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. 2014 Feb;21(2):167-74.
doi: 10.1038/nsmb.2754. Epub 2014 Jan 12.

Rapid induction of alternative lengthening of telomeres by depletion of the histone chaperone ASF1

Roderick J O'Sullivan  1 , Nausica Arnoult  1 , Daniel H Lackner  1 , Liana Oganesian  1 , Candy Haggblom  1 , Armelle Corpet  2 , Genevieve Almouzni  3 , Jan Karlseder  1
Affiliations

Rapid induction of alternative lengthening of telomeres by depletion of the histone chaperone ASF1

Roderick J O'Sullivan (V体育平台登录) et al. Nat Struct Mol Biol. 2014 Feb.

Abstract

The mechanism of activation of the alternative lengthening of telomeres (ALT) pathway of mammalian chromosome-end maintenance has been unclear. We have now discovered that co-depletion of the histone chaperones ASF1a and ASF1b in human cells induced all hallmarks of ALT in both primary and cancer cells. These included the formation of ALT-associated PML (promyelocytic leukemia) bodies (APBs), the presence of extrachromosomal telomeric DNA species, an elevated frequency of telomeric sister chromatid exchanges (t-SCE) events and intertelomeric exchange of an integrated tag. The induction of ALT characteristics in this setting led to the simultaneous suppression of telomerase. We determined that ALT induction is positively regulated by the proteins RAD17 and BLM and negatively regulated by EXO1 and DNA2. The induction of ALT phenotypes as a consequence of ASF1 depletion strongly supports the hypothesis that ALT is a consequence of histone management dysfunction VSports手机版. .

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Figures

Figure 1
Figure 1. ASF1 knockdown induces APBs and t-SCEs
(a) Confocal IF for RPA2 (red), PML (cyan) was combined with telomeric TTAGGG FISH (green) and counter-stained in DAPI (dark blue). Images are maximum intensity projections of ~10 stacks captured with a 63X objective lens. All panels include the merged channels with DAPI and enlarged sections of the merge with DAPI. siControl refers to a non-targeting scrambled siRNA sequence. siASF1 refers to transfections with siRNAs to co-deplete ASF1a and ASF1b. The scale bar is equivalent to 10 µm. (b) Quantification of APBs. Data represent means of at least 3 experiments ±SDs. The total number of APBs detected and cells counted is indicated above each column. (c) CO-FISH of metaphases from the indicated cells. Leading (red: Alexa-568), lagging (green: Alexa-488) telomeres and metaphase chromosomes (blue: DAPI) are shown. Examples of T-SCEs are indicated by white triangles. The scale bar is equivalent to 10 µm. (d) Quantification of CO-FISH analysis. Data represent means of 4 experiments ±SDs (≥10 metaphases/experiment). P-values of p<0.0001 were obtained from two-tailed Mann-Whitney test using Prism. Number of and percent of telomeres counted and scored are shown.
Figure 2
Figure 2. Copying and transfer of a telomere tag after ASF1 depletion
(a) Metaphase spread of siControl transfected HT1080 hTR cells at day 38 (~60PDs) after initial transfection. The single integrated neo tag located on chromosome Bpter is shown as green FISH signals and indicted by the adjacent white arrow. Insert shows enlarged partial metaphase of the chromosome containing the neo tag. The scale bar is equivalent to 10 µm. (b-d) Metaphase spreads showing copying and transfer of the neo tag after siASF1 transfected HT1080 hTR cells at day 38 (~60PDs) after initial transfection. (b) The metaphase spread shows the original tag on Bpter and the newly acquired tag on Cpter (white arrows). Insert shows enlarged partial sections of the metaphase with the chromosomes involved. (c) The metaphase spread shows the newly acquired tag on Fter (green arrow). Insert shows enlarged partial sections of the metaphase with the chromosome involved. (d) Intra-telomeric duplication of the newly acquired neo tag on the p and q arms of chromosome Fter (green arrows). A total of 332 and 297 metaphases from siControl and siASF1 transfected HT1080 hTR cells were scored and analyzed, respectively. All chromosomes were counterstained with DAPI but shown in red for clarity.
Figure 3
Figure 3. ASF1 knockdown generates ECTR
(a) Telomere Restriction Fragment (TRF) analysis of telomeric DNA. Left: Digested DNA (10µg) from independent prolonged ASF1 knockdowns (days 17 and 14 post transfection) in HeLa LT was resolved on a 0.7% TBE agarose gel. DNA from siControl transfected cells, Saos-2 and U2OS cells was resolved in parallel. Right: Lines traces of siControl (black) and siASF1 (red) gel lanes. (b, c) C-circle assays in siControl and siASF1 transfected IMR90-hTERT and HeLa LT. The ALT positive U2OS control is on the left. Negative controls are reactions lacking φ29 or DNA. (d) Quantification of C-circles in siRNA and drug treated HeLa LT (light grey bar) and U2OS (dark grey bar). In both (c) and (d), C-circle levels are calculated relative to those of U2OS cells (dark grey bar, far left of graph). Data represent means ±SDs of at least 3 experiments. *** indicates p-value <0.005, ** indicates p-value <0.05 (two tailed paired students t test). (e) Increasing amounts (0.25, 0.5 and 1 µg) of digested genomic DNA from control and ASF1 depleted IMR90-hTERT and HeLa LT cells were incubated +/- φ29. Arrows indicate T-circle products. (f) Quantification of C-circles in Untreated, DMSO and BIBR-1532 (TERTi) treated control and ASF1 depleted HeLa LT (light grey bar) and IMR90-hTERT cells (dark grey bar). C-circle levels are calculated as relative percentages to those of untreated cells. Data represent means ±SDs of 3 experiments.
Figure 4
Figure 4. ASF1 knockdown leads to hTERT repression
(a) qPCR quantification of hTERT and hTR gene expression in HeLa LT, ST, VST and aphidicolin treated HeLa LT cells. Gene expression is calculated relative to RPLO expression and siControl treated cells. Error bars indicate means of at least 3 experiments ±SDs. (b) TRAP assay of siControl and siASF1 treated HeLa LT cells. hTERT activity from decreasing amounts of protein inputs is shown. hTERT activity is quantified from at least 3 independent experiments. *** indicates p-value <0.005 (two tailed paired students t test).
Figure 5
Figure 5. ALT phenotypes arise from replicative stress due to ASF1 depletion
(a) Quantification of C-circles in siRNA transfected HeLa LT (light grey bar). C-circle levels are calculated relative to those of U2OS cells (dark grey bar, far left of graph). Data represent means ±SDs of at least 3 experiments. *** indicates p-value <0.005 (two tailed paired students t test). ASF1 refers to co-depletion of ASF1a and ASF1b. CAF1 refers to co-depletion of CAF1a and CAF1b. (b) Western analysis of ASF1, RPA2 and phospho-RPA2 S4/S8 following ASF1 knockdown and drug treatments. (c) Quantitation of C-circle production. Percentage values are calculated relative to ASF1 depleted cells that were left untreated following transfection. Data represent means ±SDs of at least 3 experiments. *** indicates p-value <0.005, ** indicates p-value <0.05 (two tailed paired students t test). Original blots can be found in Supplementary Figure 8. (d) Quantitation of APBs. Percentage values are calculated relative to untreated ASF1 depleted cells. Data represent means ±SDs of at least 3 experiments. The total numbers of cells counted are indicated between brackets above each column. (e) Quantification of C-circle levels in HeLa LT (light grey bar) and U2OS (dark grey bar) after prolonged S-phase arrest, replicative stress and DDR. C-circle levels are calculated as relative percentages to those of untreated cells. Data represent means ±SDs of at least 3 experiments. *** indicates p-value <0.005, ** indicates p-value <0.05 (two tailed paired students t test). (f) Quantification of C-circle levels in U2OS cells after treatment with hydroxyurea. Line of best fit from linear regression analysis is shown in red. C-circle half-life (t=1/2) is also indicated.
Figure 6
Figure 6. Kinetics of ALT induction and maintenance after ASF1 depletion
(a) C-Circle analysis during prolonged ASF1 depletion and recovery in HeLa LT cells. (b) Western analysis of phospho-RPA2 S4/S8 and γH2AX during prolonged (12 days) ASF1 knockdown. Cells were allowed to recover for 12 days after final transfection (Recovery). * indicate day of siRNA delivery. Original blots can be found in Supplementary Figure 8. (c) Quantification of C-circle levels during prolonged ASF1 depletion and recovery. The red line represents the projected degradation kinetics based on the half-life calculations in Fig. 4f. (d) qPCR analysis for ASF1a, ASF1b, hTERT and hTR expression in HeLa LT during prolonged ASF1 knockdown and recovery. Gene expression is calculated relative to RPLPO expression and siControl treated cells. Error bars indicate means of at least 3 experiments ±SDs.
Figure 7
Figure 7. Identification of factors required for induction of APBs and C-circles
(a) Confirmation of shRNA knockdown efficiency by western analysis of extracts from siControl and siASF1 transfected HeLa LT cells infected with shScramble and shRNAs against target proteins. * indicates the individual PML isoforms. Original blots can be found in Supplementary Figure 8. (b) Quantitation of APBs. Percentage values are calculated relative to ASF1 depleted shScramble infected cells. Data represent means ±SDs of at least 3 experiments. The total numbers of cells counted are indicated between brackets above each column. (c) Quantitation of C-circle production. Percentage values are calculated relative to ASF1 depleted shScramble cells. Data represent means ±SDs of at least 3 experiments. *** indicates p-value <0.005, ** indicates p-value <0.05 (two tailed paired students t test). (d) Summary of shRNA analysis of factors involved in induction of C-circles, APBs and DDR. Blue, orange, red and dark red panels represent absence, low levels, normal levels and elevated levels of the corresponding ALT feature. (e) Model for ALT induction at siASF1 dependent stalled replication forks (see text).
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