Publications by authors named "Frank G Grosveld"

45 Publications

Mild dyserythropoiesis and β-like globin gene expression imbalance due to the loss of histone chaperone ASF1B.

Hum Genomics 2020 10 16;14(1):39. Epub 2020 Oct 16.

Laboratory of Pharmacogenomics and Individualized Therapy, Department of Pharmacy, University of Patras School of Health Sciences, Patras, Greece.

The expression of the human β-like globin genes follows a well-orchestrated developmental pattern, undergoing two essential switches, the first one during the first weeks of gestation (ε to γ), and the second one during the perinatal period (γ to β). The γ- to β-globin gene switching mechanism includes suppression of fetal (γ-globin, HbF) and activation of adult (β-globin, HbA) globin gene transcription. In hereditary persistence of fetal hemoglobin (HPFH), the γ-globin suppression mechanism is impaired leaving these individuals with unusual elevated levels of fetal hemoglobin (HbF) in adulthood. Recently, the transcription factors KLF1 and BCL11A have been established as master regulators of the γ- to β-globin switch. Previously, a genomic variant in the KLF1 gene, identified by linkage analysis performed on twenty-seven members of a Maltese family, was found to be associated with HPFH. However, variation in the levels of HbF among family members, and those from other reported families carrying genetic variants in KLF1, suggests additional contributors to globin switching. ASF1B was downregulated in the family members with HPFH. Here, we investigate the role of ASF1B in γ- to β-globin switching and erythropoiesis in vivo. Mouse-human interspecies ASF1B protein identity is 91.6%. By means of knockdown functional assays in human primary erythroid cultures and analysis of the erythroid lineage in Asf1b knockout mice, we provide evidence that ASF1B is a novel contributor to steady-state erythroid differentiation, and while its loss affects the balance of globin expression, it has no major role in hemoglobin switching.
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http://dx.doi.org/10.1186/s40246-020-00283-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566067PMC
October 2020

Targeted chromatin conformation analysis identifies novel distal neural enhancers of ZEB2 in pluripotent stem cell differentiation.

Hum Mol Genet 2020 Aug;29(15):2535-2550

Department of Cell Biology, Erasmus University Medical Center, Rotterdam, CN 3015, The Netherlands.

The transcription factor zinc finger E-box binding protein 2 (ZEB2) controls embryonic and adult cell fate decisions and cellular maturation in many stem/progenitor cell types. Defects in these processes in specific cell types underlie several aspects of Mowat-Wilson syndrome (MOWS), which is caused by ZEB2 haplo-insufficiency. Human ZEB2, like mouse Zeb2, is located on chromosome 2 downstream of a ±3.5 Mb-long gene-desert, lacking any protein-coding gene. Using temporal targeted chromatin capture (T2C), we show major chromatin structural changes based on mapping in-cis proximities between the ZEB2 promoter and this gene desert during neural differentiation of human-induced pluripotent stem cells, including at early neuroprogenitor cell (NPC)/rosette state, where ZEB2 mRNA levels increase significantly. Combining T2C with histone-3 acetylation mapping, we identified three novel candidate enhancers about 500 kb upstream of the ZEB2 transcription start site. Functional luciferase-based assays in heterologous cells and NPCs reveal co-operation between these three enhancers. This study is the first to document in-cis Regulatory Elements located in ZEB2's gene desert. The results further show the usability of T2C for future studies of ZEB2 REs in differentiation and maturation of multiple cell types and the molecular characterization of newly identified MOWS patients that lack mutations in ZEB2 protein-coding exons.
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http://dx.doi.org/10.1093/hmg/ddaa141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7471508PMC
August 2020

Multifaceted actions of Zeb2 in postnatal neurogenesis from the ventricular-subventricular zone to the olfactory bulb.

Development 2020 05 26;147(10). Epub 2020 May 26.

Laboratory of Developmental Neurobiology, Department of Biology, KU Leuven, Leuven 3000, Belgium

The transcription factor Zeb2 controls fate specification and subsequent differentiation and maturation of multiple cell types in various embryonic tissues. It binds many protein partners, including activated Smad proteins and the NuRD co-repressor complex. How Zeb2 subdomains support cell differentiation in various contexts has remained elusive. Here, we studied the role of Zeb2 and its domains in neurogenesis and neural differentiation in the young postnatal ventricular-subventricular zone (V-SVZ), in which neural stem cells generate olfactory bulb-destined interneurons. Conditional knockouts and separate acute loss- and gain-of-function approaches indicated that Zeb2 is essential for controlling apoptosis and neuronal differentiation of V-SVZ progenitors before and after birth, and we identified as a potential downstream target gene of Zeb2. genetic inactivation impaired the differentiation potential of the V-SVZ niche in a cell-autonomous fashion. We also provide evidence that its normal function in the V-SVZ also involves non-autonomous mechanisms. Additionally, we demonstrate distinct roles for Zeb2 protein-binding domains, suggesting that Zeb2 partners co-determine neuronal output from the mouse V-SVZ in both quantitative and qualitative ways in early postnatal life.
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http://dx.doi.org/10.1242/dev.184861DOI Listing
May 2020

Integrative and perturbation-based analysis of the transcriptional dynamics of TGFβ/BMP system components in transition from embryonic stem cells to neural progenitors.

Stem Cells 2020 02 13;38(2):202-217. Epub 2019 Dec 13.

Department of Cell Biology, Erasmus University Medical Center, Rotterdam, The Netherlands.

Cooperative actions of extrinsic signals and cell-intrinsic transcription factors alter gene regulatory networks enabling cells to respond appropriately to environmental cues. Signaling by transforming growth factor type β (TGFβ) family ligands (eg, bone morphogenetic proteins [BMPs] and Activin/Nodal) exerts cell-type specific and context-dependent transcriptional changes, thereby steering cellular transitions throughout embryogenesis. Little is known about coordinated regulation and transcriptional interplay of the TGFβ system. To understand intrafamily transcriptional regulation as part of this system's actions during development, we selected 95 of its components and investigated their mRNA-expression dynamics, gene-gene interactions, and single-cell expression heterogeneity in mouse embryonic stem cells transiting to neural progenitors. Interrogation at 24 hour intervals identified four types of temporal gene transcription profiles that capture all stages, that is, pluripotency, epiblast formation, and neural commitment. Then, between each stage we performed esiRNA-based perturbation of each individual component and documented the effect on steady-state mRNA levels of the remaining 94 components. This exposed an intricate system of multilevel regulation whereby the majority of gene-gene interactions display a marked cell-stage specific behavior. Furthermore, single-cell RNA-profiling at individual stages demonstrated the presence of detailed co-expression modules and subpopulations showing stable co-expression modules such as that of the core pluripotency genes at all stages. Our combinatorial experimental approach demonstrates how intrinsically complex transcriptional regulation within a given pathway is during cell fate/state transitions.
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http://dx.doi.org/10.1002/stem.3111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7027912PMC
February 2020

The dynamic emergence of GATA1 complexes identified in embryonic stem cell differentiation and mouse fetal liver.

Haematologica 2020 07 3;105(7):1802-1812. Epub 2019 Oct 3.

Department of Cell Biology, ErasmusMC, Rotterdam, the Netherlands

GATA1 is an essential transcriptional regulator of myeloid hematopoietic differentiation towards red blood cells. During erythroid differentiation, GATA1 forms different complexes with other transcription factors such as LDB1, TAL1, E2A and LMO2 ("the LDB1 complex") or with FOG1. The functions of GATA1 complexes have been studied extensively in definitive erythroid differentiation; however, the temporal and spatial formation of these complexes during erythroid development is unknown. We applied proximity ligation assay (PLA) to detect, localize and quantify individual interactions during embryonic stem cell differentiation and in mouse fetal liver (FL) tissue. We show that GATA1/LDB1 interactions appear before the proerythroblast stage and increase in a subset of the CD71/TER119 cells to activate the terminal erythroid differentiation program in 12.5 day FL. Using and knockdown FL cells, we studied the functional contribution of the GATA1/LDB1 complex during differentiation. This shows that the active LDB1 complex appears quite late at the proerythroblast stage of differentiation and confirms the power of PLA in studying the dynamic interaction of proteins in cell differentiation at the single cell level. We provide dynamic insight into the temporal and spatial formation of the GATA1 and LDB1 transcription factor complexes during hematopoietic development and differentiation.
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http://dx.doi.org/10.3324/haematol.2019.216010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7327653PMC
July 2020

Forces driving the three-dimensional folding of eukaryotic genomes.

Mol Syst Biol 2018 06 1;14(6):e8214. Epub 2018 Jun 1.

Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany

The last decade has radically renewed our understanding of higher order chromatin folding in the eukaryotic nucleus. As a result, most current models are in support of a mostly hierarchical and relatively stable folding of chromosomes dividing chromosomal territories into A- (active) and B- (inactive) compartments, which are then further partitioned into topologically associating domains (TADs), each of which is made up from multiple loops stabilized mainly by the CTCF and cohesin chromatin-binding complexes. Nonetheless, the structure-to-function relationship of eukaryotic genomes is still not well understood. Here, we focus on recent work highlighting the biophysical and regulatory forces that contribute to the spatial organization of genomes, and we propose that the various conformations that chromatin assumes are not so much the result of a linear hierarchy, but rather of both converging and conflicting dynamic forces that act on it.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6024091PMC
http://dx.doi.org/10.15252/msb.20188214DOI Listing
June 2018

The detailed 3D multi-loop aggregate/rosette chromatin architecture and functional dynamic organization of the human and mouse genomes.

Epigenetics Chromatin 2016 24;9:58. Epub 2016 Dec 24.

Cell Biology, Department Cell Biology and Genetics, Erasmus MC, Dr. Molewaterplein 50, 3015 GE, Rotterdam, The Netherlands.

Background: The dynamic three-dimensional chromatin architecture of genomes and its co-evolutionary connection to its function-the storage, expression, and replication of genetic information-is still one of the central issues in biology. Here, we describe the much debated 3D architecture of the human and mouse genomes from the nucleosomal to the megabase pair level by a novel approach combining selective high-throughput high-resolution chromosomal interaction capture (), polymer simulations, and scaling analysis of the 3D architecture and the DNA sequence.

Results: The genome is compacted into a chromatin quasi-fibre with ~5 ± 1 nucleosomes/11 nm, folded into stable ~30-100 kbp loops forming stable loop aggregates/rosettes connected by similar sized linkers. Minor but significant variations in the architecture are seen between cell types and functional states. The architecture and the DNA sequence show very similar fine-structured multi-scaling behaviour confirming their co-evolution and the above.

Conclusions: This architecture, its dynamics, and accessibility, balance stability and flexibility ensuring genome integrity and variation enabling gene expression/regulation by self-organization of (in)active units already in proximity. Our results agree with the heuristics of the field and allow "architectural sequencing" at a genome mechanics level to understand the inseparable systems genomic properties.
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http://dx.doi.org/10.1186/s13072-016-0089-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5192698PMC
December 2016

Exploiting native forces to capture chromosome conformation in mammalian cell nuclei.

Mol Syst Biol 2016 Dec 9;12(12):891. Epub 2016 Dec 9.

Center for Molecular Medicine, University of Cologne, Cologne, Germany

Mammalian interphase chromosomes fold into a multitude of loops to fit the confines of cell nuclei, and looping is tightly linked to regulated function. Chromosome conformation capture (3C) technology has significantly advanced our understanding of this structure-to-function relationship. However, all 3C-based methods rely on chemical cross-linking to stabilize spatial interactions. This step remains a "black box" as regards the biases it may introduce, and some discrepancies between microscopy and 3C studies have now been reported. To address these concerns, we developed "i3C", a novel approach for capturing spatial interactions without a need for cross-linking. We apply i3C to intact nuclei of living cells and exploit native forces that stabilize chromatin folding. Using different cell types and loci, computational modeling, and a methylation-based orthogonal validation method, "TALE-iD", we show that native interactions resemble cross-linked ones, but display improved signal-to-noise ratios and are more focal on regulatory elements and CTCF sites, while strictly abiding to topologically associating domain restrictions.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5199122PMC
http://dx.doi.org/10.15252/msb.20167311DOI Listing
December 2016

Binding of nuclear factor κB to noncanonical consensus sites reveals its multimodal role during the early inflammatory response.

Genome Res 2016 11 15;26(11):1478-1489. Epub 2016 Sep 15.

Center for Molecular Medicine, University of Cologne, 50931 Cologne, Germany.

Mammalian cells have developed intricate mechanisms to interpret, integrate, and respond to extracellular stimuli. For example, tumor necrosis factor (TNF) rapidly activates proinflammatory genes, but our understanding of how this occurs against the ongoing transcriptional program of the cell is far from complete. Here, we monitor the early phase of this cascade at high spatiotemporal resolution in TNF-stimulated human endothelial cells. NF-κB, the transcription factor complex driving the response, interferes with the regulatory machinery by binding active enhancers already in interaction with gene promoters. Notably, >50% of these enhancers do not encode canonical NF-κB binding motifs. Using a combination of genomics tools, we find that binding site selection plays a key role in NF-κΒ-mediated transcriptional activation and repression. We demonstrate the latter by describing the synergy between NF-κΒ and the corepressor JDP2. Finally, detailed analysis of a 2.8-Mbp locus using sub-kbp-resolution targeted chromatin conformation capture and genome editing uncovers how NF-κΒ that has just entered the nucleus exploits pre-existing chromatin looping to exert its multimodal role. This work highlights the involvement of topology in cis-regulatory element function during acute transcriptional responses, where primary DNA sequence and its higher-order structure constitute a regulatory context leading to either gene activation or repression.
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http://dx.doi.org/10.1101/gr.210005.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5088591PMC
November 2016

Zeb2 Regulates Cell Fate at the Exit from Epiblast State in Mouse Embryonic Stem Cells.

Stem Cells 2017 03 8;35(3):611-625. Epub 2016 Nov 8.

Department of Development and Regeneration, KU Leuven, Leuven, 3000, Belgium.

In human embryonic stem cells (ESCs) the transcription factor Zeb2 regulates neuroectoderm versus mesendoderm formation, but it is unclear how Zeb2 affects the global transcriptional regulatory network in these cell-fate decisions. We generated Zeb2 knockout (KO) mouse ESCs, subjected them as embryoid bodies (EBs) to neural and general differentiation and carried out temporal RNA-sequencing (RNA-seq) and reduced representation bisulfite sequencing (RRBS) analysis in neural differentiation. This shows that Zeb2 acts preferentially as a transcriptional repressor associated with developmental progression and that Zeb2 KO ESCs can exit from their naïve state. However, most cells in these EBs stall in an early epiblast-like state and are impaired in both neural and mesendodermal differentiation. Genes involved in pluripotency, epithelial-to-mesenchymal transition (EMT), and DNA-(de)methylation, including Tet1, are deregulated in the absence of Zeb2. The observed elevated Tet1 levels in the mutant cells and the knowledge of previously mapped Tet1-binding sites correlate with loss-of-methylation in neural-stimulating conditions, however, after the cells initially acquired the correct DNA-methyl marks. Interestingly, cells from such Zeb2 KO EBs maintain the ability to re-adapt to 2i + LIF conditions even after prolonged differentiation, while knockdown of Tet1 partially rescues their impaired differentiation. Hence, in addition to its role in EMT, Zeb2 is critical in ESCs for exit from the epiblast state, and links the pluripotency network and DNA-methylation with irreversible commitment to differentiation. Stem Cells 2017;35:611-625.
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http://dx.doi.org/10.1002/stem.2521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5396376PMC
March 2017

The core spliceosome as target and effector of non-canonical ATM signalling.

Nature 2015 Jul 24;523(7558):53-8. Epub 2015 Jun 24.

Department of Genetics, Cancer Genomics Netherlands, Erasmus University Medical Center, Rotterdam, 3015 CN, The Netherlands.

In response to DNA damage, tissue homoeostasis is ensured by protein networks promoting DNA repair, cell cycle arrest or apoptosis. DNA damage response signalling pathways coordinate these processes, partly by propagating gene-expression-modulating signals. DNA damage influences not only the abundance of messenger RNAs, but also their coding information through alternative splicing. Here we show that transcription-blocking DNA lesions promote chromatin displacement of late-stage spliceosomes and initiate a positive feedback loop centred on the signalling kinase ATM. We propose that initial spliceosome displacement and subsequent R-loop formation is triggered by pausing of RNA polymerase at DNA lesions. In turn, R-loops activate ATM, which signals to impede spliceosome organization further and augment ultraviolet-irradiation-triggered alternative splicing at the genome-wide level. Our findings define R-loop-dependent ATM activation by transcription-blocking lesions as an important event in the DNA damage response of non-replicating cells, and highlight a key role for spliceosome displacement in this process.
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http://dx.doi.org/10.1038/nature14512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4501432PMC
July 2015

NLS-tagging: an alternative strategy to tag nuclear proteins.

Nucleic Acids Res 2014 Dec 26;42(21). Epub 2014 Sep 26.

Department of Cell Biology, Erasmus Medical Center, Faculty building, PO Box 2040, 3000 CA Rotterdam, The Netherlands Center for Biomedical Genetics and Medical Epigenetics Consortium, Erasmus Medical Center, Faculty building, PO Box 2040, 3000 CA Rotterdam, The Netherlands Center for Biomedical Genetics, Erasmus Medical Center, Faculty building, PO Box 2040, 3000 CA Rotterdam, The Netherlands

The characterization of transcription factor complexes and their binding sites in the genome by affinity purification has yielded tremendous new insights into how genes are regulated. The affinity purification requires either the use of antibodies raised against the factor of interest itself or by high-affinity binding of a C- or N-terminally added tag sequence to the factor. Unfortunately, fusing extra amino acids to the termini of a factor can interfere with its biological function or the tag may be inaccessible inside the protein. Here, we describe an effective solution to that problem by integrating the 'tag' close to the nuclear localization sequence domain of the factor. We demonstrate the effectiveness of this approach with the transcription factors Fli-1 and Irf2bp2, which cannot be tagged at their extremities without loss of function. This resulted in the identification of novel proteins partners and a new hypothesis on the contribution of Fli-1 to hematopoiesis.
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http://dx.doi.org/10.1093/nar/gku869DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4245968PMC
December 2014

Sox2 regulates the emergence of lung basal cells by directly activating the transcription of Trp63.

Am J Respir Cell Mol Biol 2014 Aug;51(2):311-22

Departments of 1 Pediatric Surgery of the Erasmus MC-Sophia Children's Hospital.

Lung development is determined by the coordinated expression of several key genes. Previously, we and others have shown the importance of the sex determining region Y-box 2 (Sox2) gene in lung development. Transgenic expression of Sox2 during lung development resulted in cystic airways, and here we show that modulating the timing of ectopic Sox2 expression in the branching regions of the developing lung results in variable cystic lesions resembling the spectrum of the human congenital disorder congenital cystic adenomatoid malformation (CCAM). Sox2 dominantly differentiated naive epithelial cells into the proximal lineage irrespective of the presence of Fgf10. Sox2 directly induced the expression of Trp63, the master switch toward the basal cell lineage and induced the expression of Gata6, a factor involved in the emergence of bronchoalveolar stem cells. We showed that SOX2 and TRP63 are coexpressed in the lungs of human patients with type II CCAM. The combination of premature differentiation toward the proximal cell lineage and the induction of proliferation resulted in the cyst-like structures. Thus, we show that Sox2 is directly responsible for the emergence of two lung progenitor cells: basal cells by regulating the master gene Trp63 and bronchoalveolar stem cells by regulating Gata6.
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http://dx.doi.org/10.1165/rcmb.2013-0419OCDOI Listing
August 2014

Transcription in the context of the 3D nucleus.

Curr Opin Genet Dev 2014 Apr 15;25:62-7. Epub 2014 Feb 15.

Department of Cell Biology, Erasmus Medical Center, Rotterdam, The Netherlands.

Gene activity is not only determined by processes occurring very close to or at the gene, such as transcription factor or RNA Polymerase II (PolII) binding. A multitude of past observations such as the localization of inactive chromatin to the nuclear periphery and active chromatin in the centre of the nucleus, the clustering of highly transcribed genes at transcriptional hotspots as well as the looping of active genes out of the chromosome territory made clear that the 'context matters' and the 3-dimensional organization of the chromatin fibre is fundamental for genome function. Here we want to review whether and how the different architectural levels that were recently identified by high-throughput chromatin conformation capturing techniques influence transcription.
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http://dx.doi.org/10.1016/j.gde.2013.11.020DOI Listing
April 2014

Cohesin and CTCF differentially affect chromatin architecture and gene expression in human cells.

Proc Natl Acad Sci U S A 2014 Jan 13;111(3):996-1001. Epub 2013 Dec 13.

Department of Cell Biology, Biophysical Genomics, Department of Cell Biology, Center for Biomics, Cancer Genomics Center, Erasmus Medical Center, 3015 GE, Rotterdam, The Netherlands.

Recent studies of genome-wide chromatin interactions have revealed that the human genome is partitioned into many self-associating topological domains. The boundary sequences between domains are enriched for binding sites of CTCC-binding factor (CTCF) and the cohesin complex, implicating these two factors in the establishment or maintenance of topological domains. To determine the role of cohesin and CTCF in higher-order chromatin architecture in human cells, we depleted the cohesin complex or CTCF and examined the consequences of loss of these factors on higher-order chromatin organization, as well as the transcriptome. We observed a general loss of local chromatin interactions upon disruption of cohesin, but the topological domains remain intact. However, we found that depletion of CTCF not only reduced intradomain interactions but also increased interdomain interactions. Furthermore, distinct groups of genes become misregulated upon depletion of cohesin and CTCF. Taken together, these observations suggest that CTCF and cohesin contribute differentially to chromatin organization and gene regulation.
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http://dx.doi.org/10.1073/pnas.1317788111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3903193PMC
January 2014

Identification of multiple subsets of ventral interneurons and differential distribution along the rostrocaudal axis of the developing spinal cord.

PLoS One 2013 15;8(8):e70325. Epub 2013 Aug 15.

Université catholique de Louvain, Institute of Neuroscience, Laboratory of Neural Differentiation, Brussels, Belgium.

The spinal cord contains neuronal circuits termed Central Pattern Generators (CPGs) that coordinate rhythmic motor activities. CPG circuits consist of motor neurons and multiple interneuron cell types, many of which are derived from four distinct cardinal classes of ventral interneurons, called V0, V1, V2 and V3. While significant progress has been made on elucidating the molecular and genetic mechanisms that control ventral interneuron differentiation, little is known about their distribution along the antero-posterior axis of the spinal cord and their diversification. Here, we report that V0, V1 and V2 interneurons exhibit distinct organizational patterns at brachial, thoracic and lumbar levels of the developing spinal cord. In addition, we demonstrate that each cardinal class of ventral interneurons can be subdivided into several subsets according to the combinatorial expression of different sets of transcription factors, and that these subsets are differentially distributed along the rostrocaudal axis of the spinal cord. This comprehensive molecular profiling of ventral interneurons provides an important resource for investigating neuronal diversification in the developing spinal cord and for understanding the contribution of specific interneuron subsets on CPG circuits and motor control.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0070325PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3744532PMC
September 2014

r3Cseq: an R/Bioconductor package for the discovery of long-range genomic interactions from chromosome conformation capture and next-generation sequencing data.

Nucleic Acids Res 2013 Jul 13;41(13):e132. Epub 2013 May 13.

Computational Biology Unit, Uni Computing, Uni Research AS, N-5020 Bergen, Norway.

The coupling of chromosome conformation capture (3C) with next-generation sequencing technologies enables the high-throughput detection of long-range genomic interactions, via the generation of ligation products between DNA sequences, which are closely juxtaposed in vivo. These interactions involve promoter regions, enhancers and other regulatory and structural elements of chromosomes and can reveal key details of the regulation of gene expression. 3C-seq is a variant of the method for the detection of interactions between one chosen genomic element (viewpoint) and the rest of the genome. We present r3Cseq, an R/Bioconductor package designed to perform 3C-seq data analysis in a number of different experimental designs. The package reads a common aligned read input format, provides data normalization, allows the visualization of candidate interaction regions and detects statistically significant chromatin interactions, thus greatly facilitating hypothesis generation and the interpretation of experimental results. We further demonstrate its use on a series of real-world applications.
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http://dx.doi.org/10.1093/nar/gkt373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3711450PMC
July 2013

In vivo live imaging of RNA polymerase II transcription factories in primary cells.

Genes Dev 2013 Apr;27(7):767-77

Department of Cell Biology, Erasmus Medical Center, 3015GE Rotterdam, the Netherlands.

Transcription steps are marked by different modifications of the C-terminal domain of RNA polymerase II (RNAPII). Phosphorylation of Ser5 and Ser7 by cyclin-dependent kinase 7 (CDK7) as part of TFIIH marks initiation, whereas phosphorylation of Ser2 by CDK9 marks elongation. These processes are thought to take place in localized transcription foci in the nucleus, known as "transcription factories," but it has been argued that the observed clusters/foci are mere fixation or labeling artifacts. We show that transcription factories exist in living cells as distinct foci by live-imaging fluorescently labeled CDK9, a kinase known to associate with active RNAPII. These foci were observed in different cell types derived from CDK9-mCherry knock-in mice. We show that these foci are very stable while highly dynamic in exchanging CDK9. Chromatin immunoprecipitation (ChIP) coupled with deep sequencing (ChIP-seq) data show that the genome-wide binding sites of CDK9 and initiating RNAPII overlap on transcribed genes. Immunostaining shows that CDK9-mCherry foci colocalize with RNAPII-Ser5P, much less with RNAPII-Ser2P, and not with CDK12 (a kinase reported to be involved in the Ser2 phosphorylation) or with splicing factor SC35. In conclusion, transcription factories exist in living cells, and initiation and elongation of transcripts takes place in different nuclear compartments.
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http://dx.doi.org/10.1101/gad.216200.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3639417PMC
April 2013

Next generation sequencing of SNPs for non-invasive prenatal diagnosis: challenges and feasibility as illustrated by an application to β-thalassaemia.

Eur J Hum Genet 2013 Dec 10;21(12):1403-10. Epub 2013 Apr 10.

Molecular Genetics Thalassemia Department, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus.

β-Thalassaemia is one of the most common autosomal recessive single-gene disorder worldwide, with a carrier frequency of 12% in Cyprus. Prenatal tests for at risk pregnancies use invasive methods and development of a non-invasive prenatal diagnostic (NIPD) method is of paramount importance to prevent unnecessary risks inherent to invasive methods. Here, we describe such a method by assessing a modified version of next generation sequencing (NGS) using the Illumina platform, called 'targeted sequencing', based on the detection of paternally inherited fetal alleles in maternal plasma. We selected four single-nucleotide polymorphisms (SNPs) located in the β-globin locus with a high degree of heterozygosity in the Cypriot population. Spiked genomic samples were used to determine the specificity of the platform. We could detect the minor alleles in the expected ratio, showing the specificity of the platform. We then developed a multiplexed format for the selected SNPs and analysed ten maternal plasma samples from pregnancies at risk. The presence or absence of the paternal mutant allele was correctly determined in 27 out of 34 samples analysed. With haplotype analysis, NIPD was possible on eight out of ten families. This is the first study carried out for the NIPD of β-thalassaemia using targeted NGS and haplotype analysis. Preliminary results show that NGS is effective in detecting paternally inherited alleles in the maternal plasma.
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http://dx.doi.org/10.1038/ejhg.2013.47DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3831067PMC
December 2013

Balancing of histone H3K4 methylation states by the Kdm5c/SMCX histone demethylase modulates promoter and enhancer function.

Cell Rep 2013 Apr 28;3(4):1071-9. Epub 2013 Mar 28.

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

The functional organization of eukaryotic genomes correlates with specific patterns of histone methylations. Regulatory regions in genomes such as enhancers and promoters differ in their extent of methylation of histone H3 at lysine-4 (H3K4), but it is largely unknown how the different methylation states are specified and controlled. Here, we show that the Kdm5c/Jarid1c/SMCX member of the Kdm5 family of H3K4 demethylases can be recruited to both enhancer and promoter elements in mouse embryonic stem cells and in neuronal progenitor cells. Knockdown of Kdm5c deregulates transcription via local increases in H3K4me3. Our data indicate that by restricting H3K4me3 modification at core promoters, Kdm5c dampens transcription, but at enhancers Kdm5c stimulates their activity. Remarkably, an impaired enhancer function activates the intrinsic promoter activity of Kdm5c-bound distal elements. Our results demonstrate that the Kdm5c demethylase plays a crucial and dynamic role in the functional discrimination between enhancers and core promoters.
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http://dx.doi.org/10.1016/j.celrep.2013.02.030DOI Listing
April 2013

Directed migration of cortical interneurons depends on the cell-autonomous action of Sip1.

Neuron 2013 Jan;77(1):70-82

Laboratory of Molecular Biology (Celgen), Department of Development and Regeneration, University of Leuven, 3000 Leuven, Belgium.

GABAergic interneurons mainly originate in the medial ganglionic eminence (MGE) of the embryonic ventral telencephalon (VT) and migrate tangentially to the cortex, guided by membrane-bound and secreted factors. We found that Sip1 (Zfhx1b, Zeb2), a transcription factor enriched in migrating cortical interneurons, is required for their proper differentiation and correct guidance. The majority of Sip1 knockout interneurons fail to migrate to the neocortex and stall in the VT. RNA sequencing reveals that Sip1 knockout interneurons do not acquire a fully mature cortical interneuron identity and contain increased levels of the repulsive receptor Unc5b. Focal electroporation of Unc5b-encoding vectors in the MGE of wild-type brain slices disturbs migration to the neocortex, whereas reducing Unc5b levels in Sip1 knockout slices and brains rescues the migration defect. Our results reveal that Sip1, through tuning of Unc5b levels, is essential for cortical interneuron guidance.
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http://dx.doi.org/10.1016/j.neuron.2012.11.009DOI Listing
January 2013

KLF10 gene expression is associated with high fetal hemoglobin levels and with response to hydroxyurea treatment in β-hemoglobinopathy patients.

Pharmacogenomics 2012 Oct;13(13):1487-500

Erasmus University Medical Center, Department of Cell Biology, Rotterdam, The Netherlands.

Aim: In humans, fetal hemoglobin (HbF) production is controlled by many intricate mechanisms that, to date, remain only partly understood.

Patients & Methods: Pharmacogenomic analysis of the effects of hydroxyurea (HU) on HbF production was undertaken in a collection of Hellenic β-thalassemia and sickle cell disease (SCD) compound heterozygotes and a collection of healthy and KLF1-haploinsufficient Maltese adults, to identify genomic signatures that follow high HbF patterns.

Results: KLF10 emerged as a top candidate. Moreover, genotype analysis of β-thalassemia major and intermedia patients and an independent cohort of β-thalassemia/SCD compound heterozygous patients that do or do not respond to HU treatment showed that the homozygous mutant state of a tagSNP in the KLF10 3'UTR is not present in β-thalassemia intermedia patients and is underrepresented in β-thalassemia/SCD compound heterozygous patients that respond well to HU treatment.

Conclusion: These data suggest that KLF10 may constitute a pharmacogenomic marker to discriminate between response and nonresponse to HU treatment.
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http://dx.doi.org/10.2217/pgs.12.125DOI Listing
October 2012

The mammalian gene function resource: the International Knockout Mouse Consortium.

Mamm Genome 2012 Oct 12;23(9-10):580-6. Epub 2012 Sep 12.

The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1HH, UK.

In 2007, the International Knockout Mouse Consortium (IKMC) made the ambitious promise to generate mutations in virtually every protein-coding gene of the mouse genome in a concerted worldwide action. Now, 5 years later, the IKMC members have developed high-throughput gene trapping and, in particular, gene-targeting pipelines and generated more than 17,400 mutant murine embryonic stem (ES) cell clones and more than 1,700 mutant mouse strains, most of them conditional. A common IKMC web portal (www.knockoutmouse.org) has been established, allowing easy access to this unparalleled biological resource. The IKMC materials considerably enhance functional gene annotation of the mammalian genome and will have a major impact on future biomedical research.
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http://dx.doi.org/10.1007/s00335-012-9422-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3463800PMC
October 2012

RTTN mutations link primary cilia function to organization of the human cerebral cortex.

Am J Hum Genet 2012 Sep 30;91(3):533-40. Epub 2012 Aug 30.

Department of Clinical Genetics, Erasmus University Medical Center (Erasmus MC), P.O. Box 2040, 3000 CA Rotterdam, The Netherlands.

Polymicrogyria is a malformation of the developing cerebral cortex caused by abnormal organization and characterized by many small gyri and fusion of the outer molecular layer. We have identified autosomal-recessive mutations in RTTN, encoding Rotatin, in individuals with bilateral diffuse polymicrogyria from two separate families. Rotatin determines early embryonic axial rotation, as well as anteroposterior and dorsoventral patterning in the mouse. Human Rotatin has recently been identified as a centrosome-associated protein. The Drosophila melanogaster homolog of Rotatin, Ana3, is needed for structural integrity of centrioles and basal bodies and maintenance of sensory neurons. We show that Rotatin colocalizes with the basal bodies at the primary cilium. Cultured fibroblasts from affected individuals have structural abnormalities of the cilia and exhibit downregulation of BMP4, WNT5A, and WNT2B, which are key regulators of cortical patterning and are expressed at the cortical hem, the cortex-organizing center that gives rise to Cajal-Retzius (CR) neurons. Interestingly, we have shown that in mouse embryos, Rotatin colocalizes with CR neurons at the subpial marginal zone. Knockdown experiments in human fibroblasts and neural stem cells confirm a role for RTTN in cilia structure and function. RTTN mutations therefore link aberrant ciliary function to abnormal development and organization of the cortex in human individuals.
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http://dx.doi.org/10.1016/j.ajhg.2012.07.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3511998PMC
September 2012

Super-resolution imaging reveals three-dimensional folding dynamics of the β-globin locus upon gene activation.

J Cell Sci 2012 Oct 5;125(Pt 19):4630-9. Epub 2012 Jul 5.

Department of Cell Biology and Genetics and Center for Biomedical Genetics, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands.

The chromatin architecture is constantly changing because of cellular processes such as proliferation, differentiation and changes in the expression profile during gene activation or silencing. Unravelling the changes that occur in the chromatin structure during these processes has been a topic of interest for many years. It is known that gene activation of large gene loci is thought to occur by means of an active looping mechanism. It was also shown for the β-globin locus that the gene promoter interacts with an active chromatin hub by means of an active looping mechanism. This means that the locus changes in three-dimensional (3D) nuclear volume and chromatin shape. As a means of visualizing and measuring these dynamic changes in chromatin structure of the β-globin locus, we used a 3D DNA-FISH method in combination with 3D image acquisition to volume render fluorescent signals into 3D objects. These 3D chromatin structures were geometrically analysed, and results prior to and after gene activation were quantitatively compared. Confocal and super-resolution imaging revealed that the inactive locus occurs in several different conformations. These conformations change in shape and surface structure upon cell differentiation into a more folded and rounded structure that has a substantially smaller size and volume. These physical measurements represent the first non-biochemical evidence that, upon gene activation, an actively transcribing chromatin hub is formed by means of additional chromatin looping.
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http://dx.doi.org/10.1242/jcs.108522DOI Listing
October 2012

Enhancers and silencers: an integrated and simple model for their function.

Epigenetics Chromatin 2012 Jan 9;5(1). Epub 2012 Jan 9.

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK.

Regulatory DNA elements such as enhancers, silencers and insulators are embedded in metazoan genomes, and they control gene expression during development. Although they fulfil different roles, they share specific properties. Herein we discuss some examples and a parsimonious model for their function is proposed. All are transcription units that tether their target promoters close to, or distant from, transcriptional hot spots (or 'factories').
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http://dx.doi.org/10.1186/1756-8935-5-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3281776PMC
January 2012

Sox2 cooperates with Chd7 to regulate genes that are mutated in human syndromes.

Nat Genet 2011 Jun 1;43(6):607-11. Epub 2011 May 1.

Department of Cell Biology, Erasmus Medical Center (MC), Rotterdam, The Netherlands.

The HMG-box transcription factor Sox2 plays a role throughout neurogenesis and also acts at other stages of development, as illustrated by the multiple organs affected in the anophthalmia syndrome caused by SOX2 mutations. Here we combined proteomic and genomic approaches to characterize gene regulation by Sox2 in neural stem cells. Chd7, a chromatin remodeling ATPase associated with CHARGE syndrome, was identified as a Sox2 transcriptional cofactor. Sox2 and Chd7 physically interact, have overlapping genome-wide binding sites and regulate a set of common target genes including Jag1, Gli3 and Mycn, genes mutated in Alagille, Pallister-Hall and Feingold syndromes, which show malformations also associated with SOX2 anophthalmia syndrome or CHARGE syndrome. Regulation of disease-associated genes by a Sox2-Chd7 complex provides a plausible explanation for several malformations associated with SOX2 anophthalmia syndrome or CHARGE syndrome. Indeed, we found that Chd7-haploinsufficient embryos showed severely reduced expression of Jag1 in the developing inner ear.
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http://dx.doi.org/10.1038/ng.825DOI Listing
June 2011

The Gata3 transcription factor is required for the survival of embryonic and adult sympathetic neurons.

J Neurosci 2010 Aug;30(32):10833-43

Research Group Developmental Neurobiology, Max-Planck-Institute for Brain Research, 60528 Frankfurt/M, Germany.

The transcription factor Gata3 is essential for the development of sympathetic neurons and adrenal chromaffin cells. As Gata3 expression is maintained up to the adult stage, we addressed its function in differentiated sympathoadrenal cells at embryonic and adult stages by conditional Gata3 elimination. Inactivation of Gata3 in embryonic DBH-expressing neurons elicits a strong reduction in neuron numbers due to apoptotic cell death and reduced proliferation. No selective effect on noradrenergic gene expression (TH and DBH) was observed. Interestingly, Gata3 elimination in DBH-expressing neurons of adult animals also results in a virtually complete loss of sympathetic neurons. In the Gata3-deficient population, the expression of anti-apoptotic genes (Bcl-2, Bcl-xL, and NFkappaB) is diminished, whereas the expression of pro-apoptotic genes (Bik, Bok, and Bmf) was increased. The expression of noradrenergic genes (TH and DBH) is not affected. These results demonstrate that Gata3 is continuously required for maintaining survival but not differentiation in the sympathetic neuron lineage up to mature neurons of adult animals.
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http://dx.doi.org/10.1523/JNEUROSCI.0175-10.2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6634692PMC
August 2010

Haploinsufficiency for the erythroid transcription factor KLF1 causes hereditary persistence of fetal hemoglobin.

Nat Genet 2010 Sep 1;42(9):801-5. Epub 2010 Aug 1.

Laboratory of Molecular Genetics, Department of Physiology and Biochemistry, University of Malta, Msida, Malta.

Hereditary persistence of fetal hemoglobin (HPFH) is characterized by persistent high levels of fetal hemoglobin (HbF) in adults. Several contributory factors, both genetic and environmental, have been identified but others remain elusive. HPFH was found in 10 of 27 members from a Maltese family. We used a genome-wide SNP scan followed by linkage analysis to identify a candidate region on chromosome 19p13.12-13. Sequencing revealed a nonsense mutation in the KLF1 gene, p.K288X, which ablated the DNA-binding domain of this key erythroid transcriptional regulator. Only family members with HPFH were heterozygous carriers of this mutation. Expression profiling on primary erythroid progenitors showed that KLF1 target genes were downregulated in samples from individuals with HPFH. Functional assays suggested that, in addition to its established role in regulating adult globin expression, KLF1 is a key activator of the BCL11A gene, which encodes a suppressor of HbF expression. These observations provide a rationale for the effects of KLF1 haploinsufficiency on HbF levels.
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http://dx.doi.org/10.1038/ng.630DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2930131PMC
September 2010

Differential proteomics based on 18O labeling to determine the cyclin dependent kinase 9 interactome.

J Proteome Res 2010 Sep;9(9):4464-75

Proteomics Center and Department of Cell Biology, Erasmus University Medical Center, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands.

Although enzyme catalyzed 18O labeling has been used as a tool in quantitative proteomics, this type of labeling has not yielded the same impact yet as alternative techniques for quantitation like SILAC or labeling with chemical mass tags. The practical difficulties involved in 18O labeling, most importantly the occurrence of incomplete labeling and, as a result, the difficulties in data analysis and interpretation have hampered its implementation in high-throughput comparative proteomics protocols. In this paper, we have optimized the 18O labeling procedure to such an extent that complete labeling can be achieved in a routine manner. We have implemented this approach into a protein-protein interaction analysis pipeline to differentiate between bona fide interaction partners of the low-level expressing cell cycle regulator cyclin-dependent kinase 9 (Cdk9) and nonspecifically binding or background proteins. Previously known as well as novel interaction partners of Cdk9 were found, among which most notably the Mediator complex and several other proteins involved in transcriptional regulation. We show here that a differential proteomics approach based on 18O labeling provides a valuable method for high-confidence determination of protein interaction partners and is easily implemented in protein network analysis workflows.
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http://dx.doi.org/10.1021/pr100217dDOI Listing
September 2010