Genome-wide comparison of DNA methylation between life cycle stages ofDrosophila melanogasterusing high-throughput sequencing techniques

Abstract

28Drosophila melanogaster undergoes holometabolous development, has very low levels of 29 DNA methylation, and is known to possess a single known methyltransferase, dDNMT2. 30 This study compares the DNA methylation patterns between the two life cycle stages of D. 31 melanogaster using a combination of DNA immunoprecipitation and high throughput 32 sequencing techniques. 33 Our results indicate, a change in the chromosomal distribution of the sparse DNA 34 methylation concerning genes and natural transposable elements between in the embryo and 35 the adult stages of D. melanogaster. The differentially methylated regions localised on genes 36 involved in the regulation of cell cycle processes of mitotic cell divisions and chromosomal 37 segregation. dDNMT2 knockout flies exhibited altered patterns of DNA methylation. The 38 observed differences in DNA methylation were in genes involved in cellular communication 39 and cytoskeletal functions. The variation in DNA methylation between the two life cycle 40 stages is indicative that it could have a role in regulatory processes during development and, 41 dDNMT2 may have a role as a co-factor for the hitherto undiscovered DNA 42 methyltransferase in D. melanogaster. 43 44 Keywords: differential DNA methylation, 5-methylcytosine, DNMT2, asymmetric DNA 45 methylation, non-CpG methylation 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60DNA methylation in Drosophila melanogaster was first reported in the embryos by a 61 combination of photoacoustic spectroscopy and polyclonal antibodies to be 0.008 mol percent 62 of 5-methylcytosine (5mC) 1,2 . These results were later corroborated by enzymatic digestion 63 of DNA with McrBC, high-performance liquid chromatography (HPLC) and 2D-Thin layer 64 chromatography approximately to report the presence of one 5mC molecule in 1,000-2,000 65 cytosines molecules in adult fly 3 . There was no sequence-specific data in Drosophila 66 genome until the analysis of the early embryos followed by sequencing of select bisulfite 67 modified regions which revealed methylation to be predominant in non-CpG dinucleotides 4 . 68Subsequently, high-throughput sequencing of sodium bisulfite-treated 69 immunoprecipitated fragments bearing methylation from stage 5 embryos with 10,000X 70 coverage reported less than 1% of genome-wide DNA methylation in the stage-5 embryos 5 . 71DNA samples of D. melanogaster in a mixed population of adult males and females (w 1118 72 strain) was subjected to liquid chromatography selective reaction monitoring (LC-SRM) 73 method to report 0.034% of methylated cytosines in the genome which is 10−100 fold below 74 the suggested detection limit of bisulphite sequencing 6 whereas the female adult (Oregon-R 75 strain) was reported to harbour 0.002% methylation of cytosines 7 . The whole genome 76 bisulfite-sequencing also correlated with the 55% increment in DNA methylation of the 77 infected testes; however, this did not show any correlation with cytoplasmic incompatibility 8 . 78The changes in dietary specification also...

Dynamic changes of DNA modifications: 5-hydroxymethyluracil and N6-methyladenine in the Drosophila melanogaster life cycle and in response to environmental factors

“…Subsequently, the samples were also homogenized with an ultrasonic homogenizer (SONOPULS UW 2070, BANDELIN electronic GmbH&Co. KG) two times for 10 s on ice. 45 µL of sample was mixed with 50 µL of 10% trichloroacetic acid and 5 µL of 100 µM labelled internal standard solution (L-Ascorbic Acid- 13 C 6 ,Toronto Research Chemicals) and incubated for 20 min. Then, the samples were vortexed and centrifuged at 24 400x g for 20 min at 4 ℃.…”

“…They show the de novo DNA methyltransferase activity in adult D. melanogaster and the presence of CpC methylation in selected genomic regions 12 . Deshmukh et al observed the presence of methylation in ies with a dDNMT2 knockout, although the methylation pro le was altered 13 . Methylation is also observed in the RNA of many species.…”

Dynamic changes in genomic 5-hydroxymethyluracil and N6-methyladenine levels in the Drosophila melanogaster life cycle and in response to different temperature conditions

“…These authors showed de novo DNA methyltransferase activity in adult D. melanogaster and the presence of CpC methylation in selected genomic regions 12 . Deshmukh et al observed the presence of methylation in flies with a dDNMT2 knockout, although the methylation profile was altered 13 .…”