Figure 6.
Chromatin Access of SPRTN Is Controlled by a DPC-Triggered Ubiquitin Switch
(A) Mono-ubiquitinated SPRTN is excluded from chromatin. Doxycycline-inducible YFP-SPRTN-Strep HeLa Flp-In TRex cells were either lysed directly in SDS-containing loading dye (total) or subjected to fractionation in soluble and chromatin components.
(B) Formaldehyde treatment induces deubiquitination of SPRTN coinciding with a complete relocalization to chromatin. Doxycycline-inducible YFP-SPRTN-Strep HeLa Flp-In TRex cells were treated with 1 mM formaldehyde (FA) for 2 hr.
(C) SPRTN is deubiquitinated upon formaldehyde exposure in a dose-dependent manner. Doxycycline-inducible YFP-SPRTN-Strep HeLa Flp-In TRex cells were treated for 2 hr with the indicated dose of formaldehyde.
(D) Endogenous SPRTN is deubiquitinated and relocalizes to chromatin formaldehyde exposure. U2OS cells were treated with 1 mM formaldehyde (FA) for 2 hr. Asterisk indicates an unspecific band.
(E) Deubiquitination of SPRTN is specifically triggered by DNA-protein crosslinks. Doxycycline-inducible YFP-SPRTN-Strep HeLa Flp-In TRex cells were treated with formaldehyde (FA, 1 mM, 2 hr), UVC light (UV, 20 J/m2, 2 hr before lysis), aphidicolin (Aph, 1 μM, 2 hr), or IR (3 Gy, 2 hr before lysis).
(F) SPRTN is differentially recruited to chromatin depending on the type of DNA damage. Doxycycline-induced YFP-SPRTN-Strep HeLa Flp-In TRex cells were treated with formaldehyde (FA, 0.5 mM) or UV (20 J/m2) and subjected to pre-extraction, prior to fixation and immunofluorescence.
(G) SPRTN deubiquitination and chromatin recruitment upon DPC induction is independent of binding to PCNA, p97, or ubiquitin. Doxycycline-inducible YFP-SPRTN-Strep HeLa Flp-In TRex cells expressing the indicated SPRTN variants were treated with 1 mM formaldehyde (FA) for 2 hr.
(H) SPRTN-ΔC displays an aberrant subcellular localization. Doxycycline-induced YFP-SPRTN-Strep HeLa Flp-In TRex cells were analyzed using immunofluorescence.
(I) SPRTN-ΔC is recruited to chromatin upon the induction of DPCs. Doxycycline-induced YFP-SPRTN-Strep HeLa Flp-In TRex cells were treated with formaldehyde (FA, 0.5 mM) and subjected to pre-extraction, prior to fixation and immunofluorescence.
(J) SPRTN-ΔC complements the formaldehyde sensitivity of SPRTN-deficient cells only partially. HeLa Flp-In TRex cells bearing the indicated doxycycline-inducible YFP-SPRTN-Strep alleles were transfected with siRNA against endogenous SPRTN and incubated in the absence or presence of doxycycline for 48 hr. Cells were then treated for 48 hr with 100 μM formaldehyde, and cell numbers were determined after an additional 4-day incubation. Values indicate cell numbers relative to untreated cells. Error bars represent SD of two to four replicates. Knockdown and doxycycline induction were confirmed by western blotting. Please note that autocleavage bands appear at similar positions as endogenous SPRTN in cells expressing WT YFP-SPRTN.
See also Figure S5.