Schizosaccharomyces pombe Rtf2 mediates site-specific replication termination by inhibiting replication restart.

Here, we identify a phylogenetically conserved Schizosaccharomyces pombe factor, named Rtf2, as a key requirement for efficient replication termination at the site-specific replication barrier RTS1. We show that Rtf2, a proliferating cell nuclear antigen-interacting protein, promotes termination at RTS1 by preventing replication restart; in the absence of Rtf2, we observe the establishment of "slow-moving" Srs2-dependent replication forks. Analysis of the pmt3 (SUMO) and rtf2 mutants establishes that pmt3 causes a reduction in RTS1 barrier activity, that rtf2 and pmt3 are nonadditive, and that pmt3 (SUMO) partly suppresses the rtf2-dependent replication restart. Our results are consistent with a model in which Rtf2 stabilizes the replication fork stalled at RTS1 until completion of DNA synthesis by a converging replication fork initiated at a flanking origin.

A linear programming approach for estimating the structure of a sparse linear genetic network from transcript profiling data.

BACKGROUND: A genetic network can be represented as a directed graph in which a node corresponds to a gene and a directed edge specifies the direction of influence of one gene on another. The reconstruction of such networks from transcript profiling data remains an important yet challenging endeavor. A transcript profile specifies the abundances of many genes in a biological sample of interest. Prevailing strategies for learning the structure of a genetic network from high-dimensional transcript profiling data assume sparsity and linearity. Many methods consider relatively small directed graphs, inferring graphs with up to a few hundred nodes. This work examines large undirected graphs representations of genetic networks, graphs with many thousands of nodes where an undirected edge between two nodes does not indicate the direction of influence, and the problem of estimating the structure of such a sparse linear genetic network (SLGN) from transcript profiling data. RESULTS: The structure learning task is cast as a sparse linear regression problem which is then posed as a LASSO (l1-constrained fitting) problem and solved finally by formulating a Linear Program (LP). A bound on the Generalization Error of this approach is given in terms of the Leave-One-Out Error. The accuracy and utility of LP-SLGNs is assessed quantitatively and qualitatively using simulated and real data. The Dialogue for Reverse Engineering Assessments and Methods (DREAM) initiative provides gold standard data sets and evaluation metrics that enable and facilitate the comparison of algorithms for deducing the structure of networks. The structures of LP-SLGNs estimated from the INSILICO1, INSILICO2 and INSILICO3 simulated DREAM2 data sets are comparable to those proposed by the first and/or second ranked teams in the DREAM2 competition. The structures of LP-SLGNs estimated from two published Saccharomyces cerevisae cell cycle transcript profiling data sets capture known regulatory associations. In each S. cerevisiae LP-SLGN, the number of nodes with a particular degree follows an approximate power law suggesting that its degree distributions is similar to that observed in real-world networks. Inspection of these LP-SLGNs suggests biological hypotheses amenable to experimental verification. CONCLUSION: A statistically robust and computationally efficient LP-based method for estimating the topology of a large sparse undirected graph from high-dimensional data yields representations of genetic networks that are biologically plausible and useful abstractions of the structures of real genetic networks. Analysis of the statistical and topological properties of learned LP-SLGNs may have practical value; for example, genes with high random walk betweenness, a measure of the centrality of a node in a graph, are good candidates for intervention studies and hence integrated computational - experimental investigations designed to infer more realistic and sophisticated probabilistic directed graphical model representations of genetic networks. The LP-based solutions of the sparse linear regression problem described here may provide a method for learning the structure of transcription factor networks from transcript profiling and transcription factor binding motif data.

Homeostatic imbalance between apoptosis and cell renewal in the liver of premature aging Xpd mice.

Unrepaired or misrepaired DNA damage has been implicated as a causal factor in cancer and aging. Xpd(TTD) mice, harboring defects in nucleotide excision repair and transcription due to a mutation in the Xpd gene (R722W), display severe symptoms of premature aging but have a reduced incidence of cancer. To gain further insight into the molecular basis of the mutant-specific manifestation of age-related phenotypes, we used comparative microarray analysis of young and old female livers to discover gene expression signatures distinguishing Xpd(TTD) mice from their age-matched wild type controls. We found a transcription signature of increased apoptosis in the Xpd(TTD) mice, which was confirmed by in situ immunohistochemical analysis and found to be accompanied by increased proliferation. However, apoptosis rate exceeded the rate of proliferation, resulting in homeostatic imbalance. Interestingly, a metabolic response signature was observed involving decreased energy metabolism and reduced IGF-1 signaling, a major modulator of life span. We conclude that while the increased apoptotic response to endogenous DNA damage contributes to the accelerated aging phenotypes and the reduced cancer incidence observed in the Xpd(TTD) mice, the signature of reduced energy metabolism is likely to reflect a compensatory adjustment to limit the increased genotoxic stress in these mutants. These results support a general model for premature aging in DNA repair deficient mice based on cellular responses to DNA damage that impair normal tissue homeostasis.

A human breast cell model of preinvasive to invasive transition.

A crucial step in human breast cancer progression is the acquisition of invasiveness. There is a distinct lack of human cell culture models to study the transition from preinvasive to invasive phenotype as it may occur "spontaneously" in vivo. To delineate molecular alterations important for this transition, we isolated human breast epithelial cell lines that showed partial loss of tissue polarity in three-dimensional reconstituted basement membrane cultures. These cells remained noninvasive; however, unlike their nonmalignant counterparts, they exhibited a high propensity to acquire invasiveness through basement membrane in culture. The genomic aberrations and gene expression profiles of the cells in this model showed a high degree of similarity to primary breast tumor profiles. The xenograft tumors formed by the cell lines in three different microenvironments in nude mice displayed metaplastic phenotypes, including squamous and basal characteristics, with invasive cells exhibiting features of higher-grade tumors. To find functionally significant changes in transition from preinvasive to invasive phenotype, we performed attribute profile clustering analysis on the list of genes differentially expressed between preinvasive and invasive cells. We found integral membrane proteins, transcription factors, kinases, transport molecules, and chemokines to be highly represented. In addition, expression of matrix metalloproteinases MMP9, MMP13, MMP15, and MMP17 was up-regulated in the invasive cells. Using small interfering RNA-based approaches, we found these MMPs to be required for the invasive phenotype. This model provides a new tool for dissection of mechanisms by which preinvasive breast cells could acquire invasiveness in a metaplastic context.

MPHASYS: a mouse phenotype analysis system.

BACKGROUND: Systematic, high-throughput studies of mouse phenotypes have been hampered by the inability to analyze individual animal data from a multitude of sources in an integrated manner. Studies generally make comparisons at the level of genotype or treatment thereby excluding associations that may be subtle or involve compound phenotypes. Additionally, the lack of integrated, standardized ontologies and methodologies for data exchange has inhibited scientific collaboration and discovery. RESULTS: Here we introduce a Mouse Phenotype Analysis System (MPHASYS), a platform for integrating data generated by studies of mouse models of human biology and disease such as aging and cancer. This computational platform is designed to provide a standardized methodology for working with animal data; a framework for data entry, analysis and sharing; and ontologies and methodologies for ensuring accurate data capture. We describe the tools that currently comprise MPHASYS, primarily ones related to mouse pathology, and outline its use in a study of individual animal-specific patterns of multiple pathology in mice harboring a specific germline mutation in the DNA repair and transcription-specific gene Xpd. CONCLUSION: MPHASYS is a system for analyzing multiple data types from individual animals. It provides a framework for developing data analysis applications, and tools for collecting and distributing high-quality data. The software is platform independent and freely available under an open-source license 1.

NuMA influences higher order chromatin organization in human mammary epithelium.

The coiled-coil protein NuMA is an important contributor to mitotic spindle formation and stabilization. A potential role for NuMA in nuclear organization or gene regulation is suggested by the observations that its pattern of nuclear distribution depends upon cell phenotype and that it interacts and/or colocalizes with transcription factors. To date, the precise contribution of NuMA to nuclear function remains unclear. Previously, we observed that antibody-induced alteration of NuMA distribution in growth-arrested and differentiated mammary epithelial structures (acini) in three-dimensional culture triggers the loss of acinar differentiation. Here, we show that in mammary epithelial cells, NuMA is present in both the nuclear matrix and chromatin compartments. Expression of a portion of the C terminus of NuMA that shares sequence similarity with the chromatin regulator HPC2 is sufficient to inhibit acinar differentiation and results in the redistribution of NuMA, chromatin markers acetyl-H4 and H4K20m, and regions of deoxyribonuclease I-sensitive chromatin compared with control cells. Short-term alteration of NuMA distribution with anti-NuMA C-terminus antibodies in live acinar cells indicates that changes in NuMA and chromatin organization precede loss of acinar differentiation. These findings suggest that NuMA has a role in mammary epithelial differentiation by influencing the organization of chromatin.

Taking U out, with two nucleases?

BACKGROUND: REX1 and REX2 are protein components of the RNA editing complex (the editosome) and function as exouridylylases. The exact roles of REX1 and REX2 in the editosome are unclear and the consequences of the presence of two related proteins are not fully understood. Here, a variety of computational studies were performed to enhance understanding of the structure and function of REX proteins in Trypanosoma and Leishmania species. RESULTS: Sequence analysis and homology modeling of the Endonuclease/Exonuclease/Phosphatase (EEP) domain at the C-terminus of REX1 and REX2 highlights a common active site shared by all EEP domains. Phylogenetic analysis indicates that REX proteins contain a distinct subfamily of EEP domains. Inspection of three-dimensional models of the EEP domain in Trypanosoma brucei REX1 and REX2, and Leishmania major REX1 suggests variations of previously characterized key residues likely to be important in catalysis and determining substrate specificity. CONCLUSION: We have identified features of the REX EEP domain that distinguish it from other family members and hence subfamily specific determinants of catalysis and substrate binding. The results provide specific guidance for experimental investigations about the role(s) of REX proteins in RNA editing.

The high binding affinity of human ribosomal protein S3 to 7,8-dihydro-8-oxoguanine is abrogated by a single amino acid change.

Previous studies have shown that human ribosomal protein S3 (hS3) has a high apparent binding affinity for 7,8-dihydro-8-oxoguanine (8-oxoG) residues in DNA and interacts with the human base excision repair (BER) proteins OGG1 and APE/Ref-1. We used a combination of computational and experimental approaches to understand the role of hS3 in BER and its potential to hinder repair of 8-oxoG lesions by OGG1 and APE/Ref-1. Sequence analysis was employed to identify hS3 residues likely to be involved in binding to 8-oxoG. One putative site, lysine 132 (K132), located in a helix-hairpin-helix DNA binding motif, was mutated to alanine (K132A). The hS3-K132A mutant retained the ability to cleave abasic DNA, but its capacity to bind 8-oxoG was abrogated completely. The ability of OGG1 to cleave an 8-oxoG-oligonucleotide substrate pre-incubated with hS3 or hS3-K132A was also tested. Pre-incubations with wild-type hS3 and 8-oxoG-containing oligonucleotides completely prevented the subsequent removal of 8-oxoG by OGG1. On the other hand, OGG1 incubations combined with hS3-K132A stimulated cleavage of 8-oxoG in excess of two-fold, confirming previous observations that hS3 positively interacts with OGG1, but only under conditions in which the binding of hS3 to 8-oxoG is limited. Overall, the ability of OGG1 to repair 8-oxoG is compromised when hS3 is bound to 8-oxoG sites. Conversely, in the absence of DNA binding, hS3 interacts positively with OGG1 to produce a more robust removal of 8-oxoG residues in DNA.

The spectrum of WRN mutations in Werner syndrome patients.

The International Registry of Werner syndrome (www.wernersyndrome.org) has been providing molecular diagnosis of the Werner syndrome (WS) for the past decade. The present communication summarizes, from among 99 WS subjects, the spectrum of 50 distinct mutations discovered by our group and by others since the WRN gene (also called RECQL2 or REQ3) was first cloned in 1996; 25 of these have not previously been published. All WRN mutations reported thus far have resulted in the elimination of the nuclear localization signal at the C-terminus of the protein, precluding functional interactions in the nucleus; thus, all could be classified as null mutations. We now report two new mutations in the N-terminus that result in instability of the WRN protein. Clinical data confirm that the most penetrant phenotype is bilateral ocular cataracts. Other cardinal signs were seen in more than 95% of the cases. The median age of death, previously reported to be in the range of 46-48 years, is 54 years. Lymphoblastoid cell lines (LCLs) have been cryopreserved from the majority of our index cases, including material from nuclear pedigrees. These, as well as inducible and complemented hTERT (catalytic subunit of human telomerase) immortalized skin fibroblast cell lines are available to qualified investigators.

Evaluation of normalization methods for cDNA microarray data by k-NN classification.

BACKGROUND: Non-biological factors give rise to unwanted variations in cDNA microarray data. There are many normalization methods designed to remove such variations. However, to date there have been few published systematic evaluations of these techniques for removing variations arising from dye biases in the context of downstream, higher-order analytical tasks such as classification. RESULTS: Ten location normalization methods that adjust spatial- and/or intensity-dependent dye biases, and three scale methods that adjust scale differences were applied, individually and in combination, to five distinct, published, cancer biology-related cDNA microarray data sets. Leave-one-out cross-validation (LOOCV) classification error was employed as the quantitative end-point for assessing the effectiveness of a normalization method. In particular, a known classifier, k-nearest neighbor (k-NN), was estimated from data normalized using a given technique, and the LOOCV error rate of the ensuing model was computed. We found that k-NN classifiers are sensitive to dye biases in the data. Using NONRM and GMEDIAN as baseline methods, our results show that single-bias-removal techniques which remove either spatial-dependent dye bias (referred later as spatial effect) or intensity-dependent dye bias (referred later as intensity effect) moderately reduce LOOCV classification errors; whereas double-bias-removal techniques which remove both spatial- and intensity effect reduce LOOCV classification errors even further. Of the 41 different strategies examined, three two-step processes, IGLOESS-SLFILTERW7, ISTSPLINE-SLLOESS and IGLOESS-SLLOESS, all of which removed intensity effect globally and spatial effect locally, appear to reduce LOOCV classification errors most consistently and effectively across all data sets. We also found that the investigated scale normalization methods do not reduce LOOCV classification error. CONCLUSION: Using LOOCV error of k-NNs as the evaluation criterion, three double-bias-removal normalization strategies, IGLOESS-SLFILTERW7, ISTSPLINE-SLLOESS and IGLOESS-SLLOESS, outperform other strategies for removing spatial effect, intensity effect and scale differences from cDNA microarray data. The apparent sensitivity of k-NN LOOCV classification error to dye biases suggests that this criterion provides an informative measure for evaluating normalization methods. All the computational tools used in this study were implemented using the R language for statistical computing and graphics.

A nuclear targeting determinant for SATB1, a genome organizer in the T cell lineage.

SATB1 is a nuclear protein, which acts as a cell-type specific genome organizer and gene regulator essential for T cell differentiation and activation. Several functional domains of SATB1 have been identified. However, the region required for nuclear localization remains unknown. To delineate this region, we employed sequence analysis to identify phylogenetically diverse members of the SATB1 protein family, and used hidden Markov model (HMM)-based analysis to define conserved regions and motifs in this family. One of the regions conserved in SATB1- and SATB2-like proteins in mammals, fish, frog and bird, is located near the N-terminus of family members. We found that the N-terminus of human SATB1 was essential for the nuclear localization of the protein. Furthermore, fusing residues 20-40 to a cytoplasmic green fluorescence protein (GFP) fused to pyruvate kinase (PK) was sufficient to quantitatively translocate the pyruvate kinase into the nucleus. The nuclear targeting sequence of human SATB1 (residues 20-40) is novel and does not contain clusters of basic residues, typically found in 'classical' nuclear localization signals (NLSs). We investigated the importance of four well-conserved residues (Lys29, Arg32, Glu34, and Asn36) in this nuclear targeting sequences. Remarkably, full-length SATB1 harboring a single point mutation at either Lys29 or Arg32, but not Glu34 or Asn36, did not enter the nucleus. Our results indicate that SATB1 N-terminal residues 20-40 represent a novel determinant of nuclear targeting.

SKI pathways inducing progression of human melanoma.

The proteins SKI and SnoN are implicated in processes as diverse as differentiation, transformation and tumor progression. Until recently, SKI was solely viewed as a nuclear protein with a principal function of inhibiting TGF-beta signaling through its association with the Smad proteins. However, new studies suggest that SKI plays additional roles not only inside but also outside the nucleus. In normal melanocytes and primary non-invasive melanomas, SKI localizes predominantly in the nucleus, whereas in primary invasive melanomas SKI displays both nuclear and cytoplasmic localization. Intriguingly, metastatic melanoma tumors display nuclear and cytoplasmic or predominantly cytoplasmic SKI distribution. Cytoplasmic SKI is functional, as it associates with Smad3 and prevents its nuclear localization mediated by TGF-beta. SKI can also function as a transcriptional activator, targeting the beta -catenin pathway and activating MITF and NrCAM, two proteins involved in survival, migration and invasion. Intriguingly, SKI appears to live a dual life, one as a tumor suppressor and another as a transforming protein. Loss of one copy of mouse ski increases susceptibility to tumorigenesis in mice, whereas its overexpression is associated with cancer progression of human melanoma, esophageal, breast and colon. The molecular reasons for such dramatic change in SKI function appear to result from new acquired activities. In this review, we discuss the mechanisms by which SKI regulates crucial pathways involved in the progression of human malignant melanoma.

The C terminus of the nuclear protein NuMA: phylogenetic distribution and structure.

The C terminus of the nuclear protein NuMA, NuMA-CT, has a well-known function in mitosis via its proximal segment, but it seems also involved in the control of differentiation. To further investigate the structure and function of NuMA, we exploited established computational techniques and tools to collate and characterize proteins with regions similar to the distal portion of NuMA-CT (NuMA-CTDP). The phylogenetic distribution of NuMA-CTDP was examined by PSI-BLAST- and TBLASTN-based analysis of genome and protein sequence databases. Proteins and open reading frames with a NuMA-CTDP-like region were found in a diverse set of vertebrate species including mammals, birds, amphibia, and early teleost fish. The potential structure of NuMA-CTDP was investigated by searching a database of protein sequences of known three-dimensional structure with a hidden Markov model (HMM) estimated using representative (human, frog, chicken, and pufferfish) sequences. The two highest scoring sequences that aligned to the HMM were the extracellular domains of beta3-integrin and Her2, suggesting that NuMA-CTDP may have a primarily beta fold structure. These data indicate that NuMA-CTDP may represent an important functional sequence conserved in vertebrates, where it may act as a receptor to coordinate cellular events.

Exploratory differential gene expression analysis in microarray experiments with no or limited replication.

We describe an exploratory, data-oriented approach for identifying candidates for differential gene expression in cDNA microarray experiments in terms of alpha-outliers and outlier regions, using simultaneous tolerance intervals relative to the line of equivalence (Cy5 = Cy3). We demonstrate the improved performance of our approach over existing single-slide methods using public datasets and simulation studies.

A conserved telomerase motif within the catalytic domain of telomerase reverse transcriptase is specifically required for repeat addition processivity.

Telomerase is a ribonucleoprotein reverse transcriptase responsible for the maintenance of one strand of the telomere terminal repeats. The catalytic protein subunit of the telomerase complex, known as TERT, possesses a reverse transcriptase (RT) domain that mediates nucleotide addition. The RT domain of TERT is distinguishable from retroviral and retrotransposon RTs in having a sizable insertion between conserved motifs A and B', within the so-called fingers domain. Sequence analysis revealed the existence of conserved residues in this region, named IFD (insertion in fingers domain). Mutations of some of the conserved residues in Saccharomyces cerevisiae TERT (Est2p) abolished telomerase function in vivo, testifying to their importance. Significant effects of the mutations on telomerase activity in vitro were observed, with most of the mutants exhibiting a uniform reduction in activity regardless of primer sequence. Remarkably, one mutant manifested a primer-specific defect, being selectively impaired in extending primers that form short hybrids with telomerase RNA. This mutant also accumulated products that correspond to one complete round of repeat synthesis, implying an inability to effect the repositioning of the DNA product relative to the RNA template that is necessary for multiple repeat addition. Our results suggest that the ability to stabilize short RNA-DNA hybrids is crucial for telomerase function in vivo and that this ability is mediated in part by a more elaborate fingers domain structure.

Comparative analysis of editosome proteins in trypanosomatids.

Detailed comparisons of 16 editosome proteins from Trypanosoma brucei, Trypanosoma cruzi and Leishmania major identified protein motifs associated with catalysis and protein or nucleic acid interactions that suggest their functions in RNA editing. Five related proteins with RNase III-like motifs also contain a U1-like zinc finger and either dsRBM or Pumilio motifs. These proteins may provide the endoribonuclease function in editing. Two other related proteins, at least one of which is associated with U-specific 3' exonuclease activity, contain two putative nuclease motifs. Thus, editosomes contain a plethora of nucleases or proteins presumably derived from nucleases. Five additional related proteins, three of which have zinc fingers, each contain a motif associated with an OB fold; the TUTases have C-terminal folds reminiscent of RNA binding motifs, thus indicating the presence of numerous nucleic acid and/or protein binding domains, as do the two RNA ligases and a RNA helicase, which provide for additional catalytic steps in editing. These data indicate that trypanosomatid RNA editing is orchestrated by a variety of domains for catalysis, molecular interaction and structure. These domains are generally conserved within other protein families, but some are found in novel combinations in the editosome proteins.

LMNA mutations in atypical Werner's syndrome.

BACKGROUND: Werner's syndrome is a progeroid syndrome caused by mutations at the WRN helicase locus. Some features of this disorder are also present in laminopathies caused by mutant LMNA encoding nuclear lamin A/C. Because of this similarity, we sequenced LMNA in individuals with atypical Werner's syndrome (wild-type WRN). METHODS: Of 129 index patients referred to our international registry for molecular diagnosis of Werner's syndrome, 26 (20%) had wildtype WRN coding regions and were categorised as having atypical Werner's syndrome on the basis of molecular criteria. We sequenced all exons of LMNA in these individuals. Mutations were confirmed at the mRNA level by RT-PCR sequencing. In one patient in whom an LMNA mutation was detected and fibroblasts were available, we established nuclear morphology and subnuclear localisation. FINDINGS: In four (15%) of 26 patients with atypical Werner's syndrome, we noted heterozygosity for novel missense mutations in LMNA, specifically A57P, R133L (in two people), and L140R. The mutations altered relatively conserved residues within lamin A/C. Fibroblasts from the patient with the L140R mutation had a substantially enhanced proportion of nuclei with altered morphology and mislocalised lamins. Individuals with atypical Werner's syndrome with mutations in LMNA had a more severe phenotype than did those with the disorder due to mutant WRN. INTERPRETATION: Our findings indicate that Werner's syndrome is molecularly heterogeneous, and a subset of the disorder can be judged a laminopathy.

Conserved N-terminal motifs of telomerase reverse transcriptase required for ribonucleoprotein assembly in vivo.

Telomerase is a ribonucleoprotein (RNP) reverse transcriptase responsible for the maintenance of one strand of the telomere terminal repeats. The key protein subunit of the telomerase complex, known as TERT, possesses reverse transcriptase (RT)-like motifs that directly mediate nucleotide addition. The RT motifs are located in the C-terminal region of the polypeptide. Sequence alignments also revealed the existence of four conserved motifs (named GQ, CP, QFP, and T) in the N-terminal region of TERT. The GQ motif of yeast TERT has been demonstrated previously to be essential for telomerase catalysis and may participate in RNP formation. In this report, we show that substitution of conserved residues in the CP, QFP, and T motifs of yeast TERT also impairs both telomere maintenance and telomerase activity, thus confirming the validity of the sequence alignment. The extent of telomere shortening correlates with the extent of reduction in the level of telomerase activity, TERT protein, and TERT-associated TLC1 RNA. Overexpression of the mutant proteins does not result in telomere shortening, implying that assembly rather than catalytic function was affected. This notion was further supported by comparing the efficiency of RNP formation in the wild type and the overexpression strains. Taken together, our results show that three of the four N-terminal motifs are required for efficient telomerase RNP formation in vivo but not for the enzymatic function of telomerase. We also show that the majority of telomerase-associated TLC1 RNA has a more upstream 3' end than previously reported, consistent with additional processing events during RNP maturation.