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

Abstract

Fanconi anemia (FA) is a genetic disease characterized by congenital defects, bone marrow failure, and cancer susceptibility. Cells from patients with FA exhibit genomic instability and hypersensitivity to DNA cross linking agents such as mitomycin C. Despite the identification of seven complementation groups and the cloning of six genes, the function of the encoded gene products remains elusive. The FancA (Fanconi anemia complementation group A), FancC, and FancG proteins have been detected within a nuclear complex, but no change in level, binding, or localization has been reported as a result of drug treatment or cell cycle. We show that in immunofluorescence studies, FancA appears as a non-nucleolar nuclear protein that is excluded from condensed, mitotic chromosomes. Biochemical fractionation reveals that the FA proteins are found in nuclear matrix and chromatin and that treatment with mitomycin C results in increase of the FA proteins in nuclear matrix and chromatin fractions. This induction occurs in wild-type cells and mutant FA-D (Fanconi complementation group D) cells but not in mutant FA-A cells. Immunoprecipitation of FancA protein in chromatin demonstrates the coprecipitation of FancA, FancC, and FancG, showing that the FA proteins move together as a complex. Also, fractionation of mitotic cells confirms the lack of FA proteins in chromatin or the nuclear matrix. Furthermore, phosphorylation of FancG was found to be temporally correlated with exit of the FA complex from chromosomes at mitosis. Taken together, these findings suggest a role for FA proteins in chromatin and nuclear matrix. Fanconi anemia (FA) is a genetic disease characterized by congenital defects, bone marrow failure, and cancer susceptibility. Cells from patients with FA exhibit genomic instability and hypersensitivity to DNA cross linking agents such as mitomycin C. Despite the identification of seven complementation groups and the cloning of six genes, the function of the encoded gene products remains elusive. The FancA (Fanconi anemia complementation group A), FancC, and FancG proteins have been detected within a nuclear complex, but no change in level, binding, or localization has been reported as a result of drug treatment or cell cycle. We show that in immunofluorescence studies, FancA appears as a non-nucleolar nuclear protein that is excluded from condensed, mitotic chromosomes. Biochemical fractionation reveals that the FA proteins are found in nuclear matrix and chromatin and that treatment with mitomycin C results in increase of the FA proteins in nuclear matrix and chromatin fractions. This induction occurs in wild-type cells and mutant FA-D (Fanconi complementation group D) cells but not in mutant FA-A cells. Immunoprecipitation of FancA protein in chromatin demonstrates the coprecipitation of FancA, FancC, and FancG, showing that the FA proteins move together as a complex. Also, fractionation of mitotic cells confirms the lack of FA proteins in chromatin or the nuclear matrix. Furthermore, phosphorylation of FancG was found to be temporally correlated with exit of the FA complex from chromosomes at mitosis. Taken together, these findings suggest a role for FA proteins in chromatin and nuclear matrix. Fanconi anemia Fanconi anemia complementation group A -C, -D, -F, -G, Fanconi complementation groups A, C, D, F, G fetal bovine serum phosphate-buffered solution 4′,6-diamidino-2-phenylindole Tris-buffered saline polyacrylamide gel electrophoresis mitomycin C topoisomerase II Fanconi anemia (FA)1 is a genetic disease of cancer susceptibility marked by congenital defects, bone marrow failure, and myeloid leukemia (1Alter B.P. Young N.S. Nathan D.G. Oski F.A. 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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 (24Kupfer G.M. Naf D. Suliman A. Pulsipher M. D'Andrea A.D. Nat. Genet. 1997; 17: 487-490Crossref PubMed Scopus (159) Google Scholar, 25Naf D. Kupfer G.M. Suliman A. Lambert K. D'Andrea A.D. Mol. Cell. Biol. 1998; 18: 5952-5960Crossref PubMed Scopus (107) Google Scholar, 26Yamashita T. Kupfer G. Naf D. Suliman A. Joenje H. Asano S. D'Andrea A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13085-13090Crossref PubMed Scopus (107) Google Scholar). Recent work has found the FancG and FancF proteins in the complex as well (27Christianson T.A. Bagby G.C. Blood. 2000; 95: 725-726Crossref PubMed Google Scholar, 28de Winter J.P. van Der Weel L. de Groot J. Stone S. Waisfisz Q. Arwert F. Scheper R.J. Kruyt F.A. Hoatlin M.E. Joenje H. Hum. Mol. Genet. 2000; 9: 2665-2674Crossref PubMed Scopus (172) Google Scholar, 29Garcia-Higuera I. Kuang Y. Naf D. Wasik J. D'Andrea A.D. Mol. Cell. Biol. 1999; 19: 4866-4873Crossref PubMed Scopus (199) Google Scholar, 30Waisfisz Q. de Winter J.P. Kruyt F.A. de Groot J. van der Weel L. Dijkmans L.M. Zhi Y. Arwert F. Scheper R.J. Youssoufian H. Hoatlin M.E. Joenje H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10320-10325Crossref PubMed Scopus (124) Google Scholar). A large complex is suggested by our recent work 2F. Qiao, A. Moss, and G. M. Kupfer, unpublished data. , 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 (24Kupfer G.M. Naf D. Suliman A. Pulsipher M. D'Andrea A.D. Nat. Genet. 1997; 17: 487-490Crossref PubMed Scopus (159) Google Scholar, 25Naf D. Kupfer G.M. Suliman A. Lambert K. D'Andrea A.D. Mol. Cell. Biol. 1998; 18: 5952-5960Crossref PubMed Scopus (107) Google Scholar, 26Yamashita T. Kupfer G. Naf D. Suliman A. Joenje H. Asano S. D'Andrea A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13085-13090Crossref PubMed Scopus (107) Google Scholar, 29Garcia-Higuera I. Kuang Y. Naf D. Wasik J. D'Andrea A.D. Mol. Cell. Biol. 1999; 19: 4866-4873Crossref PubMed Scopus (199) Google Scholar). 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 (31Heinrich M.C. Silvey K.V. Stone S. Zigler A.J. Griffith D.J. Montalto M. Chai L. Zhi Y. Hoatlin M.E. Blood. 2000; 95: 3970-3977Crossref PubMed Google Scholar, 32Kupfer G. Yamashita T. Naf D. Suliman A. Asano S. D'Andrea A.D. Blood. 1997; 90: 1047-1054Crossref PubMed Google Scholar). 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 (33Muchardt C. Reyes J.C. Bourachot B. Leguoy E. Yaniv M. EMBO J. 1996; 15: 3394-3402Crossref PubMed Scopus (194) Google Scholar, 34Muchardt C. Yaniv M. J. Mol. Biol. 1999; 293: 187-198Crossref PubMed Scopus (162) Google Scholar, 35Reyes J.C. Muchardt C. Yaniv M. J. Cell Biol. 1997; 137: 263-274Crossref PubMed Scopus (200) Google Scholar). 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. Cells were grown at 37 °C in a 5% CO2 incubator. FA fibroblasts cell lines were grown in Dulbecco's modified Eagle's medium supplemented with 15% (v/v) fetal bovine serum (FBS). HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% FBS. FA-C mutant cells (PD4) were grown in RPMI + 15% FBS. Mutant and corrected pairs of cell lines were prepared as previously described: GM6914 (ATCC) ± and ± the M. Thayer M. Reifsteck C. Olson S. Smith L. Jakobs P.M. Leach R. Naylor S. Joenje H. Grompe M. Nat. 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Genet. 1997; 17: 487-490Crossref PubMed Scopus (159) Google Scholar). was by gel in and were for in 5% bovine serum in and were in at were in with secondary and by (24Kupfer G.M. Naf D. Suliman A. Pulsipher M. D'Andrea A.D. Nat. Genet. 1997; 17: 487-490Crossref PubMed Scopus (159) Google Scholar). were from M.A. L.M. J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). FancG was of cell as were with for at °C in of and of in the or of were by of of and were to and In of to FA protein we the of the FancA Immunofluorescence was FA-A mutant cells no FancA or corrected with of FancA with serum corrected FA-A cells nuclear and no in the FancA was excluded from condensed chromosomes in mitotic cells mitotic as by with Mutant cells were by This is similar to of and nuclear matrix proteins, such as and which are involved in DNA of genomic and regulation (33Muchardt C. Reyes J.C. Bourachot B. Leguoy E. Yaniv M. EMBO J. 1996; 15: 3394-3402Crossref PubMed Scopus (194) Google Scholar, 34Muchardt C. Yaniv M. J. Mol. Biol. 1999; 293: 187-198Crossref PubMed Scopus (162) Google Scholar, 35Reyes J.C. Muchardt C. Yaniv M. J. Cell Biol. 1997; 137: 263-274Crossref PubMed Scopus (200) Google Scholar). Immunofluorescence suggests that FA proteins are associated with nuclear matrix and chromatin. is in with DNA C.D. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, Cell Biol. PubMed Scopus Google Scholar). To FA proteins are associated with HeLa cells were with or for at FA proteins were only with This result that FA proteins are and are suggesting an with and nuclear proteins are involved in activities such as transcription, replication, DNA and of genomic the of FancA is of chromatin and nuclear matrix proteins, we the localization of FancA, FancC, and FancG by biochemical fractionation of from HeLa cells and mutant and corrected pairs of FA-A and FA-D cells that FancA, FancC, and FancG were all found in the chromatin and nuclear matrix from mutant FA-C cells no FancC was as a Mutant FA-A cells the of chromatin and nuclear matrix localization of FancA, FancC, and FancG proteins. Our fractionation the immunofluorescence that FA proteins are found in chromatin and the nuclear matrix of the However, work has of the FA proteins only that by proteins. To chromatin localization and to demonstrate of the FA proteins, were chromatin prepared from the cell lines the coprecipitation of FancC and FancG with This result was in the FA-A corrected and FA-D corrected as well as the FA-D mutant in which FancA, FancC, and FancG have been to and to the nucleus. The FA protein complex appears in a of cytoplasm and nuclear prepared However, these show that the complex, in also be detected in a chromatin In work no of the FA proteins in or has been in to agents these agents hypersensitivity in mutant FA cells (31Heinrich M.C. Silvey K.V. Stone S. Zigler A.J. Griffith D.J. Montalto M. Chai L. Zhi Y. Hoatlin M.E. Blood. 2000; 95: 3970-3977Crossref PubMed Google Scholar, 32Kupfer G. Yamashita T. Naf D. Suliman A. Asano S. D'Andrea A.D. Blood. 1997; 90: 1047-1054Crossref PubMed Google Scholar). However, these studies not chromatin or the nuclear matrix. we the localization of FancA, FancC, and FancG in to HeLa cells were for with cell in mutant FA 10% in wild-type in and The cells were to fractionation as FA proteins in chromatin and nuclear matrix with at The of the fractionation and was by the topoisomerase II and Cells were also with and at and of drug FA proteins were found in chromatin and nuclear matrix D) by with by HeLa cells were also with of and and In to of chromatin and nuclear matrix no increase in FA proteins. HeLa cells were with and at no increase in FA protein levels in chromatin and nuclear matrix The induction of FA protein localization by consistent with the that the cell the DNA during S phase as in recent work Reifsteck C.A. Olson S.B. Grompe M. Mol. Cell. Biol. 2000; 20: PubMed Scopus Google Scholar). cell lines were as FA-A mutant and corrected cells were by treatment with was of FA-A mutant cells no the corrected cells localization of FA proteins to chromatin and nuclear matrix FA-D mutant and corrected cells were also by treatment with In this both the mutant and corrected cells FA protein localization to chromatin and nuclear matrix are consistent with previously reported that FA proteins are from the nucleus in all the complementation groups except in which mutant cells FancA, FancC, and FancG in a nuclear complex. FA proteins to chromatin and nuclear matrix of the immunofluorescence that exit of FA proteins at mitosis, we the of the cell cycle HeLa cells were to at the the cells were and were to was by not FA proteins or no change in the chromatin or nuclear matrix at the S or G2 phase However, at mitosis, as by the lack of in the nuclear matrix no FA proteins were detected in chromatin or the nuclear matrix suggesting exit of the FA proteins from condensed chromosomes. and the nuclear matrix to the nuclear as topoisomerase II was in both fractions. A of FancG was at mitosis in the This nuclear is consistent with the of FancA and from condensed chromosomes at mitosis as in 1. The FA proteins from condensed chromosomes at mitosis as by biochemical FancG as a at mitosis. To the of the of FancG at mitosis, cell was prepared from mitotic and HeLa cells and with or that of FancG with and with FancA in mitotic only of To demonstrate that the of FancG are were with The of FancG were by at at °C or with the in of both the of only the of the This the regulation and shift in localization of other proteins such as the human swi-snf protein complex. The FA proteins are of function have been to to the a localization which is to of the FA DNA In mutant FA proteins in the cytoplasm and not interact in a complex except in FA-D cells. In this study, we show that the FA proteins FancA, FancC, and FancG associate with the nuclear matrix and chromatin. The proteins shift to chromatin and nuclear matrix in an inducible by to which FA cells are FA proteins are similar to other chromatin and nuclear proteins in by and from condensed chromosomes during mitosis. This is temporally to phosphorylation of FancG, which remains to the other FA proteins. this study, has that has an inducible of FA proteins to drug treatment or cell cycle. have variations in FancC (31Heinrich M.C. Silvey K.V. Stone S. Zigler A.J. Griffith D.J. Montalto M. Chai L. Zhi Y. Hoatlin M.E. Blood. 2000; 95: 3970-3977Crossref PubMed Google Scholar, 32Kupfer G. Yamashita T. Naf D. Suliman A. Asano S. D'Andrea A.D. Blood. 1997; 90: 1047-1054Crossref PubMed Google but change in the of FA protein does not to To these have been in of and nuclear proteins. However, we have not any increase in the of or nuclear matrix FA proteins as a result of HeLa cells levels of FA proteins in these we are to the complex from the nuclear matrix of the for the of localization, protein level, and shift in to drug treatment and cell cycle of the FA proteins at least In to the of nuclear matrix proteins, recent the FA protein A FancA and FancC binding to a nuclear protein, human has been described A. S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar). This complex has been to with L.W. Lambert J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). One is that the that is of is for the mechanical in to DNA that for the FA complex to to the chromatin DNA be In addition, defective has been to be in a for DNA binding L.W. L.M. Lambert 1999; 20: PubMed Scopus Google Scholar, M. S. Moustacchi E. D. Cell Mol. Genet. 1997; PubMed Scopus Google Scholar, M. Buchwald M. P.A. Moustacchi E. D. J. Mol. 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Yamashita T. Naf D. Suliman A. Asano S. D'Andrea A.D. Blood. 1997; 90: 1047-1054Crossref PubMed Google Scholar). at of FA protein be in biochemical We for critical of the and and for of