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Studies of Cellular Regulatory Mechanisms: from Genetic Switches to Cell Migration
KTH, School of Computer Science and Communication (CSC), Computational Biology, CB.
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cellular behaviour depends ultimately on the transcription of genes. If we know how transcription is controlled we have a better chance of understanding cellular processes. This thesis presents six studies, all concerning cellular regulatory mechanisms. One study is purely experimental and five are computational studies.

A large part of the research concerns the Epstein-Barr virus (EBV). We investigate the latency programme switching of EBV, with an equilibrium statistical mechanics model that describes the transcription activities of two central viral promoters. We demonstrate that this system is bistable and predict promoter activities that correlate well with experimental data. Further we study the switching efficiency of one of the promoters, highlighting how competitive binding of transcription factors generates a more efficient geneticswitch.

The EBV protein EBNA1 is known to affect cellular gene expression. With a dinucleotide position weight matrix we search the complete human genome for regions with multiple EBNA1 binding sites. 40 potential binding regions are identified, with several of particular interest in relation to EBV infections. The final study on EBV is purely experimental, in which we demonstrate an interaction between the Syk kinase and integrin β4. Moreover, we show how reduced levels of these proteins affect migration of epithelial LMP2a positive cells, and hypothesise that these effects are due to the Syk-β4 interaction.

The two remaining studies presented in this thesis concern other cellular systems. Dynamic properties of two different regulatory feedback mechanisms for transport and metabolism of small molecules are investigated. The synergetic effect of adding a regulatory loop is exemplified with the iron metabolism in bacteria. The final project concerns the λ phage. With the equilibrium statistical mechanics method for describing promoter activities we characterise the equilibrium properties of λ mutants and compare with experimental findings. We argue that the observed differences between model and experiment are due to a larger perturbation of the genetic circuit than presumed.

The research presented in this thesis shed light on the properties of several regulatory mechanisms. As computational studies they add perspective to the experimental research in this field and provide new hypothesis for further research.

Place, publisher, year, edition, pages
Stockholm: KTH , 2010. , xiv, 80 p.
Series
Trita-CSC-A, ISSN 1653-5723 ; 2010:02
National Category
Fusion, Plasma and Space Physics
Identifiers
URN: urn:nbn:se:kth:diva-12096OAI: oai:DiVA.org:kth-12096DiVA: diva2:301348
Public defence
2010-03-18, Sal FB53, Roslagstullsbacken 21, AlbaNova, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC20100720Available from: 2010-03-03 Created: 2010-03-03 Last updated: 2010-07-20Bibliographically approved
List of papers
1. Epstein-Barr virus latency switch in human B-cells: a physico-chemical model
Open this publication in new window or tab >>Epstein-Barr virus latency switch in human B-cells: a physico-chemical model
Show others...
2007 (English)In: BMC SYST BIOL, ISSN 1752-0509, Vol. 1Article in journal (Refereed) Published
Abstract [en]

Background: The Epstein-Barr virus is widespread in all human populations and is strongly associated with human disease, ranging from infectious mononucleosis to cancer. In infected cells the virus can adopt several different latency programs, affecting the cells' behaviour. Experimental results indicate that a specific genetic switch between viral latency programs, reprograms human B-cells between proliferative and resting states. Each of these two latency programs makes use of a different viral promoter, C-P and Q(P), respectively. The hypothesis tested in this study is that this genetic switch is controlled by both human and viral transcription factors; Oct-2 and EBNA-1. We build a physico-chemical model to investigate quantitatively the dynamical properties of the promoter regulation and experimentally examine protein level variations between the two latency programs. Results: Our experimental results display significant differences in EBNA-1 and Oct-2 levels between resting and proliferating programs. With the model we identify two stable latency programs, corresponding to a resting and proliferating cell. The two programs differ in robustness and transcriptional activity. The proliferating state is markedly more stable, with a very high transcriptional activity from its viral promoter. We predict the promoter activities to be mutually exclusive in the two different programs, and our relative promoter activities correlate well with experimental data. Transitions between programs can be induced, by affecting the protein levels of our transcription factors. Simulated time scales are in line with experimental results. Conclusion: We show that fundamental properties of the Epstein-Barr virus involvement in latent infection, with implications for tumor biology, can be modelled and understood mathematically. We conclude that EBNA-1 and Oct-2 regulation of C-P and Q(P) is sufficient to establish mutually exclusive expression patterns. Moreover, the modelled genetic control predict both mono-and bistable behavior and a considerable difference in transition dynamics, based on program stability and promoter activities. Both these phenomena we hope can be further investigated experimentally, to increase the understanding of this important switch. Our results also stress the importance of the little known regulation of human transcription factor Oct-2.

Keyword
MULTIPLE EBNA1-BINDING SITES, BURKITTS-LYMPHOMA CELLS, NUCLEAR ANTIGEN 1, TRANSCRIPTION FACTORS, GENE-TRANSCRIPTION, EBNA-1 GENE, C PROMOTER, BINDING-PROTEIN, DNA-BINDING, I LATENCY
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-12078 (URN)10.1186/1752-0509-1-40 (DOI)000252362900001 ()2-s2.0-37749007113 (Scopus ID)
Note
QC20100720Available from: 2010-03-02 Created: 2010-03-02 Last updated: 2010-07-20Bibliographically approved
2. Cooperative action in eukaryotic gene regulation: Physical properties of a viral example
Open this publication in new window or tab >>Cooperative action in eukaryotic gene regulation: Physical properties of a viral example
2007 (English)In: Physical Review E. Statistical, Nonlinear, and Soft Matter Physics: Statistical Physics, Plasmas, Fluids, and Related Interdisciplinary Topics, ISSN 1063-651X, E-ISSN 1095-3787, Vol. 76, no 6Article in journal (Refereed) Published
Abstract [en]

The Epstein-Barr virus (EBV) infects more than 90% of the human population, and causes glandular fever as well as several more serious diseases. It is a tumor virus, and has been widely studied as a model system for cell transformation in humans. A central feature of the EBV life cycle is its ability to persist in human B cells in different latency states, denoted latency I, II, and III. In latency III the host cell is driven to cell proliferation and hence expansion of the viral population without entering the lytic pathway, while the latency I state is almost completely dormant. We here study the effective cooperativity of the viral C promoter, active in latency III EBV cell lines. We show that the unusually large number of binding sites of two competing transcription factors, one viral and one from the host, serves to make the switch sharper (higher Hill coefficient), either by cooperative binding between molecules of the same species when they bind, or by competition between the two species if there is sufficient steric hindrance.

Keyword
EPSTEIN-BARR-VIRUS, MULTIPLE EBNA1-BINDING SITES, NUCLEAR ANTIGEN-1, TRANSCRIPTIONAL REGULATION, C PROMOTER, DNA, ENHANCER, ORIGIN, REPLICATION, ACTIVATION
National Category
Other Physics Topics Fusion, Plasma and Space Physics
Identifiers
urn:nbn:se:kth:diva-12089 (URN)10.1103/PhysRevE.76.061909 (DOI)000251985600071 ()2-s2.0-37249031032 (Scopus ID)
Note
2007 Licentiatavhandling Acceppted QC20100720Available from: 2010-03-03 Created: 2010-03-03 Last updated: 2017-12-12Bibliographically approved
3. Dynamics of Uptake and Metabolism of Small Molecules in Cellular Response Systems
Open this publication in new window or tab >>Dynamics of Uptake and Metabolism of Small Molecules in Cellular Response Systems
2009 (English)In: PLOS ONE, ISSN 1932-6203, Vol. 4, no 3Article in journal (Refereed) Published
Abstract [en]

Background: Proper cellular function requires uptake of small molecules from the environment. In response to changes in extracellular conditions cells alter the import and utilization of small molecules. For a wide variety of small molecules the cellular response is regulated by a network motif that combines two feedback loops, one which regulates the transport and the other which regulates the subsequent metabolism.

Results: We analyze the dynamic behavior of two widespread but logically distinct two-loop motifs. These motifs differ in the logic of the feedback loop regulating the uptake of the small molecule. Our aim is to examine the qualitative features of the dynamics of these two classes of feedback motifs. We find that the negative feedback to transport is accompanied by overshoot in the intracellular amount of small molecules, whereas a positive feedback to transport removes overshoot by boosting the final steady state level. On the other hand, the negative feedback allows for a rapid initial response, whereas the positive feedback is slower. We also illustrate how the dynamical deficiencies of one feedback motif can be mitigated by an additional loop, while maintaining the original steady-state properties.

Conclusions: Our analysis emphasizes the core of the regulation found in many motifs at the interface between the metabolic network and the environment of the cell. By simplifying the regulation into uptake and the first metabolic step, we provide a basis for elaborate studies of more realistic network structures. Particularly, this theoretical analysis predicts that FeS cluster formation plays an important role in the dynamics of iron homeostasis.

Keyword
Biology; Multidisciplinary Sciences
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-12091 (URN)10.1371/journal.pone.0004923 (DOI)000265496500017 ()2-s2.0-62849127949 (Scopus ID)
Note
QC20100720Available from: 2010-03-03 Created: 2010-03-03 Last updated: 2010-07-20Bibliographically approved
4. A computational study of lambda-lac mutants
Open this publication in new window or tab >>A computational study of lambda-lac mutants
2009 (English)In: PHYS BIOL, ISSN 1478-3967, Vol. 6, no 4Article in journal (Refereed) Published
Abstract [en]

We present a comprehensive, computational study of the properties of bacteriophage lambda mutants designed by Atsumi and Little (2006 Proc. Natl. Acad. Sci. 103 4558-63). These phages underwent a genetic reconstruction where Cro was replaced by a dimeric form of the Lac repressor. To clarify the theoretical characteristics of these mutants, we built a detailed thermodynamic model. The mutants all have a different genetic wiring than the wild-type. lambda One group lacks regulation of P-RM by the lytic protein. These mutants only exhibit the lysogenic equilibrium, with no transiently active P-R. The other group lacks the negative feedback from CI. In this group, we identify a handful of bi-stable mutants, although the majority only exhibit the lysogenic equilibrium. The experimental identification of functional phages differs from our predictions. From a theoretical perspective, there is no reason why only 4 out of 900 mutants should be functional. The differences between theory and experiment can be explained in two ways. Either, the view of the lambda phage as a bi-stable system needs to be revised, or the mutants have in fact not undergone a modular replacement, as intended by Atsumi and Little, but constitute instead a wider systemic change.

Keyword
BACTERIOPHAGE-LAMBDA, ESCHERICHIA-COLI, CI REPRESSOR, TRANSCRIPTIONAL REGULATION, PROPHAGE INDUCTION, GENE-REGULATION, PHAGE-LAMBDA, OPERATOR DNA, CRO, BINDING
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-12092 (URN)10.1088/1478-3975/6/4/046007 (DOI)000272190400007 ()2-s2.0-70350722026 (Scopus ID)
Note
QC20100720Available from: 2010-03-03 Created: 2010-03-03 Last updated: 2010-07-20Bibliographically approved
5. FR-like EBNA1 binding repeats in the human genome
Open this publication in new window or tab >>FR-like EBNA1 binding repeats in the human genome
2010 (English)In: Virology, ISSN 0042-6822, E-ISSN 1096-0341, Vol. 405, no 2, 524-529 p.Article in journal (Refereed) Published
Abstract [en]

Epstein Barr Virus (EBV) is widely spread in the human population. EBV nuclear antigen 1 (EBNA1) is a transcription factor that activates viral genes and is necessary for viral replication and partitioning, which binds the EBV genome cooperatively. We identify similar EBNA1 repeat binding sites in the human genome using a nearest-neighbour positional weight matrix. Previously experimentally verified EBNA1 sites in the human genome are successfully recovered by our approach. Most importantly, 40 novel regions are identified in the human genome, constituted of tandemly repeated binding sites for EBNA1. Genes located in vicinity of these regions are presented as possible targets for EBNA1-mediated regulation. Among these, four are discussed in more detail: IQCB1, IMPG1, IRF2BP and TPO. Incorporating the cooperative actions of EBNA1 is essential when identifying regulatory regions in the human genome and we believe the findings presented here are highly valuable for the understanding of EBV-induced phenotypic changes.

Keyword
EBNA1, Epstein-Barr virus, Family of Repeats, Human binding sites
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:kth:diva-12093 (URN)10.1016/j.virol.2010.06.040 (DOI)000281130500028 ()20655080 (PubMedID)2-s2.0-77955661979 (Scopus ID)
Note
QC 20100720. Updated from submitted to published 20120326Available from: 2010-03-03 Created: 2010-03-03 Last updated: 2017-12-12Bibliographically approved
6. Integrin β4 Interacts with Syk: Effects on Migration of Epithelial Cells
Open this publication in new window or tab >>Integrin β4 Interacts with Syk: Effects on Migration of Epithelial Cells
(English)Manuscript (preprint) (Other academic)
Identifiers
urn:nbn:se:kth:diva-12094 (URN)
Note
QC20100720Available from: 2010-03-03 Created: 2010-03-03 Last updated: 2010-07-20Bibliographically approved

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