Citation
- Authors: Huguet F. et al.
- Year: 2022
- Journal: Open Biol 12 220017
- Applications: in vitro / DNA, siRNA / jetPRIME
- Cell types:
- Name: HCT 116
Description: Human colon carcinoma cells
Known as: HCT116 - Name: HeLa
Description: Human cervix epitheloid carcinoma cells
- Name: HCT 116
Method
For plasmid transfection, HeLa cells were seeded in 10 cm dish at 30% confluency 2 days prior transfection. Cells were transfected with 5 µg DNA of GFP alone, GFP-Repo-ManWT or GFPRepo-ManIRCE constructs for 24 h with JetPRIME (Polyplus Transfection).
For siRNA transfection, cells were seeded 24 h prior to transfection at 30% confluency and transfected with 50 nM siRNA for 48 h with JetPRIME.
HeLa cells were transfected with 400 ng lamin GFP and 20 mM of either control siRNA or Repo-Man siRNA using jetPRIME transfection reagent (Polyplus transfection) for 48 h as recommended in the manufacturer’s instructions.
Repo-Man-FKBP-FRB cell line was generated from HCT116 expressing TET-OsTIR1. Cells were seeded in six-well plate 2 days prior to transfection. Cells were transfected with the CRISPR/Cas9 plasmid and the two donor plasmids at equal ratio by using JetPRIME. After 48 h, cells were diluted and transferred in 10 cm dishes. Cells were grown for 15 days with media containing 700 µg ml−1 G418 and 100 µg ml−1 hygromycin B. The medium was changed every 4 days.
Abstract
Lamin A phosphorylation/de-phosphorylation is an important process during cells division as it allows for nuclear envelope (NE) disassembly at mitotic entry and its re-assembly during mitotic exit. Several kinases have been identified as responsible for these phosphorylations, but no protein phosphatase has been implicated in their reversal. One of the mitotic phosphosites in lamin A responsible for its dynamic behaviour is serine 22 (S22) which is de-phosphorylated during mitotic exit. Recent evidence has also linked the nuclear pool of lamin A S22ph in interphase to gene expression regulation. Previous work suggested that the phosphatase responsible for lamin A S22 de-phosphorylation is chromatin bound and interacts with lamin A via SUMO-SIM motives. We have previously reported that Repo-Man/protein phosphatase 1 (PP1) is a chromatin-associated phosphatase that regulates NE reformation. Here we propose that Repo-Man/PP1 phosphatase mediates lamin A S22 de-phosphorylation. We indeed show that depletion of Repo-Man leads to NE defects, causes hyperphosphorylation of lamin A S22 that can be rescued by a wild-type but not a SUMOylation-deficient mutant. Lamin A and Repo-Man interact in vivo and in vitro, and the interaction is mediated by SUMOylation. Moreover, the localization of Repo-Man/PP1 to the chromatin is essential for lamin A S22 de-phosphorylation.