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Loss of CENP-F results in distinct microtubule-related defects without chromosomal abnormalities.
Pfaltzgraff ER, Roth GM, Miller PM, Gintzig AG, Ohi R, Bader DM
(2016) Mol Biol Cell 27: 1990-9
MeSH Terms: Animals, Cell Cycle, Centromere, Chromosomal Proteins, Non-Histone, Chromosome Aberrations, Chromosome Segregation, Fibroblasts, Interphase, Kinetochores, Mice, Mice, Knockout, Microfilament Proteins, Microtubules, Mitosis, Protein Binding
Show Abstract · Added March 29, 2017
Microtubule (MT)-binding centromere protein F (CENP-F) was previously shown to play a role exclusively in chromosome segregation during cellular division. Many cell models of CENP-F depletion show a lag in the cell cycle and aneuploidy. Here, using our novel genetic deletion model, we show that CENP-F also regulates a broader range of cellular functions outside of cell division. We characterized CENP-F(+/+) and CENP-F(-/-) mouse embryonic fibroblasts (MEFs) and found drastic differences in multiple cellular functions during interphase, including cell migration, focal adhesion dynamics, and primary cilia formation. We discovered that CENP-F(-/-) MEFs have severely diminished MT dynamics, which underlies the phenotypes we describe. These data, combined with recent biochemical research demonstrating the strong binding of CENP-F to the MT network, support the conclusion that CENP-F is a powerful regulator of MT dynamics during interphase and affects heterogeneous cell functions.
© 2016 Pfaltzgraff et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
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15 MeSH Terms
The SUMO deconjugating peptidase Smt4 contributes to the mechanism required for transition from sister chromatid arm cohesion to sister chromatid pericentromere separation.
Stephens AD, Snider CE, Bloom K
(2015) Cell Cycle 14: 2206-18
MeSH Terms: Cell Cycle Proteins, Centromere, Chromatids, Chromosomal Proteins, Non-Histone, Chromosome Segregation, DNA Topoisomerases, Type II, Endopeptidases, Lac Operon, Mitosis, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Spindle Apparatus, Sumoylation
Show Abstract · Added January 25, 2016
The pericentromere chromatin protrudes orthogonally from the sister-sister chromosome arm axis. Pericentric protrusions are organized in a series of loops with the centromere at the apex, maximizing its ability to interact with stochastically growing and shortening kinetochore microtubules. Each pericentromere loop is ∼50 kb in size and is organized further into secondary loops that are displaced from the primary spindle axis. Cohesin and condensin are integral to mechanisms of loop formation and generating resistance to outward forces from kinesin motors and anti-parallel spindle microtubules. A major unanswered question is how the boundary between chromosome arms and the pericentromere is established and maintained. We used sister chromatid separation and dynamics of LacO arrays distal to the pericentromere to address this issue. Perturbation of chromatin spring components results in 2 distinct phenotypes. In cohesin and condensin mutants sister pericentric LacO arrays separate a defined distance independent of spindle length. In the absence of Smt4, a peptidase that removes SUMO modifications from proteins, pericentric LacO arrays separate in proportion to spindle length increase. Deletion of Smt4, unlike depletion of cohesin and condensin, causes stretching of both proximal and distal pericentromere LacO arrays. The data suggest that the sumoylation state of chromatin topology adjusters, including cohesin, condensin, and topoisomerase II in the pericentromere, contribute to chromatin spring properties as well as the sister cohesion boundary.
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13 MeSH Terms
Inherited variants in the inner centromere protein (INCENP) gene of the chromosomal passenger complex contribute to the susceptibility of ER-negative breast cancer.
Kabisch M, Lorenzo Bermejo J, Dünnebier T, Ying S, Michailidou K, Bolla MK, Wang Q, Dennis J, Shah M, Perkins BJ, Czene K, Darabi H, Eriksson M, Bojesen SE, Nordestgaard BG, Nielsen SF, Flyger H, Lambrechts D, Neven P, Peeters S, Weltens C, Couch FJ, Olson JE, Wang X, Purrington K, Chang-Claude J, Rudolph A, Seibold P, Flesch-Janys D, Peto J, dos-Santos-Silva I, Johnson N, Fletcher O, Nevanlinna H, Muranen TA, Aittomäki K, Blomqvist C, Schmidt MK, Broeks A, Cornelissen S, Hogervorst FB, Li J, Brand JS, Humphreys K, Guénel P, Truong T, Menegaux F, Sanchez M, Burwinkel B, Marmé F, Yang R, Bugert P, González-Neira A, Benitez J, Pilar Zamora M, Arias Perez JI, Cox A, Cross SS, Reed MW, Andrulis IL, Knight JA, Glendon G, Tchatchou S, Sawyer EJ, Tomlinson I, Kerin MJ, Miller N, kConFab Investigators, Australian Ovarian Cancer Study Group, Haiman CA, Schumacher F, Henderson BE, Le Marchand L, Lindblom A, Margolin S, Hooning MJ, Hollestelle A, Kriege M, Koppert LB, Hopper JL, Southey MC, Tsimiklis H, Apicella C, Slettedahl S, Toland AE, Vachon C, Yannoukakos D, Giles GG, Milne RL, McLean C, Fasching PA, Ruebner M, Ekici AB, Beckmann MW, Brenner H, Dieffenbach AK, Arndt V, Stegmaier C, Ashworth A, Orr N, Schoemaker MJ, Swerdlow A, García-Closas M, Figueroa J, Chanock SJ, Lissowska J, Goldberg MS, Labrèche F, Dumont M, Winqvist R, Pylkäs K, Jukkola-Vuorinen A, Grip M, Brauch H, Brüning T, Ko YD, GENICA Network, Radice P, Peterlongo P, Scuvera G, Fortuzzi S, Bogdanova N, Dörk T, Mannermaa A, Kataja V, Kosma VM, Hartikainen JM, Devilee P, Tollenaar RA, Seynaeve C, Van Asperen CJ, Jakubowska A, Lubinski J, Jaworska-Bieniek K, Durda K, Zheng W, Shrubsole MJ, Cai Q, Torres D, Anton-Culver H, Kristensen V, Bacot F, Tessier DC, Vincent D, Luccarini C, Baynes C, Ahmed S, Maranian M, Simard J, Chenevix-Trench G, Hall P, Pharoah PD, Dunning AM, Easton DF, Hamann U
(2015) Carcinogenesis 36: 256-71
MeSH Terms: 3' Untranslated Regions, Aurora Kinase B, Breast Neoplasms, Case-Control Studies, Cell Cycle Proteins, Chromosomal Proteins, Non-Histone, European Continental Ancestry Group, Female, Genetic Predisposition to Disease, Genome-Wide Association Study, Humans, Inhibitor of Apoptosis Proteins, Polymorphism, Single Nucleotide, Receptors, Estrogen, Risk, Survivin
Show Abstract · Added September 28, 2015
The chromosomal passenger complex (CPC) plays a pivotal role in the regulation of cell division. Therefore, inherited CPC variability could influence tumor development. The present candidate gene approach investigates the relationship between single nucleotide polymorphisms (SNPs) in genes encoding key CPC components and breast cancer risk. Fifteen SNPs in four CPC genes (INCENP, AURKB, BIRC5 and CDCA8) were genotyped in 88 911 European women from 39 case-control studies of the Breast Cancer Association Consortium. Possible associations were investigated in fixed-effects meta-analyses. The synonymous SNP rs1675126 in exon 7 of INCENP was associated with overall breast cancer risk [per A allele odds ratio (OR) 0.95, 95% confidence interval (CI) 0.92-0.98, P = 0.007] and particularly with estrogen receptor (ER)-negative breast tumors (per A allele OR 0.89, 95% CI 0.83-0.95, P = 0.0005). SNPs not directly genotyped were imputed based on 1000 Genomes. The SNPs rs1047739 in the 3' untranslated region and rs144045115 downstream of INCENP showed the strongest association signals for overall (per T allele OR 1.03, 95% CI 1.00-1.06, P = 0.0009) and ER-negative breast cancer risk (per A allele OR 1.06, 95% CI 1.02-1.10, P = 0.0002). Two genotyped SNPs in BIRC5 were associated with familial breast cancer risk (top SNP rs2071214: per G allele OR 1.12, 95% CI 1.04-1.21, P = 0.002). The data suggest that INCENP in the CPC pathway contributes to ER-negative breast cancer susceptibility in the European population. In spite of a modest contribution of CPC-inherited variants to the total burden of sporadic and familial breast cancer, their potential as novel targets for breast cancer treatment should be further investigated.
© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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16 MeSH Terms
The kinetochore protein, CENPF, is mutated in human ciliopathy and microcephaly phenotypes.
Waters AM, Asfahani R, Carroll P, Bicknell L, Lescai F, Bright A, Chanudet E, Brooks A, Christou-Savina S, Osman G, Walsh P, Bacchelli C, Chapgier A, Vernay B, Bader DM, Deshpande C, O' Sullivan M, Ocaka L, Stanescu H, Stewart HS, Hildebrandt F, Otto E, Johnson CA, Szymanska K, Katsanis N, Davis E, Kleta R, Hubank M, Doxsey S, Jackson A, Stupka E, Winey M, Beales PL
(2015) J Med Genet 52: 147-56
MeSH Terms: Animals, Centrioles, Chromosomal Proteins, Non-Histone, Cilia, Exome, Female, Fetus, Genetics, Medical, HEK293 Cells, High-Throughput Nucleotide Sequencing, Humans, Male, Mice, Microcephaly, Microfilament Proteins, Mutation, NIH 3T3 Cells, Pedigree, Pregnancy, Zebrafish
Show Abstract · Added September 28, 2015
BACKGROUND - Mutations in microtubule-regulating genes are associated with disorders of neuronal migration and microcephaly. Regulation of centriole length has been shown to underlie the pathogenesis of certain ciliopathy phenotypes. Using a next-generation sequencing approach, we identified mutations in a novel centriolar disease gene in a kindred with an embryonic lethal ciliopathy phenotype and in a patient with primary microcephaly.
METHODS AND RESULTS - Whole exome sequencing data from a non-consanguineous Caucasian kindred exhibiting mid-gestation lethality and ciliopathic malformations revealed two novel non-synonymous variants in CENPF, a microtubule-regulating gene. All four affected fetuses showed segregation for two mutated alleles [IVS5-2A>C, predicted to abolish the consensus splice-acceptor site from exon 6; c.1744G>T, p.E582X]. In a second unrelated patient exhibiting microcephaly, we identified two CENPF mutations [c.1744G>T, p.E582X; c.8692 C>T, p.R2898X] by whole exome sequencing. We found that CENP-F colocalised with Ninein at the subdistal appendages of the mother centriole in mouse inner medullary collecting duct cells. Intraflagellar transport protein-88 (IFT-88) colocalised with CENP-F along the ciliary axonemes of renal epithelial cells in age-matched control human fetuses but did not in truncated cilia of mutant CENPF kidneys. Pairwise co-immunoprecipitation assays of mitotic and serum-starved HEKT293 cells confirmed that IFT88 precipitates with endogenous CENP-F.
CONCLUSIONS - Our data identify CENPF as a new centriolar disease gene implicated in severe human ciliopathy and microcephaly related phenotypes. CENP-F has a novel putative function in ciliogenesis and cortical neurogenesis.
Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.
1 Communities
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20 MeSH Terms
SMARCB1 expression in epithelioid sarcoma is regulated by miR-206, miR-381, and miR-671-5p on Both mRNA and protein levels.
Papp G, Krausz T, Stricker TP, Szendrői M, Sápi Z
(2014) Genes Chromosomes Cancer 53: 168-76
MeSH Terms: Chromosomal Proteins, Non-Histone, DNA-Binding Proteins, Gene Expression Regulation, Neoplastic, Humans, MicroRNAs, RNA Interference, RNA, Messenger, SMARCB1 Protein, Sarcoma, Transcription Factors
Show Abstract · Added March 28, 2014
Proximal type epithelioid sarcoma shares similarities with malignant rhabdoid tumor, including the lack of nuclear immunoreactivity of SMARCB1. Biallelic mutation of SMARCB1 has been convincingly established as the cause of loss of protein expression in rhabdoid tumor, but the cause in epithelioid sarcoma remains unknown. In our previous work, we demonstrated that DNA hypermethylation and post-translational modification mechanisms were not involved. In this current work, we explored the hypothesis that miRNAs regulate SMARCB1 gene expression in epithelioid sarcomas. In silico target prediction analysis revealed eight candidate miRNAs, and quantitative PCR-in 32 formalin-fixed, paraffin-embedded tumor samples comprising 30 epithelioid sarcomas and two malignant rhabdoid tumors-demonstrated significant (P < 0.001) overexpression of four miRNAs in epithelioid sarcomas: miR-206, miR-381, miR-671-5p, and miR-765. Two human tumors (fibrosarcoma and colon adenocarcinoma) and a normal cell line (human dermal fibroblast) with retained SMARCB1 expression were cultured for miRNA transient transfection (electroporation) experiments. SMARCB1 mRNA expression was analyzed by quantitative real-time PCR and immunostaining of SMARCB1 was performed to examine the effect of miRNAs transfections on both RNA and protein levels. Only three of the overexpressed miRNAs (miR-206, miR-381, and miR-671-5p) could silence the SMARCB1 mRNA expression in cell cultures; most effectively miR-206. Transfection of miR-206, miR-381, miR-671-5p, and some combination of them also eliminated SMARCB1 nuclear staining, demonstrating a strong effect on not only mRNA but also protein levels. Our results suggest loss of SMARCB1 protein expression in epithelioid sarcoma is due to the epigenetic mechanism of gene silencing by oncomiRs.
Copyright © 2013 Wiley Periodicals, Inc.
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10 MeSH Terms
Individual pericentromeres display coordinated motion and stretching in the yeast spindle.
Stephens AD, Snider CE, Haase J, Haggerty RA, Vasquez PA, Forest MG, Bloom K
(2013) J Cell Biol 203: 407-16
MeSH Terms: Adenosine Triphosphatases, Cell Cycle Proteins, Centromere, Chromatids, Chromatin, Chromosomal Proteins, Non-Histone, Chromosome Segregation, DNA-Binding Proteins, Kinesin, Kinetochores, Microtubules, Mitosis, Multiprotein Complexes, Nuclear Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Spindle Apparatus, Stress, Physiological
Show Abstract · Added January 25, 2016
The mitotic segregation apparatus composed of microtubules and chromatin functions to faithfully partition a duplicated genome into two daughter cells. Microtubules exert extensional pulling force on sister chromatids toward opposite poles, whereas pericentric chromatin resists with contractile springlike properties. Tension generated from these opposing forces silences the spindle checkpoint to ensure accurate chromosome segregation. It is unknown how the cell senses tension across multiple microtubule attachment sites, considering the stochastic dynamics of microtubule growth and shortening. In budding yeast, there is one microtubule attachment site per chromosome. By labeling several chromosomes, we find that pericentromeres display coordinated motion and stretching in metaphase. The pericentromeres of different chromosomes exhibit physical linkage dependent on centromere function and structural maintenance of chromosomes complexes. Coordinated motion is dependent on condensin and the kinesin motor Cin8, whereas coordinated stretching is dependent on pericentric cohesin and Cin8. Linking of pericentric chromatin through cohesin, condensin, and kinetochore microtubules functions to coordinate dynamics across multiple attachment sites.
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18 MeSH Terms
Posttranslational modification of CENP-A influences the conformation of centromeric chromatin.
Bailey AO, Panchenko T, Sathyan KM, Petkowski JJ, Pai PJ, Bai DL, Russell DH, Macara IG, Shabanowitz J, Hunt DF, Black BE, Foltz DR
(2013) Proc Natl Acad Sci U S A 110: 11827-32
MeSH Terms: Autoantigens, Cell Cycle Proteins, Cell Line, Centromere, Centromere Protein A, Chromatin, Chromatography, High Pressure Liquid, Chromosomal Proteins, Non-Histone, Epigenesis, Genetic, Guanine Nucleotide Exchange Factors, Humans, Mass Spectrometry, Methylation, Molecular Conformation, Nuclear Proteins, Phosphorylation, Protein Processing, Post-Translational, Ultracentrifugation
Show Abstract · Added March 7, 2014
Centromeres are chromosomal loci required for accurate segregation of sister chromatids during mitosis. The location of the centromere on the chromosome is not dependent on DNA sequence, but rather it is epigenetically specified by the histone H3 variant centromere protein A (CENP-A). The N-terminal tail of CENP-A is highly divergent from other H3 variants. Canonical histone N termini are hotspots of conserved posttranslational modification; however, no broadly conserved modifications of the vertebrate CENP-A tail have been previously observed. Here, we report three posttranslational modifications on human CENP-A N termini using high-resolution MS: trimethylation of Gly1 and phosphorylation of Ser16 and Ser18. Our results demonstrate that CENP-A is subjected to constitutive initiating methionine removal, similar to other H3 variants. The nascent N-terminal residue Gly1 becomes trimethylated on the α-amino group. We demonstrate that the N-terminal RCC1 methyltransferase is capable of modifying the CENP-A N terminus. Methylation occurs in the prenucleosomal form and marks the majority of CENP-A nucleosomes. Serine 16 and 18 become phosphorylated in prenucleosomal CENP-A and are phosphorylated on asynchronous and mitotic nucleosomal CENP-A and are important for chromosome segregation during mitosis. The double phosphorylation motif forms a salt-bridged secondary structure and causes CENP-A N-terminal tails to form intramolecular associations. Analytical ultracentrifugation of phospho-mimetic CENP-A nucleosome arrays demonstrates that phosphorylation results in greater intranucleosome associations and counteracts the hyperoligomerized state exhibited by unmodified CENP-A nucleosome arrays. Our studies have revealed that the major modifications on the N-terminal tail of CENP-A alter the physical properties of the chromatin fiber at the centromere.
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18 MeSH Terms
INI1/hSNF5-interaction defective HIV-1 IN mutants exhibit impaired particle morphology, reverse transcription and integration in vivo.
Mathew S, Nguyen M, Wu X, Pal A, Shah VB, Prasad VR, Aiken C, Kalpana GV
(2013) Retrovirology 10: 66
MeSH Terms: Cell Line, Chromosomal Proteins, Non-Histone, DNA-Binding Proteins, HIV Integrase, HIV-1, Host-Pathogen Interactions, Humans, Microscopy, Electron, Transmission, Reverse Transcription, SMARCB1 Protein, Transcription Factors, Virion, Virus Assembly, Virus Integration
Show Abstract · Added May 27, 2014
BACKGROUND - Retroviral integrase catalyzes integration of viral DNA into the host genome. Integrase interactor (INI)1/hSNF5 is a host factor that binds to HIV-1 IN within the context of Gag-Pol and is specifically incorporated into HIV-1 virions during assembly. Previous studies have indicated that INI1/hSNF5 is required for late events in vivo and for integration in vitro. To determine the effects of disrupting the IN-INI1 interaction on the assembly and infectivity of HIV-1 particles, we isolated mutants of IN that are defective for binding to INI1/hSNF5 and tested their effects on HIV-1 replication.
RESULTS - A reverse yeast two-hybrid system was used to identify INI1-interaction defective IN mutants (IID-IN). Since protein-protein interactions depend on the surface residues, the IID-IN mutants that showed high surface accessibility on IN crystal structures (K71R, K111E, Q137R, D202G, and S147G) were selected for further study. In vitro interaction studies demonstrated that IID-IN mutants exhibit variable degrees of interaction with INI1. The mutations were engineered into HIV-1(NL4-3) and HIV-Luc viruses and tested for their effects on virus replication. HIV-1 harboring IID-IN mutations were defective for replication in both multi- and single-round infection assays. The infectivity defects were correlated to the degree of INI1 interaction of the IID-IN mutants. Highly defective IID-IN mutants were blocked at early and late reverse transcription, whereas partially defective IID-IN mutants proceeded through reverse transcription and nuclear localization, but were partially impaired for integration. Electron microscopic analysis of mutant particles indicated that highly interaction-defective IID-IN mutants produced morphologically aberrant virions, whereas the partially defective mutants produced normal virions. All of the IID-IN mutant particles exhibited normal capsid stability and reverse transcriptase activity in vitro.
CONCLUSIONS - Our results demonstrate that a severe defect in IN-INI1 interaction is associated with production of defective particles and a subsequent defect in post-entry events. A partial defect in IN-INI1 interaction leads to production of normal virions that are partially impaired for early events including integration. Our studies suggest that proper interaction of INI1 with IN within Gag-Pol is necessary for proper HIV-1 morphogenesis and integration.
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14 MeSH Terms
The loss of the BH3-only Bcl-2 family member Bid delays T-cell leukemogenesis in Atm-/- mice.
Biswas S, Shi Q, Wernick A, Aiello A, Zinkel SS
(2013) Cell Death Differ 20: 869-77
MeSH Terms: Animals, Annexin A5, Apoptosis, Ataxia Telangiectasia Mutated Proteins, BH3 Interacting Domain Death Agonist Protein, Cell Cycle Proteins, Checkpoint Kinase 1, Chromosomal Proteins, Non-Histone, DNA Damage, DNA-Binding Proteins, Disease Models, Animal, Female, Leukemia, T-Cell, Male, Mice, Mice, Knockout, Protein Kinases, Protein-Serine-Threonine Kinases, T-Lymphocytes, Thymocytes, Tumor Suppressor Proteins, Tumor Suppressor p53-Binding Protein 1
Show Abstract · Added August 22, 2013
Multicellular organisms maintain genomic integrity and resist tumorigenesis through a tightly regulated DNA damage response (DDR) that prevents propagation of deleterious mutations either through DNA repair or programmed cell death. An impaired DDR leads to tumorigenesis that is accelerated when programmed cell death is prevented. Loss of the ATM (ataxia telangiectasia mutated)-mediated DDR in mice results in T-cell leukemia driven by accumulation of DNA damage accrued during normal T-cell development. Pro-apoptotic BH3-only Bid is a substrate of Atm, and Bid phosphorylation is required for proper cell cycle checkpoint control and regulation of hematopoietic function. In this report, we demonstrate that, surprisingly, loss of Bid increases the latency of leukemogenesis in Atm-/- mice. Bid-/-Atm-/- mice display impaired checkpoint control and increased cell death of DN3 thymocytes. Loss of Bid thus inhibits T-cell tumorigenesis by increasing clearance of damaged cells, and preventing propagation of deleterious mutations.
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22 MeSH Terms
SMARCB1 protein and mRNA loss is not caused by promoter and histone hypermethylation in epithelioid sarcoma.
Papp G, Changchien YC, Péterfia B, Pecsenka L, Krausz T, Stricker TP, Khoor A, Donner L, Sápi Z
(2013) Mod Pathol 26: 393-403
MeSH Terms: Adolescent, Adult, Aged, Aged, 80 and over, Child, Chromosomal Proteins, Non-Histone, DNA Methylation, DNA Mutational Analysis, DNA-Binding Proteins, Down-Regulation, Enhancer of Zeste Homolog 2 Protein, Female, Gene Deletion, Gene Expression Regulation, Neoplastic, Genetic Predisposition to Disease, Histones, Humans, Immunohistochemistry, In Situ Hybridization, Fluorescence, Laser Capture Microdissection, Male, Middle Aged, Mutation, Phenotype, Poland, Polycomb Repressive Complex 2, Promoter Regions, Genetic, RNA, Messenger, Real-Time Polymerase Chain Reaction, Retrospective Studies, Reverse Transcriptase Polymerase Chain Reaction, SMARCB1 Protein, Sarcoma, Soft Tissue Neoplasms, Transcription Factors, United States, Young Adult
Show Abstract · Added March 28, 2014
About 10% of epithelioid sarcomas have biallelic mutation of the SMARCB1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily b, member 1) gene resulting in a lack of this nuclear protein. It has been suggested that SMARCB1 may be silenced by epigenetic changes in the remaining 90% of tumors. Thus, we hypothesized that the promoter of SMARCB1 is hypermethylated. We also examined SMARCB1 mRNA level to determine if a post-translational change was possible. Thirty-six cases of epithelioid sarcomas were studied. Immunohistochemistry and mutation analysis of the SMARCB1 gene were performed to select appropriate cases. Methylation status was assessed by methylation-specific PCR. Laser capture microdissection of tumor cells followed by real-time PCR was applied to examine the expression of SMARCB1 mRNA. Of 36 epithelioid sarcomas, 31 (86%) displayed a lack of SMARCB1 nuclear protein. In all, 4 (13%) of 31 SMARCB1-negative cases harbored biallelic deletion while 9 (33%) cases showed single-allelic deletion. One (4%) frameshift deletion of exon 3 and one point mutation of exon 7 were also found. In 16 (59%) cases, both alleles were intact. Altogether, 25/31 (81%) SMARCB1-negative cases had at least one intact allele. None of these cases demonstrated promoter hypermethylation. Low levels of SMARCB1 mRNA were found in all cases with tumor tissue extracted RNA (because of the minimal normal cell contamination) but no mRNA could be detected in laser dissected cases (containing only tumor cells). Enhancer of zeste homolog 2 (EZH2) overexpression was not characteristic of epithelioid sarcoma. Thus, loss of SMARCB1 expression in epithelioid sarcoma is caused neither by DNA hypermethylation nor by post-translational modifications. Most likely it is the microRNA destruction of SMARCB1 mRNA but further investigations are needed to elucidate this issue.
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37 MeSH Terms