|
|
Epigenetic factors CTCF a SMARCA5 control expression of hematopoietic transcription factor SPI1 in cells of acute myeloid leukemia and myelodysplastic syndrome.
Dluhošová, Martina ; Stopka, Tomáš (advisor) ; Machová Poláková, Kateřina (referee) ; Kozák, Tomáš (referee)
CCCTC-binding factor (CTCF) can both activate as well as inhibit transcription by forming chromatin loops between regulatory regions and promoters. In this regard, Ctcf binding on the non-methylated DNA and its interaction with the Cohesin complex results in differential regulation of the H19/Igf2 locus. Similarly, a role for CTCF has been established in normal hematopoietic development; however its involvement, despite mutations in CTCF and Cohesin complex were identified in leukemia, remains elusive. CTCF regulates transcription dependently on DNA methylation status and can if bound block interactions of enhancers and promoters. Here, we show that in hematopietic cells CTCF binds to the imprinting control region of H19/Igf2 and found that chromatin remodeller Smarca5, which also associates with the Cohesin complex, facilitates Ctcf binding and regulatory effects. Furthermore, Smarca5 supports CTCF functionally and is needed for enhancer-blocking effect at imprinting control region. We identified new CTCF-recognized locus near hematopoietic regulator SPI1 (PU.1) in normally differentiating myeloid cells together with members of the Cohesin complex. Due to DNA methylation, CTCF binding to the SPI1 gene is reduced in AML blasts and this effect was reversible by DNA methylation inhibitor 5-azacitidine.
|
|
|
Mammalian proteins carrying zinc finger arrays
Vobruba, Adam ; Svoboda, Petr (advisor) ; Mašek, Jan (referee)
A zinc finger is a small peptide motif stabilised by a single zinc ion, best known for its capability to specifically bind a 3-nucleotide sequence of DNA, depending on the exact amino acids present in the DNA-binding positions. Zinc fingers are unique in their ability to freely link together and form longer tandem arrays, which can bind DNA targets of any length and sequence determined by the combination of individual fingers. These arrays can easily mutate and be rebuilt to change binding specificity, allowing great flexibility and helping zinc fingers to their widespread presence in numerous endogenous proteins of various functions. This property of zinc finger arrays also made them a suitable tool for the creation of custom DNA- binding domains for genetic engineering. This thesis provides an overview of the discovery, structure and function of these domains and then reviews and discusses selected naturally occurring mammalian zinc finger proteins and their properties, showcasing diverse uses zinc finger arrays have been adapted for throughout evolution. The history and future of zinc fingers in artificial proteins created for gene therapy and research are discussed as well. Keywords: zinc finger, ZnF, KRAB, KZFP, CTCF, PRDM9, ZFN, mammals
|
|
|
Epigenetic Aspects of normal and malignant hematopoiesis: role of chromatin remodeling ISWIATPase.
Zikmund, Tomáš ; Stopka, Tomáš (advisor) ; Dráber, Peter (referee) ; Otáhal, Pavel (referee)
Chromatin remodeling protein Smarca5 participates on many cellular processes, which are important for tissue development and tumorigenesis. Among these processes utilizing ATPase activity of Smarca5 belong also transcription, replication and DNA repair. We hypothesized that Smarca5 represents essential molecule for chromatin modulation primarily at early developmental stages at the level of fast-dividing progenitors of many origins, in whose the ATPase is highly expressed. To such tissues may belong also hematopoiesis, in which the Smarca5 has highest expression. The subject of my doctoral thesis is therefore analysis of the effect Smarca5 depletion on proliferation and differentiation of hematopoietic progenitors in vivo and a search for mechanisms behind the resulted developmental defects. We utilized conditionally knockout allele of Smarca5 in blood precursors to study in a mouse model how depletion of the ISWI ATPase causes accumulation of earliest progenitors inhibited from further maturation to erythroid and other myeloid lines. The proerythroblasts became dysplastic and the majority of basophilic erythroblasts ceased cycling around the G2/M stage. An expected mechanism for observed changes appeared the activation of stress pathway of protein p53 that is often associated with unrepaired DNA...
|
|
|
Epigenetic factors CTCF a SMARCA5 control expression of hematopoietic transcription factor SPI1 in cells of acute myeloid leukemia and myelodysplastic syndrome.
Dluhošová, Martina ; Stopka, Tomáš (advisor) ; Machová Poláková, Kateřina (referee) ; Kozák, Tomáš (referee)
CCCTC-binding factor (CTCF) can both activate as well as inhibit transcription by forming chromatin loops between regulatory regions and promoters. In this regard, Ctcf binding on the non-methylated DNA and its interaction with the Cohesin complex results in differential regulation of the H19/Igf2 locus. Similarly, a role for CTCF has been established in normal hematopoietic development; however its involvement, despite mutations in CTCF and Cohesin complex were identified in leukemia, remains elusive. CTCF regulates transcription dependently on DNA methylation status and can if bound block interactions of enhancers and promoters. Here, we show that in hematopietic cells CTCF binds to the imprinting control region of H19/Igf2 and found that chromatin remodeller Smarca5, which also associates with the Cohesin complex, facilitates Ctcf binding and regulatory effects. Furthermore, Smarca5 supports CTCF functionally and is needed for enhancer-blocking effect at imprinting control region. We identified new CTCF-recognized locus near hematopoietic regulator SPI1 (PU.1) in normally differentiating myeloid cells together with members of the Cohesin complex. Due to DNA methylation, CTCF binding to the SPI1 gene is reduced in AML blasts and this effect was reversible by DNA methylation inhibitor 5-azacitidine.
|
|
|
Multifunctional protein CTCF and its role in regulation of gene expression
Pokorná, Linda ; Vacík, Tomáš (advisor) ; Vopálenský, Václav (referee)
CTCF is a ubiquitously expressed nuclear protein that binds to DNA through its central zinc finger domain. Thousands of CTCF binding sites have been identified throughout the human genome at gene promoters, in intergenic regions or in non-coding sequences. CTCF can function either as a positive or as a negative regulator of gene expression and is also involved in creating and maintaining long-range chromosomal interactions. Various developmentally important genes have been shown to be regulated by CTCF and its malfunction is frequently associated with developmental defects or diseases. CTCF undergoes various posttranslational modifications such as phosphorylation or SUMOylation which also affect its function in the regulation of gene expression. Keywords: CTCF, three dimensional genome, cohesin, regulation of gene expression, insulation, HOX genes
|
guest ::
