Human Molecular Genetics 2013 22:17; pp. 3498–3507; doi:10.1093/hmg/ddt204

Since publication of the above manuscript, the authors found errors in the legends to Figure 3, Figure 4, Supplementary Figure 2 and Supplementary Figure 3

The correct legends are reproduced below with changes indicated in bold.

Figure 3. Mutations in dyskerin SUMOylation consensus sites lead to telomere shortening and increased SFEs (A) Histograms of telomere length distribution in cells expressing a control shRNA (control) or in cells knocked down for endogenous dyskerin and transfected with vector, FLAG-dyskerinWT, L37del, K39R or K43R dyskerin variants (late PD). Telomere lengths are expressed as arbitrary units of fluorescence (a.u.f.). A shift to the left in the length distribution of dyskerin mutant cells indicates telomere shortening. The total number of telomeres analyzed (ranging from 2880 to 3312), mean telomere length ± standard error of the mean are indicated. A nonparametric ANOVA test, the Kruskall–wallis test (P < 0.0001), indicated telomere shortening in the vector, L37del, K39R and K43R mutants compared with the control and WT. This was confirmed in post-hoc between-group analysis with Bonferroni-corrected Mann-Whitney's U test which indicated that the differences were significant (P < 0.0001) for each pair of comparisons. The P-values in the figure refer to comparisons of the different variants with the control or dyskerinWT. Note the greater abundance of short (<100 a.u.f.) telomeres in the vector, L37del, K39R and K43R mutants compared with control and greater abundance of long (>300 a.u.f.) telomeres in the FLAG-dyskerinWT rescue cells. Telomeres were hybridized with a Cy3-conjugated PNA probe (Cy3-[CCCTAA]3). A minimum of nine metaphases per clone were analyzed. (B) Telomere SFEs are increased in the lysine-to-arginine dyskerin mutants. Expression of WT dyskerin rescues the SFEs in dyskerin knockdown cells. Error bars indicate standard deviation. (***P < 0.001, **P < 0.01, *P < 0.05 and n.s. P > 0.05).

Figure 4. Downregulation of hTR in dyskerin knockdown cells can be rescued by a SUMO-fused dyskerin mutant and dyskerin stability is increased by SUMOylation. (A) Analysis of telomerase activity by TRAP in HEK293 cells expressing a control shRNA or in HEK293 cells knocked down for endogenous dyskerin and transfected with a vector or expressing FLAG-dyskerinWT, or FLAG-dyskerin variants, L37del, K39R or K43R. Two clones (late PD) for each vector, dyskerinWT and dyskerin variants were analyzed. IC internal control. Neg. negative control. (B) Quantification of telomerase activity calculated as the intensity of the telomerase products relative to the intensity of the PCR amplification of the IC from three different experiments ± S.E.M. (C) qPCR analysis of hTR transcripts in HEK293 cells expressing a control shRNA or in cells knocked down for endogenous dyskerin and transfected with a vector, or expressing FLAG-dyskerinWT or FLAG-dyskerin variants, L37del, K39R or K43R. Two clones (late PD) for each vector, dyskerinWT and dyskerin variants were analyzed. Both vector clones have lost dyskerin shRNA knockdown expression (Supplementary Material, Fig. S1C, data not shown). Data are normalized to ActB and relative to control from three different experiments ± S.E.M. (D) A schematic of the human dyskerin protein illustrating the SUMOylation at the K39 residue (top), the lysine to arginine mutation at Position 39 (middle) and the N-terminal SUMO3 fusion of the dyskerinK39R variant (bottom). Circles (S) represent SUMOylation. Predicted sizes of FLAG-dyskerin and SUMO-dyskerin are 58 and 70kDa, respectively. (E) qPCR analysis of hTR transcripts in HEK293 transfected with pcDNA3.1, HEK293 cells expressing dyskerin shRNA and transfected with vector (early PD), HEK293 dyskerin knockdown cells stably expressing dyskerinK39R (early PD) and transiently expressing FLAG-dyskerinK39R, HEK293 cells transiently expressing SUMO3-dyskerinK39R and HEK293 dyskerin knockdown cells stably expressing dyskerinK39R (early PD) and transiently expressing SUMO3-dyskerinK39R. Data are normalized to ActB and relative to control from a minimum of three different experiments +/− S.E.M. (F) HEK293 cells were transiently transfected with FLAG-dyskerinWT and vector, 6His-SUMO1 or 6His-SUMO3 plasmids. Cells were treated with CHX, harvested at various times (0, 2, 4 and 6 h) and extracted proteins were separated by SDS-PAGE and immunoblotted using anti-dyskerin (upper panel) or anti-tubulin antibodies (lower panel).

Supplementary Fig 2. Telomere shortening in lysine-to-arginine dyskerin mutants. Histograms of telomere length distribution in cells expressing a control shRNA (control) or in a second clone for cells knocked down for endogenous dyskerin (late PD) and transfected with vector, FLAG-dyskerinWT, L37del, K39R or K43R dyskerin variants (as in Fig 3).

Supplementary Fig 3. Telomere shortening and increased telomere signal-free ends in lysine-to-arginine dyskerin mutants. Representative Q-FISH images for telomere length in cells expressing a control shRNA (control) or in a second clone for cells knocked down for endogenous dyskerin (late PD) and transfected with vector, FLAG-dyskerinWT, L37del, K39R or K43R dyskerin variants. Note the brighter telomeric signal in dyskerinWT expressing cells. Examples of signal-free ends are represented by white arrowheads. Telomeres are labeled with a Cy3-conjugated PNA probe (Cy3-[CCCTAA]3) (red) and chromosomes are labeled with DAPI.