Publications by authors named "Frank C P Holstege"

101 Publications

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

Single-cell atlas of developing murine adrenal gland reveals relation of Schwann cell precursor signature to neuroblastoma phenotype.

Proc Natl Acad Sci U S A 2021 Feb;118(5)

Oncode Institute, Princess Máxima Center for Pediatric Oncology, 3584 CS Utrecht, The Netherlands;

Neuroblastoma is the most common extracranial solid tumor and accounts for ∼10% of pediatric cancer-related deaths. The exact cell of origin has yet to be elucidated, but it is generally accepted that neuroblastoma derives from the neural crest and should thus be considered an embryonal malignancy. About 50% of primary neuroblastoma tumors arise in the adrenal gland. Here, we present an atlas of the developing mouse adrenal gland at a single-cell level. Five main cell cluster groups (medulla, cortex, endothelial, stroma, and immune) make up the mouse adrenal gland during fetal development. The medulla group, which is of neural crest origin, is further divided into seven clusters. Of interest is the Schwann cell precursor ("SCP") and the "neuroblast" cluster, a highly cycling cluster that shares markers with sympathoblasts. The signature of the medullary SCP cluster differentiates neuroblastoma patients based on disease phenotype: The SCP signature score anticorrelates with ALK and MYCN expression, two indicators of poor prognosis. Furthermore, a high SCP signature score is associated with better overall survival rates. This study provides an insight into the developing adrenal gland and introduces the SCP gene signature as being of interest for further research in understanding neuroblastoma phenotype.
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http://dx.doi.org/10.1073/pnas.2022350118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7865168PMC
February 2021

Suboptimal Global Transcriptional Response Increases the Harmful Effects of Loss-of-Function Mutations.

Mol Biol Evol 2021 03;38(3):1137-1150

HCEMM-BRC Metabolic Systems Biology Lab, Szeged, Hungary.

The fitness impact of loss-of-function mutations is generally assumed to reflect the loss of specific molecular functions associated with the perturbed gene. Here, we propose that rewiring of the transcriptome upon deleterious gene inactivation is frequently nonspecific and mimics stereotypic responses to external environmental change. Consequently, transcriptional response to gene deletion could be suboptimal and incur an extra fitness cost. Analysis of the transcriptomes of ∼1,500 single-gene deletion Saccharomyces cerevisiae strains supported this scenario. First, most transcriptomic changes are not specific to the deleted gene but are rather triggered by perturbations in functionally diverse genes. Second, gene deletions that alter the expression of dosage-sensitive genes are especially harmful. Third, by elevating the expression level of downregulated genes, we could experimentally mitigate the fitness defect of gene deletions. Our work shows that rewiring of genomic expression upon gene inactivation shapes the harmful effects of mutations.
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http://dx.doi.org/10.1093/molbev/msaa280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7947755PMC
March 2021

An Optimized Chromatin Immunoprecipitation Protocol for Quantification of Protein-DNA Interactions.

STAR Protoc 2020 Jun 19;1(1):100020. Epub 2020 Jun 19.

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

Transcription factors are important regulators of cell fate and function. Knowledge about where transcription factors are bound in the genome is crucial for understanding their function. A common method to study protein-DNA interactions is chromatin immunoprecipitation (ChIP). Here, we present a revised ChIP protocol to determine protein-DNA interactions for the yeast . We optimized several aspects of the procedure, including cross-linking and quenching, cell lysis, and immunoprecipitation steps. This protocol facilitates sensitive and reproducible quantitation of protein-DNA interactions. For complete details on the use and execution of this protocol, please refer to (de Jonge et al., 2019).
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http://dx.doi.org/10.1016/j.xpro.2020.100020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7357673PMC
June 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.
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http://dx.doi.org/10.1038/s41467-020-15155-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7066173PMC
March 2020

Regulation of Skn7-dependent, oxidative stress-induced genes by the RNA polymerase II-CTD phosphatase, Fcp1, and Mediator kinase subunit, Cdk8, in yeast.

J Biol Chem 2019 11 10;294(44):16080-16094. Epub 2019 Sep 10.

Centre for Molecular Medicine and Therapeutics, BC Children's Hospital Research Institute, Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia V5Z 4H4, Canada

Fcp1 is a protein phosphatase that facilitates transcription elongation and termination by dephosphorylating the C-terminal domain of RNA polymerase II. High-throughput genetic screening and gene expression profiling of mutants revealed a novel connection to Cdk8, the Mediator complex kinase subunit, and Skn7, a key transcription factor in the oxidative stress response pathway. Briefly, Skn7 was enriched as a regulator of genes whose mRNA levels were altered in and Δ mutants and was required for the suppression of mutant growth defects by loss of under oxidative stress conditions. Targeted analysis revealed that mutating decreased Skn7 mRNA and protein levels as well as its association with target gene promoters but paradoxically increased the mRNA levels of Skn7-dependent oxidative stress-induced genes ( and ) under basal and induced conditions. The latter was in part recapitulated via chemical inhibition of transcription in WT cells, suggesting that a combination of transcriptional and posttranscriptional effects underscored the increased mRNA levels of and observed in the mutant. Interestingly, loss of robustly normalized the mRNA levels of Skn7-dependent genes in the mutant background and also increased Skn7 protein levels by preventing its turnover. As such, our work suggested that loss of could overcome transcriptional and/or posttranscriptional alterations in the mutant through its regulatory effect on Skn7. Furthermore, our work also implicated and in the broader response to environmental stressors in yeast.
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http://dx.doi.org/10.1074/jbc.RA119.008515DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6827322PMC
November 2019

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

Dot1 promotes H2B ubiquitination by a methyltransferase-independent mechanism.

Nucleic Acids Res 2018 11;46(21):11251-11261

Division of Gene Regulation, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.

The histone methyltransferase Dot1 is conserved from yeast to human and methylates lysine 79 of histone H3 (H3K79) on the core of the nucleosome. H3K79 methylation by Dot1 affects gene expression and the response to DNA damage, and is enhanced by monoubiquitination of the C-terminus of histone H2B (H2Bub1). To gain more insight into the functions of Dot1, we generated genetic interaction maps of increased-dosage alleles of DOT1. We identified a functional relationship between increased Dot1 dosage and loss of the DUB module of the SAGA co-activator complex, which deubiquitinates H2Bub1 and thereby negatively regulates H3K79 methylation. Increased Dot1 dosage was found to promote H2Bub1 in a dose-dependent manner and this was exacerbated by the loss of SAGA-DUB activity, which also caused a negative genetic interaction. The stimulatory effect on H2B ubiquitination was mediated by the N-terminus of Dot1, independent of methyltransferase activity. Our findings show that Dot1 and H2Bub1 are subject to bi-directional crosstalk and that Dot1 possesses chromatin regulatory functions that are independent of its methyltransferase activity.
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http://dx.doi.org/10.1093/nar/gky801DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6265471PMC
November 2018

Sequence-Directed Action of RSC Remodeler and General Regulatory Factors Modulates +1 Nucleosome Position to Facilitate Transcription.

Mol Cell 2018 07;71(1):89-102.e5

Department of Molecular Biology and Institute of Genetics and Genomics in Geneva (iGE3), 30 quai Ernest-Ansermet, 1211 Geneva 4, Switzerland. Electronic address:

Accessible chromatin is important for RNA polymerase II recruitment and transcription initiation at eukaryotic promoters. We investigated the mechanistic links between promoter DNA sequence, nucleosome positioning, and transcription. Our results indicate that positioning of the transcription start site-associated +1 nucleosome in yeast is critical for efficient TBP binding and is driven by two key factors, the essential chromatin remodeler RSC and a small set of ubiquitous general regulatory factors (GRFs). Our findings indicate that the strength and directionality of RSC action on promoter nucleosomes depends on the arrangement and proximity of two specific DNA motifs. This, together with the effect on nucleosome position observed in double depletion experiments, suggests that, despite their widespread co-localization, RSC and GRFs predominantly act through independent signals to generate accessible chromatin. Our results provide mechanistic insight into how the promoter DNA sequence instructs trans-acting factors to control nucleosome architecture and stimulate transcription initiation.
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http://dx.doi.org/10.1016/j.molcel.2018.05.030DOI Listing
July 2018

FOXO Transcription Factors Both Suppress and Support Breast Cancer Progression.

Cancer Res 2018 05 12;78(9):2356-2369. Epub 2018 Feb 12.

Center for Molecular Medicine, Molecular Cancer Research, University Medical Center Utrecht, Utrecht University, the Netherlands.

FOXO transcription factors are regulators of cellular homeostasis and putative tumor suppressors, yet the role of FOXO in cancer progression remains to be determined. The data on FOXO function, particularly for epithelial cancers, are fragmentary and come from studies that focused on isolated aspects of cancer. To clarify the role of FOXO in epithelial cancer progression, we characterized the effects of inducible FOXO activation and loss in a mouse model of metastatic invasive lobular carcinoma. Strikingly, either activation or loss of FOXO function suppressed tumor growth and metastasis. We show that the multitude of cellular processes critically affected by FOXO function include proliferation, survival, redox homeostasis, and PI3K signaling, all of which must be carefully balanced for tumor cells to thrive. FOXO proteins are not solely tumor suppressors, but also support tumor growth and metastasis by regulating a multitude of cellular processes essential for tumorigenesis. .
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http://dx.doi.org/10.1158/0008-5472.CAN-17-2511DOI Listing
May 2018

The ribosome assembly gene network is controlled by the feedback regulation of transcription elongation.

Nucleic Acids Res 2017 Sep;45(16):9302-9318

Instituto de Biomedicina de Sevilla (IBiS), Hospital Virgen del Rocío-CSIC-Universidad de Sevilla, and Departamento de Genética, Universidad de Sevilla, 41013 Seville, Spain.

Ribosome assembly requires the concerted expression of hundreds of genes, which are transcribed by all three nuclear RNA polymerases. Transcription elongation involves dynamic interactions between RNA polymerases and chromatin. We performed a synthetic lethal screening in Saccharomyces cerevisiae with a conditional allele of SPT6, which encodes one of the factors that facilitates this process. Some of these synthetic mutants corresponded to factors that facilitate pre-rRNA processing and ribosome biogenesis. We found that the in vivo depletion of one of these factors, Arb1, activated transcription elongation in the set of genes involved directly in ribosome assembly. Under these depletion conditions, Spt6 was physically targeted to the up-regulated genes, where it helped maintain their chromatin integrity and the synthesis of properly stable mRNAs. The mRNA profiles of a large set of ribosome biogenesis mutants confirmed the existence of a feedback regulatory network among ribosome assembly genes. The transcriptional response in this network depended on both the specific malfunction and the role of the regulated gene. In accordance with our screening, Spt6 positively contributed to the optimal operation of this global network. On the whole, this work uncovers a feedback control of ribosome biogenesis by fine-tuning transcription elongation in ribosome assembly factor-coding genes.
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http://dx.doi.org/10.1093/nar/gkx529DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737610PMC
September 2017

EBV MicroRNA BART16 Suppresses Type I IFN Signaling.

J Immunol 2017 05 17;198(10):4062-4073. Epub 2017 Apr 17.

Department of Medical Microbiology, University Medical Center Utrecht, 3584 CX Utrecht, the Netherlands;

Type I IFNs play critical roles in orchestrating the antiviral defense by inducing direct antiviral activities and shaping the adaptive immune response. Viruses have evolved numerous strategies to specifically interfere with IFN production or its downstream mediators, thereby allowing successful infection of the host to occur. The prototypic human gammaherpesvirus EBV, which is associated with infectious mononucleosis and malignant tumors, harbors many immune-evasion proteins that manipulate the adaptive and innate immune systems. In addition to proteins, the virus encodes >40 mature microRNAs for which the functions remain largely unknown. In this article, we identify EBV-encoded miR-BART16 as a novel viral immune-evasion factor that interferes with the type I IFN signaling pathway. miR-BART16 directly targets CREB-binding protein, a key transcriptional coactivator in IFN signaling, thereby inducing CREB-binding protein downregulation in EBV-transformed B cells and gastric carcinoma cells. miR-BART16 abrogates the production of IFN-stimulated genes in response to IFN-α stimulation and it inhibits the antiproliferative effect of IFN-α on latently infected BL cells. By obstructing the type I IFN-induced antiviral response, miR-BART16 provides a means to facilitate the establishment of latent EBV infection and enhance viral replication.
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http://dx.doi.org/10.4049/jimmunol.1501605DOI Listing
May 2017

Growth condition dependency is the major cause of non-responsiveness upon genetic perturbation.

PLoS One 2017 3;12(3):e0173432. Epub 2017 Mar 3.

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

Investigating the role and interplay between individual proteins in biological processes is often performed by assessing the functional consequences of gene inactivation or removal. Depending on the sensitivity of the assay used for determining phenotype, between 66% (growth) and 53% (gene expression) of Saccharomyces cerevisiae gene deletion strains show no defect when analyzed under a single condition. Although it is well known that this non-responsive behavior is caused by different types of redundancy mechanisms or by growth condition/cell type dependency, it is not known what the relative contribution of these different causes is. Understanding the underlying causes of and their relative contribution to non-responsive behavior upon genetic perturbation is extremely important for designing efficient strategies aimed at elucidating gene function and unraveling complex cellular systems. Here, we provide a systematic classification of the underlying causes of and their relative contribution to non-responsive behavior upon gene deletion. The overall contribution of redundancy to non-responsive behavior is estimated at 29%, of which approximately 17% is due to homology-based redundancy and 12% is due to pathway-based redundancy. The major determinant of non-responsiveness is condition dependency (71%). For approximately 14% of protein complexes, just-in-time assembly can be put forward as a potential mechanistic explanation for how proteins can be regulated in a condition dependent manner. Taken together, the results underscore the large contribution of growth condition requirement to non-responsive behavior, which needs to be taken into account for strategies aimed at determining gene function. The classification provided here, can also be further harnessed in systematic analyses of complex cellular systems.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0173432PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5336285PMC
August 2017

Mapping of a FEB3 homologous febrile seizure locus on mouse chromosome 2 containing candidate genes Scn1a and Scn3a.

Eur J Neurosci 2016 12;44(11):2950-2957

Brain Center Rudolf Magnus, Department of Translational Neuroscience, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG, Utrecht, The Netherlands.

Febrile seizures (FS) are the most common seizure type in children. Recurrent FS are a risk factor for developing temporal lobe epilepsy later in life and are known to have a strong genetic component. Experimental FS (eFS) can be elicited in mice by warm-air induced hyperthermia. We used this model to screen the chromosome substitution strain (CSS) panel derived from C57BL/6J and A/J for FS susceptibility and identified C57BL/6J-Chr2 /NaJ (CSS2), as the strain with the strongest FS susceptibility phenotype. The aim of this study was to map FS susceptibility loci and select candidate genes on mouse chromosome 2. We generated an F population by intercrossing the hybrids (F ) that were derived from CSS2 and C57BL/6J mice. All CSS2-F individuals were genotyped and phenotyped for eFS susceptibility, and QTL analysis was performed. Candidate gene selection was based on bioinformatics analyses and differential brain expression between CSS2 and C57BL/6J strains determined by microarray analysis. Genetic mapping of the eFS susceptibility trait identified two significant loci: FS-QTL2a (LOD-score 3.6) and FS-QTL2b (LOD-score 6.2). FS-QTL2a contained 44 genes expressed in the brain at post natal day 14. Four of these (Arl6ip6, Cytip, Fmnl2 Ifih1) contained a non-synonymous SNP comparing CSS2 and C57BL/6J, six genes (March7, Nr4a2, Gpd2, Grb14, Scn1a, Scn3a) were differentially expressed between these strains. A region within FS-QTL2a is homologous to the human FEB3 locus. The fact that we identify mouse FS-QTL2a with high FEB3 homology is strong support for the validity of the eFS mouse model to study genetics of human FS.
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http://dx.doi.org/10.1111/ejn.13420DOI Listing
December 2016

An endometrial gene expression signature accurately predicts recurrent implantation failure after IVF.

Sci Rep 2016 Jan 22;6:19411. Epub 2016 Jan 22.

Department of Reproductive Medicine and Gynaecology, University Medical Center Utrecht, Utrecht, The Netherlands.

The primary limiting factor for effective IVF treatment is successful embryo implantation. Recurrent implantation failure (RIF) is a condition whereby couples fail to achieve pregnancy despite consecutive embryo transfers. Here we describe the collection of gene expression profiles from mid-luteal phase endometrial biopsies (n = 115) from women experiencing RIF and healthy controls. Using a signature discovery set (n = 81) we identify a signature containing 303 genes predictive of RIF. Independent validation in 34 samples shows that the gene signature predicts RIF with 100% positive predictive value (PPV). The strength of the RIF associated expression signature also stratifies RIF patients into distinct groups with different subsequent implantation success rates. Exploration of the expression changes suggests that RIF is primarily associated with reduced cellular proliferation. The gene signature will be of value in counselling and guiding further treatment of women who fail to conceive upon IVF and suggests new avenues for developing intervention.
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http://dx.doi.org/10.1038/srep19411DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4726345PMC
January 2016

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

Expression Profiling after Prolonged Experimental Febrile Seizures in Mice Suggests Structural Remodeling in the Hippocampus.

PLoS One 2015 18;10(12):e0145247. Epub 2015 Dec 18.

Brain Center Rudolf Magnus, Department of Translational Neuroscience, University Medical Center Utrecht, Utrecht, the Netherlands.

Febrile seizures are the most prevalent type of seizures among children up to 5 years of age (2-4% of Western-European children). Complex febrile seizures are associated with an increased risk to develop temporal lobe epilepsy. To investigate short- and long-term effects of experimental febrile seizures (eFS), we induced eFS in highly febrile convulsion-susceptible C57BL/6J mice at post-natal day 10 by exposure to hyperthermia (HT) and compared them to normotherm-exposed (NT) mice. We detected structural re-organization in the hippocampus 14 days after eFS. To identify molecular candidates, which entrain this structural re-organization, we investigated temporal changes in mRNA expression profiles eFS 1 hour to 56 days after eFS. We identified 931 regulated genes and profiled several candidates using in situ hybridization and histology at 3 and 14 days after eFS. This is the first study to report genome-wide transcriptome analysis after eFS in mice. We identify temporal regulation of multiple processes, such as stress-, immune- and inflammatory responses, glia activation, glutamate-glutamine cycle and myelination. Identification of the short- and long-term changes after eFS is important to elucidate the mechanisms contributing to epileptogenesis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0145247PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4684321PMC
June 2016

Identification of Srp9 as a febrile seizure susceptibility gene.

Ann Clin Transl Neurol 2014 Apr 12;1(4):239-50. Epub 2014 Mar 12.

Department of Translational Neuroscience, Brain Center Rudolf Magnus, University Medical Center Utrecht Utrecht, The Netherlands.

Objective: Febrile seizures (FS) are the most common seizure type in young children. Complex FS are a risk factor for mesial temporal lobe epilepsy (mTLE). To identify new FS susceptibility genes we used a forward genetic strategy in mice and subsequently analyzed candidate genes in humans.

Methods: We mapped a quantitative trait locus (QTL1) for hyperthermia-induced FS on mouse chromosome 1, containing the signal recognition particle 9 (Srp9) gene. Effects of differential Srp9 expression were assessed in vivo and in vitro. Hippocampal SRP9 expression and genetic association were analyzed in FS and mTLE patients.

Results: Srp9 was differentially expressed between parental strains C57BL/6J and A/J. Chromosome substitution strain 1 (CSS1) mice exhibited lower FS susceptibility and Srp9 expression than C57BL/6J mice. In vivo knockdown of brain Srp9 reduced FS susceptibility. Mice with reduced Srp9 expression and FS susceptibility, exhibited reduced hippocampal AMPA and NMDA currents. Downregulation of neuronal Srp9 reduced surface expression of AMPA receptor subunit GluA1. mTLE patients with antecedent FS had higher SRP9 expression than patients without. SRP9 promoter SNP rs12403575(G/A) was genetically associated with FS and mTLE.

Interpretation: Our findings identify SRP9 as a novel FS susceptibility gene and indicate that SRP9 conveys its effects through endoplasmic reticulum (ER)-dependent synthesis and trafficking of membrane proteins, such as glutamate receptors. Discovery of this new FS gene and mechanism may provide new leads for early diagnosis and treatment of children with complex FS at risk for mTLE.
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http://dx.doi.org/10.1002/acn3.48DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4292741PMC
April 2014

The genomic landscape of compensatory evolution.

PLoS Biol 2014 Aug 26;12(8):e1001935. Epub 2014 Aug 26.

Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Center, Szeged, Hungary.

Adaptive evolution is generally assumed to progress through the accumulation of beneficial mutations. However, as deleterious mutations are common in natural populations, they generate a strong selection pressure to mitigate their detrimental effects through compensatory genetic changes. This process can potentially influence directions of adaptive evolution by enabling evolutionary routes that are otherwise inaccessible. Therefore, the extent to which compensatory mutations shape genomic evolution is of central importance. Here, we studied the capacity of the baker's yeast genome to compensate the complete loss of genes during evolution, and explored the long-term consequences of this process. We initiated laboratory evolutionary experiments with over 180 haploid baker's yeast genotypes, all of which initially displayed slow growth owing to the deletion of a single gene. Compensatory evolution following gene loss was rapid and pervasive: 68% of the genotypes reached near wild-type fitness through accumulation of adaptive mutations elsewhere in the genome. As compensatory mutations have associated fitness costs, genotypes with especially low fitnesses were more likely to be subjects of compensatory evolution. Genomic analysis revealed that as compensatory mutations were generally specific to the functional defect incurred, convergent evolution at the molecular level was extremely rare. Moreover, the majority of the gene expression changes due to gene deletion remained unrestored. Accordingly, compensatory evolution promoted genomic divergence of parallel evolving populations. However, these different evolutionary outcomes are not phenotypically equivalent, as they generated diverse growth phenotypes across environments. Taken together, these results indicate that gene loss initiates adaptive genomic changes that rapidly restores fitness, but this process has substantial pleiotropic effects on cellular physiology and evolvability upon environmental change. Our work also implies that gene content variation across species could be partly due to the action of compensatory evolution rather than the passive loss of genes.
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http://dx.doi.org/10.1371/journal.pbio.1001935DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4144845PMC
August 2014

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

Aberrant expression and distribution of enzymes of the urea cycle and other ammonia metabolizing pathways in dogs with congenital portosystemic shunts.

PLoS One 2014 19;9(6):e100077. Epub 2014 Jun 19.

Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands.

The detoxification of ammonia occurs mainly through conversion of ammonia to urea in the liver via the urea cycle and glutamine synthesis. Congenital portosystemic shunts (CPSS) in dogs cause hyperammonemia eventually leading to hepatic encephalopathy. In this study, the gene expression of urea cycle enzymes (carbamoylphosphate synthetase (CPS1), ornithine carbamoyltransferase (OTC), argininosuccinate synthetase (ASS1), argininosuccinate lyase (ASL), and arginase (ARG1)), N-acetylglutamate synthase (NAGS), Glutamate dehydrogenase (GLUD1), and glutamate-ammonia ligase (GLUL) was evaluated in dogs with CPSS before and after surgical closure of the shunt. Additionally, immunohistochemistry was performed on urea cycle enzymes and GLUL on liver samples of healthy dogs and dogs with CPSS to investigate a possible zonal distribution of these enzymes within the liver lobule and to investigate possible differences in distribution in dogs with CPSS compared to healthy dogs. Furthermore, the effect of increasing ammonia concentrations on the expression of the urea cycle enzymes was investigated in primary hepatocytes in vitro. Gene-expression of CPS1, OTC, ASL, GLUD1 and NAGS was down regulated in dogs with CPSS and did not normalize after surgical closure of the shunt. In all dogs GLUL distribution was localized pericentrally. CPS1, OTC and ASS1 were localized periportally in healthy dogs, whereas in CPSS dogs, these enzymes lacked a clear zonal distribution. In primary hepatocytes higher ammonia concentrations induced mRNA levels of CPS1. We hypothesize that the reduction in expression of urea cycle enzymes, NAGS and GLUD1 as well as the alterations in zonal distribution in dogs with CPSS may be caused by a developmental arrest of these enzymes during the embryonic or early postnatal phase.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0100077PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4063766PMC
March 2015

Insulin-FOXO3 signaling modulates circadian rhythms via regulation of clock transcription.

Curr Biol 2014 Jun 22;24(11):1248-55. Epub 2014 May 22.

Department of Translational Neuroscience, Brain Center Rudolf Magnus, University Medical Center Utrecht, PO Box 85060, 3508 TA Utrecht, the Netherlands; Swammerdam Institute of Life Sciences, University of Amsterdam, PO Box 94232, 1090 GE Amsterdam, the Netherlands. Electronic address:

Circadian rhythms are responsive to external and internal cues, light and metabolism being among the most important. In mammals, the light signal is sensed by the retina and transmitted to the suprachiasmatic nucleus (SCN) master clock [1], where it is integrated into the molecular oscillator via regulation of clock gene transcription. The SCN synchronizes peripheral oscillators, an effect that can be overruled by incoming metabolic signals [2]. As a consequence, peripheral oscillators can be uncoupled from the master clock when light and metabolic signals are not in phase. The signaling pathways responsible for coupling metabolic cues to the molecular clock are being rapidly uncovered [3-5]. Here we show that insulin-phosphatidylinositol 3-kinase (PI3K)-Forkhead box class O3 (FOXO3) signaling is required for circadian rhythmicity in the liver via regulation of Clock. Knockdown of FoxO3 dampens circadian amplitude, an effect that is rescued by overexpression of Clock. Subsequently, we show binding of FOXO3 to two Daf-binding elements (DBEs) located in the Clock promoter area, implicating Clock as a transcriptional target of FOXO3. Transcriptional oscillation of both core clock and output genes in the liver of FOXO3-deficient mice is affected, indicating a disrupted hepatic circadian rhythmicity. Finally, we show that insulin, a major regulator of FOXO activity [6-9], regulates Clock levels in a PI3K- and FOXO3-dependent manner. Our data point to a key role of the insulin-FOXO3-Clock signaling pathway in the modulation of circadian rhythms.
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http://dx.doi.org/10.1016/j.cub.2014.04.018DOI 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.
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http://dx.doi.org/10.1016/j.cell.2014.02.054DOI Listing
April 2014

Suppression of Mediator is regulated by Cdk8-dependent Grr1 turnover of the Med3 coactivator.

Proc Natl Acad Sci U S A 2014 Feb 3;111(7):2500-5. Epub 2014 Feb 3.

College of Medicine, Swansea University, Swansea, Wales SA2 8PP, United Kingdom.

Mediator, an evolutionary conserved large multisubunit protein complex with a central role in regulating RNA polymerase II-transcribed genes, serves as a molecular switchboard at the interface between DNA binding transcription factors and the general transcription machinery. Mediator subunits include the Cdk8 module, which has both positive and negative effects on activator-dependent transcription through the activity of the cyclin-dependent kinase Cdk8, and the tail module, which is required for positive and negative regulation of transcription, correct preinitiation complex formation in basal and activated transcription, and Mediator recruitment. Currently, the molecular mechanisms governing Mediator function remain largely undefined. Here we demonstrate an autoregulatory mechanism used by Mediator to repress transcription through the activity of distinct components of different modules. We show that the function of the tail module component Med3, which is required for transcription activation, is suppressed by the kinase activity of the Cdk8 module. Med3 interacts with, and is phosphorylated by, Cdk8; site-specific phosphorylation triggers interaction with and degradation by the Grr1 ubiquitin ligase, thereby preventing transcription activation. This active repression mechanism involving Grr1-dependent ubiquitination of Med3 offers a rationale for the substoichiometric levels of the tail module that are found in purified Mediator and the corresponding increase in tail components seen in cdk8 mutants.
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http://dx.doi.org/10.1073/pnas.1307525111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3932902PMC
February 2014

Uterine selection of human embryos at implantation.

Sci Rep 2014 Feb 6;4:3894. Epub 2014 Feb 6.

1] Department for Reproductive Medicine and Gynecology, University Medical Center Utrecht, PO Box 85500, 3508 GA, Utrecht, The Netherlands [2] Division of Developmental Origins of Adult Diseases (DOHaD), University of Southampton, Coxford Road, Southampton SO16 5YA, UK.

Human embryos frequently harbor large-scale complex chromosomal errors that impede normal development. Affected embryos may fail to implant although many first breach the endometrial epithelium and embed in the decidualizing stroma before being rejected via mechanisms that are poorly understood. Here we show that developmentally impaired human embryos elicit an endoplasmic stress response in human decidual cells. A stress response was also evident upon in vivo exposure of mouse uteri to culture medium conditioned by low-quality human embryos. By contrast, signals emanating from developmentally competent embryos activated a focused gene network enriched in metabolic enzymes and implantation factors. We further show that trypsin, a serine protease released by pre-implantation embryos, elicits Ca(2+) signaling in endometrial epithelial cells. Competent human embryos triggered short-lived oscillatory Ca(2+) fluxes whereas low-quality embryos caused a heightened and prolonged Ca(2+) response. Thus, distinct positive and negative mechanisms contribute to active selection of human embryos at implantation.
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http://dx.doi.org/10.1038/srep03894DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3915549PMC
February 2014

Translation of branched-chain aminotransferase-1 transcripts is impaired in cells haploinsufficient for ribosomal protein genes.

Exp Hematol 2014 May 23;42(5):394-403.e4. Epub 2014 Jan 23.

Hubrecht Institute, Koninklijke Nederlandse Akademie van Wetenschappen and University Medical Center Utrecht, Utrecht, The Netherlands. Electronic address:

Diamond-Blackfan anemia (DBA) is a bone marrow failure syndrome linked to mutations in ribosomal protein (RP) genes that result in the impaired proliferation of hematopoietic progenitor cells. The etiology of DBA is not completely understood; however, the ribosomal nature of the genes involved has led to speculation that these mutations may alter the landscape of messenger RNA (mRNA) translation. Here, we performed comparative microarray analysis of polysomal mRNA transcripts isolated from lymphoblastoid cell lines derived from DBA patients carrying various haploinsufficient mutations in either RPS19 or RPL11. Different spectrums of changes were observed depending on the mutant gene, with large differences found in RPS19 cells and very few in RPL11 cells. However, we find that the small number of altered transcripts in RPL11 overlap for the most part with those altered in RPS19 cells. We show specifically that levels of branched-chain aminotransferase-1 (BCAT1) transcripts are significantly decreased on the polysomes of both RPS19 and RPL11 cells and that translation of BCAT1 protein is especially impaired in cells with small RP gene mutations, and we provide evidence that this effect may be due in part to the unusually long 5'UTR of the BCAT1 transcript. The BCAT1 enzyme carries out the final step in the biosynthesis and the first step of degradation of the branched-chain amino acids leucine, isoleucine, and valine. Interestingly, several animal models of DBA have reported that leucine ameliorates the anemia phenotypes generated by RPS19 loss. Our study suggests that RP mutations affect the synthesis of specific proteins involved in regulating amino acid levels that are important for maintaining the normal proliferative capacity of hematopoietic cells.
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http://dx.doi.org/10.1016/j.exphem.2013.12.010DOI Listing
May 2014

Suppression of intragenic transcription requires the MOT1 and NC2 regulators of TATA-binding protein.

Nucleic Acids Res 2014 Apr 22;42(7):4220-9. Epub 2014 Jan 22.

Department of Molecular Cancer Research, University Medical Center Utrecht, 3584 CG, Utrecht, The Netherlands and Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.

Chromatin structure in transcribed regions poses a barrier for intragenic transcription. In a comprehensive study of the yeast chromatin remodelers and the Mot1p-NC2 regulators of TATA-binding protein (TBP), we detected synthetic genetic interactions indicative of suppression of intragenic transcription. Conditional depletion of Mot1p or NC2 in absence of the ISW1 remodeler, but not in the absence of other chromatin remodelers, activated the cryptic FLO8 promoter. Likewise, conditional depletion of Mot1p or NC2 in deletion backgrounds of the H3K36 methyltransferase Set2p or the Asf1p-Rtt106p histone H3-H4 chaperones, important factors involved in maintaining a repressive chromatin environment, resulted in increased intragenic FLO8 transcripts. Activity of the cryptic FLO8 promoter is associated with reduced H3 levels, increased TBP binding and tri-methylation of H3K4 and is independent of Spt-Ada-Gcn5-acetyltransferase function. These data reveal cooperation of negative regulation of TBP with specific chromatin regulators to inhibit intragenic transcription.
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http://dx.doi.org/10.1093/nar/gkt1398DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3985625PMC
April 2014

The role of Ctk1 kinase in termination of small non-coding RNAs.

PLoS One 2013 4;8(12):e80495. Epub 2013 Dec 4.

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

Transcription termination in Saccharomyces cerevisiae can be performed by at least two distinct pathways and is influenced by the phosphorylation status of the carboxy-terminal domain (CTD) of RNA polymerase II (Pol II). Late termination of mRNAs is performed by the CPF/CF complex, the recruitment of which is dependent on CTD-Ser2 phosphorylation (Ser2P). Early termination of shorter cryptic unstable transcripts (CUTs) and small nucleolar/nuclear RNAs (sno/snRNAs) is performed by the Nrd1-Nab3-Sen1 (NNS) complex that binds phosphorylated CTD-Ser5 (Ser5P) via the CTD-interacting domain (CID) of Nrd1p. In this study, mutants of the different termination pathways were compared by genome-wide expression analysis. Surprisingly, the expression changes observed upon loss of the CTD-Ser2 kinase Ctk1p are more similar to those derived from alterations in the Ser5P-dependent NNS pathway, than from loss of CTD-Ser2P binding factors. Tiling array analysis of ctk1Δ cells reveals readthrough at snoRNAs, at many cryptic unstable transcripts (CUTs) and stable uncharacterized transcripts (SUTs), but only at some mRNAs. Despite the suggested predominant role in termination of mRNAs, we observed that a CTK1 deletion or a Pol II CTD mutant lacking all Ser2 positions does not result in a global mRNA termination defect. Rather, termination defects in these strains are widely observed at NNS-dependent genes. These results indicate that Ctk1p and Ser2 CTD phosphorylation have a wide impact in termination of small non-coding RNAs but only affect a subset of mRNA coding genes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0080495PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3851182PMC
September 2014

Recruitment of TREX to the transcription machinery by its direct binding to the phospho-CTD of RNA polymerase II.

PLoS Genet 2013 Nov 14;9(11):e1003914. Epub 2013 Nov 14.

Gene Center and Munich Center for Integrated Protein Science CIPSM at the Department of Biochemistry of the Ludwig-Maximilians-University of Munich, Munich, Germany.

Messenger RNA (mRNA) synthesis and export are tightly linked, but the molecular mechanisms of this coupling are largely unknown. In Saccharomyces cerevisiae, the conserved TREX complex couples transcription to mRNA export and mediates mRNP formation. Here, we show that TREX is recruited to the transcription machinery by direct interaction of its subcomplex THO with the serine 2-serine 5 (S2/S5) diphosphorylated CTD of RNA polymerase II. S2 and/or tyrosine 1 (Y1) phosphorylation of the CTD is required for TREX occupancy in vivo, establishing a second interaction platform necessary for TREX recruitment in addition to RNA. Genome-wide analyses show that the occupancy of THO and the TREX components Sub2 and Yra1 increases from the 5' to the 3' end of the gene in accordance with the CTD S2 phosphorylation pattern. Importantly, in a mutant strain, in which TREX is recruited to genes but does not increase towards the 3' end, the expression of long transcripts is specifically impaired. Thus, we show for the first time that a 5'-3' increase of a protein complex is essential for correct expression of the genome. In summary, we provide insight into how the phospho-code of the CTD directs mRNP formation and export through TREX recruitment.
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http://dx.doi.org/10.1371/journal.pgen.1003914DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3828145PMC
November 2013