Construction of the Rap1 targeting vector
The strategy for disrupting the Rap1
locus was designed to conditionally delete E3 through a Cre-mediated excision. 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 E2 and intron 2 (). The long homology region (LA) is a 5.6 kb DNA fragment downstream the end of E3 (). The central part contains E3 flanked by two loxP
sites and a positive selection neomycin gene (PGK-Neo) flanked by two Frt
sites (). At the 3′-end of the LA a Diphteria Toxin (DTA) selection marker was cloned (). 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. Sequencing showed no polymorphisms between the C57BL/6 and 129Sv/Pas genetic backgrounds within the isolated Rap1
sequences. The targeting vector was generated by genOway (www.genoway.com
; Lyon, France).
Generation of conditional Rap1 knockout mice
129Sv/Pas ES cells were transfected with 40 μg of linearized targeting vector. Positive selection was performed by adding 200 μg/ML G418. Approximately 230 positive resistant clones were isolated and PCR screened for homologous recombination first at the 5′ end of the Rap1
locus. Six positive 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. Direct sequencing of the PCR products amplified revealed that 3 of out of 6 cloes contained the distal loxP site. They were 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). Chimeric mice were generated by microinjection of the 3 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 17
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 26
(). Heterozygous Rap1+/Δ
were crossed either to Rap1flox/flox
to generate Rap1Δ/ΔK5-Cre
. The removal of exon 3 by Cre-mediated recombination was confirmed by PCR analysis using primers F and R ( and ). Amplification of the wild type, flox and knock-out 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.
Mice were fed either with standard chow diet (Harlan Teklad LM-485), or with a high fat diet (Research Diets 12451, 45% kJ from fat) starting at 7 weeks of age. Food intake was monitored by weighting the the consumed food every three days in individually caged animals.
ChIP-sequencing analyses and identification of a putative RAP1-binding consensus sequence
Biological duplicates of ChIP samples (see above) were independently processed into sequencing libraries with a ChIP-Seq sample prep kit (Illumina) by following manufacturer instructions with some minor modification 49
. Libraries were prepared from 85-135bp DNA fractions (excluding adaptor length). Input samples from both Rap1-null and wild type MEFs were pooled and sequenced as a single library. Libraries were sequenced in an Illumina Genome Analyzer IIx (GA2) single 36-base read run. Primary data was obtained by Pipeline 1.4 analysis (PL1.4, Illumina). Raw sequences were defined as reads passing purity filter before the genome alignment.
Genome alignment was performed with PL1.4 versus the latest mouse assembly (NCBIm37/mm9, April 2007) under default settings. These settings exploit the maximum mapping specificity allowed by the aligning algorithm. PL1.4 permits alignments with more than 2 errors for 36 base reads, but with no more than 2 errors in the first 32 bases. The best alignment among alternate candidate positions is eventually chosen based on quality scores. The experimental settings, sequences and analysis protocols of the ChIP-seq experiment have been deposited in GEO under the accession number GE20867.
Only the reads having a unique alignment in the reference genome where used for the peak detection which was performed using CisGenome v1.2 50
. Briefly, uniquely aligned 36bp-length reads obtained in two independent GA2 runs were pooled into 3 datasets, corresponding to the Rap1 wild-type (WT) MEFs, Rap1-null MEFs (KO), and IP mockup. The software pipeline to analyze two-sample ChIP-seq experimental designs was applied WT reads as sample set and KO reads as negative control set using a 100bp-sliding window and a 10% FDR. To look for known genes in the neighborhood of the RAP1 binding sites, CisGenome searching method was executed against a database of mouse annotations, using a symmetrical window of 10kbp surrounding the TSS. The gene-annotated sites were cross-related with the gene labels in the microarray expression experiment by merging their expression profiles using BioConductor. Those merged genes with average
≥1 were considered as deregulated in the peak association experiments.
The dataset of ChIP-seq peaks was strongly filtered (minFDR ≤ 0.0001, max
≥6) to select the binding sites that were clearly represented. To derive any putative consensus motifs, the resulting 30 genomic sequences corresponding to the filtered peaks were processed with the de-novo motif discovery tool Weeder 1.3 34
, as described in 51, 52
. The motifs identified by the algorithm as highest ranking were selected, and the STAMP online tool 53
was used to represent the sequence logos 54
of the consensus sequences.
The pattern matching algorithms fuzznuc
were used to scan ChIP-seq binding sequences and mouse genomic sequence for permutations of telomeric repeats. To derive any putative non-TTAGGG consensus motifs, we discarded the peaks with 3 or more occurences of the telomeric repeats. We used STAMP 53
to determine the best match between the obtained motifs and known JASPAR and TRANSFAC v11.3 matrices.
Validation of ChiP-seq results
The Chip-seq results were validated by q-PCR on pulled down DNA from an independently performed chip with a rabbit polyclonal anti-RAP1 (a gift from Dr. West, CRUK, UK). Oligos were designed to amplify the DNA fragment containing the peaks corresponding to Peak Rank 1 (chr2: 57,482,074-57,482,124), Peak Rank 4 (chr2: 28,040,118-28,040,149), Peak Rank 9 (chr3: 8,246,273-8,246,509), Peak Rank 14 (chr17: 53,291,729-53,291,957), Peak Rank 27 (chr17: 22,362,598-22,362,692), Peak Rank 41 (chr11: 3,091,929-3,092,050), Peak Rank 260 (chr17: 33,920,665-33,920,732), Peak Rank 561 (chr1: 40,496,711-40,496,796), Peak Rank 707 (chr13: 16,114,008-16,114,120), Peak Rank 764 (chr1: 12,701,710-12,701,939), Peak Rank 841 (chr19: 38,200,547-38,200,616), Peak Rank 2675 (chr10: 24,308,726-24,308,800), Peak Rank 8218 (chr11: 28,766,736-28,766,797), Peak Rank 29521 (chr9: 54,613,901-54,614,084). Genomic regions containing telomeric repeats that did not render any hit in Chip-seq analysis were choosen as negative controls; negative control 1 (chr1:121,265,202-121,265,386) and negative control 2 (chr15:54,909,216-54,909,375). The primer sequences are available upon request. The amplification levels of the above-mentioned fragments were analyzed in the input DNA in each case for normalization and in the pulled down DNA. The relative level of each fragment was determined by calculating ΔCt values between the levels obtained in input DNA and that of the pulled down DNA. The results were normalized to wild type levels.
TRF2 binding to Peak Rank 1, Peak Rank 14 and to Peak Rank 29521 corresponding to Crabp1 promoter was performed as described above using pulled down DNA with polyclonal TRF2 antibody (a gift from Dr. West, CRUK, UK). The results were normalized to input levels.
DNA fragments (~300 bp) harbouring Peak Rank 2675 (chr10: 24,308,726-24,308,800), Peak Rank 19335 (chr11: 74,972,250-74,972,307) and Peak Rank 260 (chr17: 33,920,665-33,920,732) located at CTGF, HIC1 and ANGPTL4 promoter region, respectively, were PCR amplified from mouse genomic DNA. Forward and reverse primers contained a XhoI and a HindIII and their sequence are available upon request. The fragments were cloned into XhoI-HindIII sites of pGL4.28 vector containing a minimal promoter driving luciferase (Promega) and sequence verified. LT-sv40 immortalized Rap1+/+ and Rap1Δ/Δ MEF were transfected with the reporter constructs by using Fugene (Roche). A plasmid (pGL4.75, promega) containing a CMV promoter driving Renilla luciferase was cotransfected as an internal control. Cells were harvested 48 hours after transfection, and the luciferase activities of cell lysates were measured by using Dual-luciferase Reporter Assay System (Promega).
A t-student test was used to calculate the statistical significance of the observed differences in the percentage of RAP1 positive cells, γH2AX foci, 53BP1 foci and chromosomal aberrations. Wilcoxon-Mann-Whitney rank sum test was used to calculate statistical significance of the observed differences in the mean telomere length. A two-tailed Wilcoxon-rank test was used to calculate the statistical significance of the differences in gene expression levels amongst subtelomeric and non-subtelomeric genes. To determine the gene expression levels trend in subtelomeric regions between classes under comparison we applied a one-tailed Wilcoxon-rank test. P-values<0.05 were considered statistically significant. Fisher Exact test was used to estimate the differences in the presence of repetitive sequences in the raw reads. To test whether is possible to find a binging peak located in or near the genic regions just by random chance we performed an exact binomial test. Pearson’s Chi-square test was used to check the dependency between the presence of RAP1 binding peaks and expression deregulation of their associated genes. Freely distributed R software was employed for these calculations (http://www.r-project.org/