Totally asymmetric exclusion process with site-wise dynamic disorder

We propose an extension of the totally asymmetric simple exclusion process (TASEP) in which particles hopping along a lattice can be blocked by obstacles that dynamically attach/detach from lattice sites. The model can be thought as TASEP with site-wise dynamic disorder. We consider two versions of defect dynamics: (i) defects can bind to any site, irrespective of particle occupation, (ii) defects only bind to sites which are not occupied by particles (particle-obstacle exclusion). In case (i) there is a symmetric, parabolic-like relationship between the current and particle density, as in the standard TASEP. Case (ii) leads to a skewed relationship for slow defect dynamics. We also show that the presence of defects induces particle clustering, despite the translation invariance of the system. For open boundaries the same three phases as for the standard TASEP are observed, albeit the position of phase boundaries is affected by the presence of obstacles. We develop a simple mean-field theory that captures the model's quantitative behaviour for periodic and open boundary conditions and yields good estimates for the current-density relationship, mean cluster sizes and phase boundaries. Lastly, we discuss an application of the model to the biological process of gene transcription.Comment: submitted to J. Phys.

Quantitative analysis questions the role of MeCP2 as a global regulator of alternative splicing

MeCP2 is an abundant protein in mature nerve cells, where it binds to DNA sequences containing methylated cytosine. Mutations in the MECP2 gene cause the severe neurological disorder Rett syndrome (RTT), provoking intensive study of the underlying molecular mechanisms. Multiple functions have been proposed, one of which involves a regulatory role in splicing. Here we leverage the recent availability of high-quality transcriptomic data sets to probe quantitatively the potential influence of MeCP2 on alternative splicing. Using a variety of machine learning approaches that can capture both linear and non-linear associations, we show that widely different levels of MeCP2 have a minimal effect on alternative splicing in three different systems. Alternative splicing was also apparently indifferent to developmental changes in DNA methylation levels. Our results suggest that regulation of splicing is not a major function of MeCP2. They also highlight the importance of multi-variate quantitative analyses in the formulation of biological hypotheses

Quantitative modelling predicts the impact of DNA methylation on RNA polymerase II traffic

Patterns of gene expression are primarily determined by proteins that locally enhance or repress transcription. While many transcription factors target a restricted number of genes, others appear to modulate transcription levels globally. An example is MeCP2, an abundant methylated-DNA binding protein that is mutated in the neurological disorder Rett Syndrome. Despite much research, the molecular mechanism by which MeCP2 regulates gene expression is not fully resolved. Here we integrate quantitative, multidimensionalexperimental analysis and mathematical modelling to indicate that MeCP2 is a novel type of global transcriptional regulator whose binding to DNA creates "slow sites" in gene bodies. We hypothesise that waves of slowed-down RNA polymerase II formed behind these sites travel backward and indirectly affect initiation, reminiscent of defect-induced shock waves in non-equilibrium physics transport models. This mechanism differs from conventional gene regulation mechanisms, which often involve direct modulation of transcription initiation. Our findings point to a genome-wide function of DNA methylation that may account for the reversibility of Rett syndrome in mice. Moreover, our combined theoretical and experimental approach provides a general method for understanding how global gene expression patterns are choreographed

SALL4 controls cell fate in response to DNA base composition

Contains fulltext : 232790.pdf (Publisher’s version ) (Open Access

An Orphan CpG Island Drives Expression of a let-7 miRNA Precursor with an Important Role in Mouse Development.

Most human genes are associated with promoters embedded in non-methylated, G + C-rich CpG islands (CGIs). Not all CGIs are found at annotated promoters, however, raising the possibility that many serve as promoters for transcripts that do not code for proteins. To test this hypothesis, we searched for novel transcripts in embryonic stem cells (ESCs) that originate within orphan CGIs. Among several candidates, we detected a transcript that included three members of the let-7 micro-RNA family: Let-7a-1, let-7f-1, and let-7d. Deletion of the CGI prevented expression of the precursor RNA and depleted the included miRNAs. Mice homozygous for this mutation were sub-viable and showed growth and other defects. The results suggest that despite the identity of their seed sequences, members of the let-7 miRNA family exert distinct functions that cannot be complemented by other members

Neuronal non-CG methylation is an essential target for MeCP2 function

DNA methylation is implicated in neuronal biology via the protein MeCP2, the mutation of which causes Rett syndrome. MeCP2 recruits the NCOR1/2 co-repressor complexes to methylated cytosine in the CG dinucleotide, but also to sites of non-CG methylation, which are abundant in neurons. To test the biological significance of the dual-binding specificity of MeCP2, we replaced its DNA binding domain with an orthologous domain from MBD2, which can only bind mCG motifs. Knockin mice expressing the domain-swap protein displayed severe Rett-syndrome-like phenotypes, indicating that normal brain function requires the interaction of MeCP2 with sites of non-CG methylation, specifically mCAC. The results support the notion that the delayed onset of Rett syndrome is due to the simultaneous post-natal accumulation of mCAC and its reader MeCP2. Intriguingly, genes dysregulated in both Mecp2 null and domain-swap mice are implicated in other neurological disorders, potentially highlighting targets of relevance to the Rett syndrome phenotype

Endogenous single-strand DNA breaks at RNA polymerase II promoters in <i>Saccharomyces cerevisiae</i>

K

die Rolle der Transkriptionsfaktoren Rbp-j und Olig3

1 . INTRODUCTION 1-16 1.1 . Patterning of the central nervous system 1 1.1.1 . Anterior-posterior patterning 1 1.1.2 . Dorsal-ventral patterning of the spinal cord 3 1.2 . Neuronal subtypes of the dorsal spinal cord and hindbrain 5 1.2.1 . Early-born neurons of the dorsal spinal cord and hindbrain 6 1.2.2 . Late-born neurons of the dorsal spinal cord and hindbrain 8 1.3 . The role of bHLH transcription factors in the specification of neurons 10 1.3.1 . The Olig3 bHLH transcription factor 10 1.3.2 . Ptf1a function in the nervous system 11 1.4 . Functions of the Notch pathway in the neural development 13 1.4.1 . Rbp-j function in the Notch pathway 13 1.5 . Aims 16 2 . MATERIALS AND METHODS 17-35 2.1 . Abbreviations 17 2.2 . Materials 19 2.2.1 . Chemicals 19 2.2.2 . Bacterial strains 19 2.2.3 . Vectors 19 2.2.4 . Antibodies 20 2.2.5 . Riboprobes for in situ hybridization 20 2.2.6 . Cell lines 21 2.2.7 . Chicken strains 21 2.2.8 . Mouse strains 21 2.2.9 . Bacteria culture 22 2.2.10 . Cell culture media 23 2.3 . Methods 24 2.3.1 . Preparation of plasmids and DNA fragments 24 2.3.2 . Restriction hydrolysis of DNA, ligation of DNA fragments and transformation into bacteria 24 2.3.3 . Homologous recombination in bacteria 25 2.3.4 . DNA sequencing 25 2.3.5 . Polymerase chain reaction 26 2.3.6 . Isolation of genomic DNA from mouse tissue 28 2.3.7 . Isolation of genomic DNA from ES cells 28 2.3.8 . Southern blot 29 2.3.9 . Fibroblast cell culture 30 2.3.10 . ES cell culture, transfection and selection 30 2.3.11 . Blastocyst injection 31 2.3.12 . In ovo electroporation 31 2.3.13 . Preparation of riboprobes for in situ hybridization 32 2.3.14 . Preparation of frozen sections 32 2.3.15 . Immunohistochemistry 33 2.3.16 . X-Gal staining 33 2.3.17 . In situ hybridization 34 2.3.18 . Tamoxifen gavaging 35 2.3.19 . Program for counting cell nuclei 35 3 . RESULTS 36-64 3.1 . Rbp-j function in the dorsal spinal cord 36 3.1.1 . Effect of Rbp-j mutation on neurogenesis 37 3.1.2 . The role of Rbp-j in the specification of early-born neurons 40 3.1.3 . Analysis of Rbp-j function in the specification of dILA and dILB neurons 43 3.2 . Olig3 function in the hindbrain 46 3.2.1 . Generation of mouse strains that carry Cre sequences in the Olig3 locus 46 3.2.2 . Olig3 expression in the alar plate of the hindbrain and characterization of neuron subtypes arising from the Olig3+ domain 49 3.2.3 . Analysis of rhombomeres 4-7 of Olig3 mutant mice 53 3.2.4 . Derivatives of Olig3+ cells in the hindbrain and genetic lineage tracing in homozygous Olig3 mutant mice 56 3.2.5 . The role of Olig3 in the development of dA4 and dA3 neurons 60 4 . DISCUSSION 65-77 4.1 . Rbp-j functions in the dorsal spinal cord 65 4.1.1 . Two distinct roles of Rbp-j in the dorsal spinal cord 66 4.1.2 . Evolution of the Notch-independent Rbp-j function 70 4.2 . The Olig3 function in the dorsal hindbrain 72 4.2.1 . Derivatives of the Olig3+ progenitor domain 72 4.2.2 . The function of Olig3, Ptf1a and Lbx1 in the determination of dorsal neuron types 73 4.2.3 . A similar role of Olig3 in the dorsal spinal cord and hindbrain 75 4.2.4 . Major function of Olig3 in the specification of neuronal types 76 5 . SUMMARY 78-80 ZUSAMMENFASSUNG 79 6 . BIBLIOGRAPHY 81-90Correct function of neural networks depends on a balance of inhibitory and excitatory activity. The balance is initially generated through the determination of inhibitory and excitatory neuronal fates controlled by transcription factors. A first goal in my thesis was to define if Notch, which controls asymmetric divisions in other systems, plays a role in the determination of inhibitory and excitatory fates in the dorsal spinal cord. To address this, I employed genetic analysis in mice and used two transgenic mouse models. In the first, a dominant-negative variant of Mastermind-like1 (dnMaml) was expressed in an inducible manner, which inhibits the transcriptional response to Notch signaling. In the second, I employed conditional mutagenesis of Rbp-j, the major transcriptional mediator of the Notch pathway. In Rbp-j and dnMaml mutant mice, I observed a depletion of the progenitor domain in the dorsal spinal cord, consistent with the known function of Notch signaling in the maintenance of neuronal progenitors. I also found that the GABAergic neurons were not formed in Rbp-j mutant mice, but this effect was not observable in the dnMaml mutants. Independent studies in Jane Johnson’s laboratory described a similar function for the bHLH factor Ptf1a, which is a component of the PTF1 transcriptional complex. My results together with those of Jane Johnson indicate that Rbp-j functions independently of Notch in this PTF1 complex. A second goal was the analysis of the bHLH transcription factor Olig3 that is expressed in the ventricular zone of the dorsal alar plate of the hindbrain. For this, I generated two new mouse strains, Olig3Cre and Olig3CreERT2, and used these for genetic fate mapping. I found that the Olig3+ progenitor domain gives rise to several neuronal types in the dorsal alar plate of the hindbrain, which contribute to the nucleus of the solitary tract and to precerebellar nuclei. In Olig3 mutant mice, the nucleus of the solitary tract did not form, and precerebellar nuclei were absent or smaller. My further work that relies on overexpression experiments in the chick hindbrain showed that Olig3 and Ptf1a together induce the fate of climbing fiber neurons of the inferior olivary nucleus. However, analysis of Olig3/Lbx1 double mutant mice demonstrated that Olig3 exerts its role solely by suppressing Lbx1 in specification of climbing fiber neurons. In contrast, analysis of Olig3/Ptf1a double mutant mice revealed that Olig3 has an instructive role in the determination of noradrenergic neurons of the nucleus of the solitary tract.Die korrekte Funktion neuronaler Netzwerke beruht auf einem Gleichgewicht zwischen hemmenden und aktivierenden Impulsen. Dieses Gleichgewicht wird ab initio durch Transkriptionsfaktoren hergestellt, die den inhibitorischen bzw. exzitatorischen Charakter neuronaler Zellen festlegen. Als erstes Ziel meiner Promotionsarbeit habe ich im Modellorganismus Maus untersucht, ob der Notch- Rezeptor, der in anderen Systemen asymmetrische Zellteilung steuert, diese Funktion auch bei der Determinierung hemmender und aktivierender Neurone im dorsalen Rückenmark ausübt. In der ersten von zwei verwendeten transgenen Mauslinien wurde eine dominant-negative Variante von Mastermind-like1 (dnMaml) induzierbar exprimiert, um so die Notch-abhängige transkriptionelle Aktivierung zu hemmen. In der zweiten Mauslinie wurde Rbp-j, der wichtigste Transkriptionsfaktor im Notch-Signalweg, konditionell mutiert. In beiden Mauslinien fand ich, in Übereinstimmung mit der beschriebenen Rolle von Notch bei der Erhaltung von Vorläuferzellen, eine Minderung der Vorläuferdomäne im dorsalen Rückenmark. GABAerge Neurone fehlten in den Rbp-j-, nicht jedoch in den dnMaml-Mäusen. Unabhängige Untersuchungen aus dem Labor von Jane Johnson zeigten eine ähnliche Funktion für den bHLH Transkriptionsfaktor Ptf1a, einen Bestandteil des PTF1 Transkriptionsfaktorkomplexes. Zusammengenommen deuten diese Ergebnisse darauf hin, daß Rbp-j eine Notch-unabhängige Funktion im PTF1-Komplex ausübt. Ein zweites Ziel meiner Dissertation war die Analyse des bHLH Transkriptionsfaktors Olig3, der in der Ventrikulärzone der dorsalen Alarplatte des Rhombenzephalon exprimiert wird. Ich habe dafür zwei neue Mauslinien etabliert, Olig3Cre und Olig3CreERT2, die ich zur genetischen Zellschicksalsanalyse eingesetzt habe. Ich fand, daß die Olig3+ Vorläuferdomäne verschiedene Neuronentypen in der dorsalen Alarplatte des Rhombenzephalon hervorbringt, die zum Nucleus solitarius und zu präzerebellaren Kernen beitragen. In Olig3 mutanten Mäusen wurde der Nucleus solitarius nicht gebildet, die präzerebellaren Kerne waren reduziert oder fehlten. Weiterführende Arbeiten im Hühnchen zeigten, daß Olig3 und Ptf1a zusammen die Kletterfasern des Nucleus olivarius inferior induzieren. Die Analyse dieser Fasern in Olig3/Lbx1 Maus-Doppelmutanten ergab, daß Olig3 hier auschließlich über die Unterdrückung von Lbx1 wirkt. In Olig3/Ptf1a Doppelmutanten hat Olig3 hingegen eine aktive Rolle in der Determinierung noradrenerger Neurone des Tractus solitarius

Old Columbus Image data set

Microscopy data from multiple research programmes in CRM. This dataset has been deleted from the Edinburgh DataVault for administrative reasons