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Identification of new tissue-specific interaction partners of chromatin remodelling ATPase Smarca5
Arishaka, Yuliia ; Kokavec, Juraj (advisor) ; Děd, Lukáš (referee)
The regulation of chromatin structure is fundamental to a wide range of cellular processes, including transcriptional regulation, cell division, differentiation and DNA damage repair, and ATP-dependent chromatin remodeling complexes have been established as essential components of this regulatory network. Smarca5, as an ATPase/Helicase enzyme, has been shown to regulate chromatin structure by interacting with bromodomain and DDT-WHIM domain-containing partners to control the binding of chromatin-associated proteins and transcription factors to their specific DNA target sequences. In this work we identify a previously undescribed protein with a conserved N-terminal bromodomain and ISWI protein binding DDT-WHIM domain through co-immunoprecipitation and mass spectrometry in mammalian cell lines and establish it as a novel interaction partner of chromatin remodeling ATPase Smarca5. Furthermore, we have pinpointed the region required for Smarca5 interaction that corresponds to DDT-WHIM domain. We have furthermore attempted to identify additional interaction partners which may hint on the potential function of this novel chromatin complex and validated its expression in embryonic and postnatal tissues. This discovery represents a unique opportunity for further investigation into its potential function in...
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Chromatin remodeling during temperature sensing in plants
Šlesingerová, Terezie
The evolutionary conserved transcriptional co-activator Spt–Ada–Gcn5 Acetyltransferase (SAGA) complex in Arabidopsis thaliana is tightly involved in numerous cellular processes by histone posttranslational modifications. The GENERAL CONTROL NON-REPRESSED PROTEIN 5 (GCN5), a SAGA subunit, is a histone acetyltransferase that impacts chromatin remodeling. Arabidopsis mutants lacking GCN5 are sensitive to heat stress, but the molecular mechanisms implicating GCN5 in heat stress are currently unknown. To get an insight into the molecular pathways underlying the heat sensitivity of gcn5, we assessed global proteome changes triggered by heat stress in the gcn5 background. Even though most of the identified proteins responded similarly both in gcn5 and wild type plants, some of them like CONSTITUTIVE PHOTOMORPHOGENIC 11 (COP11) were unaffected by heat in the gcn5 mutant background but decreased drastically in the wild type. We further screened mutants affected in putative GCN5 interactors under heat stress and identified that lack of SAGA COMPLEX SUBUNIT 2A (SCS2A) and INHIBITOR OF GROWTH 1 (ING1) results in heat sensitivity. Taken together, our results pave the way for a more detailed understanding of the role of GCN5 in stress responses.
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Research of epigenetic aspects of hematopoietic and spermatogenesis stem cells.
Hybešová, Michaela ; Pimková, Kristýna (advisor) ; Děd, Lukáš (referee)
Stem cell differentiation is controlled by coordinated regulation of gene transcription. One of the regulatory factors is the loosening of chromatin and the accessibility of DNA to transcription factors. Chromatin remodeling is mediated by remodeling complexes. The ISWI chromatin remodeling ATPase Smarca5 (S5) is an important factor of remodeling complexes. It is a highly conserved chromatin-remodeling factor forming a catalytic subunit that can be found in several oligosubunit complexes. In these complexes, it actively regulates nucleosome structure and remodeling during DNA replication, repair and transcription. S5 has been identified as a key protein in embryonic development. Its deficiency leads to defects in hematopoiesis and male genital development. In the presented study, we focused on the role of S5 in hematopoiesis and spermatogenesis. Using a mouse model with transgenic expression of S5, co-immunoprecipitation and mass spectrometry, we identified S5 complexes in hematopoietic and testicular cells. We also studied the phenotypic consequences of S5 deficiency in mouse testes and found that it leads to impaired sperm development and male sterility. Using transcriptomic and proteomic analysis, we identified several molecular programs that could lead to reproductive disorders. Our work...
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SWI2/SNF2 ATPases with a focus on the ISWI subfamily: protein complexes and mouse models for their study
Turková, Tereza ; Stopka, Tomáš (advisor) ; Janoštiak, Radoslav (referee)
In the nucleus the DNA is packed along with proteins into a dynamic structure called chromatin. During cell cycle the chromatin structure becomes a subject to various changes. During interphase chromatin structure becomes loose while shortly before cell division it undertakes the form of highly condensed mitotic chromosomes. Structure of chromatin influences significantly mode of gene expression and its pattern. DNA-binding proteins interacting within chromatin are also necessary during this process. To gain the access to the DNA binding factors, the chromatin has to be in a loosened form. As long as the structure of the chromatin is more condensed it creates a barrier for the DNA binding proteins. Therefore it becomes obvious that the remodeling of the chromatin structure is one of the important regulators of gene expression and that the enzymes, which execute remodeling, are of great importance. One of them is ATPase Smarca5, which belongs to the protein subfamily ISWI and which creates the catalytic subunit for several different ATP-dependent chromatin remodeling complexes. Mutations of members of those complexes disturb regulation of transcription and cellular differentiation. In some cases the incorrect function of these complexes can lead to cellular transformation into a tumours state. This...
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Generation of the Mouse Model to Delineate Function of Chromatin Remodeling Gene Smarca5 (Snf2h)
Turková, Tereza ; Stopka, Tomáš (advisor) ; Dobeš, Jan (referee)
The chromatin structure, consisting of DNA and histones, changes dynamically during the cell cycle and cell differentiation. DNA can only be transcribed and replicated when it is packaged loosely, whereas tight packaging allows for more efficient storage. Chromatin remodelling is therefore one of the tools of gene expression control. The chromatin remodelling factors recognise chromatin with varying specificity and have an effect on the interaction between DNA and the histones. One of these factors is the Smarca5 protein. This study investigates the role of Smarca5; its goal is to create a mouse model with the ability to trigger Smarca5 overproduction in specific tissues. This model will be used to study the effect of a high, unregulated dose of Smarca5 on the physiological function of the protein. Previous studies have shown that non-physiological expression of a chromatin-remodelling factor can lead to malignant transformation. Our model can help to understand this process. Another goal of this study is to investigate some phenotype aspects of the mouse model with conditional deletion of Smarca5 in T and B cells, in particular the effects of this deletion on progenitor cell differentiation. Our results show that Smarca5 has an important role in lymphocyte development, and we have observed that...
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SWI2/SNF2 ATPases with a focus on the ISWI subfamily: protein complexes and mouse models for their study
Turková, Tereza ; Stopka, Tomáš (advisor) ; Janoštiak, Radoslav (referee)
In the nucleus the DNA is packed along with proteins into a dynamic structure called chromatin. During cell cycle the chromatin structure becomes a subject to various changes. During interphase chromatin structure becomes loose while shortly before cell division it undertakes the form of highly condensed mitotic chromosomes. Structure of chromatin influences significantly mode of gene expression and its pattern. DNA-binding proteins interacting within chromatin are also necessary during this process. To gain the access to the DNA binding factors, the chromatin has to be in a loosened form. As long as the structure of the chromatin is more condensed it creates a barrier for the DNA binding proteins. Therefore it becomes obvious that the remodeling of the chromatin structure is one of the important regulators of gene expression and that the enzymes, which execute remodeling, are of great importance. One of them is ATPase Smarca5, which belongs to the protein subfamily ISWI and which creates the catalytic subunit for several different ATP-dependent chromatin remodeling complexes. Mutations of members of those complexes disturb regulation of transcription and cellular differentiation. In some cases the incorrect function of these complexes can lead to cellular transformation into a tumours state. This...
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