A mouse model of RB1 imprinting: knock-in of human PPP1R26P1 into mouse Rb1
Final Report Abstract
In summary the Rb1_PPP1R26P1 mouse model for human RB1 imprinting shows that integration of the human pseudogene PPP1R26P1 is not sufficient for acquisition of DNA methylation at CpG85. We argue that the surrounding sequence is not adequate to induce DNA methylation in the germ line of the mouse. We hypothesize that human intron 2 might contain an oocytespecific promoter element, leading to expression running over CpG85 and thereby inducing DNA methylation. As intronic sequences are generally less conserved than exonic sequences, this putative element might be absent in Rb1 intron 2. To identify such elements, expression data from human oocytes are needed, putative test regions would have to be tested in reporter assays and would have to be integrated into Rb1_PPP1R26P1 ES cells to prove this hypothesis. We could demonstrate that the un-methylated CpG85 serves as promoter for an alternative Rb1 transcript also in the mouse and that integration of PPP1R26P1 and expression of transcript 2B results in repression of the regular Rb1 transcript. This shows, that the function of PPP1R26P1 is maintained in the mouse. Experiments planned but not conducted in this funding period are 5’- and 3’-RACE to determine the 5’ and 3’-ends of transcript 2B and expression analysis of the gene Lpar6. RACE experiments have not been conducted because expression of transcript 2B was low and difficult to target. For our analyses, we used a forward primer detecting all transcripts, possibly initiating at all three transcriptional start sites detected in humans. The gene Lpar6 is contained in intron 17 of the Rb1 gene in antisense orientation. This location is conserved between mice and humans. In humans, LPAR6 was shown to exhibit skewed expression as well, but in the opposite direction than RB1: the paternal allele shows a higher level of expression than the maternal allele (Tea Berulava, Institute of Human Genetics, Essen). We hypothesized that this might be due to transcriptional interference between RB1 and LPAR6: When RB1 expression is low on the paternal allele, LPAR6 expression is high and vice versa on the maternal allele. Because RB1 expression is low on the paternal allele due to expression of transcript 2B, introduction of PPP1R26P1 into intron 2 of Rb1 and expression of transcript 2B could lead to skewed expression of Lpar6 in the mouse. We planned to test and compare expression levels of Lpar6 between hybrid control animals (breeding of C57Bl/6J with 129) and Rb1_PPP1R26P1 animals using the single nucleotide polymorphism rs31363134 (G in C57Bl/6J and C in 129) in Lpar6 and SNaPshot analysis. By now we concentrated fully on the analysis of Rb1 imprinting in the mouse and missed to conduct these experiments. However, to test the influence of transcript 2B expression and skewing of Rb1 on on Lpar6 expression is still worthwhile and could be performed now using RNA samples already prepared for SNaPshot analyses of Rb1 allelic expression. The influence of Rb1 expression strength on Lpar6 might be interesting in the context that expression changes of LPAR6 have been associated with some cancers and metastatic potential.
Publications
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(2015) A Mouse Model for Imprinting of the Human Retinoblastoma Gene. PLoS One 10: e0134672
Tasiou V, Hiber M, Steenpass L
