Roles of mammalian Rap1 in TERT recruitment to chromatin, subtelomeric gene silencing and transcriptional regulation

ABSTRACT: Repressor-activator protein 1 (scRap1) is the major binding activity at Saccharomyces cerevisiae telomeres, with roles in telomere length regulation and establishment of subtelomeric silencing by recruiting the Sir proteins. scRap1 also acts as a transcription factor controlling the expression of ribosomal proteins and glycolytic enzymes. A homolog of scRap1 exist in mammals, Rap1 (also known as Terf2ip), however, its roles in telomere biology and transcriptional regulation are largely unknown. We have employed microarrays to obtain gene expression signatures related to Rap1-deleted mice when compared to the wild-type. Construction of the Rap1 targeting vector: The strategy for disrupting the Rap1 locus was designed to conditionallty delete exon 3 (E3) through a Cre-mediated excision. Deletion of E3 leads to the removal of coding sequences encoding for the RCT domain and the NLS sequence, as well as the whole 3’UTR region and poly-adenylation signal (Fig.1A,B). Deletion of E3 was expected to destabilize the Rap1 transcript and thereby to prevent translation of any truncated RAP1 protein. The targeting vector contains homology regions isogenic with the ES cell line used (129Sv/Pas). The short homology region (SA) harbors a 2.4 kb DNA fragment encompassing exon 2 (E2) and intron 2 (Fig. 1A). The long homology region (LA) is a 5.6 kb DNA fragment downstream the end of E3 (Fig. 1A). The central part contains E3 flanked by two loxP sites and a positive selection neomycin gene (PGK-Neo) flanked by two Frt sites (Fig. 1A). At the 3’-end of the LA a Diphteria Toxin (DTA) selection marker was cloned (Fig. 1A). The vector contains a unique NruI linearization site. The targeting vector was quality controlled by sequencing of the coding exons, the junctions between the homology arms and the selection cassettes, the selection cassettes and the junctions between the homology arms and the plasmid backbone. The analysis of the sequencing results showed no polymorphis between the C57BL/6 and 129Sv/Pas genetic background within the isolated Rap1 sequences. The sequence of the primers used for the PCR amplification of the different targeting vector parts and the details of the cloning steps are available upon request. The construction of the targeting vector was performed by genOway (www.genoway.com; Lyon, France). Generation of conditional Rap1 knockout mice 129Sv/Pas ES cells were transfected with 40 µg of linearized plasmid. Positive selection was started 48 hours after electroporation by addition of 200 µg/ML G418. Approximately 230 positive resistant clones were isolated and amplified. They were PCR screened for homologous recombination first at the 5’ end of the Rap1 locus. The 6 positive identified 5’ targeted ES cells were further investigated by PCR amplification over the 3’ long homology arm to amplify the region of the targeted locus containing the distal loxP site. The direct sequencing of the PCR products amplified from the 6 ES clones revealed that 3 of them contained the distal loxP site. The recombinant clones identified by PCR were further verified by Southern blot analysis of AflII and PciI restricted genomic DNA using a 5’-internal and a 3’-external probes, respectively (data not shown). The Southern blots confirmed the correct homologous integration over the 5’ and the 3’ homology arm and the absence of any additional randomly integrated copies of the targeting vector. Chimeric mice were generated by microinjection of three independently targeted ES clones into C57BL/6J host blastocyst. The resulting offspring showed a high level of chimerism as shown by coat color, and were mated to C57BL/6J mice to assess germ line transmission. The resulting heterozygous Rap1+/flox-Neo mice were then bred to transgenic mice expressing the Flpe recombinase (Rodriguez et al., 2000) to induce excision of the Neo marker. The Rap1+/flox heterozygous mice were then intercrossed to generate Rap1flox/flox, and Rap1+/flox mice. Homozygous Rap1flox/flox mice were crossed with transgenic mice expressing the Cre recombinase under the control of the keratine 5 promoter (Tarutani et al., 1997) (Fig. 5A). Heterozygous Rap1+/D K5-Cre were crossed either to Rap1flox/flox or Rap1+/flox to generate Rap1Δ/Δ K5-Cre. The removal of exon 3 by Cre-mediated recombination was confirmed by PCR analysis using primers F and R (Fig. 1A,C and Fig. 5A). Amplification of the wild type, flox and knockout alleles renders a 3.2 kb, 3.3 kb and 0.5 kb fragments, respectively. The breeding to the F1 generation and characterization of heterozygous Rap1+/flox-Neo F1 animals was performed by geneOway (www.genoway.com; Lyon, France). All mice were generated and maintained at the Spanish National Cancer Centre (CNIO) under specific pathogen-free conditions in accordance with the recommendation of the Federation of European Laboratory Animal Science Associations.

ORGANISM(S): Mus musculus

SUBMITTER: Paula Martinez 

PROVIDER: E-GEOD-19011 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress