Timeless functions independently of the Tim-Tipin complex to promote sister chromatid cohesion in normal human fibroblasts

Abstract

The Timeless-Tipin complex and Claspin are mediators of the ATR-dependent activation of Chk1 in the intra-S checkpoint response to stalled DNA replication forks. Tim-Tipin and Claspin also contribute to sister chromatid cohesion (SCC) in various organisms, likely through a replication-coupled process. Some models of the establishment of SCC posit that interactions between cohesin rings and replisomes could result in physiological replication stress requiring fork stabilization. The contributions of Timeless, Tipin, Claspin, Chk1 and ATR to SCC were investigated in genetically stable, human diploid fibroblast cell lines. Whereas Timeless, Tipin and Claspin showed similar contributions to UVC-induced activation of Chk1, siRNA-mediated knockdown of Timeless induced a 100-fold increase in sister chromatid discohesion, whereas the inductive effects of knocking down Tipin, Claspin and ATR were 4-20-fold. Knockdown of Chk1 did not significantly affect SCC. Consistent findings were obtained in two independently derived human diploid fibroblast lines and support a conclusion that SCC in human cells is strongly dependent on Timeless but independent of Chk1. Furthermore, the 10-fold difference in discohesion observed when depleting Timeless versus Tipin indicates that Timeless has a function in SCC that is independent of the Tim-Tipin complex, even though the abundance of Timeless is reduced when Tipin is targeted for depletion. A better understanding of how Timeless, Tipin and Claspin promote SCC will elucidate non-checkpoint functions of these proteins at DNA replication forks and inform models of the establishment of SCC.

Figure 1

Depletion of checkpoint/RFPC proteins attenuates UVC-induced phosphorylation of Chk1 in NHF1-hTERT. Forty-eight h after electroporation with siRNAs, NHF1-hTERT were exposed to 0 or 2.5 J/m² UVC. Cells were harvested 1 hour after exposure. (A) A representative western blot from among three independent experiments depicting siRNA-mediated protein depletion and UVC-induced phosphorylation of Chk1 at S345. (B) Quantification of the attenuation of UVC-induced P-Chk1 S345 in NHF1-hTERT depleted of checkpoint proteins. Graph depicts average percents and standard deviations from three independent experiments.

Figure 2

Metaphases from Timeless-depleted NHF1-hTERT display defective sister chromatid cohesion. (A) Metaphase from NHF1-hTERT 48 h after introduction of NTC siRNA. (B) Metaphases from Timeless-depleted NHF1-hTERT depicting range of discohesion phenotypes from partial sister chromatid separation (left panel) to complete premature anaphase (right panel). (C) Metaphase from NHF1-hTERT electroporated with NTC siRNA and analyzed by FISH. A green centromere 9 probe and a red CDKN2A probe are shown with DAPI counter-stain. (D) Premature centromere separation in a metaphase from NHF1-hTERT electroporated with Timeless siRNA and analyzed by FISH.

Figure 3

Metaphases from checkpoint/RFPC-depleted NHF1-hTERT display defective sister chromatid cohesion. NHF1-hTERT metaphases were prepared 48 h after electroporation with siRNAs. Error bars represent standard error of the mean (S.E.M.) from 25–50 determinations each from a minimum of three independent experiments per targeting siRNA and a total of 13 independent experiments (800 metaphases) for NTC siRNA (experiments were performed in randomized sets of knockdowns). * P < 0.05, ** P < 0.002, *** P < 0.0001, † = not statistically different from Timeless siRNA result.

Figure 4

Defective sister chromatid cohesion in Timeless-depleted NHF1-hTERT is not an off-target effect of Timeless siRNA. (A) Comparison of Timeless depletion by two different siRNAs (Tim-05 or Tim-06). Forty-eight h after electroporation of siRNAs, cells were sham treated or exposed to 2.5 J/m² UVC and harvested 1 h later. *Timeless, **non-specific band. (B) NHF1-hTERT depleted of Timeless by Tim-06 siRNA display defective sister chromatid cohesion. Error bars represent standard error of the mean (S.E.M.) from 25–50 determinations each from three independent experiments.

Figure 5

Metaphases from checkpoint/RFPC-depleted NHF10-hTERT display defective sister chromatid cohesion. NHF10-hTERT metaphases were prepared 48 h after electroporation with siRNAs. Error bars represent standard error of the mean (S.E.M.) from 25–50 determinations each from a minimum of three independent experiments per targeting siRNA and a total of 16 independent experiments (852 metaphases) for NTC siRNA (experiments were performed in sets of randomized knockdowns). * P < 0.05, *** P < 0.0001, † = not statistically different from Timeless siRNA.