DNA damage-induced activation of CUL4B targets HUWE1 for proteasomal degradation

The E3 ubiquitin ligase HUWE1/Mule/ARF-BP1 plays an important role in integrating/coordinating diverse cellular processes such as DNA damage repair and apoptosis. A previous study has shown that HUWE1 is required for the early step of DNA damage-induced apoptosis, by targeting MCL-1 for proteasomal degradation. However, HUWE1 is subsequently inactivated, promoting cell survival and the subsequent DNA damage repair process. The mechanism underlying its regulation during this process remains largely undefined. Here, we show that the Cullin4B-RING E3 ligase (CRL4B) is required for proteasomal degradation of HUWE1 in response to DNA damage. CUL4B is activated in a NEDD8-dependent manner, and ubiquitinates HUWE1 in vitro and in vivo. The depletion of CUL4B stabilizes HUWE1, which in turn accelerates the degradation of MCL-1, leading to increased induction of apoptosis. Accordingly, cells deficient in CUL4B showed increased sensitivity to DNA damage reagents. More importantly, upon CUL4B depletion, these phenotypes can be rescued through simultaneous depletion of HUWE1, consistent with the role of CUL4B in regulating HUWE1. Collectively, these results identify CRL4B as an essential E3 ligase in targeting the proteasomal degradation of HUWE1 in response to DNA damage, and provide a potential strategy for cancer therapy by targeting HUWE1 and the CUL4B E3 ligase.

For generating FH-CUL4B S3 HeLa stable cell line, S3 HeLa cells were trans fected with pcDNA 3.1-Flag-HA -CUL4B plasmid in 10 cm culture dishes. 24 h later, the cells were splitted in 60 mm plates and selected against hygromycin (700 μg/ml) for two weeks. Individual colonies were picked and expanded. Positive colonies were confirmed by western blotting and/or immunofluorescence with anti-HA monoclonal antibody.
Pa ge 2 / 9 Cont rol, CUL4B, ROC1, UB C12 and DDB1 siRNA were synthesized from Invitrogen Inc or Genepharma Inc. The siRNA targeting sequences used are listed in table S2. Lipofectamine RNAi MA X (Invitrogen) was used for siRNA transfection. For transfection, cells were seeded into each well of a 6-well plate and subsequently transfected with a concent ration of 100 nM siRNA using Lipofectamine RNAi MA X. 48 h post transfection, proteins were extracted, and the lysates were examined by western blotting.

Cycloheximide (CHX) Chase Assay
293T cells were trans fected with pcDNA 3.1-Flag-HA -CUL4B wild type or mut atant (K859R) constructs. 48 h after transfection, cells were treated with cycloheximide (100 μg/ml). HeLa cells were trans fected with siRNA targeting CUL4B, DDB1, ROC1 or UBC12 and treat ed as above. Cells were harvested at the indicated times, and protein levels were evaluated by western blotting.

DNA Damage Treatment
Cells were subjected to ionizing radiation using GS R-D1 137Cs gamma-irradiator (RPS Services Limited) at a dose rate of 1.8 Gy/min (8-Gy dose). For doxorubicin and etoposide treatments, cells were treated with either 0. 5 ug /ml doxorubicin (Dox) or 10uM etoposide (Eto).
The medium was changed after the treatment, and cells were incubated at 37°C to allow for DNA Repair.

Cell Viability and Apoptosis Assays
Cell viability was assessed indirectly by MTT assay . The cells were cultured in t he 96-wells plates at a density of 5×10 4 ml -1 and treated with different concentration of doxorubicin, etoposide and cisplatin for 24 h. MTT was added to each well 4 h before termination of culture and incubat ed for 4 h at 37°C in 5% CO2. 10% S DS was then added to each well, followed by overnight incubation at 37°C and 5% CO2 to dissolve the dark blue crystal product. Each sample point was assayed with 4 replica points. Absorbance at 570 nm (A570) of the solubilized formazan was measured using a Bio-Tek Instruments (KC junior, USA) microplate reader to calculate inhibition rate for cell relative viability.
As for apoptosis assays, cells were suspended in PBS buffer and then washed, suspended in 100μl Annexin V-binding buffer. FITC-conjugated Annexin V and PI were used to stain the cells in each sample for 15 min at room temperature and analyzed by flow cytometry.
Gel analysis method with Image J