Transcription Driven Somatic DNA Methylation within the Imprinted Gnas Cluster

Abstract

Differential marking of genes in female and male gametes by DNA methylation is essential to genomic imprinting. In female gametes transcription traversing differentially methylated regions (DMRs) is a common requirement for de novo methylation at DMRs. At the imprinted Gnas cluster oocyte specific transcription of a protein-coding transcript, Nesp, is needed for methylation of two DMRs intragenic to Nesp, namely the Nespas-Gnasxl DMR and the Exon1A DMR, thereby enabling expression of the Gnas transcript and repression of the Gnasxl transcript. On the paternal allele, Nesp is repressed, the germline DMRs are unmethylated, Gnas is repressed and Gnasxl is expressed. Using mutant mouse models, we show that on the paternal allele, ectopic transcription of Nesp traversing the intragenic Exon1A DMR (which regulates Gnas expression) results in de novo methylation of the Exon1A DMR and de-repression of Gnas just as on the maternal allele. However, unlike the maternal allele, methylation on the mutant paternal allele occurs post-fertilisation, i.e. in somatic cells. This, to our knowledge is the first example of transcript/transcription driven DNA methylation of an intragenic CpG island, in somatic tissues, suggesting that transcription driven de novo methylation is not restricted to the germline in the mouse. Additionally, Gnasxl is repressed on a paternal chromosome on which Nesp is ectopically expressed. Thus, a paternally inherited Gnas cluster showing ectopic expression of Nesp is “maternalised” in terms of Gnasxl and Gnas expression. We show that these mice have a phenotype similar to mutants with two expressed doses of Gnas and none of Gnasxl.

Antisense Activity across the Nesp Promoter is Required for Nespas-Mediated Silencing in the Imprinted Gnas Cluster

“…Although +/ Nespas-T int2 appeared to be normal at birth, were generated at the expected Mendelian frequency (49.6% of 244 newborns) and showed normal viability thereafter, within a few days they were seen to be thinner than their wild type litter mates and by postnatal day 14 weighed 67.3% of their wild-type litter mates ( P = 8.35 × 10 −11 ; +/+ n = 27, +/ Nespas-T int2 n = 23). Growth retardation is typical of mutants with reduction in Gnasxl levels [ 8 , 15 , 22 , 23 , 24 ]. Expression of Exon1A and Gnas was found to be unaffected ( Figure 6 a,b).…”

“…Methylation of the Nesp DMR occurs between 3.5 dpc and 8.5 dpc [ 15 , 28 ]. Nesp is not robustly expressed until 6.5 dpc [ 24 , 29 ], but Nespas is expressed much earlier, from the two cell stage onwards [ 29 ]. Expression of Nespas is associated with elevated levels of histones H3K36me3 and unmethylated H3K4 at the Nesp promoter [ 4 ].…”

“…Nespas-T int2 is the fourth mutant in which the Nesp DMR is unmethylated on paternal inheritance and Gnasxl is downregulated. Unlike Nespas-T int2 , there is expression of Nesp from the mutant paternal allele in the three other mutants, ΔNAS-DMR , Nespas-T ex1 and Nesp-T int [ 4 , 11 , 24 ]. Furthermore, Gnasxl expression is reduced even though the Gnasxl DMR is unmethylated [ 4 , 11 ].…”

“…However, whereas methylation of the Nesp DMR appears to be crucial for full paternal Gnasxl expression, it is suppression of Nesp transcription that is essential for tissue specific paternal repression of Gnas . Ectopic paternal expression of Nesp results in loss of imprinting of Gnas [ 24 ]. This occurs because ectopic transcription of Nesp results in methylation of the Exon1A DMR; the Exon1A DMR controls the imprinted expression of Gnas [ 25 , 28 ] and methylation of the Exon1A DMR results in upregulation of Gnas in tissues where Gnas shows imprinted expression [ 4 , 11 , 24 ].…”

Epigenetic Influences upon Autism Spectrum Disorder

“…If the allele from the mother is imprinted, then only the allele from the father is expressed. Forms of genomic imprinting have been demonstrated in fungi, plants and mammals with over 150 imprinted in laboratory rodents [1,2]. Through forces of epigenetics, environmental factors, such as endocrine-disrupting agents and mental stress in early life, can alter epigenetic status and gene expression.…”