Identification of a novel type of spacer element required for imprinting in fission yeast

Asymmetrical segregation of differentiated sister chromatids is thought to be important for cellular differentiation in higher eukaryotes. Similarly, in fission yeast, cellular differentiation involves the asymmetrical segregation of a chromosomal imprint. This imprint has been shown to consist of two ribonucleotides that are incorporated into the DNA during laggingstrand synthesis in response to a replication pause, but the underlying mechanism remains unknown. Here we present key novel discoveries important for unravelling this process. Our data show that cis-acting sequences within the mat1 cassette mediate pausing of replication forks at the proximity of the imprinting site, and the results suggest that this pause dictates specific priming at the position of imprinting in a sequence-independent manner. Also, we identify a novel type of cis-acting spacer region important for the imprinting process that affects where subsequent primers are put down after the replication fork is released from the pause. Thus, our data suggest that the imprint is formed by ligation of a not-fullyprocessed Okazaki fragment to the subsequent fragment. The presented work addresses how differentiated sister chromatids are established during DNA replication through the involvement of replication barriers

Fusion genes in breast cancer

Fusion genes caused by chromosomal rearrangements are a common and important feature in haematological malignancies, but have until recently been seen as unimportant in epithelial cancers. The discovery of recurrent fusion genes in prostate and lung cancer suggests that fusion genes may play an important role in epithelial carcinogenesis, and that they have been previously under-reported due to the difficulties of cytogenetic analysis of solid tumours. In particular, breast cancers often have complex, highly rearranged karyotypes which have proved difficult to analyse using classical cytogenetic techniques. The aim of this project was to search for fusion genes in breast cancer by using high-resolution mapping of chromosome rearrangements in breast cancer cell lines. Mapping the chromosome rearrangements was initially done using high-resolution DNA microarrays and fluorescence in- situ hybridisation, but moved to high-throughput sequencing as it became available. Interesting candidate genes identified from the mapped chromosome rearrangements were investigated on a larger set of cell lines and primary tumours. The complete karyotypes of two breast cancer cell lines were constructed using a combination of microarrays, fluorescence microscopy, and high-throughput sequencing. A number of potential fusion genes were identified in these two cell lines. Although no expressed fusion genes were found, the complete karyotypes gave insight into the number and mechanisms of chromosome rearrangement in breast cancer, and identified interesting candidate genes which may be of importance in tumourigenesis. Two genes which were fused in other breast cancer cell lines, BCAS3 and ODZ4, were disrupted by chromosome rearrangements and identified as interesting candidate genes in tumorigenesis. A bioinformatic pipeline to process high-throughput sequencing data was set up and validated, and shown to more accurately predict fusion genes than other methods, and can be used to investigate further cell lines and tumours for recurrent fusion genes. The pipeline was used to analyse data from 3 other breast cancer cell lines and predict chromosomal rearrangements and fusion genes, several of which were found to be expressed. Of the fusions predicted in the cell line ZR-75-30, 7 expressed fusion genes were identified, and may have functional significance in breast cancer.This work was supported by a grant from Breast Cancer Campaign

Visualizing chromosomal rearrangements caused by replication fork stalling in a single cell

Aberrant chromosome structures can promote tumors in the early stages of carcinogenesis and lead to tumor cells becoming resistant to chemotherapy, for example by changing in drug metabolism. Dicentric (containing two centromeres) and acentric (containing no centromeres) chromosomes are two abnormal chromosome structures that consider as precursors of a variety of gross chromosomal rearrangements (GCRs) generated by subsequent recombination events [1-7]. However, the mechanism of the dicentric and acentric palindromic chromosome formation and their subsequent metabolism is difficult to directly visualise. The previous results from our lab shows that replication forks stalled at a specific replication termination sequence (RTS1) can result in the formation of the dicentric and acentric palindromic chromosomes in the fission yeast Schizosaccharomyces pombe [48-52]. However, the formation of acentric and dicentric chromosomes results in a significant visability loss, due to instability and miss-segregation of the chromosomes in the yeast cells. Thus, their fate is difficult or impossible to follow. To resolve this problem, a non-essential mini-chromosome (Ch16) was developed as a novel model system in this project. The behaviour of rearranged chromosome in vivo and their subsequent fate have been visualised by integrating the lac operator (lacO) and tetracycline operator (tetO) arrays with auxotropic makers, adjacent to the RTSI locus on Ch16. The results reveal imbalanced segregation of a dicentric chromosome and subsequently undergoes a breakage event. An acentric chromosome appears to be decoupled or lost rapidly from the nucleus

A locally adaptive statistical procedure (LAP) to identify differentially expressed chromosomal regions

Abstract Motivation: The systematic integration of expression profiles and other types of gene information, such as chromosomal localization, ontological annotations and sequence characteristics, still represents a challenge in the gene expression arena. In particular, the analysis of transcriptional data in context of the physical location of genes in a genome appears promising in detecting chromosomal regions with transcriptional imbalances often characterizing cancer. Results: A computational tool named locally adaptive statistical procedure (LAP), which incorporates transcriptional data and structural information for the identification of differentially expressed chromosomal regions, is described. LAP accounts for variations in the distance between genes and in gene density by smoothing standard statistics on gene position before testing the significance of their differential levels of gene expression. The procedure smoothes parameters and computes p-values locally to account for the complex structure of the genome and to more precisely estimate the differential expression of chromosomal regions. The application of LAP to three independent sets of raw expression data allowed identifying differentially expressed regions that are directly involved in known chromosomal aberrations characteristic of tumors. Availability: Functions in R for implementing the LAP method are available at Contact: [email protected] Supplementary Information

GeneChip analyses point to novel pathogenetic mechanisms in mantle cell lymphoma

The translocation t(11;14)(q13;q32) is the genetic hallmark of mantle cell lymphoma (MCL) but is not sufficient for inducing lymphomagenesis. Here we performed genome-wide 100K GeneChip Mapping in 26 t(11;14)-positive MCL and six MCL cell lines. Partial uniparental disomy (pUPD) was shown to be a recurrent chromosomal event not only in MCL cell lines but also in primary MCL. Remarkably, pUPD affected recurrent targets of deletion like 11q, 13q and 17p. Moreover, we identified 12 novel regions of recurrent gain and loss as well as 12 high-level amplifications and eight homozygously deleted regions hitherto undescribed in MCL. Interestingly, GeneChip analyses identified different genes, encoding proteins involved in microtubule dynamics, such as MAP2, MAP6 and TP53, as targets for chromosomal aberration in MCL. Further investigation, including mutation analyses, fluorescence in situ hybridisation as well as epigenetic and expression studies, revealed additional aberrations frequently affecting these genes. In total, 19 of 20 MCL cases, which were subjected to genetic and epigenetic analyses, and five of six MCL cell lines harboured at least one aberration in MAP2, MAP6 or TP53. These findings provide evidence that alterations of microtubule dynamics might be one of the critical events in MCL lymphomagenesis contributing to chromosomal instability

Transposon variants and their effects on gene expression in arabidopsis

Transposable elements (TEs) make up the majority of many plant genomes. Their transcription and transposition is controlled through siRNAs and epigenetic marks including DNA methylation. To dissect the interplay of siRNA–mediated regulation and TE evolution, and to examine how TE differences affect nearby gene expression, we investigated genome-wide differences in TEs, siRNAs, and gene expression among three Arabidopsis thaliana accessions. Both TE sequence polymorphisms and presence of linked TEs are positively correlated with intraspecific variation in gene expression. The expression of genes within 2 kb of conserved TEs is more stable than that of genes next to variant TEs harboring sequence polymorphisms. Polymorphism levels of TEs and closely linked adjacent genes are positively correlated as well. We also investigated the distribution of 24-nt-long siRNAs, which mediate TE repression. TEs targeted by uniquely mapping siRNAs are on average farther from coding genes, apparently because they more strongly suppress expression of adjacent genes. Furthermore, siRNAs, and especially uniquely mapping siRNAs, are enriched in TE regions missing in other accessions. Thus, targeting by uniquely mapping siRNAs appears to promote sequence deletions in TEs. Overall, our work indicates that siRNA–targeting of TEs may influence removal of sequences from the genome and hence evolution of gene expression in plants

Centromere-associated topoisomerase activity in bloodstream form Trypanosoma brucei

Topoisomerase-II accumulates at centromeres during prometaphase, where it resolves the DNA catenations that represent the last link between sister chromatids. Previously, using approaches including etoposide-mediated topoisomerase-II cleavage, we mapped centromeric domains in trypanosomes, early branching eukaryotes in which chromosome segregation is poorly understood. Here, we show that in bloodstream form Trypanosoma brucei, RNAi-mediated depletion of topoisomerase-IIα, but not topoisomerase-IIβ, results in the abolition of centromere-localized activity and is lethal. Both phenotypes can be rescued by expression of the corresponding enzyme from T. cruzi. Therefore, processes which govern centromere-specific topoisomerase-II accumulation/activation have been functionally conserved within trypanosomes, despite the long evolutionary separation of these species and differences in centromeric DNA organization. The variable carboxyl terminal region of topoisomerase-II has a major role in regulating biological function. We therefore generated T. brucei lines expressing T. cruzi topoisomerase-II truncated at the carboxyl terminus and examined activity at centromeres after the RNAi-mediated depletion of the endogenous enzyme. A region necessary for nuclear localization was delineated to six residues. In other organisms, sumoylation of topoisomerase-II has been shown to be necessary for regulated chromosome segregation. Evidence that we present here suggests that sumoylation of the T. brucei enzyme is not required for centromere-specific cleavage activity