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Mining DNA elements involved in targeting of chromatin modifiers
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Per Stenberg)ORCID iD: 0000-0001-8752-0794
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 [en]
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: urn:nbn:se:umu:diva-92979ISBN: 978-91-7601-118-8 (print)OAI: oai:DiVA.org:umu-92979DiVA: diva2:745039
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
List of papers
1. Sequence signatures involved in targeting the male-specific lethal complex to X-chromosomal genes in Drosophila melanogaster
Open this publication in new window or tab >>Sequence signatures involved in targeting the male-specific lethal complex to X-chromosomal genes in Drosophila melanogaster
2012 (English)In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 13, 97- p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: In Drosophila melanogaster, the dosage-compensation system that equalizes X-linked gene expression between males and females, thereby assuring that an appropriate balance is maintained between the expression of genes on the X chromosome(s) and the autosomes, is at least partially mediated by the Male-Specific Lethal (MSL) complex. This complex binds to genes with a preference for exons on the male X chromosome with a 3' bias, and it targets most expressed genes on the X chromosome. However, a number of genes are expressed but not targeted by the complex. High affinity sites seem to be responsible for initial recruitment of the complex to the X chromosome, but the targeting to and within individual genes is poorly understood.

RESULTS: We have extensively examined X chromosome sequence variation within five types of gene features (promoters, 5' UTRs, coding sequences, introns, 3' UTRs) and intergenic sequences, and assessed its potential involvement in dosage compensation. Presented results show that: the X chromosome has a distinct sequence composition within its gene features; some of the detected variation correlates with genes targeted by the MSL-complex; the insulator protein BEAF-32 preferentially binds upstream of MSL-bound genes; BEAF-32 and MOF co-localizes in promoters; and that bound genes have a distinct sequence composition that shows a 3' bias within coding sequence.

CONCLUSIONS: Although, many strongly bound genes are close to a high affinity site neither our promoter motif nor our coding sequence signatures show any correlation to HAS. Based on the results presented here, we believe that there are sequences in the promoters and coding sequences of targeted genes that have the potential to direct the secondary spreading of the MSL-complex to nearby genes.

Place, publisher, year, edition, pages
BioMed Central, 2012
Keyword
Dosage compensation, sequence signatures, drosophila, motif discovery, MSL-complex
National Category
Bioinformatics and Systems Biology Genetics
Research subject
Genetics
Identifiers
urn:nbn:se:umu:diva-53363 (URN)10.1186/1471-2164-13-97 (DOI)000304155400002 ()22424303 (PubMedID)
Available from: 2012-03-23 Created: 2012-03-23 Last updated: 2017-12-07Bibliographically approved
2. Non-coding roX RNAs prevent the binding of the MSL-complex to heterochromatic regions
Open this publication in new window or tab >>Non-coding roX RNAs prevent the binding of the MSL-complex to heterochromatic regions
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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.

National Category
Biochemistry and Molecular Biology Genetics Bioinformatics and Systems Biology
Identifiers
urn:nbn:se:umu:diva-93046 (URN)10.1371/journal.pgen.1004865 (DOI)000346649900060 ()25501352 (PubMedID)2-s2.0-84919667251 (Scopus ID)
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
3. Male X-linked genes in Drosophila melanogaster are compensated independently of the Male-Specific Lethal complex
Open this publication in new window or tab >>Male X-linked genes in Drosophila melanogaster are compensated independently of the Male-Specific Lethal complex
2013 (English)In: Epigenetics & Chromatin, ISSN 1756-8935, E-ISSN 1756-8935, Vol. 6, no Article number: 35Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: In organisms where the two sexes have unequal numbers of X-chromosomes, the expression of X-linked genes needs to be balanced not only between the two sexes, but also between X and the autosomes. In Drosophila melanogaster, the Male-Specific Lethal (MSL) complex is believed to produce a 2-fold increase in expression of genes on the male X, thus restoring this balance.

RESULTS: Here we show that almost all the genes on the male X are effectively compensated. However, many genes are compensated without any significant recruitment of the MSL-complex. These genes are very weakly, if at all, affected by mutations or RNAi against MSL-complex components. In addition, even the genes that are strongly bound by MSL rely on mechanisms other than the MSL-complex for proper compensation. We find that long, non-ubiquitously expressed genes tend to rely less on the MSL-complex for their compensation and genes that in addition are far from High Affinity Sites tend to not bind the complex at all or very weakly.

CONCLUSIONS: We conclude that most of the compensation of X-linked genes is produced by an MSL-independent mechanism. Similar to the case of the MSL-mediated compensation we do not yet know the mechanism behind the MSL-independent compensation that appears to act preferentially on long genes. Even if we observe similarities, it remains to be seen if the mechanism is related to the buffering that is observed in autosomal aneuploidies.

Place, publisher, year, edition, pages
BioMed Central, 2013
Keyword
Buffering, Dosage compensation, Male-Specific Lethal complex
National Category
Genetics
Research subject
Genetics
Identifiers
urn:nbn:se:umu:diva-83542 (URN)10.1186/1756-8935-6-35 (DOI)000326282200001 ()24279328 (PubMedID)
Available from: 2013-12-02 Created: 2013-12-02 Last updated: 2017-12-06Bibliographically approved
4. Preferential Genome Targeting of the CBP Co-Activator by Rel and Smad Proteins in Early Drosophila melanogaster Embryos
Open this publication in new window or tab >>Preferential Genome Targeting of the CBP Co-Activator by Rel and Smad Proteins in Early Drosophila melanogaster Embryos
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2012 (English)In: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 8, no 6, e1002769- p.Article in journal (Refereed) Published
Abstract [en]

CBP and the related p300 protein are widely used transcriptional co-activators in metazoans that interact with multiple transcription factors. Whether CBP/p300 occupies the genome equally with all factors or preferentially binds together with some factors is not known. We therefore compared Drosophila melanogaster CBP (nejire) ChIP-seq peaks with regions bound by 40 different transcription factors in early embryos, and we found high co-occupancy with the Rel-family protein Dorsal. Dorsal is required for CBP occupancy in the embryo, but only at regions where few other factors are present. CBP peaks in mutant embryos lacking nuclear Dorsal are best correlated with TGF-ß/Dpp-signaling and Smad-protein binding. Differences in CBP occupancy in mutant embryos reflect gene expression changes genome-wide, but CBP also occupies some non-expressed genes. The presence of CBP at silent genes does not result in histone acetylation. We find that Polycomb-repressed H3K27me3 chromatin does not preclude CBP binding, but restricts histone acetylation at CBP-bound genomic sites. We conclude that CBP occupancy in Drosophila embryos preferentially overlaps factors controlling dorso-ventral patterning and that CBP binds silent genes without causing histone hyperacetylation.

Place, publisher, year, edition, pages
San Francisco: Public Library of Science, 2012
National Category
Genetics
Research subject
Genetics
Identifiers
urn:nbn:se:umu:diva-56930 (URN)10.1371/journal.pgen.1002769 (DOI)000305961000033 ()22737084 (PubMedID)
Available from: 2012-06-29 Created: 2012-06-29 Last updated: 2017-12-07Bibliographically approved
5. Drosophila CBP cooperates with GAGA factor to induce high levels of Pol II promoter-proximal pausing
Open this publication in new window or tab >>Drosophila CBP cooperates with GAGA factor to induce high levels of Pol II promoter-proximal pausing
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(English)Manuscript (preprint) (Other academic)
National Category
Developmental Biology Biochemistry and Molecular Biology Bioinformatics and Systems Biology Genetics
Identifiers
urn:nbn:se:umu:diva-93047 (URN)
Available from: 2014-09-11 Created: 2014-09-11 Last updated: 2014-09-11Bibliographically approved
6. CBP functions outside of promoters and enhancers in Drosophila melanogaster
Open this publication in new window or tab >>CBP functions outside of promoters and enhancers in Drosophila melanogaster
(English)Manuscript (preprint) (Other academic)
National Category
Developmental Biology Bioinformatics and Systems Biology Biochemistry and Molecular Biology Genetics
Identifiers
urn:nbn:se:umu:diva-93048 (URN)
Available from: 2014-09-11 Created: 2014-09-11 Last updated: 2014-09-11Bibliographically approved
7. Normalization of high dimensional genomics data where the distribution of the altered variables is skewed
Open this publication in new window or tab >>Normalization of high dimensional genomics data where the distribution of the altered variables is skewed
2011 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 11, e27942- p.Article in journal (Refereed) Published
Abstract [en]

Genome-wide analysis of gene expression or protein binding patterns using different array or sequencing based technologies is now routinely performed to compare different populations, such as treatment and reference groups. It is often necessary to normalize the data obtained to remove technical variation introduced in the course of conducting experimental work, but standard normalization techniques are not capable of eliminating technical bias in cases where the distribution of the truly altered variables is skewed, i.e. when a large fraction of the variables are either positively or negatively affected by the treatment. However, several experiments are likely to generate such skewed distributions, including ChIP-chip experiments for the study of chromatin, gene expression experiments for the study of apoptosis, and SNP-studies of copy number variation in normal and tumour tissues. A preliminary study using spike-in array data established that the capacity of an experiment to identify altered variables and generate unbiased estimates of the fold change decreases as the fraction of altered variables and the skewness increases. We propose the following work-flow for analyzing high-dimensional experiments with regions of altered variables: (1) Pre-process raw data using one of the standard normalization techniques. (2) Investigate if the distribution of the altered variables is skewed. (3) If the distribution is not believed to be skewed, no additional normalization is needed. Otherwise, re-normalize the data using a novel HMM-assisted normalization procedure. (4) Perform downstream analysis. Here, ChIP-chip data and simulated data were used to evaluate the performance of the work-flow. It was found that skewed distributions can be detected by using the novel DSE-test (Detection of Skewed Experiments). Furthermore, applying the HMM-assisted normalization to experiments where the distribution of the truly altered variables is skewed results in considerably higher sensitivity and lower bias than can be attained using standard and invariant normalization methods.

Keyword
skewness, normalization, genomics, array analysis, epigenetics
National Category
Bioinformatics and Systems Biology Bioinformatics (Computational Biology) Genetics
Research subject
biology
Identifiers
urn:nbn:se:umu:diva-50313 (URN)10.1371/journal.pone.0027942 (DOI)000297792400024 ()22132175 (PubMedID)
Available from: 2011-12-06 Created: 2011-12-06 Last updated: 2017-12-08Bibliographically approved

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