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.

The ability of DNA Double Strand Breaks (DSBs) to cluster in mammalian cells has been subjected to intense debate over the past few years. Here we used a high throughput chromosome conformation capture assay (Capture Hi-C) to investigate clustering of DSBs induced at defined loci in the human genome. We unambiguously found that DSBs do cluster but only when induced in transcriptionally active genes. Clustering of damaged genes mainly occurs during the G1 cell cycle phase and coincides with delayed repair. Moreover DSB clustering depends on the MRN complex, as well as the Formin 2 (FMN2) nuclear actin organizer and the LINC (LInker of Nuclear and Cytoplasmic skeleton) complex, suggesting that active mechanisms promote DSB clustering. This work reveals that when damaged, active genes exhibit a very peculiar behavior compared to the rest of the genome, being mostly left unrepaired and clustered in G1 while being repaired by homologous recombination in post-replicative cells.

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 AUGinitiated 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.

Chromatin undergoes major remodeling around DNA double-strand breaks (DSB) to promote repair and DNA damage response (DDR) activation. We recently reported a high-resolution map of γH2AX around multiple breaks on the human genome, using a new cell-based DSB inducible system. In an attempt to further characterize the chromatin landscape induced around DSBs, we now report the profile of SMC3, a subunit of the cohesin complex, previously characterized as required for repair by homologous recombination. We found that recruitment of cohesin is moderate and restricted to the immediate vicinity of DSBs in human cells. In addition, we show that cohesin controls γH2AX distribution within domains. Indeed, as we reported previously for transcription, cohesin binding antagonizes γH2AX spreading. Remarkably, depletion of cohesin leads to an increase of γH2AX at cohesin-bound genes, associated with a decrease in their expression level after DSB induction. We propose that, in agreement with their function in chromosome architecture, cohesin could also help to isolate active genes from some chromatin remodelling and modifications such as the ones that occur when a DSB is detected on the genome.

SummaryDNA double-strand breaks (DSBs) elicit the so-called DNA damage response (DDR), largely relying on ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PKcs), two members of the PI3K-like kinase family, whose respective functions during the sequential steps of the DDR remains controversial. Using the DIvA system (DSB inducible via AsiSI) combined with high-resolution mapping and advanced microscopy, we uncovered that both ATM and DNA-PKcs spread in cis on a confined region surrounding DSBs, independently of the pathway used for repair. However, once recruited, these kinases exhibit non-overlapping functions on end joining and γH2AX domain establishment. More specifically, we found that ATM is required to ensure the association of multiple DSBs within “repair foci.” Our results suggest that ATM acts not only on chromatin marks but also on higher-order chromatin organization to ensure repair accuracy and survival.

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. INTRODUCTIONSeveral factors have been proposed to play a role in the regulation of pre rRNA synthesis (2). In "in vivo" experiments, correLations have been estabLished between pre rRNA synthesis and the evolution of given parameters such as: aminoacid pool size (3, 4), nucleotide pool size (5), turn over of labile stimulatory protein (6) in particular ornithine decarboxylase (7), and phosphorylation of specific proteins (6). The recent development of "in vitro" systems in which a defined rDNA fragment containing the "in vivo" start site can be transcribed by RNA polymerase I, should allow the characterization of factors involved in the regulation of pre rRNA synthesis (8, 9).Such regulatory proteins must be localized in the nucleolus, a large subnuclear structure which contains most of the actively transcribed ribosomal genes in exponentially growing eukaryotic cells (10). The nascent transcripts are immediately associated with ribosomal proteins (11) and

A high‐molecular‐mass nucleolar protein (100‐kDa protein) is associated with nascent pre‐rRNA and preribosomes in Chinese hamster ovary cells. We have prepared antiserum against the 100‐kDa protein and we have used it to study the intracellular localization and the possible processing of this protein. Serologically related proteins were detected in the nucleolus and in ribosomes. Proteins of various subcellular fractions were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, transferred to nitrocellulose filters, reacted with serum and the protein‐immunoglobulin complexes were revealed by 125I‐labeled protein A. In the nucleolus, four proteins with molecular masses of 100 kDa, 95 kDa, 76 kDa and 70 kDa were thus visualized. In the ribosomes, two proteins (of 100 kDa and 76 kDa) gave a strong reaction while six others (of 70 kDa, 60 kDa, 50 kDa, 30 kDa, 21 kDa, 18 kDa) reacted slightly. By immunological precipitation of total cell extracts, we have shown that the 100‐kDa protein antiserum cross‐reacts with five proteins with molecular masses of 120 kDa, 100 kDa, 95 kDa, 70 kDa and 60 kDa. Specific degradation of the 100‐kDa protein into similar peptides with molecular masses of 95 kDa, 76 kDa, 70 kDa, 60 kDa and 50 kDa can be achieved by incubation of isolated nucleoli or of purified 100‐kDa protein in vitro. Cleavage of the protein is due to a thiol endoprotease which is tightly bound to the 100‐kDa protein. Possible relations between the maturation of this preribosomal protein into ribosomal proteins and the processing of preribosomal RNA into mature ribosomal RNA are discussed.

Abstract-Both 17␤-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 ϩ/ϩ mice, but not in Fgf2 Ϫ/Ϫ mice. Cell cultures from subcutaneous Matrigel plugs demonstrated that E 2 increased both migration and proliferation in endothelial cells from Fgf2 ϩ/ϩ mice, but not from in Fgf2 Ϫ/Ϫ 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 lmwϪ/Ϫ ). E 2 stimulated in vivo angiogenesis and in vitro migration in endothelial cells from Fgf2 lmwϪ/Ϫ as it did in Fgf2 ϩ/ϩ mice. E 2 increased FGF2 hmw protein abundance in endothelial cell cultures from Fgf2 ϩ/ϩ and Fgf2 lmwϪ/Ϫ 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. Key Words: mouse Ⅲ estradiol Ⅲ growth factor Ⅲ endothelium Ⅲ migration E ndothelium, being uniquely positioned at the interface between the blood and the vessel wall, plays a crucial role in the physiology of circulation by performing multiple functions. 1,2 It is involved in the regulation of coagulation, leukocyte adhesion in inflammation, transvascular flux of cells, liquids, and solutes, vessel tone, and vascular smooth muscle growth. Endothelium also constitutes a target for the sex hormone, 17␤-estradiol (E 2 ). Using a series of experimental models, E 2 has been reported to promote angiogenic activity and endothelial cell migration and proliferation. 3 The angiogenic effect is mediated through the estrogen receptor ␣ (ER␣). 4 However, the mechanisms involved downstream of ER␣ remain unclear. 5 Fibroblast growth factor-2 (FGF2) is an important mitogenic and angiogenic factor that stimulates endothelial cell growth and migration. Therefore, FGF2 could be a good candidate to be a partner of E 2 . However, FGF2 expression is complex because at least four (18, 22, 22.5, and 24 kDa) in human and three (18, 21, and 22 kDa) FGF2 isoforms in mouse are synthesized through the alternative use of translation initiation codons. 6 -9 These isoforms differ only in their NH 2 extremities, which confer a nuclear localization to the high molecular weight CUG-initiated (22, 22.5, 24 or 21, and 22 kDa) isoforms (FGF2 hmw ), whose function is mainly unknown. In contrast, the smaller 18-kDa A...