| Kruppel-like factors (KLFs) comprise a protein family whose members contain three highly conserved Cys2-His2 zinc finger DNA-binding domains close to their C-terminus. By regulating the expression of a large number of genes that have GC-rich sequences including GC-box and GT-box (also known as CACCC box) in promoters, KLF transcription regulators have been shown to have important roles in a diverse array of physiological functions including hematopoiesis, angiogenesis, cardiac remodeling, lymphocytes development, adipogenesis, tumorigenesis, gluconeogenesis, monocyte activation, bone formation, neurological development, and determination of pluripotent stem cell fate.In this study, we identified 17 members of porcine KLF family, conducted in-depth comparative analyses to elucidate the structural and functional properties of 17 KLF proteins, and mapped the KLF genes onto relevant chromosomes in pigs. We have undertaken the first comprehensive and rigorous analysis on the gene structure and phylogeny of KLF gene family. We also studied the transcriptional regulation mechanism of KLF 15 in adipocyte differentiation. The detailed results are as follows:1. We performed RT-PCR to obtain 17 full-length cDNA sequences of porcine KLF family, among which 12 have not been previously reported. The open reading frames of 17 porcine KLFs range from 735 bp to 1485 bp in size, encoding 244 to 494 amino acids. Pig KLF proteins and their corresponding human homologs have an average 88.5% identity over the whole sequence and 98.2% identity in zinc fingers domains.2. We performed genomic PCR and referred the available porcine genomic data to obtain the genomic organization of porcine KLF genes. Interspecies comparison of KLF orthologs in pigs, humans and mice revealed a high level of conservation at the genomic structure level.3. Using the INRA-IMpRH panel and available porcine genomic data, we located the porcine KLFs on chromosomes 1,2,3,4,6,7,8,10,11,15,18 and X, respectively. The chromosomal positions of porcine KLF genes are consistent with the established human-pig comparative map.4. We obtained 103 nonredundant KLF sequences from 9 representative species, and constructed a comprehensive phylogenetic analysis. Two large groups were defined in the phylogenetic tree, and two groups were divided into several subgroups including KLF1/2/4/17, KLF3/8/12, KLF5/6/7, KLF10/11, KLF9/13/14/16 and KLF15. We proposed that the KLF gene family have expanded in two phases over the course of evolution. The first phase occurred in the chordate lineage, during the early emergence of vertebrates, and in the second phase, the full complements of 17 KLF genes were generated in the mammalian lineage.5. We constructed a predictive ancestral sequence of zinc finger domain by alignment of amino acid sequences of the zinc fingers, which can accurately identify.potential KLF proteins.6. A phylogenetic analysis revealed that KLF4 first appeared in vertebrate, and is closely related to KLF2 among the KLF1/2/4/17 subgroup. The structural characteristics of KLF4 from various species, including exon and intron sizes and number variations, and pseudogenes were compared to elucidate the evolutionary pathway.7. In the process of PCR amplification, we encountered an additional longer variant of KLF4 gene in pigs. Sequence comparison revealed that the variant retains intron 3 (99 bp) which differ with the normal porcine KLF4 mRNA. We also examined the potential transcriptional and translational regulation of KLF4 gene.8. The expression pattern of KLF15 are in concert with PPARy2 during the adipocyte differentiation. We constructed the overexpression vectors of KLF15 and luciferase reporter assay system containing four various deletion constructs of the PPARy2 promoter, we found that KLF15 increases the activities of four deletion constructs of the PPARy2 promoter in varying degrees by co-expression experiments, indicating that KLF15 may function in several different sites of the promoter of PPARy2. |
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