http://repub.eur.nl/res/aut/4873/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/28249/ 2010-08-01T00:00:00Z Hereditary persistence of fetal hemoglobin (HPFH) is characterized by persistent high levels of fetal hemoglobin (HbF) in adults. Several contributory factors, both genetic and environmental, have been identified but others remain elusive. HPFH was found in 10 of 27 members from a Maltese family. We used a genome-wide SNP scan followed by linkage analysis to identify a candidate region on chromosome 19p13.12-13. Sequencing revealed a nonsense mutation in the KLF1 gene, p.K288X, which ablated the DNA-binding domain of this key erythroid transcriptional regulator. Only family members with HPFH were heterozygous carriers of this mutation. Expression profiling on primary erythroid progenitors showed that KLF1 target genes were downregulated in samples from individuals with HPFH. Functional assays suggested that, in addition to its established role in regulating adult globin expression, KLF1 is a key activator of the BCL11A gene, which encodes a suppressor of HbF expression. These observations provide a rationale for the effects of KLF1 haploinsufficiency on HbF levels. http://repub.eur.nl/res/pub/29254/ 2008-10-01T00:00:00Z Stem cell factor (SCF)-induced activation of phosphoinositide-3-kinase (PI3K) is required for transient amplification of the erythroblast compartment. PI3K stimulates the activation of mTOR (target of rapamycin) and subsequent release of the cap-binding translation initiation factor 4E (elF4E) from the 4E-binding protein 4EBP, which controls the recruitment of structured mRNAs to polysomes. Enhanced expression of elF4E renders proliferation of erythroblasts independent of PI3K. To investigate which mRNAs are selectively recruited to polysomes, we compared SCF-dependent gene expression between total and polysome-bound mRNA. This identified 111 genes primarily subject to translational regulation. For 8 of 9 genes studied in more detail, the SCF-induced polysome recruitment of transcripts exceeded 5-fold regulation and was PI3K-dependent and elF4E-sensitive, whereas total mRNA was not affected by signal transduction. One of the targets, Immunoglobulin binding protein 1 (Igbpl), is a regulatory subunit of protein phosphatase 2A (Pp2a) sustaining mTOR signaling. Constitutive expression of Igbpl impaired erythroid differentiation, maintained 4EBP and p70S6k phosphorylation, and enhanced polysome recruitment of multiple elF4E-sensitive mRNAs. Thus, PI3K-dependent polysome recruitment of Igbpl acts as a positive feedback mechanism on translation initiation underscoring the important regulatory role of selective mRNA recruitment to polysomes in the balance between proliferation and maturation of erythroblasts. http://repub.eur.nl/res/pub/29736/ 2008-09-08T00:00:00Z The alternative splicing code that controls and coordinates the transcriptome in complex multicellular organisms remains poorly understood. It has long been argued that regulation of alternative splicing relies on combinatorial interactions between multiple proteins, and that tissue-specific splicing decisions most likely result from differences in the concentration and/or activity of these proteins. However, large-scale data to systematically address this issue have just recently started to become available. Here we show that splicing factor gene expression signatures can be identified that reflect cell type and tissue-specific patterns of alternative splicing. We used a computational approach to analyze microarray-based gene expression profiles of splicing factors from mouse, chimpanzee and human tissues. Our results show that brain and testis, the two tissues with highest levels of alternative splicing events, have the largest number of splicing factor genes that are most highly differentially expressed. We further identified SR protein kinases and small nuclear ribonucleoprotein particle (snRNP) proteins among the splicing factor genes that are most highly differentially expressed in a particular tissue. These results indicate the power of generating signature-based predictions as an initial computational approach into a global view of tissue-specific alternative splicing regulation. http://repub.eur.nl/res/pub/9022/ 2007-03-07T00:00:00Z The balance between proliferation and differentiation of committed hematopoietic progenitors is under tight control to maintain the progenitor pool and ensure maturation in response to physiological demand. Whereas development of the different hematopoietic lineages requires the coordinate expression of transcription factors, the balance between proliferation and differentiation is controlled by growth factors. These include cytokines such as erythropoietin (Epo) and granulocyte-colony stimulating factor (G-CSF) required for differentiation into specific lineages, as well as growth factors that enhance proliferation and delay differentiation of progenitors such as stem cell factor (SCF) and the ligand for FMS-like tyrosine kinase 3 (Flt3- ligand). To investigate how the balance between expansion and differentiation of erythroid progenitors is controlled by growth factors, we used a model in which erythroid progenitors proliferate in the presence of Epo, SCF and glucocorticoids while they maintain the capacity to differentiate upon addition of Epo alone. SCF- driven proliferation and inhibition of differentiation is dependent on phosphoinositide- 3-kinase (PI3K) activation. The main objective of the study described in this thesis is to understand how PI3K-controlled gene expression regulates the balance between proliferation and differentiation of erythroid progenitors. In particular, we studied the role of PI3K dependent control of mRNA translation. http://repub.eur.nl/res/pub/13919/ 2005-10-01T00:00:00Z Stem cell factor (SCF) delays differentiation and enhances the expansion of erythroid progenitors. Previously, we performed expression-profiling experiments to link signaling pathways to target genes using polysome-bound mRNA. SCF-induced phosphoinositide-3-kinase (PI3K) appeared to control polysome recruitment of specific mRNAs associated with neoplastic transformation. To evaluate the role of mRNA translation in the regulation of expansion versus differentiation of erythroid progenitors, we examined the function of the eukaryote initiation factor 4E (eIF4E) in these cells. SCF induced a rapid and complete phosphorylation of eIF4E-binding protein (4E-BP). Overexpression of eIF4E did not induce factor-independent growth but specifically impaired differentiation into mature erythrocytes. Overexpression of eIF4E rendered polysome recruitment of mRNAs with structured 5' untranslated regions largely independent of growth factor and resistant to the PI3K inhibitor LY294002. In addition, overexpression of eIF4E rendered progenitors insensitive to the differentiation-inducing effect of LY294002, indicating that control of mRNA translation is a major pathway downstream of PI3K in the regulation of progenitor expansion.