4th Palermo Conference on INNOVATIVE THERAPIES FOR LYMPHOID MALIGNANCIES
Published online 22 September 2009
Haematologica, Vol 95, Issue 1, 36-46 doi:10.3324/haematol.2009.012351
Copyright © 2010 by Ferrata Storti Foundation
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Erythropoiesis

GFI1B controls its own expression binding to multiple sites

Eduardo Anguita1,2, Ana Villegas1,2, Francisco Iborra3, Aurora Hernández2

1 University Complutense, Madrid, Spain
2 Hematology Department, Hospital Clinico San Carlos, Madrid, Spain
3 Medical Research Council Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK

Correspondence: Eduardo Anguita, Hematology Department, Hospital Clinico San Carlos, 28040, Madrid, Spain. E-mail: eduardo.anguita{at}imm.ox.ac.uk

Background: Transcription factors play essential roles in both normal and malignant hematopoiesis. This is the case for the growth factor independent 1b (GFI1B) transcription factor, which is required for erythroid and megakaryocytic differentiation and over-expressed in leukemic patients and cell lines.

Design and Methods: To investigate GFI1B regulation, we searched for multispecies conserved non-coding elements between GFI1B and neighboring genes. We used a formaldehyde-assisted isolation of regulatory elements (FAIRE) assay and DNase1 hypersensitivity to assess the chromatin conformation of these sites. Next, we analyzed transcription factor binding and histone modifications at the GFI1B locus including the conserved non-coding elements by a chromatin immunoprecipitation assay. Finally, we studied the interaction of the GFI1B promoter and the conserved non-coding elements with the chromatin conformation capture technique and used immunofluorescence to evaluate GFI1B levels in individual cells.

Results: We localized several conserved non-coding elements containing multiple erythroid specific transcription factor binding sites at the GFI1B locus. In GFI1B-expressing cells a subset of these conserved non-coding elements and the promoter adopt a close spatial conformation, localize with open chromatin sites, harbor chromatin modifications associated with gene activation and bind multiple transcription factors and co-repressors.

Conclusions: Our findings indicate that GFI1B regulatory elements behave as activators and repressors. Different protein levels within a cell population suggest that cells must activate and repress GFI1B continuously to control its final level. These data are consistent with a model of GFI1B regulation in which GFI1B binds to its own promoter and to the conserved non-coding elements as its levels rise. This would attract repressor complexes that progressively down-regulate the gene. GFI1B expression would decrease until a stage at which the activating complexes predominate and expression increases.

Key words: GFI1B, controls, multiple sites.