Studies on Antimicrobial Peptides and Epigenetic Factors in Transgenic Birds

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농업생명과학대학 농생명공학부
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서울대학교 대학원
chickenquailDNA methylationgene expressionantimicrobial peptide
학위논문 (박사)-- 서울대학교 대학원 : 농생명공학부, 2013. 8. 한재용.
In this study, we investigated the relative expression of the Rous sarcoma virus (RSV) promoter-driven expression of enhanced green fluorescent protein (EGFP) in fibroblasts of transgenic quails. We analyzed the direct influence of CpG methylation of the RSV promoter on the transcriptional activity of delivered transgenes. Embryonic fibroblasts collected from homozygous trans-genic quail (TQ2) were treated with 50 μM of DNA methyltransferase inhibitor followed by 5-aza-2´-deoxy-cytidine (5-azadC) for 48 h, and changes in expression were then analyzed by flow cytometry. The results show a significant increase of EGFP expression in TQ2 embryonic fibroblasts (QEFs) (2.64% to 79.84%). Subsequent methylation-specific amplification revealed that 5-azadC significantly reduced the CpG methylation status in the RSV promoters of the QEFs (86.42 to 48.41%)
even after 5-azadC was withdrawn, CpG methylation remained decreased in expanded culture (16.28%). Further analysis showed that potential transcription factor binding sites existed in the CpG methylation site of the RSV promoter. These results may provide the basis for understanding the epigenetic mechanism responsible for transgenic animal production and genetic preservation.

DNA methylation reprograming of primordial germ cells (PGCs) in mammals establishes monoallelic expression of imprinting genes, maintains retrotransposons in an inactive state, inactivates one of the two X chromosomes, and suppresses gene expression. However, the roles of DNA methylation in chickens PGCs are unknown. In this study, we found a 1.5-fold or greater difference in the expression of 261 transcripts when comparing PGCs and chicken embryonic fibroblasts (CEFs) using an Affymetrix GeneChip Chicken Genome Array. In addition, we analyzed the methylation patterns of the regions ~5-kb upstream of 261 sorted genes, 51 of which were imprinting homologous loci and 49 of which were X-linked homologous loci in chicken using the MeDIP Array by Roche NimbleGen. Seven hypomethylated and five hypermethylated regions within the 5-kb upstream regions of 261 genes were found in PGCs when compared with CEFs. These differentially methylated regions were restrictively matched to differentially expressed genes in PGCs. We also detected 203 differentially methylated regions within imprinting and X-linked homologous regions between male PGCs and female PGCs. These differentially methylated regions may be directly or indirectly associated with gene expression during early embryonic development, and the epigenetic difference could be evolutionally conserved between mammals and birds.

The basic functions of DNA methylation include in gene silencing by methylation of specific gene promoters, defense of the host genome from retrovirus, and transcriptional suppression of transgenes. In addition, genomic imprinting, by which certain genes are expressed in a parent-of-origin-specific manner, has been observed in a wide range of plants and animals and has been associated with differential methylation. However, imprinting phenomena of DNA methylation effects have not been revealed in chickens. To analyze whether genomic imprinting occurs in chickens, methyl DNA immunoprecipitation array analysis was applied across the entire genome of germ cells in early chick embryos. A differentially methylated region (DMR) was detected in the eighth intron of the L-arginine:glycine amidinotransferase (GATM) gene. When the DMR in GATM was analyzed by bisulfite sequencing, the methylation in male primordial germ cells (PGC) of 6-d-old embryos was higher than that in female PGC (57.5 vs. 35.0%). At 8 d, the DMR methylation of GATM in male PGC was 3.7-fold higher than that in female PGC (65.0 vs. 17.5%). Subsequently, to investigate mono- or biallelic expression of the GATM gene during embryo development, we found 2 indel sequences (GTTTAATGC and CAAAAA) within the GATM 3′-untranslated region in Korean Oge (KO) and White Leghorn (WL) chickens. When individual WL and KO chickens were genotyped for indel sequences, 3 allele combinations (homozygous insertion, homozygous deletion, and heterozygotes) were detected in both breeds using a gel shift assay and high resolution melt assay. The deletion allele was predominant in KO, whereas the insertion allele was predominant in WL. Heterozygous animals were evenly distributed in both breeds (P < 0.01). Despite the different methylation status between male and female PGC, the GATM gene conclusively displayed biallelic expression in PGC as well as somatic embryonic, extraembryonic, and adult chicken tissues.

Cathelicidins are antimicrobial peptide components of the innate immune system. Four cathelicidins have been identified in the chicken: cathelicidin1 (CATH1), cathelicidin2 (CATH2), cathelicidin3 (CATH3), and cathelicidinB1 (CATHB1). The aim of this study was to characterize the antibacterial activities, structural conservation and expression patterns of these antimicrobial peptides. All had a highly conserved functional domain. The expression of CATH1, CATH2, and CATH3 mRNA was high in the bone marrow of adult female chickens. By contrast, CATHB1 mRNA was highly expressed in the thymus. The active domains of all four chicken cathelicidins were synthesized, and their antibacterial activities on cell viability, membrane damage, and colony formation of Escherichia coli were examined. After treatment of E. coli with 0.5–10 μM of each cathelicidin, CATH1, CATH2, and CATH3 reduced cell viability at all concentrations. When E. coli were treated with 5 μM of each cathelicidin, CATH2 and CATH3 demonstrated maximum damage to the cell membrane. To examine the effect on colony formation, an ampicillin-resistant E. coli strain was established and treated with 5 μM of each cathelicidin. CATH1, CATH2, and CATH3 markedly arrested colony formation, whereas CATHB1 had no effect. The present results demonstrate dose-dependent antimicrobial effects of chicken cathelicidins that were mediated by membrane damage and had a mechanism independent of that of common antibiotics. Our data suggest a novel approach for controlling drug-resistant bacteria and for producing disease-resistant animals in the livestock industry.
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College of Agriculture and Life Sciences (농업생명과학대학)Dept. of Agricultural Biotechnology (농생명공학부)Theses (Ph.D. / Sc.D._농생명공학부)
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