Isolation and analysis of sequences showing sex-specific cytosine methylation in the mealybug Planococcus lilacinus

Abstract

Genomic libraries of Planococcus lilacinus, a mealybug in which paternal chromosomes are facultatively heterochromatic and inactive in sons but not in daughters, were probed with subtraction probes in order to estimate the number of sequences displaying sex-specific cytosine methylation in CpG dinucleotides. Sequences showing male-specific methylation were found to occur approximately 2.5 times more often than those showing female-specific methylation. In order to directly isolate sequences showing sex-specific CpG methylation, we employed methylation-specific arbitrarily primed (MS-AP) polymerase chain reaction (PCR) and identified 72 sex-specific products, of which 51 were from males and 21 from females. Amplification of bisulfite-modified DNA and subsequent Southern hybridization showed that in 33 out of these 72 sex-specific products, there was differential methylation of homologous sequences; i.e., both methylated and unmethylated copies of the same sequence occurred in one sex whereas only unmethylated copies were present in the opposite sex. Sequencing of bisulfite-modified DNA showed an interspersion of CpG and non-CpG methylation among the sex-specifically methylated sequences. Sequences showing male-specific CpG methylation are organized as transcriptionally silent chromatin in males but not in females, whereas those showing female-specific CpG methylation are organized as transcriptionally silent chromatin in females but not in males. The sequences identified in this study that show differential methylation in males, but are unmethylated in females, may prove useful in the study of imprinting in the mealybug system.

5-methyl-cytosine and 5-hydroxy-methyl-cytosine in the genome of Biomphalaria glabrata, a snail intermediate host of Schistosoma mansoni

“…Some species dominantly display methylation in the context of CpG, such as C. intestinalis , S. gregaria , B. mori , M. extradentata and A. mellifera [5,9,14,47,48,50], while other species such as P. lilacinus and D. melanogaster, Pogonomyrmex barbatus display a high proportion of methylation in non-CpG methylation sites [10,13,51]. The bisulfite sequencing analysis performed in our study identified only methylation of CpG sites, suggesting that this is the dominant type of DNA methylation in B. glabrata .…”

The Genome of the Stick Insect Medauroidea extradentata Is Strongly Methylated within Genes and Repetitive DNA

“…In contrast, Drosophila shows mainly methylation in a CpT or CpA context [27] . In the mealy bug Planococcus lilacinus , methylcytosine appears to occur nearly independently of the identity of the 3′ nucleotide [28] , [29] . Thus, the sequence context of Medauroidea DNA methylation seems to be more similar to that of vertebrates than to that of mealy bugs or Drosophila.…”

An Analysis of Histone Modifications in Relation to Sex-Specific Chromatin Organization in the Mealybug <i>Maconellicoccus hirsutus</i>

“…The presence of unusual methylation like 6mA, 7mG and 5mC and non-CpG methylation was reported in P. lilacinus [Deobagkar et al, 1982;Achwal et al, 1983]. Hypermethylation of genomic DNA in males in Pseudococcus obscurus and Pseudococcus calceolariae was reported by Scarbrough et al [1984] and enrichment of CpG methylation in males of P. lilacinus was demonstrated by Mohan et al [2005], while Bongiorni et al [1999] found hypomethylation in the paternal set in P. citri . Thus there are contradicting reports on differential methylation of DNA between males and females in mealybugs.…”