Human PPP1R26P1 Functions as cis-Repressive Element in Mouse Rb1

Abstract

The human retinoblastoma gene (RB1) is imprinted; the mouse Rb1 gene is not. Imprinted expression of RB1 is due to differential methylation of a CpG island (CpG85), which is located in the pseudogene PPP1R26P1 in intron 2 of RB1. CpG85 serves as promoter for an alternative RB1 transcript, which is expressed from the unmethylated paternal allele only and is thought to suppress expression of the full-length RB1 transcript in cis. PPP1R26P1 contains another CpG island (CpG42), which is biallelically methylated. To determine the influence of PPP1R26P1 on RB1 expression, we generated an in vitro murine embryonic stem cell model by introducing human PPP1R26P1 into mouse Rb1. Next generation bisulfite sequencing of CpG85 and CpG42 revealed differences in their susceptibility to DNA methylation, gaining methylation at a median level of 4% and 18%, respectively. We showed binding of RNA polymerase II at and transcription from the unmethylated CpG85 in PPP1R26P1 and observed reduced expression of full-length Rb1 from the targeted allele. Our results identify human PPP1R26P1 as a cis-repressive element and support a connection between retrotransposition of PPP1R26P1 into human RB1 and the reduced expression of RB1 on the paternal allele.

A Mouse Model for Imprinting of the Human Retinoblastoma Gene

“…Human PPP1R26P1 was integrated into intron 2 of the Rb1 gene by homologous recombination in R1 mouse ES cells of strain 129 ( Fig 1A ). Construction of the targeting vector was described in previous work, which identified PPP1R26P1 as a cis-repressive element [ 20 ]. The 5.2 kb PPP1R26P1 fragment comprised the complete pseudogene as it is present in the human RB1 intron 2.…”

“…The definite length of the alternative transcript is not known yet. In murine ES cells, we observed transcriptional activity at CpG85, but we could not show the expression of a transcript connecting the alternative exon 2B with exon 3 of Rb1 [ 20 ]. In heterozygous Rb1_PPP1R26P1 mice we expect to observe expression of the normal Rb1 transcript from both, the wildtype and the knock-in, alleles.…”

“…In humans, RB1 expression is higher from the maternal allele, where the CpG85 promoter is methylated and silenced, than from the paternal allele, where the CpG85 promoter is active and expresses the alternative transcript 2B [ 16 ]. In murine ES cells, we observed higher expression from the non-modified, wildtype allele compared to the allele carrying the PPP1R26P1 insertion in an unmethlyated state [ 20 ]. Thus, presence of unmethylated CpG85 negatively influences RB1/Rb1 expression in cis.…”

“…We used the Rb1 locus as template to experimentally test the consequences of pseudogene integration in respect to somatic and gametic DNA methylation and transcriptional regulation of the host gene. In vitro analysis of mouse embryonic stem cells (mESCs) carrying human PPP1R26P1 showed that it also acts as cis-repressive element in mouse, resulting in skewed Rb1 expression in favor of the allele not carrying the integrated pseudogene [ 20 ]. As imprint acquisition occurs in the germ line, we generated a humanized mouse model carrying a knock-in of human PPP1R26P1 in intron 2 of Rb1 .…”

Genome-wide methylation analysis of retrocopy-associated CpG islands and their genomic environment

“…Parent-of-origin-specific methylation of CpG85 has been confirmed by other studies. [15][16][17][18] At present, nearly 100 imprinted human genes have been identified, and several studies have taken a genome-wide look at parent-of-origin-specific DNA methylation in order to identify novel imprinted genes. 18,19 Retrocopies are instructive genomic elements for studying the evolutionary dynamics and constraints of DNA methylation.…”