Figure 1

Progerin impairs 3D telomere organization. (A) Western blots of whole cell extracts of HDF cells expressing the indicated constructs. Two independent transductions are shown (TR1 and TR2). The membrane was probed with the LaminA/C antibody to detect endogenous LaminA (A), endogenous LaminC (C) and the EGFP-fusion proteins EGFP-LA (E-A) and EGFP-PG (E-P). Actin is shown as a loading control. (B) Live HDF cells expressing EGFP-LA or EGFP-PG, 3 or 6 days post-transduction. The EGFP signal (grey) is detected using a benchtop fluorescent microscope. (C) Representative immunostaining of HDF cells expressing the indicated constructs. EGFP (cyan), TRF1 (yellow), and merge. The max projection is shown. Scale bar: 10 μm. (D) Scheme of MadID-dotblot principle. Genomic DNA is extracted from cultured cells, and digested with frequent cutter restriction enzymes that do not cut the telomeres. Telomeres are isolated using biotinylated oligonucleotides complementary to the TTAGGG telomeric sequence and streptavidin beads. The purity and the amount of isolated telomeres are verified by qPCR, before they are blotted on a membrane for hybridization with a m6A-specific antibody. (E) Dotblot of increasing amount of lambda DNA isolated from Dam− (M−) or Dam+ (M+) bacterial strains. The membrane is hybridized with a m6A-specific antibody. The m6A signal intensity in the M+ DNA is indicated on the right, relative to the signal obtained with 75 ng of lambda DNA. (F) qPCR analyses of MadID using TelC/TelG primers to amplify telomeric DNA. Left panel: a fragment of 800 bp of telomeric repeats was used as a template, before or after in vitro methylation with M.EcoGII. Right panel: isolated telomeres from HDF expressing NLS-EGFP, EGFP-LA, or EGFP-PG as indicated. Enrichments are shown for three independent telomere purifications. Fold enrichments are calculated against INPUTs, i.e. genomic DNA. (G) Representative example of a MadID-dotblot experiment performed on HDF cells expressing NLS-EGFP, EGFP-LA and EGFP-PG. INPUT DNA and isolated telomeres were blotted on a membrane hybridized with a m6A-specific antibody. (H) Quantification of 3 independent MadID experiments similar to (E). The graph represents the normalized m6A intensity (normalized to the amount of isolated telomeres measured by qPCR) relative to the EGFP-NLS control. Mean with SD is shown, n = 3. Statistical significance was determined using one-way Anova (**indicates p < 0.01). (I) ChIP analyses on HDF cells expressing NLS-EGFP, EGFP-LA, and EGFP-PG. Immunoprecipitations were performed with the indicated antibodies. The graphs represent the enrichment relative to input material (left) or to H3 (right) (Mean ± SD, n = 4). Statistical significance was determined using two-way Anova (*indicates p < 0.05, **indicates p < 0.01, ***indicates p < 0.001, ****indicates p < 0.0001).