Establishment Of A Semi-Quantitative Assay Of Global DNA Methylation Status Using Methyl-CpG Binding Protein (MBD1)and Prokaryotic Expression, Purification Of Chicken Fto Protein And Polyclonal Antibody Preparation

In mammals, DNA methylation which occurs at the5-position of cytosine is the most essential epigenetic modification. Change in the level of genome-wide DNA methylation （also known as global DNA methylation） is associated with alterations in gene expression, and thus contributes to phenotypic and physiological diversity. Current technologies for detecting global DNA methylation either suffer from the low sensitivity or require sophisticated equipments. Studies on domestic animals are hampered by lacking of genomic information. Here we developed a rapid method of slot blot with recombinant methyl-CpG binding protein （MBD1） to exam the level of global DNA methylation in pigs and chickens.1An assay for detecting the global DNA methylation by using MBD1Recombinant His6-tagged MBD1proteins were purified from200mL induced BL21（DE3） cultures on Ni-NTA agarose under denatured condition and followed by on column refolding. The specificity of the MBD1protein was tested against Sss1-methylated and non-methylated bacteriophage lambda DNA （λDNA）. We blotted a serial dilution of methylated λDNA samples and measured the status of DNA methylation. Signals were obtained only in the slots containing the methylated λDNA, indicating that the MBD1protein does not cross-react with unmethylated cytosine, in addition the intensity of the signals increased as the mass of methylated λDNA increasing. A linear regression analysis of the data showed quantitative recovery across the entire methylation range （0-100%）, with a R2of0.9705and demonstrated high specificity of the assay. Then, the sensitivity of methy-CpG-binding was examined by incubating recombinant MBD1protein with membranes carrying different quantities of genomic DNA. Genomic DNA extracted from the muscle of D1chicken was used and the result suggested that the slot blot routinely allowed detection of genomic DNA to a sensitivity of0.5μg.2Application and evaluation of the assayUsing this rapid approach, we determined the methylation status in various DNA samples of a Chinese indigenous pig breed （Erhualian）, and a Western breed （Large White）, and found that the differences between muscle and the other two tissues, liver and adrenal grand were significant with p less than0.05; in addition the degree of genomic DNA methylation is higher in Erhualian piglets than in Large White in all the three tissue types, and there is a significant breed difference detected in the liver （P=0.034）. We also chose two developmental stages--day18embryo （E18） and newly hatched chicks （D1） of a Chinese indigenous chicken breed （Wen’s yellow-feathered broiler chicken）. Analysis of the relative level of methylation revealed tissue-specific differences:the kidney is more methylated than the liver at E18. Moreover, there is age-dependent variations in the level of global DNA methylation:the chicken liver, kidney, intestine, and leg muscle exhibited a decrease at D1compared to E18; the age-related drop in global DNA methylation level was significant in kidney （P=0.05）. To confirm our slot blot assay, one of the results was compared with conventional Southern blotting with commercial anti-5mc antibody. The result was consistent with that detected by our method.3Prokaryotic expression, purification of chicken FTO protein and preparation of its polyclonal antibodyFTO （fat mass and obesity-associated gene） is associated with the risk of obesity and energy metabolism. Previous study in our lab indicated that the FTO gene was expressed in chickens. However there is no antibody specific for chicken FTO protein. In order to express and purify the recombinat protein of chicken FTO, the1523bp DNA coding sequence of chicken FTO gene was obtaied from GenBank （NM₀01185147） and subsequently amplified by PCR. Then the PCR product was inserted into pColdl plasmid to construct the prokaryotic expression plasmid pColdI-FTO which was then transformed into E.coli BL21（DE3）. The rocombinant protein was expressed with the induction of Isopropyl β-D-1-thiogalactopyranoside （IPTG） and was purified by Ni-NTA agarose chromatography column. The antiserum against FTO protein was raised from the rabbits immunized with the recombined FTO protein. The titer of the antibody was determined by ELISA and the specificity of the antibody was confirmed by Western blot. The titer of the anti-FTO antibody detected by ELISA was1:51200. Western blot analysis proved that the specificity of the antibody was adequate. In addition, protein extracts from different tissues of chicken were subjected to Western blot analysis. The results showed that the FTO protein （60kD） was ubiquitously expressed in hypothalamus, kidney, leg muscle, liver, spleen and ovary. Additional bands of approximately42kD,33kD or27kD were also observed, which may represent FTO protein isoforms translated from alternatively spliced transcripts.