| 1. |
- Alkhori, Liza
(författare)
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Mechanisms of sensory neuron diversification during development and in the adult Drosophila : How to make a difference
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The nervous system contains a vast number of neurons and displays a great diversity in cell types and classes. Even though this has been known for a long time, the exact mechanism of cell specification is still poorly understood. How does a cell know what type of neuron to which it should be specified? It is important to understand cellular specification, not only for our general understanding of biological processes, but also to allow us to develop treatments for patients with destructive diseases, such as Alzheimer’s, Parkinson, cancer or stroke. To address how neuronal specification and thereby diversification is evolved, we have chosen to study a complex but defined set of neurons, the Drosophila olfactory system. Olfactory sensory neurons (OSNs) detect an enormous variety of small volatile molecules with extremely high specificity and sensitivity. The adult Drosophila olfactory system contains 34 OSN classes each defined by their expression of a specific odorant receptor (OR). In both insects and vertebrates, each OSN expresses only one OR. In mouse there are approximately 1200 and in Drosophila 60 different OR genes. Despite the range of mechanisms known to determine cell identity and that the olfactory system is remarkably conserved across the phyla, it is still unclear how an OSN chooses to express a particular OR from a large genomic repertoire. In this thesis, the specification and diversification of the final steps establishing an OSN identity is addressed. We find seven transcription factors that are continuously required in different combinations for the expression of all ORs. The TFs can in different gene context both activate and repress OR expression, making the regulation more economical and indicating that repression is crucial for correct gene expression. We further identified a repressor complex that is able to segregate OR expression between OSN classes and propose a mechanism on how one single co-repressor can specify a large number of neuron classes.Exploring the OSN we found the developmental Hh signalling pathway is expressed in the postmitotic neuron. We show several fundamental similarities between the canonical Drosophila Hh pathway and the cilia mediated Hh transduction in component function. Further investigation revealed a function of cilia mediated Hh signalling in sensory neuron modulator. The results generated here will create a greater in vivo understanding of how postmitotic processes generate neurons with different fates and contribute to the maintaining of neuron function.
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| 2. |
- Boija, Ann, 1984-
(författare)
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Transcriptional and epigenetic control of gene expression in embryo development
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- During cell specification, temporal and spatially restricted gene expression programs are set up, forming different cell types and ultimately a multicellular organism. In this thesis, we have studied the molecular mechanisms by which sequence specific transcription factors and coactivators regulate RNA polymerase II (Pol II) transcription to establish specific gene expression programs and what epigenetic patterns that follows.We found that the transcription factor Dorsal is responsible for establishing discrete epigenetic patterns in the presumptive mesoderm, neuroectoderm and dorsal ectoderm, during early Drosophila embryo development. In addition, these different chromatin states can be linked to distinct modes of Pol II regulation. Our results provide novel insights into how gene regulatory networks form an epigenetic landscape and how their coordinated actions specify cell identity.CBP/p300 is a widely used co-activator and histone acetyltransferase (HAT) involved in transcriptional activation. We discovered that CBP occupies the genome preferentially together with Dorsal, and has a specific role during development in coordinating the dorsal-ventral axis of the Drosophila embryo. While CBP generally correlates with gene activation we also found CBP in H3K27me3 repressed chromatin.Previous studies have shown that CBP has an important role at transcriptional enhancers. We provide evidence that the regulatory role of CBP does not stop at enhancers, but is extended to many genomic regions. CBP binds to insulators and regulates their activity by acetylating histones to prevent spreading of H3K27me3. We further discovered that CBP has a direct regulatory role at promoters. Using a highly potent CBP inhibitor in combination with ChIP and PRO-seq we found that CBP regulates promoter proximal pausing of Pol II. CBP promotes Pol II recruitment to promoters via a direct interaction with TFIIB, and promotes transcriptional elongation by acetylating the first nucleosome. CBP is regulating Pol II activity of nearly all expressed genes, however, either recruitment or release of Pol II is the rate-limiting step affected by CBP.Taken together, these results reveal mechanistic insights into cell specification and transcriptional control during development.
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| 3. |
- Bysani, Madhusudhan Reddy
(författare)
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Genome-Wide Studies of Transcriptional Regulation in Human Liver Cells by High-throughput Sequencing
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The human genome contains slightly more than 20 000 genes that are expressed in a tissue specific manner. Transcription factors play a key role in gene regulation. By mapping the transcription factor binding sites genome-wide we can understand their role in different biological processes. In this thesis we have mapped transcription factors and histone marks along with nucleosome positions and RNA levels. In papers I and II, we used ChIP-seq to map five liver specific transcription factors that are crucial for liver development and function. We showed that the mapped transcription factors are involved in metabolism and other cellular processes. We showed that ChIP-seq can also be used to detect protein-protein interactions and functional SNPs. Finally, we showed that the epigenetic histone mark studied in paper I is associated with transcriptional activity at promoters. In paper III, we mapped nucleosome positions before and after treatment with transforming growth factor β (TGFβ) and found that many nucleosomes changed positions when expression changed. After treatment with TGFβ, the transcription factor HNF4α was replaced by a nucleosome in some regions. In paper IV, we mapped USF1 transcription factor and three active chromatin marks in normal liver tissue and in liver tissue of patients diagnosed with alcoholic steatohepatitis. Using gene ontology, we as expected identified many metabolism related genes as active in normal samples whereas genes in cancer pathways were active in steatohepatitis tissue. Cancer is a common complication to the disease and early signs of this were found. We also found many novel and GWAS catalogue SNPs that are candidates to be functional. In conclusion, our results have provided information on location and structure of regulatory elements which will lead to better knowledge on liver function and disease.
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| 4. |
- Crona, Filip, 1977-
(författare)
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Regulators of chromatin and transcription in Drosophila
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Development of multicellular organisms is achieved by organized temporal and spatial patterns of gene expression leading to cell differentiation. Chromatin regulators control how the DNA is utilized by altering access of proteins to DNA and thereby function as co-factors in transcription. Gene regulation also involves co-factors interacting with transcription factors at regulatory sequences of DNA. In this thesis, we have studied the in vivo role of three co-factors, CBP, dKDM4A and Brakeless, in regulating chromatin and transcription using Drosophila melanogaster. The CREB binding protein (CBP) belongs to histone acetyl transferases (HATs) and facilitates gene activation by many transcription factors. Our work has demonstrated that CBP occupies the genome preferentially together with Rel and Smad proteins controlling dorsal-ventral patterning in the Drosophila embryo. CBP occupancy generally correlates with gene expression but also occurs at silent genes without resulting in histone acetylation. KDM4A belongs to a family of JmjC domain proteins and demethylates H3K36me3, a histone modification enriched in the 3’end of active genes. We generated dKDM4A mutants with a global elevation of H3K36me3 levels and identify mis-regulated genes in first instar larvae. The data indicate that dKDM4A regulates some genes by mechanisms that do not involve H3K36 methylation. Further, over-expression of dKDM4A result in male lethality and globally reduced H3K36me3 levels, indicating impaired dosage compensation of the X-chromosome. Brakeless is a conserved co-factor participating in several important processes during development. We generated mutant brakeless embryos and identify direct genomic targets of Brakeless. To our surprise, Brakeless behaves as a direct activator for some genes but repressor in other cases. We also identify an interaction of Brakeless with the Mediator subunit Med19. In summary, these studies reveal unexpected roles for co-regulators in Drosophila development. The HAT CBP can bind silent genes without leading to histone acetylation. Brakeless has the ability to function both as a direct activator and repressor of transcription.
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| 5. |
- Dahlberg, Olle, 1979-
(författare)
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Gene regulation during development by chromatin and the Super Elongation Complex
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Developmental processes are carefully controlled at the level of transcription to ensure that the fertilized egg develops into an adult organism. The mechanisms that controls transcription of protein-coding genes ultimately ensure that the Pol II machine synthesizes mRNA from the correct set of genes in every cell type. Transcriptional control involves Pol II recruitment as well as transcriptional elongation. Recent genome-wide studies shows that recruitment of Pol II is often followed by an intermediate step where Pol II is halted in a promoter-proximal paused configuration. The release of Pol II from promoter-proximal pausing is thus an additional and commonly occurring mechanism in metazoan gene regulation. The serine kinase P-TEFb is part of the Super Elongation Complex that regulates the release of paused Pol II into productive elongation. However, little is known about the role of P-TEFb mediated gene expression in development. We have investigated the function of P-TEFb in early Drosophila embryogenesis and find that P-TEFb and other Super Elongation Complex subunits are critical for activation of the most early expressed genes. We demonstrate an unexpected function for Super Elongation Complex in activation of genes with non-paused Pol II. Furthermore, the Super Elongation Complex shares phenotypes with subunits of the Mediator complex to control the activation of essential developmental genes. This raises the possibility that the Super Elongation Complex has an unappreciated role in the recruitment of Pol II to promoters. The unique chromatin landscape of each cell type is comprised of post-translational chromatin modifications such as histone methylations and acetylations. To study the function of histone modifications during development, we depleted the histone demethylase KDM4A in Drosophila to evaluate the role of KDM4A and histone H3 lysine 36 trimethylation (H3K36me3) in gene regulation. We find that KDM4A has a male-specific function and regulates gene expression both by catalytic-dependent and independent mechanisms. Furthermore, we used histone replacement to investigate the direct role of H3K14 acetylation in a multicellular organism. We show that H3K14 acetylation is essential for development, but is not cell lethal, suggesting that H3K14 acetylation has a critical role in developmental gene regulation. This work expands our knowledge of the mechanisms that precisely controls gene regulation and transcription, and in addition highlights the complexity of metazoan development.
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| 6. |
- Dai, Qi, 1976-
(författare)
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Chromatin regulators and transcriptional control of Drosophila development
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The development of a multicellular organism is programmed by complex patterns of gene expression. In eukaryotic cells, genes are packaged by histone proteins into chromatin. Chromatin regulators often function as transcription co-factors. In this study, we have investigated the function of four co-factors, dAda2b, Reptin, Ebi and Brakeless during development of the fruit fly Drosophila melanogaster. dAda2b and Reptin belong to histone acetyl transferase (HAT) complexes, a SAGA-like complex and the Tip60 complex, respectively. We generated dAda2b mutants and found that lack of dAda2b strongly affects global histone acetylation and viability. We further propose that Ada2 may be involved in DNA repair. Our studies revealed new roles of Reptin and other Tip60 complex components in Polycomb Group mediated repression and heterochromatin formation, thereby promoting generation of silent chromatin.During embryogenesis, transcriptional repressors establish localized and tissue-specific patterns of gene expression. In this thesis, we identified two novel co-repressors in the early embryo, Ebi and Brakeless. Ebi genetically and physically interacts with the Snail repressor. The Ebi-interaction motif in the Snail protein is essential for Snail function in vivo and is evolutionarily conserved in insects. We further demonstrated that Ebi associates with histone deacetylase 3 (HDAC3) and that histone deacetylation is part of the mechanism by which Snail mediates transcriptional repression. We isolated Brakeless in a genetic screen for novel regulators of gene expression during embryogenesis. We found that mutation of brakeless impairs function of the Tailless repressor. Brakeless associates with Atrophin, another Tailless corepressor, and they function together in Tailless-mediated repression. In summary, transcription co-factors, including chromatin regulators, are selectively required in distinct processes during development.
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| 7. |
- Hunt, George, 1993-
(författare)
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The interplay of transcription and chromatin regulation during embryonic development
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- A fascinating aspect of multicellular development is the production of diverse cell types from an identical DNA genome, which depends on the ability to express different complements of genes across space and time. This thesis tries to address how the genome, within the chromatin structure that envelops it, is regulated to direct this complex process. The importance of chromatin regulation to the establishment and maintenance of transcriptional states is being rapidly revealed and understanding how chromatin-modifiers influence gene activity during complex developmental processes such as embryogenesis is an important goal. This thesis explores the interplay between chromatin regulation and transcriptional states during development.The transcriptional impacts of chromatin-modifying complexes such as the p300/CBP coactivator and Polycomb group (PcG) protein repressors are well known but deciphering how these complexes interact to fine-tune the transcriptional output is not well characterized. At repressive PcG-bound chromatin in metazoans, p300/CBP often occupies but is enzymatically silent. The work in this thesis uncovers a novel role of p300/CBP that maintains PcG mediated silencing through the deployment of a subset of known functions that support RNA Polymerase II (Pol II) recruitment at silent PcG sites. This finding highlights the importance of context-dependent chromatin-modifying complex activities in establishing transcriptional states.To explore the interplay between chromatin and transcriptional states during organismal development, we comprehensively assessed the dorsoventral (DV) patterning axis during early Drosophila embryogenesis. DV patterning is a model gene regulatory network for studying the formation of differential transcriptional programs. Here, we resolved the relationship between tissue-specific chromatin and transcriptional states using genome-wide approaches that reveal new aspects of the DV network. We find that release of paused Pol II from promoters, not recruitment, is the key molecular switch for regulatory cues to drive tissue-specific transcription. We identify an active chromatin state that correlates with the transcription of DV regulated genes and demonstrate that tissue-specific recruitment of the pause releasing P-TEFb complex is a key transcriptional effector linked to the chromatin state. We also infer the transcriptional burst dynamics of DV genes to model how chromatin regulatory signals direct the transcriptional response.We uncover a novel function of the GAGA transcription factor (GAF) as a propagator of transcriptional states across cell divisions during embryogenesis by bookmarking regulatory sequences during mitosis. A novel ChIP-seq variant that profiles embryos specifically in mitosis reveals the genomic sites bound by GAF at this cell cycle stage. Live imaging of transcription in vivo across cell divisions reveals GAF is important for rapid postmitotic activation and is involved in transcriptional memory, whereby descendants of transcriptionally active nuclei undergo stronger or more rapid transcription.Overall, the results reveal the complex interactions between chromatin-modifiers that govern transcriptional effects and highlight the importance of regulation across DNA regulatory sequences, the transcription factors they recruit and the surrounding chromatin environment in orchestrating complex developmental processes.
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| 8. |
- Ueberschär, Malin, 1991-
(författare)
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BEN-solo proteins in genome architecture and function
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Eukaryotic genomes are organized in a complex manner inside the nucleus of each cell. Chromatin insulators are DNA elements that help to fold the linear DNA fiber in three-dimensional space and partition regulatory gene units. However, the mechanisms by which chromatin insulators and their associated proteins, insulator binding proteins (IBPs), determine genome organization and modulate gene expression remain elusive. This thesis investigates these mechanisms by examining the roles of a stage-specific family of insulator proteins in Drosophila, the BEN-solo proteins. The BEN proteins regulate gene expression and cell specification in animal development. We characterized the genomic binding sites of the three BEN-solo proteins Elba1, Elba2 and Insensitive (Insv), and the Elba3 protein that acts as adapter for the heterotrimeric ELBA complex. ELBA and Insv share a set of common targets at the time of their ubiquitous expression – throughout the major wave of zygotic genome activation – but also regulate distinct loci. We found that ELBA and Insv not only repress their direct target genes, but also co-localize and act in redundancy with other known insulator binding proteins. Importantly, ELBA and Insv insulate closely-spaced transcription units ensuring the correct levels of promoter transcripts.The regulatory state of paused RNA Polymerase II (Pol II) has been suggested to comprise a mechanism for insulator function. To understand how paused promoters may block enhancer interaction, we assessed a series of paused and non-paused promoters in a transgenic reporter assay. Although we observed a high likelihood of paused promoters to act as enhancer blockers, there were several exceptions. Using a paused representative promoter, we dissected the contributions of Pol II and promoter-proximal IBPs to enhancer-blocking. We found that Pol II is dispensable for insulation at this site, while the IBPs and their motifs are essential, arguing against a general function of paused Pol II as enhancer-blocking mechanism.Insulators are known to set barriers between active and silent chromatin domains. We found that chromatin accessibility at chromatin boundaries in elba and insv mutants is frequently altered, suggesting an involvement of the proteins in chromatin boundary formation. Remarkably, shifted boundaries are accompanied by a deregulation of nascent transcripts. We further assessed the initial establishment of heterochromatin domains. Using a reporter embedded in the heterochromatin of the Drosophila Y chromosome, and the GFP-tagged heterochromatin protein 1a (HP1a), we found that ELBA contributes to heterochromatin silencing while Insv appears to limit HP1a clustering on a global scale. We hypothesize that ELBA and Insv contribute to boundary formation at some common sites, while diverging in their contributions to heterochromatin domain formation.Taken together, our results identify multiple roles of the BEN-solo family as chromatin factors, and highlight the intrinsic properties of insulator elements and IBPs as the main mechanism in enhancer blocking.
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| 9. |
- 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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| 10. |
- Johnsson, Anna
(författare)
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Characterization of Gcn5 histone acetyltransferase in Schizosaccharomyces pombe
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The organisation of eukaryotic DNA into chromatin provides a natural barrier that prevents full access to the DNA thereby inhibiting events such as transcription, replication and repair. In order for these DNA-related events to occur, the chromatin needs to be modified by chromatin remodelling or, by reversible post-translational modifications. Histone acetylation is such a modification and is essential of numerous DNA related events. The enzymes involved in this event are conserved throughout evolution, underscoring their importance. This thesis describes the role of the conserved histone acetyltransferase (HAT) Gcn5 in transcriptional regulation in Schizosaccharomyces pombe. Here we show that Gcn5 plays an important role in stress response. We map genome-wide Gcn5 occupancy and show that Gcn5 is predominantly localized to coding regions of highly transcribed genes. We also map H3K14 acetylation during salt stress and show that Gcn5 collaborates antagonistically with the class-II histone deacetylase, Clr3, to modulate H3K14ac levels and transcriptional elongation. The interplay between Gcn5 and Clr3 is crucial for the regulation of many stress-response genes. Our findings suggest a new role for Gcn5 during transcriptional elongation, in addition to its known role in transcriptional initiation. We also investigate the interactions between Gcn5 and other histone deacetylases and acetyltransferases and show overlapping functionality between Gcn5 and another histone acetyltransferase, Mst2, in stress response, regulation of subtelomeric genes and DNA damage repair. Finally, we show that the role of Gcn5 in stress response is mediated by its catalytic activity and that its function in stress response is conserved among yeast species.
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