| Abstract |
DNA methylation is essential for embryonic development and in diverse biological processes. The DNA methyltransferase Dnmt1 maintains parental cell methylation patterns on daughter DNA strands in mitotic cells. DNMT1 knock out/down (KO/KD) studies in hematopoietic lineages or in mice revealed that DNMT1 drives HSCs to myeloerythroid fate. In adult-stage erythroid cells, DNMT1 represses γ-globin gene expression by methylating its promoter and interacts with proteins (TR2/TR4, BCL11A) implicated in the silencing of γ-globin expression. Publications of our laboratory showed that DNMT1 co-purified with several hematopoietic transcription factors (GATA1, FOG-1, GFI-1b) involved in erythroid differentiation forming a core complex composed by DNMT1 and transcription factors ZBP89 and ZNF143, present also in non-hematopoietic cells, which interacts with distinct hematopoietic protein subcomplexes. Additionally, a short PCNA Binding domain (PBD) of DNMT1 proved necessary and sufficient for DNMT1 interaction with these transcription factors. Further evidence suggested that DNMT1 functions as a co-repressor of ZBP-89 and GATA1 acting through upstream regulatory elements of major hematopoietic transcription factors, the PU.1 and GATA1 gene loci. Moreover, DNMT1 KD in murine erythroleukemic (MEL) cells showed a clear lack in cell cycle arrest in erythroid differentiation due to the lack of repression of genes responsible for cell proliferation. The main aim of this study is to elucidate more clearly the role of DNMT1 in erythroid cell differentiation. Towards this aim, we attempted to generate and characterize DNMT1 KO MEL cells. Furthermore, we tried to further identify the protein partners of DNMT1 and exert its function, utilizing a biotin-tagging approach coupled to mass spectrometry. Another aim of this study is to identify gene targets of DNMT1 that are suppressed by its regulatory control through a methylation dependent or independent mechanism along global DNA demethylated erythroid genome, taking into account that DNMT1 is the main DNA methyltransferase expressed throughout murine fetal liver derived erythroid differentiation. Moreover, we tried to investigate whether the short PCNA binding domain (PBD) of DNMT1 renders DNMT1 a scaffolding protein for the recruitment of additional repressive partners inducing gene silencing performing phenotypic rescue assays of DNMT1 KO cells by stably transfecting DNMT1 deletion mutants.
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Characterization of the molecular functions of the DNA methyltransferase 1, DNMT1 in erythropoiesis
