http://repub.eur.nl/res/aut/8234/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/13817/ 2005-06-01T00:00:00Z Development of red blood cells requires the correct regulation of cellular processes including changes in cell morphology, globin expression and heme synthesis. Transcription factors such as erythroid Kruppel-like factor EKLF (Klf1) play a critical role in erythropoiesis. Mice lacking EKLF die around embryonic day 14 because of defective definitive erythropoiesis, partly caused by a deficit in beta-globin expression. To identify additional target genes, we analyzed the phenotype and gene expression profiles of wild-type and EKLF null primary erythroid progenitors that were differentiated synchronously in vitro. We show that EKLF is dispensable for expansion of erythroid progenitors, but required for the last steps of erythroid differentiation. We identify EKLF-dependent genes involved in hemoglobin metabolism and membrane stability. Strikingly, expression of these genes is also EKLF-dependent in primitive, yolk sac-derived, blood cells. Consistent with lack of upregulation of these genes we find previously undetected morphological abnormalities in EKLF-null primitive cells. Our data provide an explanation for the hitherto unexplained severity of the EKLF null phenotype in erythropoiesis. http://repub.eur.nl/res/pub/7346/ 2004-10-27T00:00:00Z The human body consists of a huge number of cells. At the base of all these cells is one fertilized oocyte. By cell divisions the number of cells is increased during development, and cells specialize into various types of cells, i.e. muscle, brain and blood cells. Groups of specialized cells form the various tissues. DNA contains the blueprint of all cells. By each cell division the DNA is duplicated and equally distributed over the two daughter cells. Hence, practically all the cells contain identical genetic information. The DNA in each cell contains about 25.000 genes. Each gene contains information necessary to produce a particular protein. These proteins are involved in many processes such as energy metabolism, cell composition and gene regulation. Although all cells contain the same genetic information, they specialize into various cell types. For this, the genes are specifically activated or repressed. A tight regulation of the genes makes it possible to specialize into a particular type of cell. Transcription factors play an important role in this tight regulation of activating or repressing genes. EKLF is such a transcription factor. EKLF is mainly expressed in red blood cells. These cells give blood its red color. They carry oxygen from lungs to tissues in the body that require the oxygen for energy metabolism. Red blood cells are filled with hemoglobin that actually binds the oxygen. Each hemoglobin particle consists of two a-globin, two ß-globin and four heme molecules. The genetic blueprint for the ß-globin gene is called the ß-globin locus. In the mouse, this locus contains four globin genes. Two of them, the embryonic ß-globin genes, are active during embryonic development of the mice and the other two, the adult ß-globin genes are activated later in development and stay active during the rest of the lifespan of the mouse. Apart from the four ß-globin genes, the locus contains regulatory elements. These include the Locus Control Region (LCR), and a number of other hypersensitive sites. The LCR is required for a proper regulation of the globin genes; in absence of the LCR the genes are not activated, and mutations in the LCR can lead to impaired activation. The LCR is located relatively far from the genes it controls. However, when a globin gene is active in red blood cells, the LCR is physically in close proximity to this gene. In fact, the globin locus forms a spatial organization that consists not only of the active gene and the LCR, but also of the other hypersensitive sites that are located even further away on both sides of the genes. This structure is called the Active Chromatin Hub (ACH). Prior to the stage in which the genes are active, a substructure of this ACH is formed. Unlike the complete ACH, this substructure does not contain the globin genes and part of the LCR. In Chapter 4 we show that a similar substructure is found in red blood cells that are EKLF deficient. We demonstrated that EKLF is necessary for the completion of the ACH, a requirement for activating the genes. The transcription factor EKLF is necessary for the proper development of red blood cells; apart from the regulation of the ß-globin genes it regulates also other genes. However, it was not known which other genes. To study the genes EKLF activates (or represses), we used a culture method with erythroid progenitor cells. These progenitor cells are restricted to the red blood cell lineage, but have not yet developed to fully differentiated, hemoglobin- containing red blood cells. The culture method allows us to culture the cells as progenitors or induce them to start their terminal differentiation to red blood cells.