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Airn transcriptional overlap but not its lncRNA products induce imprinted Igf2r silencing.

ABSTRACT: To test if the imprinted long non-coding RNA (lncRNA) Airn transcription or its product silences the protein-coding Igf2r gene, we shortened the endogenous lncRNA to four different lengths by inserting polyadenylation (polyA) cassettes on the paternal chromosome via homologous recombination in ES cells. ES cell differentiation was used to recapitulate the developmental onset of Airn and Igf2r imprinted expression and polyA cassettes inserted before (T3, T16, T27) or after (T31, T51) the Igf2r promoter successfully truncated Airn, with the exception of T27. RNA hybridization to a tiling array (MIRTA) demonstrated loss of Airn upstream of the Igf2r promoter (except T27) and absence of novel spliced variants in all truncation alleles. To further test the transcriptional overlap model we moved the Airn promoter ~700bp before the Igf2r TSS in ES cells that lack an endogenous paternal Airn promoter and called this cell line FAP (Forward-Airn-Promoter). RNA hybridization to a tiling array (MIRTA) demonstrated that the moved Airn promoter expresses Airn in wildtype orientation and terminates at the wildtype 3' end.

ORGANISM(S): Mus musculus

PROVIDER: GSE41444 | GEO | 2012/12/20

SECONDARY ACCESSION(S): PRJNA177027

REPOSITORIES: GEO

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Project description:The Igf2r imprinted cluster is an epigenetic model of cis-acting silencing in which expression of a ncRNA silences multiple genes. Here, we map chromatin profiles on the maternal and paternal chromosome in a 250 kb region. We show that histone modifications associated with expressed and silent genes are mutually exclusive and localize to discrete regions. Expressed genes are modified in two ways; at promoter regions by H3K4me3+H3K4me2+H3K9Ac and on putative regulatory elements by H3K4me2+H3K9Ac. Repressed genes displayed two types of non-overlapping profiles. Type 1 localized to short regions and contained H3K9me3+H4K20me3 and sometimes HP1. Type 2 spanned a large domain containing multiple tissue-specific silent genes and contained H3K27me3 alone. These data identify different forms of repressive chromatin on chromosome arms that resemble constitutive and facultative heterochromatin but are restricted to short regions and interspersed with euchromatin. Keywords: ChIP-Chip, chromatin profile, genomic imprinting, noncoding RNA Chromatin immunoprecipitaion (ChIP) was combined with a custom PCR tiling array (Chip) to determine the profile of histone modifications on the maternal and paternal allele of the imprinted Igf2r cluster on mouse chromosome 17. ChIP-Chip experiments were carried out on mouse embryo fibroblast (13.5dpc) cell lines. The main steps in the array design were (i) design of primers to amplify single copy DNA, (ii) spotting each PCR fragment 8 times on the array.The main steps in the ChIP were: (i) preparation of chromatin by micrococcal nuclease (MNase) digestion, (ii) use of specific antibodies, (iii) amplification of input and ChIP enriched chromatin using two rounds of T7 in vitro transcription, (iv) conversion of amplified RNA to cDNA and labelling with Alexa dyes, (v) hybridisation, washing, scanning. Two biological replicates were performed for each antibody in each cell line. A dye swap was also incorporated in the second hybridisation. A novel method to test gene expression was also employed. PCR fragments lying within the exons of expressed genes gave a significant signal. This method also allowed for the detection of non-coding RNAs. A total of forty hybridizations were performed in the study.

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Airn transcriptional overlap but not its lncRNA products induce imprinted Igf2r silencing.

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