Publications by authors named "Philip Lijnzaad"

23 Publications

  • Page 1 of 1

LGR6 marks nephron progenitor cells.

Dev Dyn 2021 Apr 13. Epub 2021 Apr 13.

Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands.

Background: Nephron progenitor cells (NPCs) undergo a stepwise process to generate all mature nephron structures. Mesenchymal to epithelial transition (MET) is considered a multistep process of NPC differentiation to ensure progressive establishment of new nephrons. However, despite this important role, to date, no marker for NPCs undergoing MET in the nephron exists.

Results: Here, we identify LGR6 as a NPC marker, expressed in very early cap mesenchyme, pre-tubular aggregates, renal vesicles, and in segments of S-shaped bodies, following the trajectory of MET. By using a lineage tracing approach in embryonic explants in combination with confocal imaging and single-cell RNA sequencing, we provide evidence for the multiple fates of LGR6+ cells during embryonic nephrogenesis. Moreover, by using long-term in vivo lineage tracing, we show that postnatal LGR6+ cells are capable of generating the multiple lineages of the nephrons.

Conclusions: Given the profound early mesenchymal expression and MET signature of LGR6 cells, together with the lineage tracing of mesenchymal LGR6 cells, we conclude that LGR6+ cells contribute to all nephrogenic segments by undergoing MET. LGR6+ cells can therefore be considered an early committed NPC population during embryonic and postnatal nephrogenesis with potential regenerative capability.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/dvdy.346DOI Listing
April 2021

Tumor to normal single-cell mRNA comparisons reveal a pan-neuroblastoma cancer cell.

Sci Adv 2021 Feb 5;7(6). Epub 2021 Feb 5.

Great Ormond Street Hospital for Children (GOSH), NHS Foundation Trust, NIHR Great Ormond Street Hospital Biomedical Research Centre, WC1N 3JH London, UK.

Neuroblastoma is a childhood cancer that resembles developmental stages of the neural crest. It is not established what developmental processes neuroblastoma cancer cells represent. Here, we sought to reveal the phenotype of neuroblastoma cancer cells by comparing cancer ( = 19,723) with normal fetal adrenal single-cell transcriptomes ( = 57,972). Our principal finding was that the neuroblastoma cancer cell resembled fetal sympathoblasts, but no other fetal adrenal cell type. The sympathoblastic state was a universal feature of neuroblastoma cells, transcending cell cluster diversity, individual patients, and clinical phenotypes. We substantiated our findings in 650 neuroblastoma bulk transcriptomes and by integrating canonical features of the neuroblastoma genome with transcriptional signals. Overall, our observations indicate that a pan-neuroblastoma cancer cell state exists, which may be attractive for novel immunotherapeutic and targeted avenues.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/sciadv.abd3311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7864567PMC
February 2021

Genome-wide off-rates reveal how DNA binding dynamics shape transcription factor function.

Mol Syst Biol 2020 10;16(10):e9885

Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands.

Protein-DNA interactions are dynamic, and these dynamics are an important aspect of chromatin-associated processes such as transcription or replication. Due to a lack of methods to study on- and off-rates across entire genomes, protein-DNA interaction dynamics have not been studied extensively. Here, we determine in vivo off-rates for the Saccharomyces cerevisiae chromatin organizing factor Abf1, at 191 sites simultaneously across the yeast genome. Average Abf1 residence times span a wide range, varying between 4.2 and 33 min. Sites with different off-rates are associated with different functional characteristics. This includes their transcriptional dependency on Abf1, nucleosome positioning and the size of the nucleosome-free region, as well as the ability to roadblock RNA polymerase II for termination. The results show how off-rates contribute to transcription factor function and that DIVORSEQ (Determining In Vivo Off-Rates by SEQuencing) is a meaningful way of investigating protein-DNA binding dynamics genome-wide.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/msb.20209885DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7586999PMC
October 2020

An organoid biobank for childhood kidney cancers that captures disease and tissue heterogeneity.

Nat Commun 2020 03 11;11(1):1310. Epub 2020 Mar 11.

Oncode Institute, Princess Máxima Center for Pediatric Oncology, Heidelberglaan 25, 3584 CS, Utrecht, The Netherlands.

Kidney tumours are among the most common solid tumours in children, comprising distinct subtypes differing in many aspects, including cell-of-origin, genetics, and pathology. Pre-clinical cell models capturing the disease heterogeneity are currently lacking. Here, we describe the first paediatric cancer organoid biobank. It contains tumour and matching normal kidney organoids from over 50 children with different subtypes of kidney cancer, including Wilms tumours, malignant rhabdoid tumours, renal cell carcinomas, and congenital mesoblastic nephromas. Paediatric kidney tumour organoids retain key properties of native tumours, useful for revealing patient-specific drug sensitivities. Using single cell RNA-sequencing and high resolution 3D imaging, we further demonstrate that organoid cultures derived from Wilms tumours consist of multiple different cell types, including epithelial, stromal and blastemal-like cells. Our organoid biobank captures the heterogeneity of paediatric kidney tumours, providing a representative collection of well-characterised models for basic cancer research, drug-screening and personalised medicine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-15155-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7066173PMC
March 2020

CHETAH: a selective, hierarchical cell type identification method for single-cell RNA sequencing.

Nucleic Acids Res 2019 09;47(16):e95

Princess Máxima Center for Pediatric Oncology, Heidelberglaan 25, 3584 CS, Utrecht, The Netherlands.

Cell type identification is essential for single-cell RNA sequencing (scRNA-seq) studies, currently transforming the life sciences. CHETAH (CHaracterization of cEll Types Aided by Hierarchical classification) is an accurate cell type identification algorithm that is rapid and selective, including the possibility of intermediate or unassigned categories. Evidence for assignment is based on a classification tree of previously available scRNA-seq reference data and includes a confidence score based on the variance in gene expression per cell type. For cell types represented in the reference data, CHETAH's accuracy is as good as existing methods. Its specificity is superior when cells of an unknown type are encountered, such as malignant cells in tumor samples which it pinpoints as intermediate or unassigned. Although designed for tumor samples in particular, the use of unassigned and intermediate types is also valuable in other exploratory studies. This is exemplified in pancreas datasets where CHETAH highlights cell populations not well represented in the reference dataset, including cells with profiles that lie on a continuum between that of acinar and ductal cell types. Having the possibility of unassigned and intermediate cell types is pivotal for preventing misclassification and can yield important biological information for previously unexplored tissues.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkz543DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895264PMC
September 2019

The ability of transcription factors to differentially regulate gene expression is a crucial component of the mechanism underlying inversion, a frequently observed genetic interaction pattern.

PLoS Comput Biol 2019 05 13;15(5):e1007061. Epub 2019 May 13.

Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands.

Genetic interactions, a phenomenon whereby combinations of mutations lead to unexpected effects, reflect how cellular processes are wired and play an important role in complex genetic diseases. Understanding the molecular basis of genetic interactions is crucial for deciphering pathway organization as well as understanding the relationship between genetic variation and disease. Several hypothetical molecular mechanisms have been linked to different genetic interaction types. However, differences in genetic interaction patterns and their underlying mechanisms have not yet been compared systematically between different functional gene classes. Here, differences in the occurrence and types of genetic interactions are compared for two classes, gene-specific transcription factors (GSTFs) and signaling genes (kinases and phosphatases). Genome-wide gene expression data for 63 single and double deletion mutants in baker's yeast reveals that the two most common genetic interaction patterns are buffering and inversion. Buffering is typically associated with redundancy and is well understood. In inversion, genes show opposite behavior in the double mutant compared to the corresponding single mutants. The underlying mechanism is poorly understood. Although both classes show buffering and inversion patterns, the prevalence of inversion is much stronger in GSTFs. To decipher potential mechanisms, a Petri Net modeling approach was employed, where genes are represented as nodes and relationships between genes as edges. This allowed over 9 million possible three and four node models to be exhaustively enumerated. The models show that a quantitative difference in interaction strength is a strict requirement for obtaining inversion. In addition, this difference is frequently accompanied with a second gene that shows buffering. Taken together, these results provide a mechanistic explanation for inversion. Furthermore, the ability of transcription factors to differentially regulate expression of their targets provides a likely explanation why inversion is more prevalent for GSTFs compared to kinases and phosphatases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pcbi.1007061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6532943PMC
May 2019

Developing a strategy for computational lab skills training through Software and Data Carpentry: Experiences from the ELIXIR Pilot action.

F1000Res 2017 3;6. Epub 2017 Jul 3.

ELIXIR-UK, Software Sustainability Institute UK, School of Computer Science, University of Manchester, Manchester, M13 9PL, UK.

Quality training in computational skills for life scientists is essential to allow them to deliver robust, reproducible and cutting-edge research. A pan-European bioinformatics programme, ELIXIR, has adopted a well-established and progressive programme of computational lab and data skills training from Software and Data Carpentry, aimed at increasing the number of skilled life scientists and building a sustainable training community in this field. This article describes the Pilot action, which introduced the Carpentry training model to the ELIXIR community.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.12688/f1000research.11718.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5516217PMC
July 2017

Molecular mechanisms that distinguish TFIID housekeeping from regulatable SAGA promoters.

EMBO J 2017 02 15;36(3):274-290. Epub 2016 Dec 15.

Molecular Cancer Research, University Medical Center Utrecht, Utrecht, The Netherlands

An important distinction is frequently made between constitutively expressed housekeeping genes versus regulated genes. Although generally characterized by different DNA elements, chromatin architecture and cofactors, it is not known to what degree promoter classes strictly follow regulatability rules and which molecular mechanisms dictate such differences. We show that SAGA-dominated/TATA-box promoters are more responsive to changes in the amount of activator, even compared to TFIID/TATA-like promoters that depend on the same activator Hsf1. Regulatability is therefore an inherent property of promoter class. Further analyses show that SAGA/TATA-box promoters are more dynamic because TATA-binding protein recruitment through SAGA is susceptible to removal by Mot1. In addition, the nucleosome configuration upon activator depletion shifts on SAGA/TATA-box promoters and seems less amenable to preinitiation complex formation. The results explain the fundamental difference between housekeeping and regulatable genes, revealing an additional facet of combinatorial control: an activator can elicit a different response dependent on core promoter class.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embj.201695621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5286361PMC
February 2017

A high-resolution gene expression atlas of epistasis between gene-specific transcription factors exposes potential mechanisms for genetic interactions.

BMC Biol 2015 Dec 23;13:112. Epub 2015 Dec 23.

Molecular Cancer Research, University Medical Centre Utrecht, Universiteitsweg 100, Utrecht, The Netherlands.

Background: Genetic interactions, or non-additive effects between genes, play a crucial role in many cellular processes and disease. Which mechanisms underlie these genetic interactions has hardly been characterized. Understanding the molecular basis of genetic interactions is crucial in deciphering pathway organization and understanding the relationship between genotype, phenotype and disease.

Results: To investigate the nature of genetic interactions between gene-specific transcription factors (GSTFs) in Saccharomyces cerevisiae, we systematically analyzed 72 GSTF pairs by gene expression profiling double and single deletion mutants. These pairs were selected through previously published growth-based genetic interactions as well as through similarity in DNA binding properties. The result is a high-resolution atlas of gene expression-based genetic interactions that provides systems-level insight into GSTF epistasis. The atlas confirms known genetic interactions and exposes new ones. Importantly, the data can be used to investigate mechanisms that underlie individual genetic interactions. Two molecular mechanisms are proposed, "buffering by induced dependency" and "alleviation by derepression".

Conclusions: These mechanisms indicate how negative genetic interactions can occur between seemingly unrelated parallel pathways and how positive genetic interactions can indirectly expose parallel rather than same-pathway relationships. The focus on GSTFs is important for understanding the transcription regulatory network of yeast as it uncovers details behind many redundancy relationships, some of which are completely new. In addition, the study provides general insight into the complex nature of epistasis and proposes mechanistic models for genetic interactions, the majority of which do not fall into easily recognizable within- or between-pathway relationships.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12915-015-0222-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4690272PMC
December 2015

FOXO target gene CTDSP2 regulates cell cycle progression through Ras and p21(Cip1/Waf1).

Biochem J 2015 Jul 20;469(2):289-98. Epub 2015 May 20.

University Medical Centre Utrecht, Universiteitsweg 100 STR3.217, 3584CG Utrecht, The Netherlands

Activity of FOXO (forkhead box O) transcription factors is inhibited by growth factor-PI3K (phosphoinositide 3-kinase)-PKB (protein kinase B)/Akt signalling to control a variety of cellular processes including cell cycle progression. Through comparative analysis of a number of microarray datasets we identified a set of genes commonly regulated by FOXO proteins and PI3K-PKB/Akt, which includes CTDSP2 (C-terminal domain small phosphatase 2). We validated CTDSP2 as a genuine FOXO target gene and show that ectopic CTDSP2 can induce cell cycle arrest. We analysed transcriptional regulation after CTDSP2 expression and identified extensive regulation of genes involved in cell cycle progression, which depends on the phosphatase activity of CTDSP2. The most notably regulated gene is the CDK (cyclin-dependent kinase) inhibitor p21(Cip1/Waf1) and in the present study we show that p21(Cip1/Waf1) is partially responsible for the cell cycle arrest through decreasing cyclin-CDK activity. Our data suggest that CTDSP2 induces p21(Cip1/Waf1) through increasing the activity of Ras. As has been described previously, Ras induces p21(Cip1/Waf1) through p53-dependent and p53-independent pathways and indeed both p53 and MEK inhibition can mitigate the CTDSP2-induced p21(Cip1/Waf1) mRNA up-regulation. In support of Ras activation by CTDSP2, depletion of endogenous CTDSP2 results in reduced Ras activity and thus CTDSP2 seems to be part of a larger set of genes regulated by FOXO proteins, which increase growth factor signalling upon FOXO activation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/BJ20140831DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4613505PMC
July 2015

Cell cycle population effects in perturbation studies.

Mol Syst Biol 2014 Jun 21;10:732. Epub 2014 Jun 21.

Molecular Cancer Research, University Medical Center Utrecht, Utrecht, the Netherlands

Growth condition perturbation or gene function disruption are commonly used strategies to study cellular systems. Although it is widely appreciated that such experiments may involve indirect effects, these frequently remain uncharacterized. Here, analysis of functionally unrelated Saccharyomyces cerevisiae deletion strains reveals a common gene expression signature. One property shared by these strains is slower growth, with increased presence of the signature in more slowly growing strains. The slow growth signature is highly similar to the environmental stress response (ESR), an expression response common to diverse environmental perturbations. Both environmental and genetic perturbations result in growth rate changes. These are accompanied by a change in the distribution of cells over different cell cycle phases. Rather than representing a direct expression response in single cells, both the slow growth signature and ESR mainly reflect a redistribution of cells over different cell cycle phases, primarily characterized by an increase in the G1 population. The findings have implications for any study of perturbation that is accompanied by growth rate changes. Strategies to counter these effects are presented and discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4265054PMC
http://dx.doi.org/10.15252/msb.20145172DOI Listing
June 2014

Large-scale genetic perturbations reveal regulatory networks and an abundance of gene-specific repressors.

Cell 2014 Apr;157(3):740-52

Molecular Cancer Research, University Medical Centre Utrecht, Universiteitsweg 100, Utrecht 3584 CG, the Netherlands. Electronic address:

To understand regulatory systems, it would be useful to uniformly determine how different components contribute to the expression of all other genes. We therefore monitored mRNA expression genome-wide, for individual deletions of one-quarter of yeast genes, focusing on (putative) regulators. The resulting genetic perturbation signatures reflect many different properties. These include the architecture of protein complexes and pathways, identification of expression changes compatible with viability, and the varying responsiveness to genetic perturbation. The data are assembled into a genetic perturbation network that shows different connectivities for different classes of regulators. Four feed-forward loop (FFL) types are overrepresented, including incoherent type 2 FFLs that likely represent feedback. Systematic transcription factor classification shows a surprisingly high abundance of gene-specific repressors, suggesting that yeast chromatin is not as generally restrictive to transcription as is often assumed. The data set is useful for studying individual genes and for discovering properties of an entire regulatory system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2014.02.054DOI Listing
April 2014

A novel microscopy-based high-throughput screening method to identify proteins that regulate global histone modification levels.

J Biomol Screen 2014 Feb 11;19(2):287-96. Epub 2013 Dec 11.

1Department of Molecular Cancer Research, University Medical Centre, Utrecht, The Netherlands.

Posttranslational modifications of histones play an important role in the regulation of gene expression and chromatin structure in eukaryotes. The balance between chromatin factors depositing (writers) and removing (erasers) histone marks regulates the steady-state levels of chromatin modifications. Here we describe a novel microscopy-based screening method to identify proteins that regulate histone modification levels in a high-throughput fashion. We named our method CROSS, for Chromatin Regulation Ontology SiRNA Screening. CROSS is based on an siRNA library targeting the expression of 529 proteins involved in chromatin regulation. As a proof of principle, we used CROSS to identify chromatin factors involved in histone H3 methylation on either lysine-4 or lysine-27. Furthermore, we show that CROSS can be used to identify chromatin factors that affect growth in cancer cell lines. Taken together, CROSS is a powerful method to identify the writers and erasers of novel and known chromatin marks and facilitates the identification of drugs targeting epigenetic modifications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/1087057113515024DOI Listing
February 2014

Centromere binding and a conserved role in chromosome stability for SUMO-dependent ubiquitin ligases.

PLoS One 2013 13;8(6):e65628. Epub 2013 Jun 13.

Molecular Cancer Research, University Medical Centre Utrecht, Universiteitsweg 100, Utrecht, The Netherlands.

The Saccharomyces cerevisiae Slx5/8 complex is the founding member of a recently defined class of SUMO-targeted ubiquitin ligases (STUbLs). Slx5/8 has been implicated in genome stability and transcription, but the precise contribution is unclear. To characterise Slx5/8 function, we determined genome-wide changes in gene expression upon loss of either subunit. The majority of mRNA changes are part of a general stress response, also exhibited by mutants of other genome integrity pathways and therefore indicative of an indirect effect on transcription. Genome-wide binding analysis reveals a uniquely centromeric location for Slx5. Detailed phenotype analyses of slx5Δ and slx8Δ mutants show severe mitotic defects that include aneuploidy, spindle mispositioning, fish hooks and aberrant spindle kinetics. This is associated with accumulation of the PP2A regulatory subunit Rts1 at centromeres prior to entry into anaphase. Knockdown of the human STUbL orthologue RNF4 also results in chromosome segregation errors due to chromosome bridges. The study shows that STUbLs have a conserved role in maintenance of chromosome stability and links SUMO-dependent ubiquitination to a centromere-specific function during mitosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0065628PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3681975PMC
January 2014

The specificity and topology of chromatin interaction pathways in yeast.

Mol Cell 2011 May;42(4):536-49

Molecular Cancer Research, University Medical Centre Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands.

Packaging of DNA into chromatin has a profound impact on gene expression. To understand how changes in chromatin influence transcription, we analyzed 165 mutants of chromatin machinery components in Saccharomyces cerevisiae. mRNA expression patterns change in 80% of mutants, always with specific effects, even for loss of widespread histone marks. The data are assembled into a network of chromatin interaction pathways. The network is function based, has a branched, interconnected topology, and lacks strict one-to-one relationships between complexes. Chromatin pathways are not separate entities for different gene sets, but share many components. The study evaluates which interactions are important for which genes and predicts additional interactions, for example between Paf1C and Set3C, as well as a role for Mediator in subtelomeric silencing. The results indicate the presence of gene-dependent effects that go beyond context-dependent binding of chromatin factors and provide a framework for understanding how specificity is achieved through regulating chromatin.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2011.03.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4435841PMC
May 2011

Gcn4 misregulation reveals a direct role for the evolutionary conserved EKC/KEOPS in the t6A modification of tRNAs.

Nucleic Acids Res 2011 Aug 1;39(14):6148-60. Epub 2011 Apr 1.

LEA Laboratory of Nuclear RNA metabolism, Centre de Génétique Moléculaire, CNRS-FRE3144, 1 av de la Terrasse, 91190 Gif sur Yvette, France.

The EKC/KEOPS complex is universally conserved in Archaea and Eukarya and has been implicated in several cellular processes, including transcription, telomere homeostasis and genomic instability. However, the molecular function of the complex has remained elusive so far. We analyzed the transcriptome of EKC/KEOPS mutants and observed a specific profile that is highly enriched in targets of the Gcn4p transcriptional activator. GCN4 expression was found to be activated at the translational level in mutants via the defective recognition of the inhibitory upstream ORFs (uORFs) present in its leader. We show that EKC/KEOPS mutants are defective for the N6-threonylcarbamoyl adenosine modification at position 37 (t(6)A(37)) of tRNAs decoding ANN codons, which affects initiation at the inhibitory uORFs and provokes Gcn4 de-repression. Structural modeling reveals similarities between Kae1 and bacterial enzymes involved in carbamoylation reactions analogous to t(6)A(37) formation, supporting a direct role for the EKC in tRNA modification. These findings are further supported by strong genetic interactions of EKC mutants with a translation initiation factor and with threonine biosynthesis genes. Overall, our data provide a novel twist to understanding the primary function of the EKC/KEOPS and its impact on several essential cellular functions like transcription and telomere homeostasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkr178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3152333PMC
August 2011

Functional overlap and regulatory links shape genetic interactions between signaling pathways.

Cell 2010 Dec;143(6):991-1004

University Medical Centre Utrecht, The Netherlands.

To understand relationships between phosphorylation-based signaling pathways, we analyzed 150 deletion mutants of protein kinases and phosphatases in S. cerevisiae using DNA microarrays. Downstream changes in gene expression were treated as a phenotypic readout. Double mutants with synthetic genetic interactions were included to investigate genetic buffering relationships such as redundancy. Three types of genetic buffering relationships are identified: mixed epistasis, complete redundancy, and quantitative redundancy. In mixed epistasis, the most common buffering relationship, different gene sets respond in different epistatic ways. Mixed epistasis arises from pairs of regulators that have only partial overlap in function and that are coupled by additional regulatory links such as repression of one by the other. Such regulatory modules confer the ability to control different combinations of processes depending on condition or context. These properties likely contribute to the evolutionary maintenance of paralogs and indicate a way in which signaling pathways connect for multiprocess control.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2010.11.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3073509PMC
December 2010

A consensus of core protein complex compositions for Saccharomyces cerevisiae.

Mol Cell 2010 Jun;38(6):916-28

Department of Physiological Chemistry, University Medical Centre Utrecht, 3584 CG Utrecht, The Netherlands.

Analyses of biological processes would benefit from accurate definitions of protein complexes. High-throughput mass spectrometry data offer the possibility of systematically defining protein complexes; however, the predicted compositions vary substantially depending on the algorithm applied. We determine consensus compositions for 409 core protein complexes from Saccharomyces cerevisiae by merging previous predictions with a new approach. Various analyses indicate that the consensus is comprehensive and of high quality. For 85 out of 259 complexes not recorded in GO, literature search revealed strong support in the form of coprecipitation. New complexes were verified by an independent interaction assay and by gene expression profiling of strains with deleted subunits, often revealing which cellular processes are affected. The consensus complexes are available in various formats, including a merge with GO, resulting in 518 protein complex compositions. The utility is further demonstrated by comparison with binary interaction data to reveal interactions between core complexes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2010.06.002DOI Listing
June 2010

Adaptable gene-specific dye bias correction for two-channel DNA microarrays.

Mol Syst Biol 2009 28;5:266. Epub 2009 Apr 28.

Department of Physiological Chemistry, University Medical Center Utrecht, Universiteitsweg, Utrecht, The Netherlands.

DNA microarray technology is a powerful tool for monitoring gene expression or for finding the location of DNA-bound proteins. DNA microarrays can suffer from gene-specific dye bias (GSDB), causing some probes to be affected more by the dye than by the sample. This results in large measurement errors, which vary considerably for different probes and also across different hybridizations. GSDB is not corrected by conventional normalization and has been difficult to address systematically because of its variance. We show that GSDB is influenced by label incorporation efficiency, explaining the variation of GSDB across different hybridizations. A correction method (Gene- And Slide-Specific Correction, GASSCO) is presented, whereby sequence-specific corrections are modulated by the overall bias of individual hybridizations. GASSCO outperforms earlier methods and works well on a variety of publically available datasets covering a range of platforms, organisms and applications, including ChIP on chip. A sequence-based model is also presented, which predicts which probes will suffer most from GSDB, useful for microarray probe design and correction of individual hybridizations. Software implementing the method is publicly available.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/msb.2009.21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2683724PMC
June 2009

On defining the dynamics of hydrophobic patches on protein surfaces.

Proteins 2008 Jul;72(1):105-14

Department of Physiological Chemistry, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands.

We present a simple and efficient method called PATCHTRACK, for studying the dynamics of hydrophobic surface patches. It tracks the patches on snapshot structures taken from a Molecular Dynamics simulation. They are connected into so-called patch runs, which are subsequently clustered into so-called recurrent patches. The method is applied to simulations of three different proteins. Protein motion causes addition and removal of one or more atoms to a patch, resulting in size fluctuations of around 25%. The fluctuations eventually lead to the break-up of a patch, and their average life span is therefore remarkably short at around 4 ps. However, some patch runs are much more stable, lasting hundreds of picoseconds. One such case is the largest patch in amicyanin that is known to be biologically relevant. Another case, previously not reported, is found in phospholipase A(2), where the functional significance of a large recurrent patch formed by Leu58 and Phe94 seems likely. This patch appears to have been overlooked as it is relatively small in the X-ray structure, demonstrating the utility of the current method. The most frequently occurring patch size is 40-60 A(2), but sizes of up to 500 A(2) are also observed. There is no clear relation between patch run durations and their average size. However, long-lasting patch runs tend not to have large fluctuations. The recurrent patches have alternating periods of "liveness" and "dormancy"; around 25% of them is predominantly in the live state.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/prot.21924DOI Listing
July 2008

Genome-wide location of the coactivator mediator: Binding without activation and transient Cdk8 interaction on DNA.

Mol Cell 2006 Apr;22(2):179-92

Department of Physiological Chemistry, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands.

Mediator is a general coactivator of RNA polymerase II (Pol II) transcription. Genomic location analyses of different Mediator subunits indicate a uniformly composed core complex upstream of active genes but unexpectedly also upstream of inactive genes and on the coding regions of some highly active genes. The repressive Cdk8 submodule is associated with core Mediator at all sites but with a lower degree of occupancy, indicating transient interaction, regardless of promoter activity. This suggests gene-specific regulation of Cdk8 activity, rather than regulated Cdk8 recruitment. Mediator presence is not necessarily linked to transcription. This goes beyond Cdk8-repressed genes, indicating that Mediator can mark some regulatory regions ahead of additional signals. Overlap with intergenic Pol II location in stationary phase points to a role as a binding platform for inactive Pol II during quiescence. These results shed light on Cdk8 repression, suggest additional roles for Mediator, and query models of recruitment-coupled regulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2006.03.023DOI Listing
April 2006

Genome-wide analyses reveal RNA polymerase II located upstream of genes poised for rapid response upon S. cerevisiae stationary phase exit.

Mol Cell 2005 Apr;18(2):171-83

Department of Physiological Chemistry, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands.

The resting state of eukaryotic cells (G0) is relatively uncharacterized. We have applied DNA microarray expression profiling of S. cerevisiae to reveal multiple transitions during a complete 9-day growth cycle between stationary phase (SP) exit and entry. The findings include distinct waves of transcription after the diauxic shift (DS), identification of genes active in SP, and upregulation of over 2500 genes during the first minutes of lag phase. This provides a framework for analyzing large-scale reprogramming of gene expression. Despite global repression, the general transcription machinery is found to be present in quiescent cells but is largely inactive. Genome-wide location analysis by chromatin immunoprecipitation (ChIP on chip) reveals that RNA polymerase II is more predominantly bound at intergenic regions in SP, upstream of hundreds of genes immediately induced upon exit. In contrast to current models of activation-coupled recruitment, the results show that RNA polymerase II is located and maintained upstream of many inactive genes in quiescence.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2005.03.010DOI Listing
April 2005

An expression profile for diagnosis of lymph node metastases from primary head and neck squamous cell carcinomas.

Nat Genet 2005 Feb 9;37(2):182-6. Epub 2005 Jan 9.

Department of Physiological Chemistry, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, the Netherlands.

Metastasis is the process by which cancers spread to distinct sites in the body. It is the principal cause of death in individuals suffering from cancer. For some types of cancer, early detection of metastasis at lymph nodes close to the site of the primary tumor is pivotal for appropriate treatment. Because it can be difficult to detect lymph node metastases reliably, many individuals currently receive inappropriate treatment. We show here that DNA microarray gene-expression profiling can detect lymph node metastases for primary head and neck squamous cell carcinomas that arise in the oral cavity and oropharynx. The predictor, established with an 82-tumor training set, outperforms current clinical diagnosis when independently validated. The 102 predictor genes offer unique insights into the processes underlying metastasis. The results show that the metastatic state can be deciphered from the primary tumor gene-expression pattern and that treatment can be substantially improved.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ng1502DOI Listing
February 2005