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1.  CDK-mediated activation of the SCFFBXO28 ubiquitin ligase promotes MYC-driven transcription and tumourigenesis and predicts poor survival in breast cancer 
EMBO Molecular Medicine  2013;5(7):999-1018.
SCF (Skp1/Cul1/F-box) ubiquitin ligases act as master regulators of cellular homeostasis by targeting key proteins for ubiquitylation. Here, we identified a hitherto uncharacterized F-box protein, FBXO28 that controls MYC-dependent transcription by non-proteolytic ubiquitylation. SCFFBXO28 activity and stability are regulated during the cell cycle by CDK1/2-mediated phosphorylation of FBXO28, which is required for its efficient ubiquitylation of MYC and downsteam enhancement of the MYC pathway. Depletion of FBXO28 or overexpression of an F-box mutant unable to support MYC ubiquitylation results in an impairment of MYC-driven transcription, transformation and tumourigenesis. Finally, in human breast cancer, high FBXO28 expression and phosphorylation are strong and independent predictors of poor outcome. In conclusion, our data suggest that SCFFBXO28 plays an important role in transmitting CDK activity to MYC function during the cell cycle, emphasizing the CDK-FBXO28-MYC axis as a potential molecular drug target in MYC-driven cancers, including breast cancer.
doi:10.1002/emmm.201202341
PMCID: PMC3721474  PMID: 23776131
Breast cancer; CDK; F-box protein; FBXO28; MYC
2.  Library Preparation and Multiplex Capture for Massive Parallel Sequencing Applications Made Efficient and Easy 
PLoS ONE  2012;7(11):e48616.
During the recent years, rapid development of sequencing technologies and a competitive market has enabled researchers to perform massive sequencing projects at a reasonable cost. As the price for the actual sequencing reactions drops, enabling more samples to be sequenced, the relative price for preparing libraries gets larger and the practical laboratory work becomes complex and tedious. We present a cost-effective strategy for simplified library preparation compatible with both whole genome- and targeted sequencing experiments. An optimized enzyme composition and reaction buffer reduces the number of required clean-up steps and allows for usage of bulk enzymes which makes the whole process cheap, efficient and simple. We also present a two-tagging strategy, which allows for multiplex sequencing of targeted regions. To prove our concept, we have prepared libraries for low-pass sequencing from 100 ng DNA, performed 2-, 4- and 8-plex exome capture and a 96-plex capture of a 500 kb region. In all samples we see a high concordance (>99.4%) of SNP calls when comparing to commercially available SNP-chip platforms.
doi:10.1371/journal.pone.0048616
PMCID: PMC3489721  PMID: 23139805
4.  Antibody-based Protein Profiling of the Human Chromosome 21* 
Molecular & Cellular Proteomics : MCP  2011;11(3):M111.013458.
The Human Proteome Project has been proposed to create a knowledge-based resource based on a systematical mapping of all human proteins, chromosome by chromosome, in a gene-centric manner. With this background, we here describe the systematic analysis of chromosome 21 using an antibody-based approach for protein profiling using both confocal microscopy and immunohistochemistry, complemented with transcript profiling using next generation sequencing data. We also describe a new approach for protein isoform analysis using a combination of antibody-based probing and isoelectric focusing. The analysis has identified several genes on chromosome 21 with no previous evidence on the protein level, and the isoform analysis indicates that a large fraction of human proteins have multiple isoforms. A chromosome-wide matrix is presented with status for all chromosome 21 genes regarding subcellular localization, tissue distribution, and molecular characterization of the corresponding proteins. The path to generate a chromosome-specific resource, including integrated data from complementary assay platforms, such as mass spectrometry and gene tagging analysis, is discussed.
doi:10.1074/mcp.M111.013458
PMCID: PMC3316724  PMID: 22042635
5.  Defining the transcriptome and proteome in three functionally different human cell lines 
An essential question in human biology is how cells and tissues differ in gene and protein expression and how these differences delineate specific biological function. Here, we have performed a global analysis of both mRNA and protein levels based on sequence-based transcriptome analysis (RNA-seq), SILAC-based mass spectrometry analysis and antibody-based confocal microscopy. The study was performed in three functionally different human cell lines and based on the global analysis, we estimated the fractions of mRNA and protein that are cell specific or expressed at similar/different levels in the cell lines. A highly ubiquitous RNA expression was found with >60% of the gene products detected in all cells. The changes of mRNA and protein levels in the cell lines using SILAC and RNA ratios show high correlations, even though the genome-wide dynamic range is substantially higher for the proteins as compared with the transcripts. Large general differences in abundance for proteins from various functional classes are observed and, in general, the cell-type specific proteins are low abundant and highly enriched for cell-surface proteins. Thus, this study shows a path to characterize the transcriptome and proteome in human cells from different origins.
doi:10.1038/msb.2010.106
PMCID: PMC3018165  PMID: 21179022
cell lines; expression; human; proteome; transcriptome
6.  Analysis of transcript and protein overlap in a human osteosarcoma cell line 
BMC Genomics  2010;11:684.
Background
An interesting field of research in genomics and proteomics is to compare the overlap between the transcriptome and the proteome. Recently, the tools to analyse gene and protein expression on a whole-genome scale have been improved, including the availability of the new generation sequencing instruments and high-throughput antibody-based methods to analyze the presence and localization of proteins. In this study, we used massive transcriptome sequencing (RNA-seq) to investigate the transcriptome of a human osteosarcoma cell line and compared the expression levels with in situ protein data obtained in-situ from antibody-based immunohistochemistry (IHC) and immunofluorescence microscopy (IF).
Results
A large-scale analysis based on 2749 genes was performed, corresponding to approximately 13% of the protein coding genes in the human genome. We found the presence of both RNA and proteins to a large fraction of the analyzed genes with 60% of the analyzed human genes detected by all three methods. Only 34 genes (1.2%) were not detected on the transcriptional or protein level with any method. Our data suggest that the majority of the human genes are expressed at detectable transcript or protein levels in this cell line. Since the reliability of antibodies depends on possible cross-reactivity, we compared the RNA and protein data using antibodies with different reliability scores based on various criteria, including Western blot analysis. Gene products detected in all three platforms generally have good antibody validation scores, while those detected only by antibodies, but not by RNA sequencing, generally consist of more low-scoring antibodies.
Conclusion
This suggests that some antibodies are staining the cells in an unspecific manner, and that assessment of transcript presence by RNA-seq can provide guidance for validation of the corresponding antibodies.
doi:10.1186/1471-2164-11-684
PMCID: PMC3014981  PMID: 21126332
7.  Increased Throughput by Parallelization of Library Preparation for Massive Sequencing 
PLoS ONE  2010;5(4):e10029.
Background
Massively parallel sequencing systems continue to improve on data output, while leaving labor-intensive library preparations a potential bottleneck. Efforts are currently under way to relieve the crucial and time-consuming work to prepare DNA for high-throughput sequencing.
Methodology/Principal Findings
In this study, we demonstrate an automated parallel library preparation protocol using generic carboxylic acid-coated superparamagnetic beads and polyethylene glycol precipitation as a reproducible and flexible method for DNA fragment length separation. With this approach the library preparation for DNA sequencing can easily be adjusted to a desired fragment length. The automated protocol, here demonstrated using the GS FLX Titanium instrument, was compared to the standard manual library preparation, showing higher yield, throughput and great reproducibility. In addition, 12 libraries were prepared and uniquely tagged in parallel, and the distribution of sequence reads between these indexed samples could be improved using quantitative PCR-assisted pooling.
Conclusions/Significance
We present a novel automated procedure that makes it possible to prepare 36 indexed libraries per person and day, which can be increased to up to 96 libraries processed simultaneously. The yield, speed and robust performance of the protocol constitute a substantial improvement to present manual methods, without the need of extensive equipment investments. The described procedure enables a considerable efficiency increase for small to midsize sequencing centers.
doi:10.1371/journal.pone.0010029
PMCID: PMC2850305  PMID: 20386591
8.  In-Depth Transcriptome Analysis Reveals Novel TARs and Prevalent Antisense Transcription in Human Cell Lines 
PLoS ONE  2010;5(3):e9762.
Several recent studies have indicated that transcription is pervasive in regions outside of protein coding genes and that short antisense transcripts can originate from the promoter and terminator regions of genes. Here we investigate transcription of fragments longer than 200 nucleotides, focusing on antisense transcription for known protein coding genes and intergenic transcription. We find that roughly 12% to 16% of all reads that originate from promoter and terminator regions, respectively, map antisense to the gene in question. Furthermore, we detect a high number of novel transcriptionally active regions (TARs) that are generally expressed at a lower level than protein coding genes. We find that the correlation between RNA-seq data and microarray data is dependent on the gene length, with longer genes showing a better correlation. We detect high antisense transcriptional activity from promoter, terminator and intron regions of protein-coding genes and identify a vast number of previously unidentified TARs, including putative novel EGFR transcripts. This shows that in-depth analysis of the transcriptome using RNA-seq is a valuable tool for understanding complex transcriptional events. Furthermore, the development of new algorithms for estimation of gene expression from RNA-seq data is necessary to minimize length bias.
doi:10.1371/journal.pone.0009762
PMCID: PMC2845605  PMID: 20360838
9.  Genome-wide profiling of Populus small RNAs 
BMC Genomics  2009;10:620.
Background
Short RNAs, and in particular microRNAs, are important regulators of gene expression both within defined regulatory pathways and at the epigenetic scale. We investigated the short RNA (sRNA) population (18-24 nt) of the transcriptome of green leaves from the sequenced Populus trichocarpa using a concatenation strategy in combination with 454 sequencing.
Results
The most abundant size class of sRNAs were 24 nt. Long Terminal Repeats were particularly associated with 24 nt sRNAs. Additionally, some repetitive elements were associated with 22 nt sRNAs. We identified an sRNA hot-spot on chromosome 19, overlapping a region containing both the proposed sex-determining locus and a major cluster of NBS-LRR genes. A number of phased siRNA loci were identified, a subset of which are predicted to target PPR and NBS-LRR disease resistance genes, classes of genes that have been significantly expanded in Populus. Additional loci enriched for sRNA production were identified and characterised. We identified 15 novel predicted microRNAs (miRNAs), including miRNA*sequences, and identified a novel locus that may encode a dual miRNA or a miRNA and short interfering RNAs (siRNAs).
Conclusions
The short RNA population of P. trichocarpa is at least as complex as that of Arabidopsis thaliana. We provide a first genome-wide view of short RNA production for P. trichocarpa and identify new, non-conserved miRNAs.
doi:10.1186/1471-2164-10-620
PMCID: PMC2811130  PMID: 20021695
10.  Automation of cDNA Synthesis and Labelling Improves Reproducibility 
Background. Several technologies, such as in-depth sequencing and microarrays, enable large-scale interrogation of genomes and transcriptomes. In this study, we asses reproducibility and throughput by moving all laboratory procedures to a robotic workstation, capable of handling superparamagnetic beads. Here, we describe a fully automated procedure for cDNA synthesis and labelling for microarrays, where the purification steps prior to and after labelling are based on precipitation of DNA on carboxylic acid-coated paramagnetic beads. Results. The fully automated procedure allows for samples arrayed on a microtiter plate to be processed in parallel without manual intervention and ensuring high reproducibility. We compare our results to a manual sample preparation procedure and, in addition, use a comprehensive reference dataset to show that the protocol described performs better than similar manual procedures. Conclusions. We demonstrate, in an automated gene expression microarray experiment, a reduced variance between replicates, resulting in an increase in the statistical power to detect differentially expressed genes, thus allowing smaller differences between samples to be identified. This protocol can with minor modifications be used to create cDNA libraries for other applications such as in-depth analysis using next-generation sequencing technologies.
doi:10.1155/2009/396808
PMCID: PMC2763127  PMID: 19841682

Results 1-10 (10)