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Histone acetylation plays essential roles in cell cycle progression, DNA repair, gene expression and silencing. Although the knowledge regarding the roles of acetylation of histone lysine residues is rapidly growing, very little is known about the biochemical pathways providing the nucleus with metabolites necessary for physiological chromatin acetylation. Here, we show that mutations in the scheggia (sea)-encoded Sea protein, the Drosophila ortholog of the human mitochondrial citrate carrier Solute carrier 25 A1 (SLC25A1), impair citrate transport from mitochondria to the cytosol. Interestingly, inhibition of sea expression results in extensive chromosome breakage in mitotic cells and induces an ATR-dependent cell cycle arrest associated with a dramatic reduction of global histone acetylation. Notably, loss of SLC25A1 in short interfering RNA (siRNA)-treated human primary fibroblasts also leads to chromosome breaks and histone acetylation defects, suggesting an evolutionary conserved role for Sea/SLC25A1 in the regulation of chromosome integrity. This study therefore provides an intriguing and unexpected link between intermediary metabolism and epigenetic control of genome stability.

Cornelia de Lange syndrome (CdLS) is a clinically heterogeneous developmental disorder characterized by facial dysmorphia, upper limb malformations, growth and cognitive retardation. Mutations in the sister chromatid cohesion factor genes NIPBL, SMC1A and SMC3 are present in approximately 65% of CdLS patients. In addition to their canonical roles in chromosome segregation, the cohesin proteins are involved in other biological processes such as regulation of gene expression, DNA repair and maintenance of genome stability. To gain insights into the molecular basis of CdLS, we analyzed the affinity of mutated SMC1A and SMC3 hinge domains for DNA. Mutated hinge dimers bind DNA with higher affinity than wild-type proteins. SMC1A- and SMC3-mutated CdLS cell lines display genomic instability and sensitivity to ionizing radiation and interstrand crosslinking agents. We propose that SMC1A and SMC3 CdLS mutations affect the dynamic association between SMC proteins and DNA, providing new clues to the underlying molecular cause of CdLS.

Mutations in the cohesin regulators NIPBL and ESCO2 are causative of the Cornelia de Lange syndrome (CdLS) and Roberts or SC phocomelia syndrome, respectively. Recently, mutations in the cohesin complex structural component SMC1A have been identified in two probands with features of CdLS. Here, we report the identification of a mutation in the gene encoding the complementary subunit of the cohesin heterodimer, SMC3, and 14 additional SMC1A mutations. All mutations are predicted to retain an open reading frame, and no truncating mutations were identified. Structural analysis of the mutant SMC3 and SMC1A proteins indicate that all are likely to produce functional cohesin complexes, but we posit that they may alter their chromosome binding dynamics. Our data indicate that SMC3 and SMC1A mutations (1) contribute to approximately 5% of cases of CdLS, (2) result in a consistently mild phenotype with absence of major structural anomalies typically associated with CdLS, and (3) in some instances, result in a phenotype that approaches that of apparently nonsyndromic mental retardation.

Cellular processes involved in fragile site expression have been investigated by studying the effect on the replication pattern of the commonest fragile site FRA3B of RNA interference (RNAi)-mediated sister maintenance chromosome 1 (SMC1) inhibition in normal human fibroblasts. Replication timing of FRA3B in G2 was studied by bromodeoxyuridine (BrdU) labeling for the final 2h of cell culture whereas in the S phase was investigated by a fluorescence in situ hybridization (FISH)-based approach through the analysis of clones spanning the FRA3B region. Results showed that FRA3B is normally late replicated even though it is not expressed in untreated cells. On the other hand, SMC1 inhibition leads to FRA3B expression even if the percent of late replicated cells is comparable to control cells. These results obtained by analysing the commonest fragile site suggest that SMC1 plays a role in protecting late replicating regions from stresses occurring in the final steps of genome replication and that delayed replication is necessary but not sufficient for inducing fragile site expression.

Common fragile sites have been involved in neoplastic transformation, although their molecular basis is still poorly understood. Here, we demonstrate that inhibition of the SMC1 by RNAi is sufficient to induce fragile site expression. By investigating normal, ATM- and ATR-deficient cell lines, we provide evidence that the contribution of SMC1 in preventing the collapse of stalled replication fork is an Atr-dependent pathway. Using a fluorescent antibody specific for gamma-H2AX, we show that very rare discrete nuclear foci appear 1 and 2 h after exposure to aphidicolin and/or RNAi-SMC1, but became more numerous and distinct after longer treatment times. In this context, fragile sites might be viewed as an in vitro phenomenon originating from double-strand breaks formed because of a stalled DNA replication that lasted too long to be managed by physiological rescue acting through the Atr/Smc1 axis. We propose that in vivo, following an extreme replication block, rare cells could escape checkpoint mechanisms and enter mitosis with a defect in genome assembly, eventually leading to neoplastic transformation.

The relative contribution of aneuploidy and gene mutations to human tumorigenesis is not yet known. Studies in mice have demonstrated that even single point mutations in oncogenes and tumor suppressor genes can dramatically increase tumor frequency. However, models to evaluate the definitive role of aneuploidy and genomic instability are not yet available. Human fibroblast cells have long been used as a tool for investigating proliferation, senescence, immortalization, and tumorigenesis, all processes that are strongly interrelated. We have now used antisense and ribozyme-mediated temporary inhibition of BUB1 to study the consequences of mitotic checkpoint failure on the development of aneuploidy. The analysis of cell colonies selected by soft agar growth showed evidence of chromosome instability and delayed senescence, without being tumorigenic in nude mice. Our data suggest that chromosomal instability and aneuploidy are early changes that precede tumorigenicity in the multistep process leading to neoplastic transformation.