The molecular mechanism underlying Roberts syndrome involves loss of ESCO2 acetyltransferase activity

Human Molecular Genetics, Jul 2008

Roberts syndrome/SC phocomelia (RBS) is an autosomal recessive disorder with growth retardation, craniofacial abnormalities and limb reduction. Cellular alterations in RBS include lack of cohesion at the heterochromatic regions around centromeres and the long arm of the Y chromosome, reduced growth capacity, and hypersensitivity to DNA damaging agents. RBS is caused by mutations in ESCO2, which encodes a protein belonging to the highly conserved Eco1/Ctf7 family of acetyltransferases that is involved in regulating sister chromatid cohesion. We identified 10 new mutations expanding the number to 26 known ESCO2 mutations. We observed that these mutations result in complete or partial loss of the acetyltransferase domain except for the only missense mutation that occurs in this domain (c.1615T>G, W539G). To investigate the mechanism underlying RBS, we analyzed ESCO2 mutations for their effect on enzymatic activity and cellular phenotype. We found that ESCO2 W539G results in loss of autoacetyltransferase activity. The cellular phenotype produced by this mutation causes cohesion defects, proliferation capacity reduction and mitomycin C sensitivity equivalent to those produced by frameshift and nonsense mutations associated with decreased levels of mRNA and absence of protein. We found decreased proliferation capacity in RBS cell lines associated with cell death, but not with increased cell cycle duration, which could be a factor in the development of phocomelia and cleft palate in RBS. In summary, we provide the first evidence that loss of acetyltransferase activity contributes to the pathogenesis of RBS, underscoring the essential role of the enzymatic activity of the Eco1p family of proteins.

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The molecular mechanism underlying Roberts syndrome involves loss of ESCO2 acetyltransferase activity

Miriam Gordillo Hugo Vega Alison H. Trainer Fajian Hou Norio Sakai Ricardo Luque Hu lya Kayserili Seher Basaran Flemming Skovby Raoul C. M. Hennekam Maria L. Giovannucci Uzielli Rhonda E. Schnur Sylvie Manouvrier Susan Chang Edward Blair Jane A. Hurst Francesca Forzano Moritz Meins Kalle O.J. Simola Annick Raas-Rothschild Roger A. Schultz Lisa D. McDaniel Keiichi Ozono Koji Inui Hui Zou Ethylin Wang Jabs FEimrsatiAl: Roberts syndrome/SC phocomelia (RBS) is an autosomal recessive disorder with growth retardation, craniofacial abnormalities and limb reduction. Cellular alterations in RBS include lack of cohesion at the heterochromatic regions around centromeres and the long arm of the Y chromosome, reduced growth capacity, and hypersensitivity to DNA damaging agents. RBS is caused by mutations in ESCO2, which encodes a protein belonging to the highly conserved Eco1/Ctf7 family of acetyltransferases that is involved in regulating sister chromatid cohesion. We identified 10 new mutations expanding the number to 26 known ESCO2 mutations. We observed that these mutations result in complete or partial loss of the acetyltransferase domain except for the only missense mutation that occurs in this domain (c.1615T>G, W539G). To investigate the mechanism underlying RBS, we analyzed ESCO2 mutations for their effect on enzymatic activity and cellular phenotype. We found that ESCO2 W539G results in loss of autoacetyltransferase activity. The cellular # The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: - INTRODUCTION Roberts syndrome (RBS, MIM #268300) is an autosomal recessive developmental disorder described initially by John Roberts in affected siblings from a consanguineous Italian couple (1). Later, in four individuals from two families of European descent, Herrmann et al. (2) reported similar, but milder malformations which were referred to as SC phocomelia (MIM #269000). These two conditions are considered the same syndrome with varying phenotypic expression and hereafter, will be referred to as RBS (3). This syndrome is rare with approximately 100 cases described in the literature. Typical clinical features of RBS are pre-natal and post-natal growth retardation, bilateral symmetric limb reduction and craniofacial abnormalities (3,4). Survival is poor beyond the neonatal period. Of interest, four RBS patients have been reported with neoplastic processes including one malignant melanoma, one rhabdomyosarcoma and two oculomotor nerve cavernous angiomas (5 8). At the cytogenetic level, RBS chromosomes present with a rod-like morphology resulting in a railroad-track appearance due to the absence of the primary constriction at the centromeric regions and with a puffing or repulsion localized at their heterochromatin especially of chromosomes 1, 9 and 16, the acrocentrics, and the distal segment of the long arm of the Y chromosome (9 12). This phenomenon known as premature centromere separation (PCS) or heterochromatin repulsion (HR) constitutes the major diagnostic marker for RBS. Associated with PCS/HR is aneuploidy most likely due to outlying, lagging or prematurely advancing chromosomes during mitosis (11,13). RBS cells also exhibit hypersensitivity to clastogens such as mitomycin C (MMC), cisplatin, and gamma and ultraviolet radiation (14,15). In RBS fibroblasts, abnormalities in cell division include reduced cell growth, prolonged metaphase duration, abnormal cytokinesis and failure of cells to divide in mitosis (16). Cohesion of sister chromatids is essential for accurate chromosome segregation and genomic stability. Cohesion is mediated by a multimeric complex known as cohesin (17,18). Cohesin is loaded onto chromatin from telophase (in mammalian cells and fission yeast) or G1 (in budding yeast) until the next mitosis (18 20). Binding of cohesin to chromatin is not sufficient for functional cohesion. In Saccharomyces cerevisiae, cohesion is established during S phase in a process dependent on Eco1/Ctf7, the founding member of a family of highly conserved acetyltransferases (21 23). Cohesion can also be induced by DNA damage in a process that is also mediated by Eco1/Ctf7 (24,25). The causative gene for RBS is Establishment of Cohesion 1 Homolog 2 (ESCO2) with 16 different mutations reported to date (26 29). ESCO2 codes for a 601 amino acid protein belonging to the Eco1/Ctf7 family. Characteristic of members of this family, including S. cerevisiae Eco1/Ctf7, human ESCO1 (a second human homolog of Eco1/Ctf7) and ESCO2 proteins, are C2H2 zinc finger and acetyltransferase domains at the C-terminus. In some of the members of the family, this conserved C-terminal domain is fused to an N-terminal extension that is neither conserved among species nor in paralogs in the same organism (21 23,26,30 33). The ESCO2 N-terminal extension has no similarity to any other known proteins to date. Eco1/Ctf7, ESCO1 and ESCO2 exhibit in vitro autoacetyltransferase activity and it has been proposed that establishment of cohesion might be regulated directly or indirectly by the acetylation activity of these proteins (23,32,33). Based on studies of viability and fidelity of chromosome transmission in some Eco1/Ctf7 alleles defective in acetyltransferase activity in yeast, it was suggested that this enzymatic activity might not be relevant to the essential function of the protein (34). However, recent evidence indicates that reduction of Eco1/Ctf7 in vitro enzymatic activity is correlated with defects in cohesion establishment and DNA damage-induced cohesion (24,25,35). The relevance of the enzymatic activity in vertebrates is unknown. Here, analysis of ESCO2 in RBS pedigrees revealed 10 novel mutations. Most ESCO2 mutations are predicted to cause premature stop codons that may result in truncated proteins or mRNA instability due to nonsense-mediated mRNA decay (NMD). We demonstrated protein loss for three different frameshift mutations and one nonsense mutation. In contrast, the missense mutation ESCO2 W539G did not affect mRNA or protein instability, but resulted in the disruption of the in vitro autoacetyltransferase activity of the protein. Furthermore, we found that the cytogenetic and cellular phenotypes of cells with the missense mutation were indistinguishable from the phenotypes associated with nonsense and frameshift mutations. Taken together, our results indicate the importance of ESCO2 acetyltransferase activity in human cells and strongly suggest that loss of this activity is implicated in the pathogenesis of RBS. ESCO2 mutations Mutation analysis of the DNA sequence of the 11 exons of ESCO2 in 16 pedigrees with 17 individuals who were clinically and cytogenetically diagnosed with RBS revealed 15 c.294_297delGAGA c.1132-7A.Gc,f c.308_309delAA c.760delAc,f c.760_761insAc,f c.760_761insAc,f c.1132-7A.Gc,f c.764_765delTT c.875_878delACAG c.879_880delAGd,e,f c.879_880delAGd,e,f c.879_880delAGd,e,f c.879_880delAGd,e,f c.955 (...truncated)


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Miriam Gordillo, Hugo Vega, Alison H. Trainer, Fajian Hou, Norio Sakai, Ricardo Luque, Hülya Kayserili, Seher Basaran, Flemming Skovby, Raoul C. M. Hennekam, Maria L. Giovannucci Uzielli, Rhonda E. Schnur, Sylvie Manouvrier, Susan Chang, Edward Blair, Jane A. Hurst, Francesca Forzano, Moritz Meins, Kalle O.J. Simola, Annick Raas-Rothschild, Roger A. Schultz, Lisa D. McDaniel, Keiichi Ozono, Koji Inui, Hui Zou, Ethylin Wang Jabs. The molecular mechanism underlying Roberts syndrome involves loss of ESCO2 acetyltransferase activity, Human Molecular Genetics, 2008, pp. 2172-2180, 17/14, DOI: 10.1093/hmg/ddn116