| 1. |
- Dorafshan Esfahani, Eshagh, 1980-
(författare)
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Methyltransferase Ash1, histone methylation and their impact on Polycomb repression
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Antagonistic interactions between Polycomb Group (PcG) and Trithorax Group (TrxG) proteins orchestrate the expression of key developmental genes. Distinct maternally loaded repressors establish the silenced state of these genes in cells where they should not be expressed and later PcG proteins sense whether a target gene is inactive and maintain the repression throughout multiple cell divisions. PcG proteins are targeted to genes by DNA elements called Polycomb Response Elements (PREs). The proteins form two major classes of complexes, namely Polycomb Repressive Complex 1 (PRC1) and Polycomb Repressive Complex 2 (PRC2). Mechanistic details of Polycomb repression are not fully understood, however, tri-methylation of Lysine 27 of histone H3 (H3K27me3) is essential for this process. Using Drosophila cell lines deficient for either PRC1 or PRC2, I investigated the role of H3K27 methylation and the interdependence of PRC1 complexes for their recruitment to PREs. My results indicate that recruitment of PcG complexes to PREs proceed via multiple pathways and that H3K27 methylation is not needed for their targeting. However, the methylation is required to stabilize interactions of PRE-anchored PcG complexes with surrounding chromatin.TrxG proteins prevent erroneous repression of Polycomb target genes where these genes need to be expressed. Ash1 is a TrxG protein which binds Polycomb target genes when they are transcriptionally active. It contains a SET domain which methylates Lysine 36 of histone H3 (H3K36). In vitro, histone H3 methylated at K36 is a poor substrate for H3K27 methylation by PRC2. This prompted a model where Ash1 counteracts Polycomb repression through H3K36 methylation. However, this model was never tested in vivo and does not consider several experimental observations. First, in the ash1 mutant flies the bulk H3K36me2/H3K36me3 levels remain unchanged. Second, in Drosophila, there are two other H3K36-specific histone methyltransferases, NSD and Set2, which should be capable to inhibit PRC2. Third, Ash1 contains multiple evolutionary conserved domains whose roles have not been investigated. Therefore, I asked whether H3K36 methylation is critical for Ash1 to counteract Polycomb repression in vivo and whether NSD and Set2 proteins contribute to this process. I used flies lacking endogenous histone genes and complemented them with transgenic histone genes where Lysine 36 is replaced by Arginine. In these animals, I assayed erroneous repression of HOX genes as a readout for erroneous Polycomb repression. I used the same readout in the NSD or Set2 mutant flies. I also asked if other conserved domains of Ash1 are essential for its function. In addition to SET and domain, Ash1 contains three AT hook motifs as well as BAH and PHD domains. I genetically complemented ash1 loss of function animals with transgenic Ash1 variants, in each, one domain of Ash1 is deleted. I found that Ash1 is the only H3K36-specific histone methyltransferase which counteracts Polycomb repression in Drosophila. My findings suggest that the model, where Ash1 counteracts PcG repression by inhibiting PRC2 via methylation of H3K36, has to be revised. I also showed that, in vivo, Ash1 acts as a multimer and requires SET, BAH and PHD domains to counteract Polycomb repression.This work led to two main conclusions. First, trimethylation of H3K27 is not essential for targeting PcG proteins to PREs but acts afterwards to stabilize their interaction with the chromatin of the neighboring genes. Second, while SET domain is essential for Ash1 to oppose Polycomb repression, methylation of H3K36 does not play a central role in the process.
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| 2. |
- Mendoza, Patricia, et al.
(författare)
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DamID transcriptional profiling identifies the Snail/Scratch transcription factor Kahuli as an Alk target in the Drosophila visceral mesoderm
- 2021
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Ingår i: Development. - : The Company of Biologists. - 0950-1991 .- 1477-9129. ; 148:23
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Tidskriftsartikel (refereegranskat)abstract
- Development of the Drosophila visceral muscle depends on Anaplastic Lymphoma Kinase (Alk) receptor tyrosine kinase (RTK) signaling, which specifies founder cells (FCs) in the circular visceral mesoderm (VM). Although Alk activation by its ligand Jelly Belly (Jeb) is well characterized, few target molecules have been identified. Here, we used targeted DamID (TaDa) to identify Alk targets in embryos overexpressing Jeb versus embryos with abrogated Alk activity, revealing differentially expressed genes, including the Snail/Scratch family transcription factor Kahuli (Kah). We confirmed Kah mRNA and protein expression in the VM, and identified midgut constriction defects in Kah mutants similar to those of pointed (pnt). ChIP and RNA-Seq data analysis defined a Kah target-binding site similar to that of Snail, and identified a set of common target genes putatively regulated by Kah and Pnt during midgut constriction. Taken together, we report a rich dataset of Alk-responsive loci in the embryonic VM and functionally characterize the role of Kah in the regulation of embryonic midgut morphogenesis.
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| 3. |
- Figueiredo, Margarida L. A., et al.
(författare)
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Non-coding roX RNAs prevent the binding of the MSL-complex to heterochromatic regions
- 2014
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Ingår i: PLOS Genetics. - : Public Library of Science (PLoS). - 1553-7390 .- 1553-7404. ; 10:12, s. e1004865-
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 4. |
- Lindehell, Henrik, 1984-, et al.
(författare)
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Methylation of lysine 36 on histone H3 is required to control transposon activities in somatic cells
- 2023
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Ingår i: Life Science Alliance. - : NLM (Medline). - 2575-1077. ; 6:8
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Tidskriftsartikel (refereegranskat)abstract
- Transposable elements constitute a substantial portion of most eukaryotic genomes and their activity can lead to developmental and neuronal defects. In the germline, transposon activity is antagonized by the PIWI-interacting RNA pathway tasked with repression of transposon transcription and degrading transcripts that have already been produced. However, most of the genes required for transposon control are not expressed outside the germline, prompting the question: what causes deleterious transposons activity in the soma and how is it managed? Here, we show that disruptions of the Histone 3 lysine 36 methylation machinery led to increased transposon transcription in Drosophila melanogaster brains and that there is division of labour for the repression of transposable elements between the different methyltransferases Set2, NSD, and Ash1. Furthermore, we show that disruption of methylation leads to somatic activation of key genes in the PIWI-interacting RNA pathway and the preferential production of RNA from dual-strand piRNA clusters.
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| 5. |
- Philip, Philge, 1983-
(författare)
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Mining DNA elements involved in targeting of chromatin modifiers
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 6. |
- Crona, Filip, 1977-, et al.
(författare)
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Gene regulation by the lysine demethylase KDM4A in Drosophila
- 2013
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Ingår i: Developmental Biology. - : Elsevier BV. - 0012-1606 .- 1095-564X. ; 373:2, s. 453-463
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Tidskriftsartikel (refereegranskat)abstract
- Lysine methylation of histones is associated with both transcriptionally active chromatin and with silent chromatin, depending on what residue is modified. Histone methyltransferases and demethylases ensure that histone methylations are dynamic and can vary depending on cell cycle- or developmental stage. KDM4A demethylates H3K36me3, a modification enriched in the 3' end of active genes. The genomic targets and the role of KDM4 proteins in development remain largely unknown. We therefore generated KDM4A mutant Drosophila, and identified 99 mis-regulated genes in first instar larvae. Around half of these genes were down-regulated and the other half up-regulated in dKDM4A mutants. Although heterochromatin protein 1a (HP1a) can stimulate dKDM4A demethylase activity in vitro, we find that they antagonize each other in control of dKDM4A-regulated genes. Appropriate expression levels for some dKDM4A-regulated genes rely on the demethylase activity of dKDM4A, whereas others do not. Surprisingly, although highly expressed, many demethylase-dependent and independent genes are devoid of H3K36me3 in wild-type as well as in dKDM4A mutant larvae, suggesting that some of the most strongly affected genes in dKDM4A mutant animals are not regulated by H3K36 methylation. By contrast, dKDM4A over-expression results in a global decrease in H3K36me3 levels and male lethality, which might be caused by impaired dosage compensation. Our results show that a modest increase in global H3K36me3 levels is compatible with viability, fertility, and the expression of most genes, whereas decreased H3K36me3 levels are detrimental in males.
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| 7. |
- Faucillion, Marie-Line, 1989-, et al.
(författare)
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Increased expression of X-linked genes in mammals is associated with a higher stability of transcripts and an increased ribosome density
- 2015
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Ingår i: Genome Biology and Evolution. - : Oxford University Press. - 1759-6653. ; 7:4, s. 1039-1052
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Tidskriftsartikel (refereegranskat)abstract
- Mammalian sex chromosomes evolved from the degeneration of one homolog of a pair of ancestral autosomes, the proto-Y. This resulted in a gene dose imbalance that is believed to be restored (partially or fully) through up-regulation of gene expression from the single active X-chromosome in both sexes by a dosage compensatory mechanism. We analyzed multiple genome-wide RNA stability datasets and found significantly longer average half-lives for X-chromosome transcripts than for autosomal transcripts in various human cell lines, both male and female, and in mice. Analysis of ribosome profiling data shows that ribosome density is higher on X-chromosome transcripts than on autosomal transcripts in both humans and mice, suggesting that the higher stability is causally linked to a higher translation rate. Our results and observations are in accordance with a dosage compensatory upregulation of expressed X-linked genes. We therefore propose that differential mRNA stability and translation rates of the autosomes and sex chromosomes contribute to an evolutionarily conserved dosage compensation mechanism in mammals.
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| 8. |
- Johansson, Anna-Mia, 1978-, et al.
(författare)
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POF Regulates the Expression of Genes on the Fourth Chromosome in Drosophila melanogaster by Binding to Nascent RNA
- 2012
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Ingår i: Molecular and Cellular Biology. - : American Society for Microbiology. - 0270-7306 .- 1098-5549. ; 32:11, s. 2121-2134
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- In Drosophila, two chromosome-wide compensatory systems have been characterized: the dosage compensation system that acts on the male X chromosome and the chromosome-specific regulation of genes located on the heterochromatic fourth chromosome. Dosage compensation in Drosophila is accomplished by hypertranscription of the single male X chromosome mediated by the male-specific lethal (MSL) complex. The mechanism of this compensation is suggested to involve enhanced transcriptional elongation mediated by the MSL complex, while the mechanism of compensation mediated by the painting of fourth (POF) protein on the fourth chromosome has remained elusive. Here, we show that POF binds to nascent RNA, and this binding is associated with increased transcription output from chromosome 4. We also show that genes located in heterochromatic regions spend less time in transition from the site of transcription to the nuclear envelope. These results provide useful insights into the means by which genes in heterochromatic regions can overcome the repressive influence of their hostile environment.
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| 9. |
- Lindehell, Henrik, 1984-
(författare)
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Jack of all trades, master of none : the multifaceted nature of H3K36 methylation
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Post-translational modifications of histones enable differential transcriptional control of the genome between cell types and developmental stages, and in response to environmental factors. The methylation of Histone 3 Lysine 36 (H3K36) is one the most complex and well-studied histone modifications and is known to be involved in a wide range of molecular processes. Commonly associated with active genes and transcriptional elongation, H3K36 methylation also plays a key role in DNA repair, repression of cryptic transcription, and guiding additional post-translational modifications to histones, genomic DNA, and RNA. In Drosophila melanogaster, trimethylated H3K36 has also been linked to dosage compensation of the single male X chromosome as a binding substrate for the Male-Specific Lethal (MSL) complex. However, this model has been challenged by structural and biochemical studies demonstrating higher MSL complex affinity for other methylated lysines. There is an additional system of chromosome-specific gene regulation in D. melanogaster where transcription from the small heterochromatic fourth chromosome is increased by Painting of fourth (POF), a protein specifically binding nascent RNA on the fourth chromosome. The fourth chromosome is thought to have been an ancestral X chromosome that reverted into an autosome. POF mediating high transcription levels from an autosome is believed to be a remnant of an ancient sex-chromosome dosage compensation mechanism. Proximity ligation assays revealed no interaction between MSL complex components and methylated H3K36. This finding was corroborated by RNA sequencing of H3K36 methylation impaired mutants: the transcriptional output of the male X chromosome was unaffected in mutants where Lysine 36 on Histone 3 was replaced by an Arginine, abolishing methylation of this site. However, we found that knocking out Set2, which encodes the methyltransferase responsible for H3K36 trimethylation, significantly reduced X-linked transcription relative to autosomal transcription. This strongly suggests the existence of previously unrecognized alternate Set2 substrates. Interestingly, we also found that Ash1- and NSD-mediated methylation of H3K36 was required to maintain high expression from chromosome four. Recent studies have also implicated H3K36 methylation in the silencing of transposon activity in somatic cells. By analyzing the transcription of transposable elements and Piwi-interacting RNAs (piRNAs), we identified dimethylation of H3K36 by Set2 as the main methylation mark involved in this process and showed that dual-stranded piRNA clusters are preferentially activated upon disturbing the methylation machinery. These findings extends the long list of processes dependent on functional H3K36 methylation.
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| 10. |
- Chen, Sa, 1967-
(författare)
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Expression and function of Suppressor of zeste 12 in Drosophila melanogaster
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The development of animals and plants needs a higher order of regulation of gene expression to maintain proper cell state. The mechanisms that control what, when and where a gene should (or should not) be expressed are essential for correct organism development. The Polycomb group (PcG) is a family of genes responsible for maintaining gene silencing and Suppressor of zeste 12 (Su(z)12) is one of the core components in the PcG. The gene is highly conserved in organisms ranging from plants to humans, however, the specific function is not well known. The main tasks of this thesis was to investigate the function of Su(z)12 and its expression at different stages of Drosophila development. In polytene chromosomes of larval salivary glands, Su(z)12 binds to about 90 specific euchromatic sites. The binding along the chromosome arms is mostly in interbands, which are the most DNA de-condensed regions. The binding sites of Su(z)12 in polytene chromosomes correlate precisely with those of the Enhancer-of-zeste (E(z)) protein, indicating that Su(z)12 mainly exists within the Polycomb Repressive Complex 2 (PRC2). However, the binding pattern does not overlap well with Histone 3 lysine 27 tri-methylations (H3K27me3), the specific chromatin mark created by PRC2. The Su(z)12 binding to chromatin is dynamically regulated during mitotic and meiotic cell division. The two different Su(z)12 isoforms: Su(z)12-A and Su(z)12-B (resulting from alternative RNA splicing), have very different expression patterns during development. Functional analyses indicate that they also have different functions he Su(z)12-B form is the main mediator of silencing. Furthermore, a neuron specific localization pattern in larval brain and a giant larval phenotype in transgenic lines reveal a potential function of Su(z)12-A in neuron development. In some aspects the isoforms seem to be able to substitute for each other. The histone methyltransferase activity of PRC2 is due to the E(z) protein. However, Su(z)12 is also necessary for H3K27me3 methylation in vivo, and it is thus a core component of PRC2. Clonal over-expression of Su(z)12 in imaginal wing discs results in an increased H3K27me3 activity, indicating that Su(z)12 is a limiting factor for silencing. When PcG function is lost, target genes normally become de-repressed. The segment polarity gene engrailed, encoding a transcription factor, is a target for PRC2 silencing. However, we found that it was not activated when PRC2 function was deleted. We show that the Ultrabithorax protein, encoded by another PcG target gene, also acts as an inhibitor of engrailed and that de-regulation of this gene causes a continued repression of engrailed. The conclusion is that a gene can have several negative regulators working in parallel and that secondary effects have to be taken into consideration, when analyzing effects of mutants. PcG silencing affects very many cellular processes and a large quantity of knowledge is gathered on the overall mechanisms of PcG regulation. However, little is known about how individual genes are silenced and how cells “remember” their fate through cell generations.
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