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Transcriptional regulation of miR-17-92 microRNA cluster during macrophage differentiation.
Rybářová, Jana ; Stopka, Tomáš (advisor) ; Pospíšek, Martin (referee)
miR-17-92 cluster (Oncomir1) encodes seven microRNAs (miRNA, miR) regulating many biological processes including proliferation, differentiation or apoptosis. Overexpression of microRNAs encoded by miR-17-92 cluster is found in a number of tumors including acute and chronic myeloid leukemias (Dixon-McIver et al., 2008; Li et al., 2008; Venturini et al., 2007). Myeloid progenitors express miR-17-92 cluster at a high level, while macrophage differentiation associates with its downregulation. Our laboratory found, that miR-17-92 cluster is repressed by transcription factor Early growth response 2 (Egr2) upon differentiation of primary myeloid PUER progenitors, induced with transcription factor PU.1. Aim of this thesis is to further test the abovementioned data by preparing a reporter vectors set, carrying various fragments of miR-17-92 putative promoter, which enables us to study regulation of transcription of miR-17-92 cluster. This task complicated by presence of increased GC content of the miR-17-92 promoter was successfully accomplished resulting in amplification of eight fragments containing the various parts of miR-17-92 promoter including region -3.3 to 0 kb relative to the start of miR-17-5p sequence, that were inserted into pGL3 reporter vector. Transfection of pGL3 reporter vector carrying...
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The Role of oncogenic microRNA - 155 and proto - oncogen MYB in chronic lymphocytic leukemia
Vargová, Karina
(EN) Chronic lymphocytic leukemia (B-CLL) represents a disease of mature-like B-cells. Due to failed apoptosis but also due to enhanced proliferative signals, the leukemic B-cells accumulate in the peripheral blood, bone marrow, lymph nodes and spleen. The clinical course of B-CLL is very heterogeneous; in some patients B-CLL progresses very rapidly into an aggressive form. Such patients need therapy sooner while in other patients with indolent B-CLL the onset of therapy takes years. Several standard prognostic and disease progression markers are used for disease staging and monitoring, however a reliable marker that will suggest when to start therapy is unknown. Expression of small, non-coding microRNAs is often deregulated and represent important prognostic markers in variety of cancers including leukemia. Hence in our study we concentrated to miR-155, an important molecule regulating differentiation of hematopoietic cells, inflammation process and antibody production. Its aberrant expression was described in Hodgkin`s as well as in non-Hodgkin`s lymphoma, including indolent lymphoproliferations like B-CLL. Our results confirmed elevated levels of both, primary miR-155 transcript and mature form of miR-155 in our B-CLL patient samples (N=239). The aberrant expression of miR-155 in B-CLL samples...
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Delineating aggressiveness of acute myeloid leukemia in a mouse model carrying mutations of Spil (PU.1) and Trp53.
Bašová, Petra
PU.1 downregulation within haematopoietic stem and progenitor cells (HSPCs) is the primary mechanism for the development of acute myeloid leukaemia (AML) in mice with homozygous deletion of the upstream regulatory element (URE) of PU.1 gene. p53 is a well known tumor suppressor that is often mutated in human haematologic malignancies including AML and adds to their aggressiveness; however its genetic deletion does not cause AML in mouse. Deletion of p53 in the PU.1ure/ure mice (PU.1ure/ure p53-/- ) results in more aggressive AML with shortened overall survival. PU.1ure/ure p53-/- progenitors express significantly lower PU.1 levels. In addition to URE deletion we searched for other mechanisms that in absence of p53 contribute to decreased PU.1 levels in PU.1ure/ure p53-/- mice. We found involvement of Myb and miR-155 in downregulation of PU.1 in aggressive murine AML. Upon inhibition of either Myb or miR-155 in vitro the AML progenitors restore PU.1 levels and lose leukaemic cell growth similarly to PU.1 rescue. The MYB/miR-155/PU.1 axis is a target of p53 and is activated early after p53 loss as indicated by transient p53 knockdown. Furthermore, deregulation of both MYB and miR-155 coupled with PU.1 downregulation was observed in human AML, suggesting that MYB/miR-155/PU.1 mechanism may be involved...
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The role of transcription factors PU.1 a GATA-1 during leukemia differentiation.
Burda, Pavel ; Stopka, Tomáš (advisor) ; Kořínek, Vladimír (referee) ; Machová Poláková, Kateřina (referee)
Hematopoiesis is coordinated by a complex regulatory network of transcription factors among them PU.1 (Spi1, Sfpi1) and GATA-1 represent key molecules. GATA-1 and PU.1 bind each other on DNA to block each others transcriptional programs to prevent development of undesired lineage during hematopoietic commitment. Murine erythroleukemia (MEL) cells, transformed erythroid precursors that are blocked from completing the late stages of erythroid differentiation, co-express GATA-1 and PU.1 and as my and others data document, are able to respond to molecular removal (down-regulation) of PU.1 or addition (up-regulation) of GATA-1 by inducing terminal erythroid differentiation. We provide novel evidence that downregulation of GATA-1 or upregulation of PU.1 induces incompletely differentiation into cell cycle arrested monocytic-like cells. Furthermore, PU.1- dependent transcriptome is negatively regulated by GATA-1 in MEL cells, including CCAAT/enhancer binding protein alpha (Cebpa) and Core-binding factor, beta subunit (Cbfb) that encode additional key hematopoietic transcription factors. Chromatin immunoprecipitation and reporter assays identified PU.1 motif sequences near Cebpa and Cbfb that are co-occupied by PU.1 and GATA-1 in the leukemic blasts. Furthermore, transcriptional regulation of these loci by...
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Model for study of transcription regulation of granulocytic genes MPO and MMP9 by different levels of PU.1 transcription factor
Chramostová, Kamila ; Pospíšil, Vít (advisor) ; Kleibl, Zdeněk (referee)
9 Abstract Enhancers are distal cis - regulatory DNA sequences that regulate (enhance) transcription of the respective gene driven by its promoter. Enhancers are found in non-coding DNA upstream or downstream of the gene coding sequence, or in introns or coding regions that are located up to hundreds kb away from the gene. Superenhancers are newly discovered clusters of multiple enhancers that play a vital role in activating tissue-specific genes, determining cell identity and regulating differentiation. PU.1 is the transcription factor (TF) that is necessary for normal haematopoiesis, specifically for the development of myeloid and lymphoid blood lineages. Distinct levels of PU.1 induce differentiation of hematopoietic cells into different cell lineages whereby disruption of PU.1 levels leads to leukemogenesis. High PU.1 levels stimulate macrophage development, while intermediate levels stimulate the development of granulocytes. This diploma thesis seeks to contribute to addressing the interesting biological question of what are the regulatory mechanisms to ensure that granulocytic genes are activated only at the intermediate concentration of PU.1, whereas macrophage genes are activated only at its high levels. The aim of this diploma thesis was to create a series of reporter vectors carrying regulatory...
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Epigenetic control of PU.1 gene transcription during development of 5-Azacytidine resistance in acute myeloid leukemia
Křtěnová, Petra ; Burda, Pavel (advisor) ; Čuřík, Nikola (referee)
Hematopoiesis is a highly orchestrated process, in which a single hematopoietic stem cell (HSC) gives a rise to all blood cellular components. For myeloid and lymphoid development precise controlled expression of the PU.1 transcription factor is needed. Deletion of PU.1 gene in mouse is lethal and its dysregulation during hematopoietic differentiation is associated with blood malignancies including acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). MDS and AML are serious blood disorders characterized by expansion of immature blood cells and lack of differentiated functional cells. Not only genetic but also epigenetic aberrations represent a very important field for studying pathophysiology of leukemia genesis and dysregulation of the PU.1 gene represents intensively studied candidate mechanism. Modern therapy of selected MDS and subset of AML patients is based on treatment with DNA hypomethylating agent Azacytidine (AZA) interfering in PU.1 gene regulatory mechanism. However, poor response or resistance to this therapy often occurs. In this thesis we present data obtained from AZA-resistant clones of MDS/AML cell line OCI-M2. We analysed DNA methylation and DNA hydroxymethylation at the key regulatory element of the PU.1 gene (URE). We found that these epigenetic modifications at URE...
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The Role of oncogenic microRNA - 155 and proto - oncogen MYB in chronic lymphocytic leukemia
Vargová, Karina
(EN) Chronic lymphocytic leukemia (B-CLL) represents a disease of mature-like B-cells. Due to failed apoptosis but also due to enhanced proliferative signals, the leukemic B-cells accumulate in the peripheral blood, bone marrow, lymph nodes and spleen. The clinical course of B-CLL is very heterogeneous; in some patients B-CLL progresses very rapidly into an aggressive form. Such patients need therapy sooner while in other patients with indolent B-CLL the onset of therapy takes years. Several standard prognostic and disease progression markers are used for disease staging and monitoring, however a reliable marker that will suggest when to start therapy is unknown. Expression of small, non-coding microRNAs is often deregulated and represent important prognostic markers in variety of cancers including leukemia. Hence in our study we concentrated to miR-155, an important molecule regulating differentiation of hematopoietic cells, inflammation process and antibody production. Its aberrant expression was described in Hodgkin`s as well as in non-Hodgkin`s lymphoma, including indolent lymphoproliferations like B-CLL. Our results confirmed elevated levels of both, primary miR-155 transcript and mature form of miR-155 in our B-CLL patient samples (N=239). The aberrant expression of miR-155 in B-CLL samples...
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Delineating aggressiveness of acute myeloid leukemia in a mouse model carrying mutations of Spil (PU.1) and Trp53.
Bašová, Petra
PU.1 downregulation within haematopoietic stem and progenitor cells (HSPCs) is the primary mechanism for the development of acute myeloid leukaemia (AML) in mice with homozygous deletion of the upstream regulatory element (URE) of PU.1 gene. p53 is a well known tumor suppressor that is often mutated in human haematologic malignancies including AML and adds to their aggressiveness; however its genetic deletion does not cause AML in mouse. Deletion of p53 in the PU.1ure/ure mice (PU.1ure/ure p53-/- ) results in more aggressive AML with shortened overall survival. PU.1ure/ure p53-/- progenitors express significantly lower PU.1 levels. In addition to URE deletion we searched for other mechanisms that in absence of p53 contribute to decreased PU.1 levels in PU.1ure/ure p53-/- mice. We found involvement of Myb and miR-155 in downregulation of PU.1 in aggressive murine AML. Upon inhibition of either Myb or miR-155 in vitro the AML progenitors restore PU.1 levels and lose leukaemic cell growth similarly to PU.1 rescue. The MYB/miR-155/PU.1 axis is a target of p53 and is activated early after p53 loss as indicated by transient p53 knockdown. Furthermore, deregulation of both MYB and miR-155 coupled with PU.1 downregulation was observed in human AML, suggesting that MYB/miR-155/PU.1 mechanism may be involved...
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Role onkogenní mikroRNA-155 a proto-onkogenu MYB u chronické lymfatické leukémie
Vargová, Karina ; Stopka, Tomáš (advisor) ; Móciková, Heidi (referee) ; Trka, Jan (referee)
(EN) Chronic lymphocytic leukemia (B-CLL) represents a disease of mature-like B-cells. Due to failed apoptosis but also due to enhanced proliferative signals, the leukemic B-cells accumulate in the peripheral blood, bone marrow, lymph nodes and spleen. The clinical course of B-CLL is very heterogeneous; in some patients B-CLL progresses very rapidly into an aggressive form. Such patients need therapy sooner while in other patients with indolent B-CLL the onset of therapy takes years. Several standard prognostic and disease progression markers are used for disease staging and monitoring, however a reliable marker that will suggest when to start therapy is unknown. Expression of small, non-coding microRNAs is often deregulated and represent important prognostic markers in variety of cancers including leukemia. Hence in our study we concentrated to miR-155, an important molecule regulating differentiation of hematopoietic cells, inflammation process and antibody production. Its aberrant expression was described in Hodgkin`s as well as in non-Hodgkin`s lymphoma, including indolent lymphoproliferations like B- CLL. Our results confirmed elevated levels of both, primary miR-155 transcript and mature form of miR-155 in our B-CLL patient samples (N=239). The aberrant expression of miR-155 in B-CLL samples...
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Delineating aggressiveness of acute myeloid leukemia in a mouse model carrying mutations of Spil (PU.1) and Trp53.
Bašová, Petra ; Stopka, Tomáš (advisor) ; Machová Poláková, Kateřina (referee) ; Zuna, Jan (referee)
PU.1 downregulation within haematopoietic stem and progenitor cells (HSPCs) is the primary mechanism for the development of acute myeloid leukaemia (AML) in mice with homozygous deletion of the upstream regulatory element (URE) of PU.1 gene. p53 is a well known tumor suppressor that is often mutated in human haematologic malignancies including AML and adds to their aggressiveness; however its genetic deletion does not cause AML in mouse. Deletion of p53 in the PU.1ure/ure mice (PU.1ure/ure p53-/- ) results in more aggressive AML with shortened overall survival. PU.1ure/ure p53-/- progenitors express significantly lower PU.1 levels. In addition to URE deletion we searched for other mechanisms that in absence of p53 contribute to decreased PU.1 levels in PU.1ure/ure p53-/- mice. We found involvement of Myb and miR-155 in downregulation of PU.1 in aggressive murine AML. Upon inhibition of either Myb or miR-155 in vitro the AML progenitors restore PU.1 levels and lose leukaemic cell growth similarly to PU.1 rescue. The MYB/miR-155/PU.1 axis is a target of p53 and is activated early after p53 loss as indicated by transient p53 knockdown. Furthermore, deregulation of both MYB and miR-155 coupled with PU.1 downregulation was observed in human AML, suggesting that MYB/miR-155/PU.1 mechanism may be involved...
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