Depletion of A-type lamins and Lap2 alpha reduces 53BP1 accumulation at UV-induced DNA lesions and Lap2 alpha protein is responsible for compactness of irradiated chromatin
Abstrakt
We studied how deficiency in lamins A/C and lamina-associated polypeptide 2 alpha (Lap2 alpha) affects DNA repair after irradiation. A-type lamins and Lap2 alpha were not recruited to local DNA lesions and did not accumulate to gamma-irradiation-induced foci (IRIF), as it is generally observed for well-known marker of DNA lesions, 53BP1 protein.
At micro-irradiated chromatin of lmna double knockout (dn) and Lap2 alpha dn cells, 53BP1 protein levels were reduced, compared to locally irradiated wild-type counterpart. Decreased levels of 53BP1 we also observed in whole populations of lmna dn and Lap2 alpha dn cells, irradiated by UV light.
We also studied distribution pattern of 53BP1 protein in a genome outside micro-irradiated region. In Lap2 alpha deficient cells, identical fluorescence of mCherry-tagged 53BP1 protein was found at both microirradiated region and surrounding chromatin.
However, a well-known marker of double strand breaks, gamma H2AX, was highly abundant in the lesion-surrounding genome of Lap2 alpha deficient cells. Described changes, induced by irradiation in Lap2 alpha dn cells, were not accompanied by cell cycle changes.
In Lap2 alpha dn cells, we additionally performed analysis by FLIM (Fluorescence Lifetime Imaging Microscopy) that showed different dynamic behavior of mCherry-tagged 53BP1 protein pools when it was compared with wild-type (wt) fibroblasts. This analysis revealed three different fractions of mCherry-53BP1 protein.
Two of them showed identical exponential decay times (tau 1 and tau 3), but the decay rate of tau 2 and amplitudes of fluorescence decays (A1-A3) were statistically different in wt and Lap2 alpha dn fibroblasts. Moreover, gamma-irradiation weakened an interaction between A-type lamins and Lap2 alpha.
Together, our results demonstrate how depletion of Lap2 alpha affects DNA damage response (DDR) and how chromatin compactness is changed in Lap2 alpha deficient cells exposed to radiation.