Fanconi Anemia Proteins Localize to Chromatin and the Nuclear Matrix in a DNA Damage- and Cell Cycle-regulated Manner

Abstract

Fanconi anemia (FA) 1 is a genetic disease of cancer susceptibility marked by congenital defects, bone marrow failure, and myeloid leukemia (1-4). To date at least seven complementation groups have been defined (5, 6). Six genes accounting for six groups have been cloned (7-15). However, all the gene products resemble no known proteins and have no identifiable functional protein motif.Cells derived from patients with the disease exhibit characteristic hypersensitivity caused by DNA cross-linking agents and generalized decreased survival (16 -20). In addition, a well described G 2 phase cell cycle delay has also been described that is thought to be secondary to a defective S or G 2 checkpoint (21-23). However, no defined biochemical mechanism for this hypersensitivity has been elucidated. Patient and cellular phenotypes across all the complementation groups are similar, suggesting an interrelatedness or cooperativity between the FA proteins.This cooperativity has been borne out by work we have done in showing binding of FancA and FancC in a protein complex in both nucleus and cytoplasm (24 -26). Recent work has found the FancG and FancF proteins in the complex as well (27)(28)(29)(30). A large complex is suggested by our recent work 2 , and binding does not occur in any of the complementation groups except the FA-D group.One clue to FA function lies in the study of the FancA protein, which contains a classic bipartite nuclear localization signal and is phosphorylated. FancA nuclear localization, phosphorylation, and binding to FancC are abolished in all complementation groups except the FA-D group (24 -26, 29). This suggests that a nuclear event is critical to the normal function of the FA proteins, and the aberrant protein in the FA-D group may have a role downstream of the FA complex in the nucleus.To date, other than the alteration of binding, nuclear localization, and FancA phosphorylation in mutant FA complementation groups, no other consistent biochemical change has been described, although some have described variations in FancC levels in the cell cycle (31,32). In our study, we have found that the FA proteins not only reside in the nucleus but also are closely associated with the nuclear matrix and chromatin.The nuclear matrix is a loose mechanical framework of proteins that has also been implicated in enzymatic activities in the regulation of transcription, replication, and DNA repair. The nuclear matrix is intimately associated with chromatin. BRG and brm, the human swi-snf homologues, are examples of nuclear matrix proteins involved in transcriptional regulation that change phosphorylation state, shift to chromatin, and become distinct from the nuclear matrix during mitosis (33-35). Immunofluorescence of FancA reveals a similar shift away from the condensed chromosomes of mitosis, suggesting that they also interact with chromatin and the nuclear matrix. Our studies demonstrate that the FA proteins associate with chromatin and the nuclear matrix in an inducible fashion. MATERIALS AND METHODSCell Cultu...

Tumor suppressor menin: the essential role of nuclear localization signal domains in coordinating gene expression

“…expressing either wild-type menin or its NLS mutants were fractionated into WCL, soluble fraction, chromatin and nuclear matrix, as previously described with certain modifications (Qiao et al, 2001;Zaidi et al, 2001). Briefly, cell pellets were resuspended in 500 ml permeablized buffer (50 mg digitonin, 10 nM HEPES pH 7.4, 10 nM KCl, 1 nM Na 3 VO 4 , 1 Â protease inhibitors) to remove the soluble fraction.…”

Phosphorylation of Fanconi Anemia (FA) Complementation Group G Protein, FANCG, at Serine 7 Is Important for Function of the FA Pathway

“…was made as previously described (37). All PCR were done using high fidelity Taq polymerase (Amersham Biosciences).…”

“…FA-A mutant GM6914 cells, corrected GM6914 ϩ FLAG-FANCA cells, FA-D2 mutant PD20 cells, FA-D2 corrected 20 -315 cells, and FA-G mutant cells 326SV were grown in F12 ϩ 15% fetal bovine serum. 293VSV producer cells were grown as described (37). HeLa cells were also transduced with pMMP-FLAG-FANCA, and the resulting cells were selected with puromycin.…”

“…In addition, we have shown during the cell cycle that the FA proteins detach from chromatin during mitosis, and FANCG becomes phosphorylated while remaining part of the complex (37). One group has demonstrated that FANCG has a isoform seen in asynchronous cells that is phosphatase sensitive (39).…”

“…mRNA or protein levels change little in response to DNA damage or the cell cycle. Our recent work has revealed that at least a subset of the FA proteins resides in the nucleus bound to chromatin, where increased protein binding occurs in response to DNA damage (37). One of the non-core complex FA proteins, FANCD2, becomes monoubiquitinated in response to DNA damage (38).…”