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1.
PLoS One ; 12(3): e0167130, 2017.
Article in English | MEDLINE | ID: mdl-28248967

ABSTRACT

We evaluated performance characteristics of a laboratory-developed, non-invasive prenatal screening (NIPS) assay for fetal aneuploidies. This assay employs massively parallel shotgun sequencing with full automation. GC sequencing bias correction and statistical smoothing were performed to enhance discrimination of affected and unaffected pregnancies. Maternal plasma samples from pregnancies with known aneuploidy status were used for assay development, verification, and validation. Assay verification studies using 2,085 known samples (1873 unaffected, 69 trisomy 21, 20 trisomy 18, 17 trisomy 13) demonstrated complete discrimination between autosomal trisomy (Z scores >8) and unaffected (Z scores <4) singleton pregnancies. A validation study using 552 known samples (21 trisomy 21, 10 trisomy 18, 1 trisomy 13) confirmed complete discrimination. Twin pregnancies showed similar results. Follow-up of abnormal results from the first 10,000 clinical samples demonstrated PPVs of 98% (41/42) for trisomy 21, 92% (23/25) for trisomy 18, and 69% (9/13) for trisomy 13. Adjustment for causes of false-positive results identified during clinical testing (eg, maternal duplications) improved PPVs to 100% for trisomy 21 and 96% for trisomy 18. This NIPS test demonstrates excellent discrimination between trisomic and unaffected pregnancies. The PPVs obtained in initial clinical testing are substantially higher than previously reported NIPS methods.


Subject(s)
Chromosomes, Human/genetics , Mass Screening/methods , Prenatal Diagnosis/methods , Trisomy/diagnosis , Trisomy/genetics , False Positive Reactions , Female , Follow-Up Studies , Humans , Male , Pregnancy
2.
Hum Mutat ; 37(12): 1318-1328, 2016 12.
Article in English | MEDLINE | ID: mdl-27633797

ABSTRACT

As next-generation sequencing increases access to human genetic variation, the challenge of determining clinical significance of variants becomes ever more acute. Germline variants in the BRCA1 and BRCA2 genes can confer substantial lifetime risk of breast and ovarian cancer. Assessment of variant pathogenicity is a vital part of clinical genetic testing for these genes. A database of clinical observations of BRCA variants is a critical resource in that process. This article describes BRCA Share™, a database created by a unique international alliance of academic centers and commercial testing laboratories. By integrating the content of the Universal Mutation Database generated by the French Unicancer Genetic Group with the testing results of two large commercial laboratories, Quest Diagnostics and Laboratory Corporation of America (LabCorp), BRCA Share™ has assembled one of the largest publicly accessible collections of BRCA variants currently available. Although access is available to academic researchers without charge, commercial participants in the project are required to pay a support fee and contribute their data. The fees fund the ongoing curation effort, as well as planned experiments to functionally characterize variants of uncertain significance. BRCA Share™ databases can therefore be considered as models of successful data sharing between private companies and the academic world.


Subject(s)
BRCA1 Protein/genetics , BRCA2 Protein/genetics , Breast Neoplasms/genetics , Databases, Factual , Ovarian Neoplasms/genetics , Data Curation , Databases, Factual/economics , Female , Genetic Predisposition to Disease , Humans , Mutation
3.
Hum Mutat ; 37(1): 127-34, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26467025

ABSTRACT

We developed a rules-based scoring system to classify DNA variants into five categories including pathogenic, likely pathogenic, variant of uncertain significance (VUS), likely benign, and benign. Over 16,500 pathogenicity assessments on 11,894 variants from 338 genes were analyzed for pathogenicity based on prediction tools, population frequency, co-occurrence, segregation, and functional studies collected from internal and external sources. Scores were calculated by trained scientists using a quantitative framework that assigned differential weighting to these five types of data. We performed descriptive and comparative statistics on the dataset and tested interobserver concordance among the trained scientists. Private variants defined as variants found within single families (n = 5,182), were either VUS (80.5%; n = 4,169) or likely pathogenic (19.5%; n = 1,013). The remaining variants (n = 6,712) were VUS (38.4%; n = 2,577) or likely benign/benign (34.7%; n = 2,327) or likely pathogenic/pathogenic (26.9%, n = 1,808). Exact agreement between the trained scientists on the final variant score was 98.5% [95% confidence interval (CI) (98.0, 98.9)] with an interobserver consistency of 97% [95% CI (91.5, 99.4)]. Variant scores were stable and showed increasing odds of being in agreement with new data when re-evaluated periodically. This carefully curated, standardized variant pathogenicity scoring system provides reliable pathogenicity scores for DNA variants encountered in a clinical laboratory setting.


Subject(s)
Computational Biology/methods , Genetic Predisposition to Disease , Genetic Variation , Genomics/methods , Software , Humans , Observer Variation , Reproducibility of Results , Web Browser
4.
Neuromuscul Disord ; 25(12): 945-51, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26420234

ABSTRACT

We compare molecular combing to Southern blot in the analysis of the facioscapulohumeral muscular dystrophy type 1 locus (FSHD1) on chromosome 4q35-qter (chr 4q) in genomic DNA specimens sent to a clinical laboratory for FSHD testing. A de-identified set of 87 genomic DNA specimens determined by Southern blot as normal (n = 71), abnormal with D4Z4 macrosatellite repeat array contractions (n = 7), indeterminate (n = 6), borderline (n = 2), or mosaic (n = 1) was independently re-analyzed by molecular combing in a blinded fashion. The molecular combing results were identical to the Southern blot results in 75 (86%) of cases. All contractions (n = 7) and mosaics (n = 1) detected by Southern blot were confirmed by molecular combing. Of the 71 samples with normal Southern blot results, 67 (94%) had concordant molecular combing results. The four discrepancies were either mosaic (n = 2), rearranged (n = 1), or borderline by molecular combing (n = 1). All indeterminate Southern blot results (n = 6) were resolved by molecular combing as either normal (n = 4), borderline (n = 1), or rearranged (n = 1). The two borderline Southern blot results showed a D4Z4 contraction on the chr 4qA allele and a normal result by molecular combing. Molecular combing overcomes a number of technical limitations of Southern blot by providing direct visualization of D4Z4 macrosatellite repeat arrays on specific chr 4q and chr 10q alleles and more precise D4Z4 repeat sizing. This study suggests that molecular combing has superior analytical validity compared to Southern blot for determining D4Z4 contraction size, detecting mosaicism, and resolving borderline and indeterminate Southern blot results. Further studies are needed to establish the clinical validity and diagnostic accuracy of these findings in FSHD.


Subject(s)
Blotting, Southern/methods , Chromosomes, Human, Pair 4 , Molecular Diagnostic Techniques/methods , Muscular Dystrophy, Facioscapulohumeral/genetics , Sequence Analysis, DNA/methods , Humans
6.
Mol Genet Genomic Med ; 2(6): 522-9, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25614874

ABSTRACT

We report the frequency, positive rate, and type of mutations in 14 genes (PMP22, GJB1, MPZ, MFN2, SH3TC2, GDAP1, NEFL, LITAF, GARS, HSPB1, FIG4, EGR2, PRX, and RAB7A) associated with Charcot-Marie-Tooth disease (CMT) in a cohort of 17,880 individuals referred to a commercial genetic testing laboratory. Deidentified results from sequencing assays and multiplex ligation-dependent probe amplification (MLPA) were analyzed including 100,102 Sanger sequencing, 2338 next-generation sequencing (NGS), and 21,990 MLPA assays. Genetic abnormalities were identified in 18.5% (n = 3312) of all individuals. Testing by Sanger and MLPA (n = 3216) showed that duplications (dup) (56.7%) or deletions (del) (21.9%) in the PMP22 gene accounted for the majority of positive findings followed by mutations in the GJB1 (6.7%), MPZ (5.3%), and MFN2 (4.3%) genes. GJB1 del and mutations in the remaining genes explained 5.3% of the abnormalities. Pathogenic mutations were distributed as follows: missense (70.6%), nonsense (14.3%), frameshift (8.7%), splicing (3.3%), in-frame deletions/insertions (1.8%), initiator methionine mutations (0.8%), and nonstop changes (0.5%). Mutation frequencies, positive rates, and the types of mutations were similar between tests performed by either Sanger (n = 17,377) or NGS (n = 503). Among patients with a positive genetic finding in a CMT-related gene, 94.9% were positive in one of four genes (PMP22, GJB1, MPZ, or MFN2).

8.
Neurogenetics ; 12(3): 169-73, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21630033

ABSTRACT

The rate of DNA variation discovery has accelerated the need to collate, store and interpret the data in a standardised coherent way and is becoming a critical step in maximising the impact of discovery on the understanding and treatment of human disease. This particularly applies to the field of neurology as neurological function is impaired in many human disorders. Furthermore, the field of neurogenetics has been proven to show remarkably complex genotype-to-phenotype relationships. To facilitate the collection of DNA sequence variation pertaining to neurogenetic disorders, we have initiated the "Neurogenetics Consortium" under the umbrella of the Human Variome Project. The Consortium's founding group consisted of basic researchers, clinicians, informaticians and database creators. This report outlines the strategic aims established at the preliminary meetings of the Neurogenetics Consortium and calls for the involvement of the wider neurogenetic community in enabling the development of this important resource.


Subject(s)
Databases, Genetic/standards , Genetic Variation , Genetics, Medical/organization & administration , International Cooperation , Nervous System/metabolism , Algorithms , Congresses as Topic , Genetic Variation/physiology , Genetics, Medical/standards , Human Genome Project/organization & administration , Humans , Nervous System Diseases/genetics , Nervous System Diseases/metabolism , Research Report
9.
Genet Med ; 13(7): 686-94, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21673580

ABSTRACT

Spinal muscular atrophy is a common autosomal recessive neuromuscular disorder caused by mutations in the survival motor neuron (SMN1) gene, affecting approximately 1 in 10,000 live births. The disease is characterized by progressive symmetrical muscle weakness resulting from the degeneration and loss of anterior horn cells in the spinal cord and brainstem nuclei. The disease is classified on the basis of age of onset and clinical course. Two almost identical SMN genes are present on 5q13: the SMN1 gene, which is the spinal muscular atrophy-determining gene, and the SMN2 gene. The homozygous absence of the SMN1 exon 7 has been observed in the majority of patients and is being used as a reliable and sensitive spinal muscular atrophy diagnostic test. Although SMN2 produces less full-length transcript than SMN1, the number of SMN2 copies has been shown to modulate the clinical phenotype. Carrier detection relies on the accurate determination of the SMN1 gene copies. This document follows the outline format of the general Standards and Guidelines for Clinical Laboratories. It is designed to be a checklist for genetic testing professionals who are already familiar with the disease and methods of analysis.


Subject(s)
Genetic Testing/methods , Genetic Testing/standards , Guidelines as Topic , Muscular Atrophy, Spinal/genetics , Clinical Laboratory Techniques/methods , Clinical Laboratory Techniques/standards , DNA Mutational Analysis , Gene Dosage , Humans , Muscular Atrophy, Spinal/diagnosis , Mutation , Survival of Motor Neuron 1 Protein/genetics , Survival of Motor Neuron 2 Protein/genetics
10.
Brain ; 131(Pt 4): 1078-86, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18321925

ABSTRACT

Mutations in the receptor expression enhancing protein 1 (REEP1) have recently been reported to cause autosomal dominant hereditary spastic paraplegia (HSP) type SPG31. In a large collaborative effort, we screened a sample of 535 unrelated HSP patients for REEP1 mutations and copy number variations. We identified 13 novel and 2 known REEP1 mutations in 16 familial and sporadic patients by direct sequencing analysis. Twelve out of 16 mutations were small insertions, deletions or splice site mutations. These changes would result in shifts of the open-reading-frame followed by premature termination of translation and haploinsufficiency. Interestingly, we identified two disease associated variations in the 3'-UTR of REEP1 that fell into highly conserved micro RNA binding sites. Copy number variation analysis in a subset of 133 HSP index patients revealed a large duplication of REEP1 that involved exons 2-7 in an Irish family. Clinically most SPG31 patients present with a pure spastic paraplegia; rare complicating features were restricted to symptoms or signs of peripheral nerve involvement. Interestingly, the distribution of age at onset suggested a bimodal pattern with the appearance of initial symptoms of disease either before the age of 20 years or after the age of 30 years. The overall mutation rate in our clinically heterogeneous sample was 3.0%; however, in the sub-sample of pure HSP REEP1 mutations accounted for 8.2% of all patients. These results firmly establish REEP1 as a relatively frequent autosomal dominant HSP gene for which genetic testing is warranted. We also establish haploinsufficiency as the main molecular genetic mechanism in SPG31, which should initiate and guide functional studies on REEP1 with a focus on loss-of-function mechanisms. Our results should be valid as a reference for mutation frequency, spectrum of REEP1 mutations, and clinical phenotypes associated with SPG31.


Subject(s)
Membrane Transport Proteins/genetics , Mutation , Spastic Paraplegia, Hereditary/genetics , Adolescent , Adult , Age of Onset , Aged , Aged, 80 and over , Child , Child, Preschool , DNA Mutational Analysis/methods , Female , Genotype , Humans , Infant , Male , Middle Aged , Pedigree , Phenotype
11.
Am J Med Genet A ; 143A(15): 1775-7, 2007 Aug 01.
Article in English | MEDLINE | ID: mdl-17593543

ABSTRACT

Ataxia with oculomotor apraxia type 2 (AOA2) is an autosomal recessive disorder associated with mutations in the Senataxin (SETX) gene. Clinical manifestations (ataxia, peripheral neuropathy, oculomotor apraxia) of this disease have previously been limited to the nervous system. We describe a patient homozygous for a novel mutation of SETX who manifested not only ataxia but also ovarian failure.


Subject(s)
Apraxia, Ideomotor/genetics , Mutation , Primary Ovarian Insufficiency/complications , RNA Helicases/genetics , Adult , Apraxia, Ideomotor/complications , DNA/blood , DNA/genetics , DNA/isolation & purification , DNA Helicases , Female , Homozygote , Humans , Multifunctional Enzymes , Primary Ovarian Insufficiency/diagnostic imaging , Primary Ovarian Insufficiency/genetics , Radiography , alpha-Fetoproteins/metabolism
12.
J Cell Physiol ; 209(3): 706-10, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17001681

ABSTRACT

A prominent role for the execution of cell cycle and growth regulatory mechanisms within the three-dimensional context of nuclear architecture is becoming increasingly evident. Signaling pathways and regulatory networks that govern activation and suppression of genes controlling proliferation are functionally integrated for the organization and assembly of transcriptional machinery in nuclear microenvironments. The transcriptional activation of histone genes at the G1/S phase transition (S-point) is temporarily, functionally, and spatially distinct from transcriptional mechanisms at the restriction point (R-point). The spatial distinction in R-point versus S-point control is the localization of clustered histone gene loci at cajal bodies, which is modulated during the cell cycle. Histone nuclear factor P (HiNF-P), the principal factor mediating H4 histone gene transcription, is the final link in the signaling cascade that is initiated with growth factor dependent induction of cyclin E/CDK2 kinase activity at the R-point and culminates in the NPAT-mediated activation of histone H4 genes through HiNF-P at the G1/S phase cell-cycle transition.


Subject(s)
Cell Cycle/physiology , G1 Phase/physiology , S Phase/physiology , Cell Nucleus/metabolism , Cell Nucleus/ultrastructure , Gene Expression Regulation , Histones/genetics , Histones/metabolism , Humans , Transcription, Genetic
13.
J Biol Chem ; 280(45): 37400-7, 2005 Nov 11.
Article in English | MEDLINE | ID: mdl-16131487

ABSTRACT

The replication of eukaryotic genomes necessitates the coordination of histone biosynthesis with DNA replication at the onset of S phase. The multiple histone H4 genes encode identical proteins, but their regulatory sequences differ. The contributions of these individual genes to histone H4 mRNA expression have not been described. We have determined, by real-time quantitative PCR and RNase protection, that the human histone H4 genes are not equally expressed and that a subset contributes disproportionately to the total pool of H4 mRNA. Differences in histone H4 gene expression can be attributed to observed unequal activities of the H4 gene promoters, which exhibit variations in gene regulatory elements. The overall expression pattern of the histone H4 gene complement is similar in normal and cancer cells. However, H4 genes that are moderately expressed in normal cells are sporadically silenced in tumor cells with compensation of expression by other H4 gene copies. Chromatin immunoprecipitation analyses and in vitro DNA binding assays indicated that 11 of the 15 histone H4 genes interact with the cell cycle regulatory histone nuclear factor P, which forms a complex with the cyclin E/CDK2-responsive co-regulator p220(NPAT). These 11 H4 genes account for 95% of the histone H4 mRNA pool. We conclude that the cyclin E/CDK2/p220(NPAT)/histone nuclear factor P signaling pathway is the principal regulator of histone H4 biosynthesis.


Subject(s)
Gene Expression Regulation, Neoplastic , Gene Expression Regulation , Histones/genetics , S Phase/genetics , S Phase/physiology , Animals , Base Sequence , Cell Line , Consensus Sequence , DNA Replication , Gene Expression Profiling , Humans , Promoter Regions, Genetic , RNA, Messenger/metabolism , Signal Transduction
14.
Mol Cell Biol ; 25(14): 6140-53, 2005 Jul.
Article in English | MEDLINE | ID: mdl-15988025

ABSTRACT

Genome replication in eukaryotic cells necessitates the stringent coupling of histone biosynthesis with the onset of DNA replication at the G1/S phase transition. A fundamental question is the mechanism that links the restriction (R) point late in G1 with histone gene expression at the onset of S phase. Here we demonstrate that HiNF-P, a transcriptional regulator of replication-dependent histone H4 genes, interacts directly with p220(NPAT), a substrate of cyclin E/CDK2, to coactivate histone genes during S phase. HiNF-P and p220 are targeted to, and colocalize at, subnuclear foci (Cajal bodies) in a cell cycle-dependent manner. Genetic or biochemical disruption of the HiNF-P/p220 interaction compromises histone H4 gene activation at the G1/S phase transition and impedes cell cycle progression. Our results show that HiNF-P and p220 form a critical regulatory module that directly links histone H4 gene expression at the G1/S phase transition to the cyclin E/CDK2 signaling pathway at the R point.


Subject(s)
Cell Cycle Proteins/metabolism , Gene Expression Regulation , Histones/genetics , Nuclear Proteins/metabolism , Repressor Proteins/metabolism , S Phase/genetics , Amino Acid Motifs , Animals , CDC2-CDC28 Kinases/metabolism , Cell Cycle Proteins/analysis , Cell Cycle Proteins/genetics , Cell Nucleus/chemistry , Cell Nucleus/metabolism , Chromatin/metabolism , Cyclin E/metabolism , Cyclin-Dependent Kinase 2 , G1 Phase/genetics , Humans , Mutation , Nuclear Proteins/analysis , Nuclear Proteins/genetics , Promoter Regions, Genetic , Protein Interaction Mapping , Repressor Proteins/analysis , Transcription, Genetic , Transcriptional Activation
15.
Gene ; 342(1): 35-40, 2004 Nov 10.
Article in English | MEDLINE | ID: mdl-15527963

ABSTRACT

Histones are the major protein component of nucleosomes, and de novo histone synthesis is essential for packaging newly replicated DNA into chromatin. As a result, histone gene expression is exquisitely and functionally coupled with DNA replication. Vastly divergent organisms such as yeast, fly and human all demonstrate the phylogenetically conserved propensity to maintain clustering of histone genes at one or more genomic loci. Although specific mechanisms are unclear, clustering is presumed to be important for common stringent transcriptional control of these genes at the G1/S phase transition. In this study, we describe a genomic duplication of the human histone gene cluster located at chromosome 1q21, which effectively doubles the previously known size and gene number of that cluster. The duplication persists in all examined tissues and cell lines, and the duplicated genes are transcriptionally active. Levels of messenger RNAs for duplicated histone H4 genes are high relative to those for non-duplicated H4 genes. Our data suggest that transcriptionally robust histone H4 genes may have been preferentially duplicated during evolution.


Subject(s)
Histones/genetics , Multigene Family/genetics , Amino Acid Sequence , Base Sequence , Cell Line, Tumor , Chromosomes, Human, Pair 1/genetics , Gene Duplication , Genes, Duplicate/genetics , Genome, Human , HL-60 Cells , HeLa Cells , Humans , Molecular Sequence Data , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Homology, Nucleic Acid
16.
Int J Oncol ; 25(2): 503-9, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15254750

ABSTRACT

A promising family of anticancer agents, the camptothecins, is noted for their ability to induce apoptosis specifically in malignant cells. However, a major obstacle for successful cancer treatment by these and other chemotherapeutic agents is the intrinsic or acquired resistance to drug treatment. Resistance to 9NC6, a camptothecin derivative, has been modeled in vitro using a human prostate cancer cell line. To elucidate the mechanism for acquired 9NC resistance, we have used a subtractive cloning approach to identify genes whose altered expression level is reflective of 9NC resistance or susceptibility. Differential gene expression was compared between wild-type human prostate cancer cell line, DU-145, and a 9NC-resistant subline, RC1. Results were confirmed by Northern and Western blot analyses. In this report, we focus on one gene, 14-3-3zeta. An expression vector of a full-length myc-epitope-tagged 14-3-3zeta cDNA was constructed and used for transfection into DU-145 cells. We consistently observed that 14-3-3zeta message and protein levels were dramatically increased in 9NC resistant cells. The expression levels of other 14-3-3 family members were unaffected. Strikingly, ectopic overexpression of 14-3-3zeta in wild-type 9NC-susceptible prostate cancer cells decreased 9NC-induced apoptosis. Our results suggest a novel direct or indirect role of 14-3-3zeta in mediating resistance of DU-145 cells to the topoisomerase I inhibitor, 9NC. We are currently exploring whether this represents a more general pathway for drug resistance as well.


Subject(s)
14-3-3 Proteins/metabolism , Antineoplastic Agents/pharmacology , Apoptosis , Camptothecin/analogs & derivatives , Camptothecin/pharmacology , Prostatic Neoplasms/metabolism , 14-3-3 Proteins/genetics , Caspase 3 , Caspases/metabolism , Cloning, Molecular , Drug Resistance, Neoplasm/genetics , Gene Expression , Humans , Male , Poly(ADP-ribose) Polymerases/metabolism , Prostatic Neoplasms/genetics , Transfection , Tumor Cells, Cultured
17.
J Biol Chem ; 279(17): 17515-23, 2004 Apr 23.
Article in English | MEDLINE | ID: mdl-14766752

ABSTRACT

Cancer cells are more susceptible to chemotherapeutic agent-induced apoptosis than their normal counterparts. Although it has been demonstrated that the increased sensitivity results from deregulation of oncoproteins during cancer development (Evan, G. I., and Vousden, K. H. (2001) Nature 411, 342-348; Green, D. R., and Evan, G. I. (2002) Cancer Cell 1, 19-30), little is known about the signaling pathways leading to changes in the apoptotic threshold in cancer cells. Here we show that low RKIP expression levels in tumorigenic human prostate and breast cancer cells are rapidly induced upon chemotherapeutic drug treatment, sensitizing the cells to apoptosis. We show that the maximal RKIP expression correlates perfectly with the onset of apoptosis. In cancer cells resistant to DNA-damaging agents, treatment with the drugs does not up-regulate RKIP expression. However, ectopic expression of RKIP resensitizes DNA-damaging agent-resistant cells to undergo apoptosis. This sensitization can be reversed by up-regulation of survival pathways. Down-regulation of endogenous RKIP by expression of antisense and small interfering RNA (siRNA) confers resistance on sensitive cancer cells to anticancer drug-induced apoptosis. Our studies suggest that RKIP may represent a novel effector of signal transduction pathways leading to apoptosis and a prognostic marker of the pathogenesis of human cancer cells and tumors after treatment with clinically relevant chemotherapeutic drugs.


Subject(s)
Androgen-Binding Protein/physiology , Apoptosis , Breast Neoplasms/pathology , Prostatic Neoplasms/pathology , Cell Line, Tumor , Cell Survival , DNA/chemistry , Down-Regulation , Flow Cytometry , Gene Transfer Techniques , Genes, Reporter , Humans , Immunoblotting , Immunohistochemistry , Male , Phosphatidylethanolamine Binding Protein , Plasmids/metabolism , Prognosis , RNA, Antisense/chemistry , RNA, Small Interfering/chemistry , Retroviridae/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Time Factors , Transfection , Up-Regulation
18.
Trends Cell Biol ; 13(11): 584-92, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14573352

ABSTRACT

The organization and sorting of regulatory information for transcription, replication and repair depends on components of nuclear architecture. It is necessary, therefore, to understand cellular processes within the context of intranuclear microenvironments that mediate the focal assembly of the machinery for transcription, replication and repair and which facilitate the orchestration of these essential processes. Here, we discuss how nuclear anatomy supports the temporal and spatial coordination of regulatory protein recruitment for combinatorial control.


Subject(s)
Cell Nucleus/physiology , DNA Repair , DNA Replication , Gene Expression Regulation , Animals , Apoptosis/physiology , Cell Nucleus/genetics , Cell Nucleus/ultrastructure , DNA Methylation , Humans , Models, Genetic , Nuclear Matrix/genetics , Nuclear Matrix/physiology
19.
J Biol Chem ; 278(10): 8261-8, 2003 Mar 07.
Article in English | MEDLINE | ID: mdl-12475992

ABSTRACT

The ability of p53 to alter, at the transcriptional level, the gene expression of downstream targets is critical for its role as a tumor suppressor. Most models of p53 activation postulate the stepwise recruitment by p53 of coactivators, histone acetyltransferases, and/or chromatin remodeling factors to a promoter region to facilitate the subsequent access of the general transcriptional machinery required for transcriptional induction. We demonstrate here, however, that the promoter regions for the p53 target genes, p21, 14-3-3sigma, and KARP-1, exist in a constitutively open conformation that is readily accessible to DNase I. This conformation was not altered by DNA damage or by whether p53 was present or absent in the cell. In contrast, p53 response elements, which resided outside the immediate promoter regions, existed within DNase I-resistant chromatin domains. Thus, p53 activation of downstream target genes occurs without p53 inducing chromatin alterations detectable by DNase I accessibility at either the promoter or the response element. As such, these data support models of p53 activation that do not require extensive chromatin alterations to support cognate gene expression.


Subject(s)
Biomarkers, Tumor , DNA Helicases , Deoxyribonuclease I/metabolism , Neoplasm Proteins , Promoter Regions, Genetic , Tumor Suppressor Protein p53/physiology , 14-3-3 Proteins , Base Sequence , Carrier Proteins/genetics , Cell Line , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/genetics , DNA Primers , Exonucleases/genetics , Exoribonucleases , Infrared Rays , Ku Autoantigen , Protein Binding , Reverse Transcriptase Polymerase Chain Reaction , Tumor Suppressor Protein p53/metabolism , Up-Regulation/radiation effects
20.
Nucleic Acids Res ; 30(8): 1713-24, 2002 Apr 15.
Article in English | MEDLINE | ID: mdl-11937624

ABSTRACT

The human Ku86 gene and an isoform, KARP-1 (Ku86 autoantigen related protein-1), encode overlapping, but differentially regulated, transcripts. Ku86 is constitutively transcribed at high levels and, although it plays a seminal role in DNA double-strand break repair, its expression is not induced by DNA damage. KARP-1, in contrast, is expressed constitutively only at low levels and its expression is induced by DNA damage in a p53-dependent fashion. The regulatory elements promoting KARP-1 gene expression and p53 responsiveness, however, were unknown. Here, we report that a strong DNase I hypersensitive site (DHS) resides approximately 25 kb upstream from the Ku86 promoter. This DHS is encompassed by a hypomethylated CpG island. Reporter assays demonstrated that this region corresponded to a promoter(s), which promoted transcription of peroxisomal trans-2-enoyl CoA reductase in the centromeric direction and KARP-1 in the telomeric direction. KARP-1 primer extension products were mapped to this CpG island in the correct transcriptional orientation confirming that KARP-1 transcription initiates from this site. Moreover, a p53 response element within the first intron of the KARP-1 transcriptional unit was identified using chromatin immunoprecipitation and antibodies specific to activated forms of p53. These data expand our understanding of this important DNA repair locus.


Subject(s)
Carrier Proteins/genetics , CpG Islands , DNA Helicases , NADH, NADPH Oxidoreductases , Response Elements , Transcriptional Activation , Tumor Suppressor Protein p53/metabolism , 5' Flanking Region , Base Sequence , Carrier Proteins/biosynthesis , DNA Methylation , Deoxyribonuclease I/chemistry , Fatty Acid Desaturases/biosynthesis , Fatty Acid Desaturases/genetics , Humans , Ku Autoantigen , Molecular Sequence Data , Oxidoreductases Acting on CH-CH Group Donors , RNA, Messenger/biosynthesis , Radiation, Ionizing , Transcription Initiation Site , Tumor Cells, Cultured
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