2 weeks of age was the methylation percentage negatively correlated with CEBPA expression (Pearson's r = -0.8312, P = 0.0029). Of all seven tested CpGs, only two, the CpGs at -1494 and -1478 bp, displayed a significantly negative correlation with CEBPA mRNA expression. These results suggest that the CEBPA is methylated in adipose tissue and may regulate chicken early adipose development.

Identification and characterization of transcript variants of chicken peroxisome proliferator-activated receptor gamma.

Peroxisome proliferator-activated receptor gamma regulates adipogenesis. The genomic structure of the chicken peroxisome proliferator-activated receptor gamma (cPPARγ) gene has not been fully characterized, and only one cPPARγ gene mRNA sequence has been reported in genetic databases. Using 5' rapid amplification of cDNA ends, we identified five different cPPARγ mRNAs that are transcribed from three transcription initiation sites. The open reading frame analysis showed that these five cPPARγ transcript variants (cPPARγ1 to 5) could encode two cPPARγ protein isoforms (cPPARγ1 and cPPARγ2), which differ only in their N-terminal region. Quantitative real-time RT-PCR analysis showed that, of these five cPPARγ transcript variants, cPPARγ1 was ubiquitously highly expressed in various chicken tissues, including adipose tissue, liver, kidney, spleen and duodenal; cPPARγ2 was exclusively highly expressed in adipose tissue; cPPARγ3 was highly expressed in adipose tissue, kidney, spleen and liver; cPPARγ4 and cPPARγ5 were ubiquitously weakly expressed in all the tested tissues, and comparatively, cPPARγ5 was highly expressed in adipose tissue, heart, liver and kidney. The comparison of the expression of the five cPPARγ transcript variants showed that adipose tissue cPPARγ1 expression was significantly higher in the fat line than in the lean line from 2 to 7 wk of age (P < 0.05 or P < 0.01). Adipose tissue cPPARγ3 expression was significantly higher in the fat line than in the lean line at 3, 5 and 6 wk of age (P < 0.01, P < 0.05), but lower at 4 wk of age (P < 0.05). Adipose tissue cPPARγ5 expression was significantly higher in the fat line than in the lean line at 3, 4, and 6 wk of age (P < 0.01) and at 2 and 7 wk of age (P < 0.05). This is the first report of transcript variants and protein isoforms of cPPARγ gene. Our findings provided a foundation for future investigations of the function and regulation of cPPARγ gene in adipose tissue development.

E2F transcription factor 5, a new regulator in adipogenesis to mediate the role of Krüppel-like factor 7 in chicken preadipocyte differentiation and proliferation.

E2F transcription factor 5 (E2F5) gene is a transcription factor, plays an important role in the development of a variety of cells. E2F5 is expressed in human and mouse adipocytes, but its specific function in adipogenesis is unclear. Krüppel-like factor 7 (KLF7) facilitates proliferation and inhibits differentiation in chicken preadipocytes. Our previous KLF7 chromatin immunoprecipitation-sequencing analysis revealed a KLF7-binding peak in the 3' flanking region of the E2F5, indicating a regulatory role of KLF7 in this region. In the present study, we investigated E2F5 potential role, the overexpression and knockdown analyses revealed that E2F5 inhibited the differentiation and promoted the proliferation of chicken preadipocytes. Moreover, we identified enhancer activity in the 3' flanking region (nucleotides +22661/+22900) of E2F5 and found that KLF7 overexpression increased E2F5 expression and luciferase activity in this region. Deleting the putative KLF7-binding site eliminated the promoting effect of KLF7 overexpression on E2F5 expression. Further, E2F5 reversed the KLF7-induced decrease in preadipocyte differentiation and increase in preadipocyte proliferation. Taken together, our findings demonstrate that KLF7 inhibits differentiation and promotes proliferation in preadipocytes by enhancing E2F5 transcription.

Klf7 modulates the differentiation and proliferation of chicken preadipocyte.

Krüppel-like factor 7 (Klf7) has been extensively studied in the mammalian species, but its function in avian species is unclear. The objective of this study was to reveal the function of chicken Klf7 (Gallus gallus Klf7, gKlf7) in adipogenesis. The results of real-time reverse transcription polymerase chain reaction demonstrated that the relative mRNA level of chicken Klf7 (gKlf7/gß-Actin) in the abdominal adipose tissue was significantly associated with the abdominal fat content and the age of broilers (P < 0.05), and gKlf7 was more highly expressed in preadipocytes than in mature adipocytes (P < 0.05). In addition, Oil red O staining showed that gKlf7 inhibited chicken preadipocyte differentiation, and MTT assay indicated that gKlf7 overexpression promoted preadipocyte proliferation. Additionally, luciferase assays showed that gKlf7 overexpression suppressed the chicken CCAAT/enhancer-binding protein α (C/ebpα), fatty acid synthase (Fasn), and lipoprotein lipase (Lpl) promoter activities (P < 0.05), and gKlf7 knockdown increased the chicken peroxisome proliferator-activated receptor γ (Pparγ), C/ebpα and fatty acid-binding protein 4 (Fabp4) promoter activities (P < 0.05). Together, our study demonstrated that chicken Klf7 inhibits preadipocyte differentiation and promotes preadipocyte proliferation.

Genome-wide survey identifies TNNI2 as a target of KLF7 that inhibits chicken adipogenesis via downregulating FABP4.

Krüppel-like factor 7 (KLF7) negatively regulates adipocyte differentiation; however, the mechanism underlying its activity in mammals and birds remains poorly understood. To identify genome-wide KLF7-binding motifs in preadipocytes, we conducted a chromatin immunoprecipitation-sequencing analysis of immortalized chicken preadipocytes (ICP2), which revealed 11,063 specific binding sites. Intergenic binding site analysis showed that KLF7 regulates several novel factors whose functions in chicken and mammal adipogenesis are underexplored. We identified a novel regulator, troponin I2 (TNNI2), which is positively regulated by KLF7. TNNI2 is downregulated during preadipocyte differentiation and acts as an adipogenic repressor at least in part by repressing FABP4 promoter activity. In conclusion, we demonstrated that KLF7 functions through cis-regulation of TNNI2, which inhibits adipogenesis. Our findings not only provide the first genome-wide picture of KLF7 associations in preadipocytes but also identify a novel function of TNNI2.

KLF2 Inhibits Chicken Preadipocyte Differentiation at Least in Part via Directly Repressing PPARγ Transcript Variant 1 Expression.

Peroxisome proliferator-activated receptor gamma (PPARγ) is the master regulatory factor of preadipocyte differentiation. As a result of alternative splicing and alternative promoter usage, PPARγ gene generates multiple transcript variants encoding two protein isoforms. Krüppel-like factor 2 (KLF2) plays a negative role in preadipocyte differentiation. However, its underlying mechanism remains incompletely understood. Here, we demonstrated that KLF2 inhibited the P1 promoter activity of the chicken PPARγ gene. Bioinformatics analysis showed that the P1 promoter harbored a conserved putative KLF2 binding site, and mutation analysis showed that the KLF2 binding site was required for the KLF2-mediated transcription inhibition of the P1 promoter. ChIP, EMSA, and reporter gene assays showed that KLF2 could directly bind to the P1 promoter regardless of methylation status and reduced the P1 promoter activity. Consistently, histone modification analysis showed that H3K9me2 was enriched and H3K27ac was depleted in the P1 promoter upon KLF2 overexpression in ICP1 cells. Furthermore, gene expression analysis showed that KLF2 overexpression reduced the endogenous expression of PPARγ transcript variant 1 (PPARγ1), which is driven by the P1 promoter, in DF1 and ICP1 cells, and that the inhibition of ICP1 cell differentiation by KLF2 overexpression was accompanied by the downregulation of PPARγ1 expression. Taken together, our results demonstrated that KLF2 inhibits chicken preadipocyte differentiation at least inpart via direct downregulation of PPARγ1 expression.

GATA Binding Protein 3 Is a Direct Target of Kruppel-Like Transcription Factor 7 and Inhibits Chicken Adipogenesis.

Kruppel-like transcription factor 7 (KLF7) is a negative regulator of adipogenesis, however, its precise mechanism is poorly understood. Our previous KLF7 ChIP-seq analysis showed that one of the KLF7 binding peaks was present upstream of GATA binding protein 3 (GATA3) in chicken preadipocytes. In the present study, we identified GATA3 as a target of KLF7. Overexpression analysis showed KLF7 markedly enhanced the endogenous expression of GATA3 in the immortalized chicken preadipcyte cell line (ICP2), and the luciferase reporter assay showed that KLF7 overexpression increased the reporter gene activity of the cloned upstream region (-5285/-4336 relative to the translation initiation codon ATG) of GATA3 in ICP2 and DF1 cells, and mutation of the putative KLF7 binding site abolished the promotive effect of KLF7 overexpression on the reporter gene activity of the cloned GATA3 upstream region. ChIP-qPCR further demonstrated that KLF7 directly bound to the GATA3 upstream region. Gene expression analysis showed that GATA3 mRNA expression in abdominal adipose tissue was significantly higher in lean chicken line than in the fat line at 2, 3, and 6 weeks of age. In addition, GATA3 mRNA expression markedly decreased during the preadipocyte differentiation. Furthermore, a functional study showed that GATA3 overexpression inhibited the differentiation of the ICP2 cells. Taken together, our results demonstrated that KLF7 inhibits chicken adipogenesis, at least in part through direct upregulation of GATA3.