Cornelia-de Lange syndrome-associated mutations cause a DNA damage signalling and repair defect

Cornelia de Lange syndrome is a multisystem developmental disorder typically caused by mutations in the gene encoding the cohesin loader NIPBL. The associated phenotype is generally assumed to be the consequence of aberrant transcriptional regulation. Recently, we identified a missense mutation in BRD4 associated with a Cornelia de Lange-like syndrome that reduces BRD4 binding to acetylated histones. Here we show that, although this mutation reduces BRD4-occupancy at enhancers it does not affect transcription of the pluripotency network in mouse embryonic stem cells. Rather, it delays the cell cycle, increases DNA damage signalling, and perturbs regulation of DNA repair in mutant cells. This uncovers a role for BRD4 in DNA repair pathway choice. Furthermore, we find evidence of a similar increase in DNA damage signalling in cells derived from NIPBL-deficient individuals, suggesting that defective DNA damage signalling and repair is also a feature of typical Cornelia de Lange syndrome

The Cellular Phenotype of Roberts Syndrome Fibroblasts as Revealed by Ectopic Expression of ESCO2

Cohesion between sister chromatids is essential for faithful chromosome segregation. In budding yeast, the acetyltransferase Eco1/Ctf7 establishes cohesion during DNA replication in S phase and in response to DNA double strand breaks in G2/M phase. In humans two Eco1 orthologs exist: ESCO1 and ESCO2. Both proteins are required for proper sister chromatid cohesion, but their exact function is unclear at present. Since ESCO2 has been identified as the gene defective in the rare autosomal recessive cohesinopathy Roberts syndrome (RBS), cells from RBS patients can be used to elucidate the role of ESCO2. We investigated for the first time RBS cells in comparison to isogenic controls that stably express V5- or GFP-tagged ESCO2. We show that the sister chromatid cohesion defect in the transfected cell lines is rescued and suggest that ESCO2 is regulated by proteasomal degradation in a cell cycle-dependent manner. In comparison to the corrected cells RBS cells were hypersensitive to the DNA-damaging agents mitomycin C, camptothecin and etoposide, while no particular sensitivity to UV, ionizing radiation, hydroxyurea or aphidicolin was found. The cohesion defect of RBS cells and their hypersensitivity to DNA-damaging agents were not corrected by a patient-derived ESCO2 acetyltransferase mutant (W539G), indicating that the acetyltransferase activity of ESCO2 is essential for its function. In contrast to a previous study on cells from patients with Cornelia de Lange syndrome, another cohesinopathy, RBS cells failed to exhibit excessive chromosome aberrations after irradiation in G2 phase of the cell cycle. Our results point at an S phase-specific role for ESCO2 in the maintenance of genome stability

Multiple Organ System Defects and Transcriptional Dysregulation in the Nipbl+/− Mouse, a Model of Cornelia de Lange Syndrome

Cornelia de Lange Syndrome (CdLS) is a multi-organ system birth defects disorder linked, in at least half of cases, to heterozygous mutations in the NIPBL gene. In animals and fungi, orthologs of NIPBL regulate cohesin, a complex of proteins that is essential for chromosome cohesion and is also implicated in DNA repair and transcriptional regulation. Mice heterozygous for a gene-trap mutation in Nipbl were produced and exhibited defects characteristic of CdLS, including small size, craniofacial anomalies, microbrachycephaly, heart defects, hearing abnormalities, delayed bone maturation, reduced body fat, behavioral disturbances, and high mortality (75–80%) during the first weeks of life. These phenotypes arose despite a decrease in Nipbl transcript levels of only ∼30%, implying extreme sensitivity of development to small changes in Nipbl activity. Gene expression profiling demonstrated that Nipbl deficiency leads to modest but significant transcriptional dysregulation of many genes. Expression changes at the protocadherin beta (Pcdhb) locus, as well as at other loci, support the view that NIPBL influences long-range chromosomal regulatory interactions. In addition, evidence is presented that reduced expression of genes involved in adipogenic differentiation may underlie the low amounts of body fat observed both in Nipbl+/− mice and in individuals with CdLS

Highly mutagenic exocyclic DNA adducts are substrates for the human nucleotide incision repair pathway

Background: Oxygen free radicals induce lipid peroxidation (LPO) that damages and breaks polyunsaturated fatty acids in cell membranes. LPO-derived aldehydes and hydroxyalkenals react with DNA leading to formation of etheno(ε)-bases including 1,N6-ethenoadenine (εA) and 3,N4-ethenocytosine (εC). The εA and εC residues are highly mutagenic in mammalian cells and eliminated in the base excision repair (BER) pathway and/or by AlkB family proteins in the direct damage reversal process. BER initiated by DNA glycosylases is thought to be the major pathway for the removal of non-bulky endogenous base damage. Alternatively, in the nucleotide incision repair (NIR) pathway, the apurinic/apyrimidinic (AP) endonucleases can directly incise DNA duplex 5’ to a damaged base in a DNA glycosylase-independent manner. Methodology/Principal Findings: Here, we characterized the substrate specificity of human major AP endonuclease 1, APE1, towards εA, εC, thymine glycol (Tg) and 7,8-dihydro-8-oxoguanine (8oxoG) residues when present in duplex DNA. APE1 cleaves oligonucleotide duplexes containing εA, εC and Tg, but not those containing 8oxoG. The activity depends strongly on sequence context. The apparent kinetic parameters of the reactions suggest that APE1 has high affinity to DNA containing ε-bases but cleaves DNA duplex at an extremely slow rate. Consistent with this observation, the oligonucleotide duplexes containing an ε-base strongly inhibit AP site nicking activity of APE1 with IC50 values in the range of 5-10 nM. MALDI-TOF MS analysis of the reaction products demonstrated that APE1-catalyzed cleavage of εA•T and εC•G duplexes generates as expected DNA fragments containing 5’-terminal ε-base residue. Conclusions/Significance: The fact that ε-bases and Tg in duplex DNA are recognized and cleaved by APE1 in vitro, suggest that NIR may act as a backup pathway to BER one to remove a large variety of genotoxic base lesions in human cells

Intragenic and large NIPBL rearrangements revealed by MLPA in Cornelia de Lange patients

Cornelia de Lange syndrome (CdLS) is a rare multisystemic congenital anomaly disorder that is characterised by intellectual disability and growth retardation, congenital heart defects, intestinal anomalies, facial dysmorphism (including synophyris and high arched eyebrows) and limb reduction defects. Mutations in three cohesin-associated genes encoding a key regulator (NIPBL, chr 5p13.2) and one structural component of the cohesin ring (SMC1A, chr Xp11) occur in about 65% of CdLS patients. NIPBL is the major causative gene, and accounts for 40-60% of CdLS patients as shown by a number of mutational screening studies that indicate a wide mutational repertoire of mainly small deletions and point mutations. Only a few data are available concerning the occurrence of large NIPBL rearrangements or intragenic deletions or duplications involving whole exons. We used multiplex ligation-dependent probe amplification (MLPA) to study 132 CdLS patients negative to the standard mutation NIPBL test out of a cohort of 200 CdLS patients. A total of 7 out of 132 patients were found to carry NIPBL alterations, including two large gene deletions extending beyond the gene, four intragenic multi- or single-exon deletions and one single-exon duplication. These findings show that MLPA leads to a 5.3% increase in the detection of mutations when used in addition to the standard NIPBL scan, and contributes per se to the molecular diagnosis of 3.5% (7/200) of clinically diagnosed CdLS patients. It is recommended that MLPA be included in the CdLS diagnostic flow chart.European Journal of Human Genetics advance online publication, 22 February 2012; doi:10.1038/ejhg.2012.7