Fig 1: Alterations in the expression and interaction of shelterin protein complexes. (A) A schematic figure of the shelterin protein complex. (B and C) Western blot for six shelterin proteins and quantification in endothelial cells and vascular smooth muscle cells from both control and Hutchinson–Gilford progeria syndrome; n = 4–6. (D) Co-immunoprecipitation of ?H2A.X and TIN2 with RAP1 in both endothelial cells and vascular smooth muscle cells from control and Hutchinson–Gilford progeria syndrome; n = 3 (E) Co-immunoprecipitation of TRF2 with lamin A in both endothelial cells and vascular smooth muscle cells from control and Hutchinson–Gilford progeria syndrome; n = 3. C, control; HGPS, Hutchinson–Gilford progeria syndrome. Statistical analysis was performed by an unpaired t-test when comparing two groups. A value of P < 0.05 was considered statistically significant. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig 2: Composition and architecture of the shelterin complex.(A) Cartoon representation of the full shelterin complex POT1-TPP1-TIN2-TRF2(2×)-RAP1(2×) bound to a telomeric double-stand/singe-stand junction. (B) Interaction map for the shelterin subcomplex POT1-TPP1-TIN2. The OB3/HJR domain of POT1 (orange) interacts with the PDB of TPP1 (blue), while the TBM of TPP1 (blue) interacts with TIN2 through the TRFH domain of TIN2 (green). TIN2 also has a TBM and a DC motif. (C) Published crystal structures of domains of the POT1-TPP1-TIN2 shelterin subcomplex: POT1 (OB1 & OB2) (yellow) bound to the minimal telomeric sequence TTAGGGTTAG (red) (PDB ID: 1XJV) (top left), POT1 (OB3 & HJRD) (yellow) bound by the PBD of TPP1 (E266-L326) (blue) (PDB ID: 5UN7, 5H65) (top right), TPP1 OB domain (blue) (PDB ID: 2I46) (bottom left), and TIN2(green)-TPP1(blue)-TRF2(grey) interface complex (PDB ID: 5XYF) (bottom right).
Fig 3: TRF1 levels respond to extracellular signals in a PI3K/AKT dependent manner.A. Representative western blot images of TRF1, SMC1, pAKT(S473) and total AKT (top) and quantification (bottom) of TRF1 protein levels in TRF1+/+, TRF1T358A/T358A and TRF1T273A/T273A cells under normal fed conditions in DMEM+FBS, starved for 24 hours in DMEM without FBS and either refed for 5 hours with FBS or with FBS plus PI3K inhibitor (ETP47037). B. Representative western blot images of TRF1, SMC1, pAKT(S473), total AKT and TIN2 (top) and quantification (bottom) of TRF1 protein levels in TRF1+/+, TRF1T358A/T358A and TRF1T273A/T273A cells starved for 24 hours in DMEM without FBS and either refed for 5 hours with insulin, with insulin plus AKT inhibitor (MK22-06) or with insulin plus PI3K inhibitor (ETP47037). Bars and error bars represent mean values ± SE, n = number of independent experiments. Student’s t-test was used for the statistical analysis; *p < 0.05, **p < 0.01, ***p < 0.001 ns, no significant.
Fig 4: AKT-dependent TRF1 phosphorylation is required for proper telomere binding but dispensable for the interaction with other shelterins.A. Representative western blot images (left) and quantification (right) of TRF1, TIN2, POT1, TRF2 and RAP1 protein levels in TRF1+/+, TRF1T358A/T358A and TRF1T273A/T273A cells. B. Representative western blot images (left) and quantification (right) of TRF1, TIN2, POT1, TRF2 and RAP1 protein levels in TRF1+/+, TRF1T358A/T358A and TRF1T273A/T273A cells expressing FLAG-TRF1-WT. C. Total TRF1 levels in wild type HEK293T cells expressing either Flag-TRF1, Flag-TRF1-T273A or Flag-TRF1-T358A by western blot. D. Flag-TRF1, Flag-TRF1-T273A and Flag-TRF1-T358A co-immunoprecipitate with endogenous TIN2 and POT1. Co-immunoprecipitation was performed from lysates of HEK293T cells expressing either the empty vector or the indicated Flag-TRF1 alleles. Quantification of TIN2 and POT1 pulled down with anti-Flag. E-F. Representative images of chromatin immunoprecipitation (ChIP) of telomeric DNA and of ALU sequences with anti-Flag (E) and with anti-TRF2 (F) of HEK293T cells expressing the indicated Flag-TRF1 alleles. DNA input signal is also shown. Quantification of telomeric DNA pulled down with anti-Flag (E) or with anti-TRF2 (F) is shown to the right. ChIP values are normalized by the input of each individual sample. Bars and error bars represent mean values ± SE, n = number of independent experiments. Student’s t-test was used for the statistical analysis; *p < 0.05, **p < 0.01, ***p < 0.001 ns, no significant.
Fig 5: EMSA demonstrate that binding of telomeric ssDNA to the POT1 OB1/OB2 domain is not limited to the minimal binding sequence, but to sequences separated by spacers as well.(A) Cartoon representation of the POT1 OB1/2 domains and its possible binding conformations that can allow binding to the minimal tight binding sequence TTAGGGTTAG (top), or the same sequence separated by either a single telomeric repeat (center) or a ~6-nt long non-telomeric spacer (bottom). (B) Horizontal scatter plot with median marking of the apparent binding affinity of various telomeric ligands for the POT1-TPP1-TIN2(1–354) subcomplex. Each individual Kd value was computed from the EMSA data shown in S6B–S6H Fig. The binding experiments confirm that telo64 is truly the minimal tight-binding sequence. Both shorter and longer oligos, with the notable exception of telo65 ligand, present reduced affinities for the subcomplex. (C) Horizontal scatter plot with median marking of the apparent binding affinity of various non-contiguous telomeric ligands for the POT1-TPP1-TIN2(1–354) subcomplex. Each individual Kd value was computed from the EMSA data shown in S6I–S6Q Fig. The binding experiments show that the minimal tight-binding sequence ligands that have been interrupted with various non-telomeric spacers retain binding affinity comparable to that of the contiguous telo64 ligand.
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