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.

Mammalian imprinted genes, which are expressed from only one of the parental alleles, have a tendency to form clusters and are regulated by long-range mechanisms. Nuclear matrix-attachment regions (MARs), the anchor points of loop domains, are involved in coordination of gene expression and could play a role in regulation of imprinted domains. We have identified and mapped a total of 52 MARs in a 1-Mb imprinted domain on mouse distal chromosome 7 using our cosmid contigs and an in vitro MAR assay. We find two MAR clusters (comprising 20 and 19 MARs), one of which is mapped in the Th-Ins2 intergenic region, coincident with the boundary between the two imprinted subdomains. However, the imprinted/non-imprinted boundaries are not associated with a MAR. Based on the sequence information, we find that many of the MARs are rich in long interspersed nuclear elements. In addition, comparisons of the results obtained with several MAR-prediction software programs reveal good performance of ChrClass in terms of both sensitivity and specificity. This study presents the first large-scale mapping of MARs in an imprinted domain and provides a platform for understanding the roles of MARs in imprinting.

Mammalian genes subject to genomic imprinting often form clusters and are regulated by long-range mechanisms. The distal imprinted domain of mouse chromosome 7 is orthologous to the Beckwith-Wiedemann syndrome domain in human chromosome 11p15.5 and contains at least 13 imprinted genes. This domain consists of two subdomains, which are respectively regulated by an imprinting center. We here report the finished-quality sequence of a 0.6-Mb region encompassing the more centromeric subdomain. The sequence contains four imprinted genes (Ascl2/Mash2, Ins2, Igf2 and H19) and reveals previously unidentified CpG islands and tandem repeats, which may be features of imprinted genes. Most interestingly, a unique 210-kb segment consisting almost exclusively of tandem repeats and retroelements is identified. This segment, located between Th and Ins2, has features of heterochromatin-forming DNA and is highly methylated at CpG sites. The segment exhibits asynchronous replication on the parental chromosomes, a feature of the imprinted domains. We propose that this repeat segment could serve either as a boundary between the two subdomains or as a target for epigenetic chromatin modifications that regulate imprinting.