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Purification and characterization of GATA-1 related transcription factor complexes and target genes by in vivo biotinylation tagging

Projectomschrijving

Rode bloedcellen ontwikkelen zich uit voorlopercellen. Specifieke sets van genen worden aan- en uitgeschakeld zodat de cellen de vereiste ontwikkeling en differentiatie doormaken. Dat aan- en uitschakelen geschiedt onder invloed van specifieke factoren (transcriptiefactoren) in de cel. Een sleutelrol speelt de transcriptiefactor GATA-1. Deze is betrokken bij de vorming van verschillende eiwitcomplexen die zich aan het erfelijk materiaal in de cel hechten en zo de differentiatie sturen. Met een specifieke, door de onderzoekers ontwikkelde, techniek zijn diverse eiwitcomplexen en hun bindingsplaatsen op het genoom gekarakteriseerd in zebravissen en muizen. Nieuwe wegen van regulatie in bloedcellen zijn ontdekt, evenals een snelle techniek om grote hoeveelheden eiwitcomplexen te onderzoeken. Dat is wezenlijk voor het begrijpen van de cellulaire ontwikkeling in zoogdieren en daarmee samenhangende ziekten.

Producten

Titel: Isolation and characterization of hematopoietic transcription factor complexes by in vivo biotinylation tagging and mass spectrometry
Magazine: Annals of the New York Acadamy of Sciences
Titel: TFT Mapper: A BLAST search tool for identification of Transcription Factors Target Genes
Magazine: BMC Bioinformatics
Titel: Novel binding partners of Ldb1 are required for haematopoietic development.
Auteur: Rodriguez P, Braun H, Kolodziej KE, de Boer E, Campbell J, Bonte E, Grosveld F, Philipsen S, Strouboulis J.
Titel: ETO2 coordinates cellular proliferation and differentiation during erythropoiesis
Magazine: EMBO Journal
Titel: Multiple interactions between regulatory regions are required to stabilize an active chromatin hub.
Auteur: de Boer E, Rodriguez P, Bonte E, Krijgsveld J, Katsantoni E, Heck A, Grosveld F, Strouboulis J.
Titel: Characterization of megakaryocyte GATA1-interacting proteins: the corepressor ETO2 and GATA1 interact to regulate terminal megakaryocyte maturation.
Auteur: Sánchez C, Sánchez I, Demmers JA, Rodriguez P, Strouboulis J, Vidal M.
Titel: The genome-wide dynamics of the binding of Ldb1 complexes during erythroid differentiation.
Auteur: Soler E, Andrieu-Soler C, de Boer E, Bryne JC, Thongjuea S, Stadhouders R, Palstra RJ, Stevens M, Kockx C, van Ijcken W, Hou J, Steinhoff C, Rijkers E, Lenhard B, Grosveld F.
Magazine: Genes and Development
Titel: GATA-1 forms distinct activating and repressive complexes in erythroid cells.
Auteur: Patrinos GP, de Krom M, de Boer E, Langeveld A, Imam AM, Strouboulis J, de Laat W, Grosveld FG.
Titel: Isolation and characterization of hematopoietic transcription factor complexes by in vivo biotinylation tagging and mass spectrometry.
Auteur: Schuh AH, Tipping AJ, Clark AJ, Hamlett I, Guyot B, Iborra FJ, Rodriguez P, Strouboulis J, Enver T, Vyas P, Porcher C.
Titel: ETO-2 associates with SCL in erythroid cells and megakaryocytes and provides repressor functions in erythropoiesis.
Magazine: Molecular and Cellular Biology
Titel: Proteomics analysis of Ring1B/Rnf2 interactors identifies a novel complex with the Fbxl10/Jhdm1B histone demethylase and the Bcl6 interacting corepressor.
Auteur: Meier N, Krpic S, Rodriguez P, Strouboulis J, Monti M, Krijgsveld J, Gering M, Patient R, Hostert A, Grosveld F.
Titel: ETO2 coordinates cellular proliferation and differentiation during erythropoiesis.
Auteur: Grosveld F, Rodriguez P, Meier N, Krpic S, Pourfarzad F, Papadopoulos P, Kolodziej K, Patrinos GP, Hostert A, Strouboulis J.
Titel: Novel binding partners of Ldb1 are required for hematopoietic development
Magazine: Development
Titel: Isolation of transcription factor complexes by in vivo biotinylation tagging and direct binding to streptavidin beads.
Auteur: Horsman S, Moorhouse MJ, de Jager VC, van der Spek P, Grosveld F, Strouboulis J, Katsantoni EZ.
Titel: GATA-1 forms distinct activating and repressive complexes in erythroid cells
Magazine: EMBO Journal
Titel: A generic tool for biotinylation of tagged proteins in transgenic mice
Magazine: Transgenic Research
Titel: TF Target Mapper: a BLAST search tool for the identification of Transcription Factor target genes.
Auteur: Goardon N, Lambert JA, Rodriguez P, Nissaire P, Herblot S, Thibault P, Dumenil D, Strouboulis J, Romeo PH, Hoang T.
Titel: Isolation of transcription factor complexes by in vivo biotinylation tagging and direct binding to streptavidin beads
Magazine: Methods Mol Biol
Titel: A generic tool for biotinylation of tagged proteins in transgenic mice.
Auteur: Rodriguez P, Bonte E, Krijgsveld J, Kolodziej KE, Guyot B, Heck AJ, Vyas P, de Boer E, Grosveld F, Strouboulis J.
Titel: ETO-2 associates with SCL in erythroid cells and megakaryocytes and provides repressor functions in erythropoiesis.
Auteur: Driegen S, Ferreira R, van Zon A, Strouboulis J, Jaegle M, Grosveld F, Philipsen S, Meijer D.

Verslagen


Eindverslag

Het doel van dit project is de ontwikkeling en differentiatie van erythroide cellen te leren begrijpen, op basis van de functie van sleutel-transcriptiefactoren in hematopoiese, nl. GATA-1 en interactieve partners.
Er zijn 3 'key objectives':
1. De isolatie en karakterisering van eiwitcomplexen die gevormd worden door GATA-1 en interactieve partners in erythroide cellen.
2. De identificatie van erythroide gen doelen voor GATA-1 en interactieve factoren.
3. De functionele karakterisering van nieuw geidentificeerde eiwitpartners door genetische en biochemische middelen.

Het einddoel van dit project is het proberen te doorgronden van de ontwikkeling en differentiatie van erythroide cellen m.b.v. de 'sleutel' transcriptiefactor-functies in hematopoiese, nl. GATA-1 en zijn interactieve partners.

Samenvatting van de aanvraag

The overall aim of this project is to understand the development and differentiation of erythroid cells on the basis of key transcription factor (TF) functions in hematopoiesis. Specific TFs serve to execute lineage decision, commitment and differentiation programs in hematopoiesis by regulating the expression of target genes. The ultimate goal of hematopoiesis is the continuous replenishment of the many different hematopoietic cell types in a constantly ongoing process. Genetic evidence indicates that the functions of key TFs such as TAL-1, GATA-1 and EKLF represent essential early-to-late developmental decisions in hematopoiesis (reviewed in ref. 1). Loss of GATA-1 in mice results in arrest at the pro-erythroblast stage and apoptosis2 while GATA-1 overexpression results in a block at the final stage of differentiation due to an absence of cell cycle arrest3. These data show a requirement for GATA-1 for differentiation past the pro-erythroblast stage as well as a role for GATA-1 protein levels in cell cycle regulation and terminal erythroid cell differentiation. GATA-1 interacts with TAL-1, the latter fulfilling an early requirement in the development of the hematopoietic system since lack of TAL-1 in mice leads to a complete absence of blood4. TAL-1 also interacts with the widely expressed factor Ldb15, which is the mammalian homolog of the Drosophila Chip gene thought to mediate long-range interactions between distant regulatory elements and target promoters6. Lack of EKLF in mice results in lethal anemia at the fetal liver stage due to a deficiency in b globin synthesis and failure in the production of normal red blood cells7-9. Binding sites for both GATA-1 and EKLF appear clustered together at many sites in the b globin locus (e.g. ref. 10), as well as in many other erythroid genes, thus suggesting that the two factors act in concert in regulating erythroid-specific transcription. The evidence outlined above is indicative of a network of overlapping and distinct transcription factor functions in erythropoiesis. However, despite a wealth of information regarding these factors, it is still not clear what role which particular complex plays in gene regulation (i.e. chromatin remodeling/modification and/or transcriptional activation/repression). It is evident that these factors act as multiprotein complexes, but only some interactions have been characterized (with little functional evidence). For example, GATA-1 forms several different complexes with potentially different subnuclear localization and function(s) (section 4b and ref. 11) but the role of these complexes is unknown. Beginning with GATA-1, we propose to gain insight into the function of these factors in hematopoiesis by taking a combined biochemical, cellular and genetic approach to characterize the protein complexes formed by GATA-1 and interacting proteins and to identify the genes targeted by these complexes in hematopoiesis. This aim translates into the following key objectives:i. the isolation and characterization of GATA-1 related transcription factor protein complexes from erythroid and/or precursor cellsii. the identification of erythroid gene targets for GATA-1 and interacting factors, e.g. TAL-1, EKLFiii. the functional characterization of newly identified protein partners and gene targets of hematopoietic transcription factors, primarily by genetic means (i.e.knockouts, RNAi).

Onderwerpen

Kenmerken

Projectnummer:
91204041
Looptijd: 100%
Looptijd: 100 %
2004
2008
Onderdeel van programma:
Gerelateerde subsidieronde:
Projectleider en penvoerder:
Dr. I. Strouboulis PhD
Verantwoordelijke organisatie:
Erasmus MC