Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 283 (15), 9844-51

Tip60 Is Required for DNA Interstrand Cross-Link Repair in the Fanconi Anemia Pathway

Affiliations

Tip60 Is Required for DNA Interstrand Cross-Link Repair in the Fanconi Anemia Pathway

James Hejna et al. J Biol Chem.

Abstract

The disease Fanconi anemia is a genome instability syndrome characterized by cellular sensitivity to DNA interstrand cross-linking agents, manifest by decreased cellular survival and chromosomal aberrations after such treatment. There are at least 13 proteins acting in the pathway, with the FANCD2 protein apparently functioning as a late term effecter in the maintenance of genome stability. We find that the chromatin remodeling protein, Tip60, interacts directly with the FANCD2 protein in a yeast two-hybrid system. This interaction has been confirmed by co-immunoprecipitation and co-localization using both endogenous and epitope-tagged FANCD2 and Tip60 from human cells. The observation of decreased cellular survival after exposure to mitomycin C in normal fibroblasts depleted for Tip60 indicates a direct function in interstrand cross-link repair. The coincident function of Tip60 and FANCD2 in one pathway is supported by the finding that depletion of Tip60 in Fanconi anemia cells does not increase sensitivity to DNA cross-links. However, depletion of Tip60 did not reduce monoubiquitination of FANCD2 or its localization to nuclear foci following DNA damage. The observations indicate that Fanconi anemia proteins act in concert with chromatin remodeling functions to maintain genome stability after DNA cross-link damage.

Figures

FIGURE 1.
FIGURE 1.
The FANCD2-Tip60 interaction is independent of FANCD2 Lys561 but requires the Tip60 acetyl-CoA-binding site. A, subcloning of FANCD2 amino acid 55–971 into pGBKT7. BD is the binding domain. B, diploid yeast plated on -Leu-Trp-His-Ade with X-α-Gal. Library clones, as well as full-length Tip60β, interact with FANCD2 in either binding or activation domain constructs (FANCD2–5′ denotes the subclone encoding amino acid 55–971) as indicated by blue. The interaction of FANCG and FANCA is shown for comparison. C, liquidβ-galactosidase assay of diploid yeast with indicated constructs. D, mutant proteins were expressed in diploid yeast, as shown by immunoblotting. FANCD2 and Tip60 fusions were probed withα-Myc and α-hemagglutinin, respectively, for the epitope tag fusion from the yeast vector. WT, wild type; d.m., double mutant.
FIGURE 2.
FIGURE 2.
Interacting subclones. A, subclones of FANCD2, with interaction with Tip60 indicated as +. The K561R mutation prevents monoubiqutination of FANCD2. B, subclones of Tip60, with interaction with FANCD2 indicated as +. d.m., double mutant.
FIGURE 3.
FIGURE 3.
Immunoblot blot of PD20 clones transfected with FANCD2 expression constructs. In clone name, C indicates C terminus tag; N indicates N terminus tag. PD20R3 was transfected with a pIRESneo-FANCD2 nontagged construct. GM639 and PD20 whole cell extracts are shown for comparison.
FIGURE 4.
FIGURE 4.
FANCD2 and Tip60 co-immunoprecipitate. A, GST-Tip60β and GST alone bound to glutathione-agarose beads, Coomassie-stained. B, GST-Tip60β pulldown of FANCD2. Lane WCE, GM639 cleared whole cell lysate; lane U, unbound fraction; lane W, wash 3; lane P, fraction bound to beads. Electrophoresis on 8% SDS-PAGE (panel i) demonstrated pulldown of FANCD2, but did not separate FANCD2-Ub (FANCD2-L) from unmodified FANCD2 (FANCD2-S). Electrophoresis on a 3–8% denaturing gradient gel (panel ii) resolved the two forms. C, co-immunoprecipitation (IP) of FANCD2 with FLAG-tagged Tip60. HEK293 cells were transiently transfected with FLAG-Tip60, harvested, and immunoprecipitated with anti-FLAG antibody and protein A/G-agarose beads. Lane WCE, input HEK293 whole cell cleared lysate; lane U, unbound fraction; lanes W1, W2, and W3, successive washes; lane P, immunoprecipitate. The control was an anti-FLAG immunoprecipitation with nontransfected cell extract. D, co-immunoprecipitation of BRCA2 and Tip60 with FANCD2-V5His. PD20 cells functionally complemented with FANCD2-V5His, were lysed and immunoprecipitated with α-V5 antibody and protein A/G-Sepharose beads. Lane WCE, cleared lysate; lane U, unbound; lanes W1, W2, and W3, washes 1, 2, and 3, respectively; lane P, final pellet. Control was the same anti-V5 immunoprecipitation with PD20 (FA-D2) nontransfected whole cell extracts, probed with anti-Tip60 antibody. E, co-immunoprecipitation of Tip60 with anti-FANCD2 antibody. Immunoprecipitations from whole cell extracts used normal mouse serum (control) or anti-FANCD2 antibody, and the blots were probed with anti-FANCD2 and anti-Tip60 antibody.
FIGURE 5.
FIGURE 5.
Co-localization of Tip60 with FANCD2 in nuclear foci after DNA damage. Upper panels, the corrected PD20C1 cells were either untreated or treated with 80 ng/ml MMC for 12 h, and the foci were visualized with anti-V5 (red) and anti-Tip60 (green) antibodies. Lower panels, Tip60 siRNA was used to deplete Tip60 in PD20C1 cells; cells were then either untreated or damaged with 80 ng/ml MMC for 12 h and visualized. DAPI, 4′,6′-diamino-2-phenylindole.
FIGURE 6.
FIGURE 6.
Depletion of Tip60 and Tip49. A, immunoblot showing Tip60 depletion 72 h post-transfection. The lower band is a nonspecific band that serves as a loading control. B, colony-forming assay with depletion of Tip60 and MMC treatment. C, Western blot of whole cell extracts, showing FANCD2-Ub formation following MMC treatment in Tip60-depleted GM639 cells. D, colony-forming assay with depletion of Tip60 in (FA-C) cells (PD331) and MMC treatment. E, immunoblot of mock-depleted and Tip49-depleted GM639 fibroblasts, 48 h post-transfection. F, colony-forming assay of GM639 cells with Tip49 depletion and MMC treatment.

Similar articles

See all similar articles

Cited by 12 PubMed Central articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback