| Group: Denise P. Barlow Quanah Hudson | Postdoc Floiran Pauler | Postdoc Martha Koerner | PhD Student Ru Huang | PhD Student Irena Vlatkovic | PhD Student Basak Senergin | PhD Student Federica Santoro | PhD Student Philipp Günzl | PhD Student Stefan Huetter | Dipl. Student Lucas Rudigier | Dipl. Student Romana Bittner | Technician Katarzyna Warczok | Technician Angelika Eisner | Administration |
The function of the macro Airn non-coding RNA in imprinted gene silencing
This project investigates the epigenetic mechanism responsible for Genomic Imprinting in mammalian cells (see box below). In particular we will investigate how an unusual macro noncoding (nc) RNA named Airn induces imprinted expression of a small cluster of genes in the mouse Igf2r imprinted gene cluster. Macro ncRNAs are provisionally defined as ‘ncRNAs that can be as short as a few hundred nucleotides or as long as several hundred-thousand nucleotides, whose function does not depend on processing into short or micro RNAs’.
| Genomic imprinting - basic biology & history: Mammals are diploid organisms whose cells contain two matched sets of chromosomes, one inherited from the mother and one from the father. Thus, mammals have two copies of every gene with the same potential to be expressed in any cell. Genomic imprinting is known to depend on a cis-acting epigenetic mechanism as both the silent and active parental alleles are contained in the same nucleus. Approximately 1% of genes show imprinted expression which restricts their expression early in development, to one of the two parental chromosomes. Genes showing parental-specific expression were hypothesized to exist in mammals following a series of landmark experiments that started thirty years ago. These included the failure of embryos to develop by parthenogenesis in the absence of fertilization, the phenotype of embryos inheriting two copies of one parental chromosome in the absence of the other parental copy, and, the inability of oocyte nuclear transfer experiments to generate viable embryos containing only two maternal or two paternal pronuclei. This hypothesis was supported in 1991 with the discovery of three imprinted genes: the maternally-expressed Igf2r gene, the paternally-expressed Igf2 gene and the maternally-expressed H19 ncRNA. |
Imprinted genes cluster together with a macro ncRNA: In mammals, most imprinted genes are grouped into small clusters containing from 2–12 imprinted genes that spread over 80–3,700 kilobase pairs. 26 regions in mice and humans have been shown to contain clustered imprinted genes. Two interesting findings are that at least one gene in each imprinted cluster is a macro ncRNA, and, that while most genes in a cluster show concordant parental-specific expression, the ncRNA is the odd-one-out and is expressed from the other parental chromosome. A common observation seen in several imprinted clusters is that the macro ncRNA represses a larger number of genes in cis in the placenta compared to the embryo. Six well-studied imprinted clusters have now been shown to express a ncRNA from the parental allele that silences the cluster of imprinted mRNA genes. Only 3 ncRNAs, all of which show reciprocal parental-specific expression have been functionally tested. The H19 ncRNA plays no role in silencing the upstream Igf2 gene instead a methyl-sensitive insulator controls imprinted expression of Igf2 & H19. In contrast the Airn and Kcnq1ot1 ncRNAs are required for paternal-specific silencing of all clustered mRNA genes respectively in the Igf2r and Kcnq1 imprinted clusters. Notably, the Airn and Kcnq1ot1 ncRNAs silence genes in embryo/adult and in placenta. Thus imprinted gene clusters offer an important model for macro ncRNAs that induce cis-regulatory effects that silence overlapped and nonoverlapped mRNA genes.
The 108kb long Airn ncRNA is only expressed from the paternal chromosome where it silences in cis, the flanking Igf2r, Slc22a2 and Slc22a3 genes. Notably, Airn overlaps the Igf2r promoter in an antisense orientation, but lies 200 upstream from the Slc22a2 and Slc22a3. Our previous data (Seidl et al., 2006, Regha et al., 2007) indicated the possibility of a model whereby Airn transcription but not the Airn ncRNA itself, induced silencing of the overlapped Igf2r gene However, we have also proposed a transcriptional interference model for the silencing of non-overlapped genes, if they were controlled by enhancers lying within the Airn gene body (Seidl et al., 2006, Pauler et al., 2006). This SFB project will test this model by focusing on three main areas: (1) Testing the ability of Airn truncated to different lengths to silence Igf2r in cis, (2) the role of the Airn promoter in regulating its repressor functions, and (3) the role of cis-acting transcription activator elements in mediating imprinted Igf2r expression. In the first two projects we are using a newly developed in vitro embryonic stem cell imprinting model (Latos et al., 2009) combined with homologous recombination to introduce changes into the Airn ncRNA that will truncate it to different lengths and delete and/or replace the endogenous promoter. The 3rd project requires the identification of Igf2r cis-regulatory elements that lie within the body of the 108kb Airn transcript in . As a 1st step towards this goal, we will map all DNase1 hypersensitive sites (DHS) inside the Airn gene body in mouse extra-embryonic tissues that show imprinted expression of the Slc22a2 and Slc22a3 genes.
Koerner MV, Pauler FM, Huang R, Barlow DP. The function of non-coding RNAs in genomic imprinting. Development 136, (2009) in press. Latos PA, Barlow DP. Regulation of imprinted expression by macro non-coding RNAs. RNA Biol. 2009 Apr 14;6(2).
Latos PA, Stricker SH, Steenpass L, Pauler FM, Huang R, Senergin BH, Regha K, Koerner MV, Warczok KE, Unger C, Barlow DP. An in vitro ES cell imprinting model shows that imprinted expression of the Igf2r gene arises from an allele-specific expression bias. Development. 2009 Feb;136(3):437-48.
Pauler FM, Sloane MA, Huang R, Regha K, Koerner MV, Tamir I, Sommer A, Aszodi A, Jenuwein T, Barlow DP. H3K27me3 forms BLOCs over silent genes and intergenic regions and specifies a histone banding pattern on a mouse autosomal chromosome. Genome Res. 2009 Feb;19(2):221- 33.
Stricker SH, Steenpass L, Pauler FM, Santoro F, Latos PA, Huang R, Koerner MV, Sloane MA, Warczok KE, Barlow DP. Silencing and transcriptional properties of the imprinted Airn ncRNA are independent of the endogenous promoter. EMBO J. 2008 Dec 3;27(23):3116-28.
Yotova IY, Vlatkovic IM, Pauler FM, Warczok KE, Ambros PF, Oshimura M, Theussl HC, Gessler M, Wagner EF, Barlow DP. Identification of the human homolog of the imprinted mouse Air noncoding RNA. Genomics. 2008 Dec;92(6):464-73.