Browsing by Subject "transcription"

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  • Kyheröinen, Salla (Helsingfors universitet, 2016)
    Actin is known as abundant cytoplasmic protein, which functions as a component of the cytoskeleton and in cell motility together with motor protein myosins. However, actin is also present in the nucleus, where it has been shown to take part in the control of gene expression, both independently and as part of chromatin remodeling complexes. An important aspect in the study of nuclear actin is to identify other nuclear proteins interacting with actin and to confirm these interactions in biochemical experiments. Through these interactions actin can be linked to various nuclear processes. The aim of this master’s thesis study was to express and purify five nuclear proteins that have been suggested to bind actin and to study the binding in detail both with actin filaments and monomers. The proteins of interest include four actin-related proteins (ARPs) Arp4, Arp5, Arp6 and Arp8 as well as RNA polymerase II subunit Rpb8. Out of the proteins selected for this study, the expression and purification of Arp4 and Rpb8 was successful. Neither one did bind monomeric actin with high affinity, but interestingly Rpb8 did bind actin filaments. On the other hand, Rpb8 did not have any effect on actin polymerization. These results provide new insights into nuclear actin function. It has been suggested earlier that Arp4 would form a heterocomplex with actin, but the results of this study do not support this. The binding between Rpb8 and actin in RNA polymerase II complex has not been extensively studied before, so the results provide new information about the function of actin in the polymerase complex.
  • Lamichane, Nicole (Helsingin yliopisto, 2019)
    Over the past years sugar consumption has seen great increases worldwide, together with a rise in the prevalence of metabolic diseases. There is a growing need for a comprehensive characterisation of the genes involved in sugar metabolism, yet the mechanisms by which cells sense and respond to sugars in vivo have remained incompletely understood. Here, I analyse members of a protein family best known for their regulation of differentiation during development with regards to their role in sugar metabolism. The Hairy and Enhancer of Split (HES) protein family are a group of basic helix-loop-helix (bHLH) transcription factors that function as major downstream effectors of the Notch signalling pathway. In mammals, the HES proteins have mostly been studied for their role in cell differentiation, but HES1 has been implicated in metabolic control. Drosophila has several transcription factors belonging to the HES family, including Hairy and seven bHLH transcription factors located in the Enhancer of split complex (E(spl)-C). The E(spl)-C bHLH transcription factors display high homology and are considered to be genetically redundant, and therefore little is known about their individual functions. The other HES family members in Drosophila have not previously been linked to metabolic regulation, but Hairy has been shown to repress the tricarboxylic acid cycle. In light of the findings implicating HES1 and Hairy in the regulation of metabolism, I systematically investigated the role of the HES transcription factors in sugar metabolism. By using the GAL4/UAS system in Drosophila melanogaster, I knocked down gene expression of each of the family members, and raised the flies on diets varying in sugar content to identify possible sugar intolerance phenotypes. Here, I show that knockdown of one of the E(spl)-C bHLH genes led to severe sugar intolerance that affected both survival and organismal growth, but did not alter the levels of circulating carbohydrates and storage lipids as measured with colorimetric assays and lipid staining. Furthermore, I identify the tissues in which this transcription factor functions to provide sugar tolerance. Using analysis of publically available chromatin-immunoprecipitation sequencing data coupled with quantitative RT-PCR, I uncover mTOR target Thor/4E-BP as a putative target gene. Additionally, I show that Hairy is similarly required for complete sugar tolerance, but that the mechanism differs from the E(spl)-C bHLH transcription factor. Hairy binds to and positively regulates expression of genes involved in glycolysis and the pentose phosphate pathway, suggestive of a cooperation with earlier known regulators of sugar sensing. In conclusion, I have shown that only two HES family members are involved in the regulation of sugar metabolism and that their regulatory mechanisms are distinct, implying that the HES family members have more diverse roles than previously assumed.
  • Savilammi, Tiina; Papakostas, Spiros; Leder, Erica H.; Vollestad, L. Asbjorn; Debes, Paul V.; Primmer, Craig R. (2021)
    Temperature is a key environmental parameter affecting both the phenotypes and distributions of organisms, particularly ectotherms. Rapid organismal responses to thermal environmental changes have been described for several ectotherms; however, the underlying molecular mechanisms often remain unclear. Here, we studied whole genome cytosine methylation patterns of European grayling (Thymallus thymallus) embryos from five populations with contemporary adaptations of early life history traits at either 'colder' or 'warmer' spawning grounds. We reared fish embryos in a common garden experiment using two temperatures that resembled the 'colder' and 'warmer' conditions of the natal natural environments. Genome-wide methylation patterns were similar in populations originating from colder thermal origin subpopulations, whereas single nucleotide polymorphisms uncovered from the same data identified strong population structure among isolated populations, but limited structure among interconnected populations. This was surprising because the previously studied gene expression response among populations was mostly plastic, and mainly influenced by the developmental temperature. These findings support the hypothesis of the magnified role of epigenetic mechanisms in modulating plasticity. The abundance of consistently changing methylation loci between two warmer-to-colder thermal origin population pairs suggests that local adaptation has shaped the observed methylation patterns. The dynamic nature of the methylomes was further highlighted by genome-wide and site-specific plastic responses. Our findings support both the presence of a plastic response in a subset of CpG loci, and the evolutionary role of methylation divergence between populations adapting to contrasting thermal environments.
  • Kuuluvainen, Emilia; Domenech-Moreno, Eva; Niemela, Elina H.; Makela, Tomi P. (2018)
    In cancer, oncogene activation is partly mediated by acquired superenhancers, which therefore represent potential targets for inhibition. Superenhancers are enriched for BRD4 and Mediator, and both BRD4 and the Mediator MED12 subunit are disproportionally required for expression of superenhancer-associated genes in stem cells. Here we show that depletion of Mediator kinase module subunit MED12 or MED13 together with MED13L can be used to reduce expression of cancer-acquired superenhancer genes, such as the MYC gene, in colon cancer cells, with a concomitant decrease in proliferation. Whereas depletion of MED12 or MED13/MED13L caused a disproportional decrease of superenhancer gene expression, this was not seen with depletion of the kinases cyclin-dependent kinase 9 (CDK8) and CDK19. MED12-MED13/MED13L-dependent superenhancer genes were coregulated by beta-catenin, which has previously been shown to associate with MED12. Importantly, beta-catenin depletion caused reduced binding of MED12 at the MYC superenhancer. The effect of MED12 or MED13/MED13L depletion on cancer-acquired superenhancer gene expression was more specific than and partially distinct from that of BRD4 depletion, with the most efficient inhibition seen with combined targeting. These results identify a requirement of MED12 and MED13/MED13L for expression of acquired superenhancer genes in colon cancer, implicating these Mediator subunits as potential therapeutic targets for colon cancer, alone or together with BRD4.
  • Andjelkovic, Ana; Mordas, Amelia; Bruinsma, Lyon; Ketola, Annika; Cannino, Giuseppe; Giordano, Luca; Dhandapani, Praveen K.; Szibor, Marten; Dufour, Eric; Jacobs, Howard T. (2018)
    Downregulation of Jun N-terminal kinase (JNK) signaling inhibits cell migration in diverse model systems. In Drosophila pupal development, attenuated JNK signaling in the thoracic dorsal epithelium leads to defective midline closure, resulting in cleft thorax. Here we report that concomitant expression of the Ciona intestinalis alternative oxidase (AOX) was able to compensate for JNK pathway downregulation, substantially correcting the cleft thorax phenotype. AOX expression also promoted wound-healing behavior and single-cell migration in immortalized mouse embryonic fibroblasts (iMEFs), counteracting the effect of JNK pathway inhibition. However, AOX was not able to rescue developmental phenotypes resulting from knockdown of the AP-1 transcription factor, the canonical target of JNK, nor its targets and had no effect on AP-1-dependent transcription. The migration of AOX-expressing iMEFs in the wound-healing assay was differentially stimulated by antimycin A, which redirects respiratory electron flow through AOX, altering the balance between mitochondrial ATP and heat production. Since other treatments affecting mitochondrial ATP did not stimulate wound healing, we propose increased mitochondrial heat production as the most likely primary mechanism of action of AOX in promoting cell migration in these various contexts.
  • Sokolova, Maria; Moore, Henna M.; Prajapati, Bina; Dopie, Joseph; Meriläinen, Leena; Honkanen, Mikko; Matos, Rita Cerejeira; Poukkula, Minna; Hietakangas, Ville; Vartiainen, Maria K. (2018)
    Actin has been linked to processes spanning the whole gene expression cascade, from regulating specific transcription factors, such as myocardin-related transcription factor (Mrtf), to chromatin remodelling and RNA polymerase (Pol) function. However, whether actin controls transcription of only specific genes or has a global role in gene expression has remained elusive. Our genome-wide analysis reveals, for the first time, that actin interacts with essentially all transcribed genes in Drosophila ovaries. Actin co-occupies majority of gene promoters together with Pol II, and on highly expressed genes these two proteins also associate with gene bodies. Mechanistically, actin is required for Pol II recruitment to gene bodies and manipulation of nuclear transport factors for actin leads to decreased expression of egg shell genes. Collectively, these results uncover a global role for actin in transcription, and demonstrate the in vivo importance of balanced nucleo-cytoplasmic shuttling of actin in transcriptional control of a developmental process.
  • Sidorenko, Ekaterina; Vartiainen, Maria K. (2019)
    Myocardin-related transcription factor A (MRTF-A) and serum response factor (SRF) form an essential transcriptional complex that regulates the expression of many cytoskeletal genes in response to dynamic changes in the actin cytoskeleton. The nucleoskeleton, a "dynamic network of networks," consists of numerous proteins that contribute to nuclear shape and to its various functions, including gene expression. In this review, we will discuss recent work that has identified many nucleoskeletal proteins, such as nuclear lamina and lamina-associated proteins, nuclear actin, and the linker of the cytoskeleton and nucleoskeleton complex as important regulators of MRTF-A/SRF transcriptional activity, especially in the context of mechanical control of transcription. Impact statement Regulation of gene expression is a fundamental cellular process that ensures the appropriate response of a cell to its surroundings. Alongside biochemical signals, mechanical cues, such as substrate rigidity, have been recognized as key regulators of gene expression. Nucleoskeletal components play an important role in mechanoresponsive transcription, particularly in controlling the activity of MRTF-A/SRF transcription factors. This ensures that the cell can balance the internal and external mechanical forces by fine-tuning the expression of cytoskeletal genes.
  • Hyrskyluoto, Alise; Vartiainen, Maria K. (2020)
    Actin has essential functions both in the cytoplasm and in the nucleus, where it has been linked to key nuclear processes, from transcription to DNA damage response. The multifunctional nature of actin suggests that the cell must contain mechanisms to accurately control the cellular actin balance. Indeed, recent results have demonstrated that nuclear actin levels fluctuate to regulate the transcriptional activity of the cell and that controlled nuclear actin polymerization is required for transcription activation, cell cycle progression, and DNA repair. Intriguingly, aberrant nuclear actin regulation has been observed, for example, in cancer, signifying the importance of this process for cellular homeostasis. This review discussed the latest research on how nuclear actin is regulated, and how this influences actin-dependent nuclear processes.
  • Wei, Ting; Najmi, Saman M.; Liu, Hester; Peltonen, Karita; Kucerova, Alena; Schneider, David A.; Laiho, Marikki (2018)
    Summary Inhibition of RNA polymerase I (Pol I) is a promising strategy for modern cancer therapy. BMH-21 is a first-in-class small molecule that inhibits Pol I transcription and induces degradation of the enzyme, but how this exceptional response is enforced is not known. Here, we define key elements requisite for the response. We show that Pol I preinitiation factors and polymerase subunits (e.g., RPA135) are required for BMH-21-mediated degradation of RPA194. We further find that Pol I inhibition and induced degradation by BMH-21 are conserved in yeast. Genetic analyses demonstrate that mutations that induce transcription elongation defects in Pol I result in hypersensitivity to BMH-21. Using a fully reconstituted Pol I transcription assay, we show that BMH-21 directly impairs transcription elongation by Pol I, resulting in long-lived polymerase pausing. These studies define a conserved regulatory checkpoint that monitors Pol I transcription and is activated by therapeutic intervention.
  • Liu, Ying; Mattila, Jaakko; Hietakangas, Ville (2020)
    Insulin/insulin-like growth factor signaling (IIS) is a conserved mechanism to regulate animal physiology in response to nutrition. IIS activity controls gene expression, but only a subset of transcriptional regulators (TRs) targeted by the IIS pathway is currently known. Here we report the results of an unbiased screen forDrosophilaTRs phosphorylated in an IIS-dependent manner. To conduct the screen, we built a library of 857 V5/Strep-tagged TRs under the control of Copper-inducible metallothionein promoter (pMt). The insulin-induced phosphorylation changes were detected by using Phos-tag SDS-PAGE and Western blotting. Eight proteins were found to display increased phosphorylation after acute insulin treatment. In each case, the insulin-induced phosphorylation was abrogated by mTORC1 inhibitor rapamycin. The hits included two components of the NURF complex (NURF38 and NURF55), bHLHZip transcription factor Max, as well as theDrosophilaortholog of human proliferation-associated 2G4 (dPA2G4). Subsequent experiments revealed that the expression of thedPA2G4gene was promoted by the mTOR pathway, likely through transcription factor Myc. Furthermore, NURF38 was found to be necessary for growth in larvae, consistent with the role of IIS/mTOR pathway in growth control.
  • Bradley, Jeremy; Skribnik, Elena (2021)
    The paper at hand presents the recently published COPIUS Orthographic Toolset’s Mansi module. This open-source software, part of the COPIUS drive to create necessary international infrastructures for teaching/learning and researching Uralic languages, allows for rule-based transcription between four basic writing systems historically used for Mansi: the Cyrillic alphabet, the Latin-based Unified Northern Alphabet (UNA), Finno-Ugric Transcription (FUT), and the International Phonetic Alphabet (IPA). The software aims to take variation in the usage of these respective writing systems into consideration as best possible in a purely rule-based approach currently lacking lexical support. Section 1 will give a short summary of the history of Mansi literacy and aims to elucidate how changing trends, both local and Russia-wide, influenced the manner in which Mansi was captured in writing by scientists and speakers throughout history. Section 2 will give an overview of (Northern) Mansi phonology and discuss how difficult aspects of it are handled in the writing systems under consideration. Finally, Section 3 will illustrate the transcription software, in its current version, in action, with a sample text transcribed from each of the four writing systems under consideration into the three other ones.
  • Lidschreiber, Katja; Jung, Lisa A.; von der Emde, Henrik; Dave, Kashyap; Taipale, Jussi; Cramer, Patrick; Lidschreiber, Michael (2021)
    The growth of human cancer cells is driven by aberrant enhancer and gene transcription activity. Here, we use transient transcriptome sequencing (TT-seq) to map thousands of transcriptionally active putative enhancers in fourteen human cancer cell lines covering seven types of cancer. These enhancers were associated with cell type-specific gene expression, enriched for genetic variants that predispose to cancer, and included functionally verified enhancers. Enhancer-promoter (E-P) pairing by correlation of transcription activity revealed similar to 40,000 putative E-P pairs, which were depleted for housekeeping genes and enriched for transcription factors, cancer-associated genes, and 3D conformational proximity. The cell type specificity and transcription activity of target genes increased with the number of paired putative enhancers. Our results represent a rich resource for future studies of gene regulation by enhancers and their role in driving cancerous cell growth.