Comparative Analysis of Genomic Imprinting and DNA Methylation between Mammals and Chickens

　Genomic imprinting, the parent-of-origin-specific expression of genes, has been observed in a variety of eutherian mammals but its precise mechanisms and the reasons for its evolution are largely unknown. Previous studies revealed that IGF2 and MPR/IGF2R are imprinted in eutherian mammals and marsupials but not in monotremes or birds. As a step to understand how genomic imprinting evolved and is regulated, I studied chicken (Gallus gallus), which is one of the best model animals among oviparous vertebrate species. First, I examined whether the chicken orthologues of mammalian imprinted genes other than IGF2 and MPR/IGF2R show parent-of-origin-specific expression. I found that chicken ASCL2/CASH4 and INS are biallelically expressed, further supporting the notion that imprinting evolved after the divergence of mammals and birds. Second, I carried out a comparative analysis of the ASCL2/CASH4-INS-IGF2-H19 region for cis-regulatory elements involved in imprinting, between mammals and chickens. The H19 imprinting center (IC) and many of the local regulatory elements identified in mammals were not found in chickens. Third, I attempted to clone and characterize the chicken DNA methyltransferase (DNMT) genes because DNA methylation has been shown to be involved in imprinting in mammals. I found that DNMT3A, a de novo methyltransferase gene essential for imprint establishment, is highly conserved in mammals and chickens. However, DNMT3L, a germ line-specific DNMT3-like protein essential for imprint establishment, was not present in chickens. Thus, many of the cis-regulatory elements involved in imprinting and some of the proteins essential for imprint establishment were missing in chickens. This study presents that the critical factors associated with imprinting probably evolved after the divergence of mammals and birds

Structural and functional analysis of a 0.5-Mb chicken region orthologous to the imprinted mammalian Ascl2/Mash2–Igf2–H19 region

Previous studies revealed that Igf2 and Mpr/Igf2r are imprinted in eutherian mammals and marsupials but not in monotremes or birds. Igf2 lies in a large imprinted cluster in eutherians, and its imprinting is regulated by long-range mechanisms. As a step to understand how the imprinted cluster evolved, we have determined a 490-kb chicken sequence containing the orthologs of mammalian Ascl2/Mash2, Ins2 and Igf2. We found that most of the genes in this region are conserved between chickens and mammals, maintaining the same transcriptional polarities and exon–intron structures. However, H19, an imprinted noncoding transcript, was absent from the chicken sequence. Chicken ASCL2/CASH4 and INS, the orthologs of the imprinted mammalian genes, showed biallelic expression, further supporting the notion that imprinting evolved after the divergence of mammals and birds. The H19 imprinting center and many of the local regulatory elements identified in mammals were not found in chickens. Also, a large segment of tandem repeats and retroelements identified between the two imprinted subdomains in mice was not found in chickens. Our findings show that the imprinted genes were clustered before the emergence of imprinting and that the elements associated with imprinting probably evolved after the divergence of mammals and birds