Abstract
A CpG island (CGI) lies at the 5′ end of the Airn macro non-protein-coding (nc) RNA that represses the flanking Igf2r promoter in cis on paternally inherited chromosomes. In addition to being modified on maternally inherited chromosomes by a DNA methylation imprint, the Airn CGI shows two unusual organization features: its position immediately downstream of the Airn promoter and transcription start site and a series of tandem direct repeats (TDRs) occupying its second half. The physical separation of the Airn promoter from the CGI provides a model to investigate if the CGI plays distinct transcriptional and epigenetic roles. We used homologous recombination to generate embryonic stem cells carrying deletions at the endogenous locus of the entire CGI or just the TDRs. The deleted Airn alleles were analyzed by using an ES cell imprinting model that recapitulates the onset of Igf2r imprinted expression in embryonic development or by using knock-out mice. The results show that the CGI is required for efficient Airn initiation and to maintain the unmethylated state of the Airn promoter, which are both necessary for Igf2r repression on the paternal chromosome. The TDRs occupying the second half of the CGI play a minor role in Airn transcriptional elongation or processivity, but are essential for methylation on the maternal Airn promoter that is necessary for Igf2r to be expressed from this chromosome. Together the data indicate the existence of a class of regulatory CGIs in the mammalian genome that act downstream of the promoter and transcription start.
Highlights
Atypical CpG-rich regions known as CpG islands (CGIs) overlap 60–70% of mammalian transcription start sites [1]
CGIs can lie up- and downstream of the transcription start site (TSS) We first examined if the position of the Airn CGI that lies downstream of the TSS, represents a rare exception or a common occurrence in the mouse genome (Figure 1C)
For each CGI annotated by the UCSC genome browser, if a known protein-coding or non-protein-coding gene taken from the NCBI RNA reference sequences collection (RefSeq), has its TSS within the CGI or in the DNA region representing half the length of the CGI up- or downstream. 57% of all RefSeq genes were associated with a CGI, of which 88.5% including Igf2r, have their TSSs within the body of the CGI, with the majority lying in the first half. 11.5% of CGI-associated RefSeq genes have their TSS located outside of the annotated CGI, 8.7% of these have their TSS upstream and 2.8% have a TSS downstream of the CGI
Summary
Atypical CpG-rich regions known as CpG islands (CGIs) overlap 60–70% of mammalian transcription start sites [1]. A recent study used a biochemical purification strategy to identify a large number of novel CGIs not associated with annotated promoters, in the body of coding genes or in intergenic regions [2] While this could indicate the mammalian genome has many transcripts still to be identified, it is possible that CGIs have additional functions in addition to promoter regulation. CGIs within an ICE are similar to classic promoter-associated CGIs as they show a CpG density higher than the genome average and lack sequence conservation, even between homologous mouse and human elements [7]. They differ in several ways [8,9]. In six imprinted clusters the ICE has been shown by deletion experiments that include the CGI, to control repression of all Author Summary
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