Owen J. Marshall

PerlPrimer: cross-platform, graphical primer design for standard, bisulphite and real-time PCR

UNLABELLED PerlPrimer is a cross-platform graphical user interface application for the design of primers for standard, bisulphite and real-time PCR, and sequencing. The program incorporates highly accurate melting-temperature and primer-dimer prediction algorithms with powerful tools such as sequence retrieval from Ensembl and the ability to BLAST search primer pairs. It aims to automate and simplify the process of primer design. AVAILABILITY Open-source and freely available from http://perlprimer.sourceforge.net.

Neocentromeres: New Insights into Centromere Structure, Disease Development, and Karyotype Evolution

Since the discovery of the first human neocentromere in 1993, these spontaneous, ectopic centromeres have been shown to be an astonishing example of epigenetic change within the genome. Recent research has focused on the role of neocentromeres in evolution and speciation, as well as in disease development and the understanding of the organization and epigenetic maintenance of the centromere. Here, we review recent progress in these areas of research and the significant insights gained.

Active transcription and essential role of RNA polymerase II at the centromere during mitosis

Transcription of the centromeric regions has been reported to occur in G1 and S phase in different species. Here, we investigate whether transcription also occurs and plays a functional role at the mammalian centromere during mitosis. We show the presence of actively transcribing RNA polymerase II (RNAPII) and its associated transcription factors, coupled with the production of centromere satellite transcripts at the mitotic kinetochore. Specific inhibition of RNAPII activity during mitosis leads to a decrease in centromeric α-satellite transcription and a concomitant increase in anaphase-lagging cells, with the lagging chromosomes showing reduced centromere protein C binding. These findings demonstrate an essential role of RNAPII in the transcription of α-satellite DNA, binding of centromere protein C, and the proper functioning of the mitotic kinetochore.

Contrasting roles of condensin I and condensin II in mitotic chromosome formation

In vertebrates, two condensin complexes exist, condensin I and condensin II, which have differing but unresolved roles in organizing mitotic chromosomes. To dissect accurately the role of each complex in mitosis, we have made and studied the first vertebrate conditional knockouts of the genes encoding condensin I subunit CAP-H and condensin II subunit CAP-D3 in chicken DT40 cells. Live-cell imaging reveals highly distinct segregation defects. CAP-D3 (condensin II) knockout results in masses of chromatin-containing anaphase bridges. CAP-H (condensin I)-knockout anaphases have a more subtle defect, with chromatids showing fine chromatin fibres that are associated with failure of cytokinesis and cell death. Super-resolution microscopy reveals that condensin-I-depleted mitotic chromosomes are wider and shorter, with a diffuse chromosome scaffold, whereas condensin-II-depleted chromosomes retain a more defined scaffold, with chromosomes more stretched and seemingly lacking in axial rigidity. We conclude that condensin II is required primarily to provide rigidity by establishing an initial chromosome axis around which condensin I can arrange loops of chromatin.

Cell-type-specific profiling of gene expression and chromatin binding without cell isolation: assaying RNA Pol II occupancy in neural stem cells

Summary Cell-type-specific transcriptional profiling often requires the isolation of specific cell types from complex tissues. We have developed “TaDa,” a technique that enables cell-specific profiling without cell isolation. TaDa permits genome-wide profiling of DNA- or chromatin-binding proteins without cell sorting, fixation, or affinity purification. The method is simple, sensitive, highly reproducible, and transferable to any model system. We show that TaDa can be used to identify transcribed genes in a cell-type-specific manner with considerable temporal precision, enabling the identification of differential gene expression between neuroblasts and the neuroepithelial cells from which they derive. We profile the genome-wide binding of RNA polymerase II in these adjacent, clonally related stem cells within intact Drosophila brains. Our data reveal expression of specific metabolic genes in neuroepithelial cells, but not in neuroblasts, and highlight gene regulatory networks that may pattern neural stem cell fates.

The Architecture and Evolution of Cancer Neochromosomes

We isolated and analyzed, at single-nucleotide resolution, cancer-associated neochromosomes from well- and/or dedifferentiated liposarcomas. Neochromosomes, which can exceed 600 Mb in size, initially arise as circular structures following chromothripsis involving chromosome 12. The core of the neochromosome is amplified, rearranged, and corroded through hundreds of breakage-fusion-bridge cycles. Under selective pressure, amplified oncogenes are overexpressed, while coamplified passenger genes may be silenced epigenetically. New material may be captured during punctuated chromothriptic events. Centromeric corrosion leads to crisis, which is resolved through neocentromere formation or native centromere capture. Finally, amplification terminates, and the neochromosome core is stabilized in linear form by telomere capture. This study investigates the dynamic mutational processes underlying the life history of a special form of cancer mutation.

Three-dimensional localization of CENP-A suggests a complex higher order structure of centromeric chromatin

The histone H3 variant centromere protein A (CENP-A) is central to centromere formation throughout eukaryotes. A long-standing question in centromere biology has been the organization of CENP-A at the centromere and its implications for the structure of centromeric chromatin. In this study, we describe the three-dimensional localization of CENP-A at the inner kinetochore plate through serial-section transmission electron microscopy of human mitotic chromosomes. At the kinetochores of normal centromeres and at a neocentromere, CENP-A occupies a compact domain at the inner kinetochore plate, stretching across two thirds of the length of the constriction but encompassing only one third of the constriction width and height. Within this domain, evidence of substructure is apparent. Combined with previous chromatin immunoprecipitation results (Saffery, R., H. Sumer, S. Hassan, L.H. Wong, J.M. Craig, K. Todokoro, M. Anderson, A. Stafford, and K.H.A. Choo. 2003. Mol. Cell. 12:509–516; Chueh, A.C., L.H. Wong, N. Wong, and K.H.A. Choo. 2005. Hum. Mol. Genet. 14:85–93), our data suggest that centromeric chromatin is arranged in a coiled 30-nm fiber that is itself coiled or folded to form a higher order structure.

Identification of an interaction between SOX9 and HSP70

The campomelic dysplasia/autosomal sex reversal protein SOX9 is an important developmental transcription factor, required for correct bone and testis formation. Through in vitro and in vivo studies we have identified the heat shock protein HSP70 as an interacting partner for SOX9 in chondrocyte and testicular cell lines. HSP70 forms a ternary complex with DNA‐bound SOX9. The interaction between HSP70 and SOX9 is ATP‐independent and involves a highly conserved region of SOX9 hitherto of unknown function and the C‐terminal region of HSP70. Our results implicate HSP70–SOX9 interactions in the assembly of multi‐protein complexes during SOX9‐mediated transcription.

damidseq_pipeline: an automated pipeline for processing DamID sequencing datasets

Summary: DamID is a powerful technique for identifying regions of the genome bound by a DNA-binding (or DNA-associated) protein. Currently, no method exists for automatically processing next-generation sequencing DamID (DamID-seq) data, and the use of DamID-seq datasets with normalization based on read-counts alone can lead to high background and the loss of bound signal. DamID-seq thus presents novel challenges in terms of normalization and background minimization. We describe here damidseq_pipeline, a software pipeline that performs automatic normalization and background reduction on multiple DamID-seq FASTQ datasets. Availability and implementation: Open-source and freely available from http://owenjm.github.io/damidseq_pipeline. The damidseq_pipeline is implemented in Perl and is compatible with any Unix-based operating system (e.g. Linux, Mac OSX). Contact: o.marshall@gurdon.cam.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online.

Graphical design of primers with PerlPrimer.

PerlPrimer is a cross-platform application for the design of standard PCR, bisulfite PCR, real-time PCR, and sequencing primers. The program combines accurate primer-design algorithms with powerful interfaces to commonly used Internet databases, such as sequence retrieval from the Ensembl genome databases and the ability to BLAST search primer pairs. The use of PerlPrimer for designing primers is described, together with a synopsis of the primer search and primer-dimer algorithms.