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Drosophila CBP cooperates with GAGA factor to induce high levels of Pol II promoter-proximal pausing
Dept. of Molecular Biosciences, the Wenner-Gren Institute, Stockholm University, SE-10691 Stockholm, Sweden. (Mattias Mannervik)
Dept. of Molecular Biosciences, the Wenner-Gren Institute, Stockholm University, SE-10691 Stockholm, Sweden. (Mattias Mannervik)
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Computational Life Science Cluster (CLiC), Umeå, Sweden. (Per Stenberg)ORCID iD: 0000-0001-8752-0794
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Computational Life Science Cluster (CLiC), Umeå, Sweden. (Per Stenberg)
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(English)Manuscript (preprint) (Other academic)
National Category
Developmental Biology Biochemistry and Molecular Biology Bioinformatics and Systems Biology Genetics
Identifiers
URN: urn:nbn:se:umu:diva-93047OAI: oai:DiVA.org:umu-93047DiVA: diva2:745810
Available from: 2014-09-11 Created: 2014-09-11 Last updated: 2014-09-11Bibliographically approved
In thesis
1. Mining DNA elements involved in targeting of chromatin modifiers
Open this publication in new window or tab >>Mining DNA elements involved in targeting of chromatin modifiers
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Background: In all higher organisms, the nuclear DNA is condensed into nucleosomes that consist of DNA wrapped around a core of highly conserved histone proteins. DNA bound to histones and other structural proteins form the chromatin. Generally, only few regions of DNA are accessible and most of the time RNA polymerase and other DNA binding proteins have to overcome this compaction to initiate transcription. Several proteins are involved in making the chromatin more compact or open. Such chromatin-modifying proteins make distinct post-translational modifications of histones – especially in the histone tails – to alter their affinity to DNA. Aim: The main aim of my thesis work is to study the targeting of chromatin modifiers important for correct gene expression in Drosophila melanogaster (fruit flies). Primary DNA sequences, chromatin associated proteins, transcription, and non-coding RNAs are all likely to be involved in targeting mechanisms. This thesis work involves the development of new computational methods for identification of DNA motifs and protein factors involved in the targeting of chromatin modifiers. Targeting and functional analysis of two chromatin modifiers, namely male-specific lethal (MSL) complex and CREB-binding protein (CBP) are specifically studied. The MSL complex is a protein complex that mediates dosage compensation in flies. CBP protein is known as a transcriptional co-regulator in metazoans and it has histone acetyl transferase activity and CBP has been used to predict novel enhancers. Results: My studies of the binding sites of MSL complex shows that promoters and coding sequences of MSL-bound genes on the X-chromosome of Drosophila melanogaster can influence the spreading of the complex along the X-chromosome. Analysis of MSL binding sites when two non-coding roX RNAs are mutated shows that MSL-complex recruitment to high-affinity sites on the Xchromosome is independent of roX, and the role of roX RNAs is to prevent binding to repeats in autosomal sites. Functional analysis of MSL-bound genes using their dosage compensation status shows that the function of the MSL complex is to enhance the expression of short housekeeping genes, but MSL-independent mechanisms exist to achieve complete dosage compensation. Studies of the binding sites of the CBP protein show that, in early embryos, Dorsal in cooperation with GAGA factor (GAF) and factors like Medea and Dichaete target CBP to its binding sites. In the S2 cell line, GAF is identified as the targeting factor of CBP at promoters and enhancers, and GAF and CBP together are found to induce high levels of polymerase II pausing at promoters. In another study using integrated data analysis, CBP binding sites could be classified into polycomb protein binding sites, repressed enhancers, insulator protein-bound regions, active promoters, and active enhancers, and this suggested different potential roles for CBP. A new approach was also developed to eliminate technical bias in skewed experiments. Our study shows that in the case of skewed datasets it is always better to identify non-altered variables and to normalize the data using only such variables.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2014. 76 p.
Keyword
nucleosome, histone, chromatin, chromatin modifiers, targeting, DNA motifs, protein factors, MSL
National Category
Bioinformatics and Systems Biology Biochemistry and Molecular Biology Genetics
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-92979 (URN)978-91-7601-118-8 (ISBN)
Public defence
2014-10-03, E 04 Unod R1, Norrland University Hospital, Umeå, 09:00 (English)
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Available from: 2014-09-12 Created: 2014-09-09 Last updated: 2014-09-23Bibliographically approved

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