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Non-coding roX RNAs prevent the binding of the MSL-complex to heterochromatic regions
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Jan Larsson)
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Jan Larsson)
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Computational Life Science Cluster (CLiC), Umeå University, SE-90187 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). Division of CBRN Defence and Security, FOI, Swedish Defence Research Agency, Sweden.
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2014 (English)In: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 10, no 12, e1004865- p.Article in journal (Refereed) Published
Abstract [en]

Long non-coding RNAs contribute to dosage compensation in both mammals and Drosophila by inducing changes in the chromatin structure of the X-chromosome. In Drosophila melanogaster, roX1 and roX2 are long non-coding RNAs that together with proteins form the male-specific lethal (MSL) complex, which coats the entire male X-chromosome and mediates dosage compensation by increasing its transcriptional output. Studies on polytene chromosomes have demonstrated that when both roX1 and roX2 are absent, the MSL-complex becomes less abundant on the male X-chromosome and is relocated to the chromocenter and the 4thchromosome. Here we address the role of roX RNAs in MSL-complex targeting and the evolution of dosage compensation in Drosophila. We performed ChIP-seq experiments which showed that MSL-complex recruitment to high affinity sites (HAS) on the X-chromosome is independent of roX and that the HAS sequence motif is conserved in D. simulans. Additionally, a complete and enzymatically active MSL-complex is recruited to six specific genes on the 4thchromosome. Interestingly, our sequence analysis showed that in the absence of roX RNAs, the MSL-complex has an affinity for regions enriched in Hoppel transposable elements and repeats in general. We hypothesize that roX mutants reveal the ancient targeting of the MSL-complex and propose that the role of roX RNAs is to prevent the binding of the MSL-complex to heterochromatin.

Place, publisher, year, edition, pages
2014. Vol. 10, no 12, e1004865- p.
National Category
Biochemistry and Molecular Biology Genetics Bioinformatics and Systems Biology
Identifiers
URN: urn:nbn:se:umu:diva-93046DOI: 10.1371/journal.pgen.1004865ISI: 000346649900060PubMedID: 25501352Scopus ID: 2-s2.0-84919667251OAI: oai:DiVA.org:umu-93046DiVA: diva2:745803
Funder
Swedish Research Council, 621-2012-2165
Note

Originally included in thesis in manuscript form.

Available from: 2014-09-11 Created: 2014-09-11 Last updated: 2017-12-05Bibliographically 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)
Opponent
Supervisors
Available from: 2014-09-12 Created: 2014-09-09 Last updated: 2014-09-23Bibliographically approved
2. Epigenetics and targeting mechanisms in Drosophila melanogaster
Open this publication in new window or tab >>Epigenetics and targeting mechanisms in Drosophila melanogaster
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Place, publisher, year, edition, pages
Umeå: Umeå University, 2015. 65 p.
Keyword
Drosophila, Aneuplodies, HP1a, POF, MSL, non-coding RNAs
National Category
Biological Sciences
Research subject
Genetics
Identifiers
urn:nbn:se:umu:diva-102890 (URN)978-91-7601-267-3 (ISBN)
Public defence
2015-06-04, Astrid Fagraeus Hörsal A 103 Unod R 1, NUS – Norrlands universitetssjukhus, Umeå, 09:00 (English)
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Supervisors
Available from: 2015-05-13 Created: 2015-05-09 Last updated: 2015-05-18Bibliographically approved

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Figueiredo, Margarida L. A.Kim, MariaPhilip, PhilgeStenberg, PerLarsson, Jan

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