While both Homologous recombination (HR) and Non Homologous End Joining (NHEJ) can repair DNA double Strand Breaks (DSB), the mechanisms by which one or other of these pathways is chosen remain unclear. Here we show that transcriptionally active chromatin is preferentially repaired by HR. Using chromatin immunoprecipitation-sequencing (ChIP-seq), to analyse repair of multiple DSBs induced throughout the human genome, we identify an “HRprone” subset of DSBs that recruit the HR protein RAD51, undergo resection and rely on RAD51 for efficient repair. These DSBs are located in actively transcribed genes, and targeted to HR repair via the transcription-elongation associated histone mark, histone H3 lysine 36 trimethylation (H3K36me3). In agreement, depletion of SETD2, the main H3K36 trimethyltransferase, severely impedes HR at such DSBs. Our study thereby demonstrates a primary role of the chromatin context, in which a break occurs, in DSB repair.

Cdc25B phosphatases have a key role in G2/M cell-cycle progression by activating the CDK1-cyclinB1 complexes and functioning as important targets of checkpoints. Overexpression of Cdc25B results in a bypass of the G2/M checkpoint and illegitimate entry into mitosis. It can also cause replicative stress, which leads to genomic instability. Thus, fine-tuning of the Cdc25B expression level is critical for correct cell-cycle arrest in response to DNA damage. In response to genotoxic stress, Cdc25B is mainly regulated by post-transcriptional mechanisms affecting either Cdc25B protein stability or translation. Here, we show that upon DNA damage Cdc25B can be regulated at the transcriptional level. Although ionizing radiation downregulates Cdc25B in a p53-dependent pathway, doxorubicin transcriptionally upregulates Cdc25B in p53-proficient cancer cells. We show that in the presence of wild-type p53, doxorubicin activates the Cdc25B promoter by preventing the binding of Sp1 and increasing the binding of NF-Y on the Cdc25B promoter, thus preventing p53 from downregulating this promoter. Our results highlight the mechanistically distinct regulation of the three Cdc25 phosphatases by checkpoint signalling following doxorubicin treatment.

Both 17β-estradiol (E 2 ) and fibroblast growth factor-2 (FGF2) stimulate angiogenesis and endothelial cell migration and proliferation. The first goal of this study was to explore the potential link between this hormone and this growth factor. E 2 -stimulated angiogenesis in SC Matrigel plugs in Fgf2 +/+ mice, but not in Fgf2 −/− mice. Cell cultures from subcutaneous Matrigel plugs demonstrated that E 2 increased both migration and proliferation in endothelial cells from Fgf2 +/+ mice, but not from in Fgf2 −/− mice. Several isoforms of fibroblast growth factor-2 (FGF2) are expressed: the low molecular weight 18-kDa protein (FGF2 lmw ) is secreted and activates tyrosine kinase receptors (FGFRs), whereas the high molecular weight (21 and 22 kDa) isoforms (FGF2 hmw ) remains intranuclear, but their role is mainly unknown. The second goal of this study was to explore the respective roles of FGF2 isoforms in the effects of E 2 . We thus generated mice deficient only in the FGF2 lmw ( Fgf2 lmw−/− ). E 2 stimulated in vivo angiogenesis and in vitro migration in endothelial cells from Fgf2 lmw−/− as it did in Fgf2 +/+ mice. E 2 increased FGF2 hmw protein abundance in endothelial cell cultures from Fgf2 +/+ and Fgf2 lmw−/− mice. As shown using siRNA transfection, these effects were FGFR independent but involved FGF2-Interacting Factor, an intracellular FGF2 hmw partner. This is the first report for a physiological role for the intracellular FGF2 hmw found to mediate the effect of E 2 on endothelial cell migration via an intracrine action.

Numerous evidence indicates that some of the activities of fibroblast growth factor 2 (FGF-2) depend on an intracrine mode of action. Recently, we showed that three high molecular mass (HMM) nuclear forms of FGF-2 are part of a 320-kDa protein complex while the cytoplasmic AUG-initiated form is included in a 130-kDa complex. Consequently, the characterization of FGF endogenous targets has become crucial to allow the elucidation of their endogenous activities. Through the screening of GAL4-based yeast two-hybrid expression libraries, we have isolated a gene encoding a nuclear protein of 55 kDa, FIF (FGF-2-interacting-factor), which interacts specifically with FGF-2 but not with FGF-1, FGF-3, or FGF-6. In this system, FIF interacts equally well with the NH2-extended 24-kDa FGF form as with the 18-kDa form, indicating that the FIF-binding motif is located in the last 155 amino acids of FGF-2. Nevertheless, coimmunoprecipitation experiments showed an exclusive association with HMM FGF-2. The predicted protein contains a canonical leucine zipper domain and three overlapping hydrophobic heptad repeats. The region spanning these repeats is, together with a region located in the N-terminal part of the FIF protein, implicated in the binding to FGF-2. In contrast to the full-length FIF protein, several deletion constructs were able to transactivate a lac-Z reporter gene. Furthermore, the COOH-terminal part, but not the full-length FIF protein, has previously been shown to exhibit antiapoptotic properties. Thus we discuss the possibility that these activities could reflect a physiological function of FIF through its interaction with FGF-2.

Dimerization is a prerequisite for many growth factors in their receptor activation leading to cellular response. FGF-1 and FGF-2, members of the Fibroblast Growth Factor (FGF) family, were shown to form non-covalent dimers and oligomersin vitro.Using the two-hybrid system as anin vivobinding assay we show here that of three representative members of the FGF family, only FGF-2 is able to homodimerize. Moreover the FGF-2 isoforms could heterodimerize. Two single-point mutants (T121F and W123R), defective in their dimerization capability, were isolated through random mutagenesis and were used to study the role of FGF-2 dimerization with regard to its biological activity. Remarkably, these mutant proteins were still able to induce cell differentiation, but were strongly affected in their capacity to promote cell proliferation. This study thus highlights the uncoupling between proliferation and differentiation FGF-2 signaling pathways and the crucial role of FGF-2 dimerization in the mitogenic activity of this factor.

Four forms of basic fibroblast growth factor (bFGF or FGF-2) result from an alternative initiation of translation involving one AUG (155-amino acid form) and three CUGs (210-, 201- and 196-amino acid forms). These different forms of bFGF show different intracellular biological activities. To identify their intracellular targets, the 210- and 155-amino acid forms of bFGF were independently transfected into CHO cells and their correct subcellular localizations were verified, the 155-amino acid bFGF form being essentially cytoplasmic whereas the 210-amino acid protein was nuclear. The radiation fragmentation method was used to determine the target size of the different bFGF isoforms in the transfected CHO cells and to show that the 210- and 155-amino acids bFGF isoforms were included in protein complexes of 320 and 130 kDa respectively. Similar results were obtained using the SK-Hep1 cell line, which naturally expressed all forms of bFGF. Co-immunoprecipitation assays using different chimaeric bFGF-chloramphenicol acetyltransferase proteins showed that different cellular proteins are associated with different parts of the bFGF molecule. We conclude that bFGF isoforms are involved in different molecular complexes in the cytosol and nucleus, which would reflect different functions for these proteins.

Four forms of basic fibroblast-growth factor (bFGF or FGF-2) using one AUG (155 amino acids) and three upstream CUG (210, 201 and 196 amino acids) start codons, were synthesized through an alternative use of initiation codons. The 210-amino acid form of FGF-2 (210FGF-2) was expressed in a plasmid vector under the control of a bacteriophage T7 RNA polymerase promoter system in Escherichia coli. Characterization of the purified protein was performed by electrospray mass spectrometry and Edman degradation. The recombinant 210FGF-2 produced in E. coli had a mitogenic activity similar to the 146-amino acid form extracted from tissues.

Nucleolin (713 aa), a major nucleolar protein, presents two structural domains: a N-terminus implicated in interaction with chromatin and a C-terminus containing four RNA-binding domains (RRMs) and a glycine/arginine-rich domain mainly involved in pre-rRNA packaging. Furthermore, nucleolin was shown to shuttle between cytoplasm and nucleolus. To get an insight on the nature of nuclear and nucleolar localization signals, a set of nucleolin deletion mutants in fusion with the prokaryotic chloramphenicol acetyltransferase (CAT) were constructed, and the resulting chimeric proteins were recognized by anti-CAT antibodies. First, a nuclear location signal bipartite and composed of two short basic stretches separated by eleven residues was characterized. Deletion of either motifs renders the protein cytoplasmic. Second, by deleting one or more domains implicated in nucleolin association either with DNA, RNA, or proteins, we demonstrated that nucleolar accumulation requires, in addition to the nuclear localization sequence, at least two of the five RRMs in presence or absence of N-terminus. However, in presence of only one RRM the N-terminus allowed a partial targeting of the chimeric protein to the nucleolus.

Three forms of basic fibroblast growth factor (bFGF), initiated at an AUG (18 kDa) and two CUG (21 and 22.5 kDa) start codons, were produced following transfection of COS cells with human hepatoma bFGF cDNA. The subcellular localization of the different forms was investigated directly or by using chimeric genes constructed by fusion of the bFGF and chloramphenicol acetyltransferase open reading frames. The AUG-initiated proteins were cytoplasmic, while the CUG-initiated forms were nuclear. The signal sequence responsible for the nuclear localization of bFGF is contained within 37 amino acid residues between the second CUG and the AUG start codons. Alternative initiation of translation regulates the subcellular localization of bFGF and thus could modulate its role in cell growth and differentiation control.

Annals of the New York Academy of SciencesVolume 638, Issue 1 p. 127-138 Nuclear Translocation of Basic Fibroblast Growth Factora F. AMALRIC, F. AMALRIC Center for Research in Biochemistry and Cellular Genetics of the CNRS 118, Route de Narbonne 31062 Toulouse Cédex. FranceSearch for more papers by this authorV. BALDIN, V. BALDIN Center for Research in Biochemistry and Cellular Genetics of the CNRS 118, Route de Narbonne 31062 Toulouse Cédex. FranceSearch for more papers by this authorI. BOSC-BIERNE, I. BOSC-BIERNE Center for Research in Biochemistry and Cellular Genetics of the CNRS 118, Route de Narbonne 31062 Toulouse Cédex. FranceSearch for more papers by this authorB. BUGLER, B. BUGLER Center for Research in Biochemistry and Cellular Genetics of the CNRS 118, Route de Narbonne 31062 Toulouse Cédex. FranceSearch for more papers by this authorB. COUDERC, B. COUDERC Center for Research in Biochemistry and Cellular Genetics of the CNRS 118, Route de Narbonne 31062 Toulouse Cédex. FranceSearch for more papers by this authorM. GUYADER, M. GUYADER Center for Research in Biochemistry and Cellular Genetics of the CNRS 118, Route de Narbonne 31062 Toulouse Cédex. FranceSearch for more papers by this authorV. PATRY, V. PATRY Center for Research in Biochemistry and Cellular Genetics of the CNRS 118, Route de Narbonne 31062 Toulouse Cédex. FranceSearch for more papers by this authorH. PRATS, H. PRATS Center for Research in Biochemistry and Cellular Genetics of the CNRS 118, Route de Narbonne 31062 Toulouse Cédex. FranceSearch for more papers by this authorA. M. ROMAN, A. M. ROMAN Center for Research in Biochemistry and Cellular Genetics of the CNRS 118, Route de Narbonne 31062 Toulouse Cédex. FranceSearch for more papers by this authorG. BOUCHE, G. BOUCHE Center for Research in Biochemistry and Cellular Genetics of the CNRS 118, Route de Narbonne 31062 Toulouse Cédex. FranceSearch for more papers by this author F. AMALRIC, F. AMALRIC Center for Research in Biochemistry and Cellular Genetics of the CNRS 118, Route de Narbonne 31062 Toulouse Cédex. FranceSearch for more papers by this authorV. BALDIN, V. BALDIN Center for Research in Biochemistry and Cellular Genetics of the CNRS 118, Route de Narbonne 31062 Toulouse Cédex. FranceSearch for more papers by this authorI. BOSC-BIERNE, I. BOSC-BIERNE Center for Research in Biochemistry and Cellular Genetics of the CNRS 118, Route de Narbonne 31062 Toulouse Cédex. FranceSearch for more papers by this authorB. BUGLER, B. BUGLER Center for Research in Biochemistry and Cellular Genetics of the CNRS 118, Route de Narbonne 31062 Toulouse Cédex. FranceSearch for more papers by this authorB. COUDERC, B. COUDERC Center for Research in Biochemistry and Cellular Genetics of the CNRS 118, Route de Narbonne 31062 Toulouse Cédex. FranceSearch for more papers by this authorM. GUYADER, M. GUYADER Center for Research in Biochemistry and Cellular Genetics of the CNRS 118, Route de Narbonne 31062 Toulouse Cédex. FranceSearch for more papers by this authorV. PATRY, V. PATRY Center for Research in Biochemistry and Cellular Genetics of the CNRS 118, Route de Narbonne 31062 Toulouse Cédex. FranceSearch for more papers by this authorH. PRATS, H. PRATS Center for Research in Biochemistry and Cellular Genetics of the CNRS 118, Route de Narbonne 31062 Toulouse Cédex. FranceSearch for more papers by this authorA. M. ROMAN, A. M. ROMAN Center for Research in Biochemistry and Cellular Genetics of the CNRS 118, Route de Narbonne 31062 Toulouse Cédex. FranceSearch for more papers by this authorG. BOUCHE, G. BOUCHE Center for Research in Biochemistry and Cellular Genetics of the CNRS 118, Route de Narbonne 31062 Toulouse Cédex. FranceSearch for more papers by this author First published: December 1991 https://doi.org/10.1111/j.1749-6632.1991.tb49023.xCitations: 27 a This work was supported by grants from the Centre National de la Recherche Scientifique, Fondation pour la Recherche Medicale, and Association pour le Développement de la Recherche sur le Cancer. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume638, Issue1The Fibroblast Growth Factor FamilyDecember 1991Pages 127-138 RelatedInformation

Nucleolin (C23 or 100 kDa) is a major nucleolar phosphoprotein whose primary structure has recently been determined (Lapeyre, B., Bourbon, H., and Amalric, F. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1472-1476) and found to be associated with preribosomal RNA (Herrera, A. H., and Olson, M. O. J. (1986) Biochemistry 25, 6258-6263). To identify the RNA binding region of the molecule, cyanogen bromide fragments were tested for binding of 18 S and 28 S ribosomal RNA by a "Western blotting" technique. Fragments with apparent molecular masses of 13, 33, and 47 kDa bound RNA with no preference for either 18 S or 28 S RNA. By protein sequencing, these fragments were localized in the carboxyl-terminal two-thirds of the molecule. The nucleolin sequence was searched for the ribonucleoprotein consensus sequence found in other RNA binding proteins. Four copies of a closely related 11-residue sequence were found within 80-90 residue repeats in the RNA binding region between residues 285 and 629. These results suggest that a highly conserved structure for the binding of different classes of RNA is utilized by several proteins.

The synthesis of preribosomal RNA is inhibited "in vivo" and "in vitro" by the protease inhibitor leupeptin. "In vivo" leupeptin decreases by 74% the incorporation of labeled uridine into 45S pre rRNA while the synthesis of other RNA species is only slightly decreased. "In vitro", the elongation of already initiated pre rRNA chains that is achieved by incubation of isolated nucleoli is blocked by leupeptin. On the other hand, "in vitro" leupeptin has no direct effect on RNA polymerase I, tested in a nonspecific transcriptional system with Calf thymus DNA as template and in run off experiments with a cloned DNA containing the initiation site of the rDNA gene. A 100 kDa nucleolar protein which has been shown to be endoproteolytic cleaved "in vivo" (1) acts as an inhibitor of rDNA transcription in presence of leupeptin but produces little effect on the nonspecific transcription. In absence of the drug, the 100 kDa protein is processed in specific peptides which appeared to be similar to the "in vivo" maturation products. The possible role of the 100 kDa maturation process in the regulation of rDNA transcription is discussed.

A 100 kDa nucleolar protein which is transitorely associated with preribosomes in the nucleoli of Chinese hamster ovary cells has been found to be specifically cleaved by a thiol protease. During an ‘in vitro’ incubation of nucleoli, the 100 kDa protein is processed into eight different proteins which are detected by immunoreaction with a serum raised against the 100 kDa protein. qualitative and quantitative variations in the maturation products of the 100 kDa protein are obtained by ‘in vitro’ incubation of the 60S and 80S preribosomes. The 100 kDa protein has been purified to homogeneity with the protease activity still associated. The properties of the enzyme are described and its role in the maturation of preribosomes is discussed.

FEBS LettersVolume 155, Issue 2 p. 218-222 Full-length articleFree Access Role of phosphorylation on the maturation pathways of a 100 kDa nucleolar protein H. Bourbon, H. Bourbon Centre de Recherche de Biochimie et de Génétique Cellulaires du CNRS, 118, route de Narbonne, 31062 Toulouse Cédex, FranceSearch for more papers by this authorB. Bugler, B. Bugler Centre de Recherche de Biochimie et de Génétique Cellulaires du CNRS, 118, route de Narbonne, 31062 Toulouse Cédex, FranceSearch for more papers by this authorM. Caizergues-Ferrer, M. Caizergues-Ferrer Centre de Recherche de Biochimie et de Génétique Cellulaires du CNRS, 118, route de Narbonne, 31062 Toulouse Cédex, FranceSearch for more papers by this authorF. Amalric, F. Amalric Centre de Recherche de Biochimie et de Génétique Cellulaires du CNRS, 118, route de Narbonne, 31062 Toulouse Cédex, FranceSearch for more papers by this author H. Bourbon, H. Bourbon Centre de Recherche de Biochimie et de Génétique Cellulaires du CNRS, 118, route de Narbonne, 31062 Toulouse Cédex, FranceSearch for more papers by this authorB. Bugler, B. Bugler Centre de Recherche de Biochimie et de Génétique Cellulaires du CNRS, 118, route de Narbonne, 31062 Toulouse Cédex, FranceSearch for more papers by this authorM. Caizergues-Ferrer, M. Caizergues-Ferrer Centre de Recherche de Biochimie et de Génétique Cellulaires du CNRS, 118, route de Narbonne, 31062 Toulouse Cédex, FranceSearch for more papers by this authorF. Amalric, F. Amalric Centre de Recherche de Biochimie et de Génétique Cellulaires du CNRS, 118, route de Narbonne, 31062 Toulouse Cédex, FranceSearch for more papers by this author First published: May 08, 1983 https://doi.org/10.1016/0014-5793(82)80606-6Citations: 39AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL References 1 C.S. Rubin, O.M. Rosen, Annu. Rev. Biochem., 44, (1975), 831– 887. 2 E.G. Krebs, J.A. Beavo, Annu. Rev. Biochem., 48, (1979), 923– 959. 3 H. Holzer, P. Heinrich, Annu. Rev. Biochem., 49, (1980), 63– 91. 4 L.R. Gurley, R.A. Walters, S.S. Barham, L.L. Deaven, Exp. Cell Res., 111, (1978), 373– 383. 5 G.D. Kuehn, H.U. Affolter, V.J. Atmar, T. Seebeck, U. Gubler, R. Braun, Proc. Natl. Acad. Sci. USA, 76, (1979), 2541– 2545. 6 G. Thomas, M. Siegmann, J. Gordon, Proc. Natl. Acad. Sci. USA, 76, (1979), 3952– 3956. 7 D.H. Wolf, Trends Biochem. Sci., 1, (1982), 35– 37. 8 D.F. Steiner, J.L. Clark, C. Nolan, A.H. Rubenstein, E. Margoliuh, B. Aten, P.E. Oyer, Rec. Prog. Hormone Res., 25, (1969), 207– 282. 9 G. Blobel, B. Dobberstein, J. Cell Biol., 67, (1975), 835– 851. 10 G. Blobel, Proc. Natl. Acad. Sci. USA, 77, (1980), 1496– 1500. 11 A. Husilik, E.F. Neufeld, J. Biol. Chem., 255, (1980), 4937– 4945. 12 B. Bugler, M. Caizergues-Ferrer, G. Bouche, H. Bourbon, F. Amalric, Eur. J. Biochem., 128, (1982), 475– 480. 13 H. Bourbon, B. Bugler, M. Caizergues-Ferrer, F. Amalric, J.P. Zalta, Mol. Biol. Rep., (1983), in press 14 M. Caboche, J.P. Bachellerie, Eur. J. Biochem., 74, (1977), 19– 29. 15 M. Caizergues-Ferrer, G. Bouche, F. Amalric, J.P. Zalta, Eur. J. Biochem., 108, (1980), 399– 404. 16 J.P. Bachellerie, M. Nicoloso, J.P. Zalta, Eur. J. Biochem., 79, (1977), 23– 32. 17 J. Zalta, J.P. Zalta, R. Simard, J. Cell Biol., 51, (1971), 563– 568. 18 G. Bouche, F. Amalric, M. Caizergues-Ferrer, J.P. Zalta, Nucleic Acids Res., 7, (1979), 1739– 1747. 19 M.D. Mamrack, M.O. Olson, H. Busch, Biochemistry, 18, (1979), 3381– 3386. 20 M. Olson, K. Guetsow, Biochim. Biophys. Acta, 526, (1978), 174– 185. Citing Literature Volume155, Issue2May 08, 1983Pages 218-222 ReferencesRelatedInformation