Functional profiling of the embryonic-adult erythroid switching in mouse hematopoiesis The hematopoietic system is the most characterized model for the study of lineage specification and differentiation. During this process, the hematopoietic Stem Cells and progenitors renew themselves and progressively differentiate to sustain the production of the different blood cell populations. Fate choice is the result of a complex regulation program coordinating proliferation, differentiation and programmed cell death and leading to the accumulation of specific gene products in each mature cell type.
Within this process, erythropoiesis leads to the production of terminally differentiated erythrocytes that synthetize the globin chains -embryonic, fetal, adult- required at different stages of development. Erythropoiesis requires complex interactions between extra cellular and intracellular signals converging on lineage specific transcription factors acting in conjunction with more general transcription factors. In turn, transcription factors control the transcription of genes whose products are specifically required for the function of the mature, fully differentiated, cell types.
Failure of these fine tuning regulating mechanisms is often cause of disease, such as hemoglobinopathies and leukemias.
To better understand the molecular mechanisms regulating and controlling the erythroid differentiation program our research is focused on two different aspects:
characterization of new “erythroid” partners of transcription factors already known to play an important role in erythroid differentiation and globin transcription.
In particular, we identified the ubiquitous protein CP2 as a new partner of the erythroid specific transcription factor GATA-1. Our working hypothesis is that adjacent binding sites for GATA1 and CP2 might represent a novel module for common regulation of a subset of erythroid specific genes.
identification of genes whose expression correlates with globin switch and are therefore candidates for a role in regulating globin gene expression and erythroid differentiation.
To this end, we undertook a functional genomic approach based on the analysis of the expression profile of murine cells undergoing erythroid differentiation.
In the mouse model system, the crucial events responsible for erythroid maturation and for the establishment of the definitive pattern of globin gene expression take place in the fetal liver between 10.5 and 13.5 d.p.c.
We sorted by FACS from fetal livers at 11.5, 12.5 and 13.5 d.p.c. homogenous populations of multipotent progenitors, erythroid committed immature progenitors and erythroid precursors undergoing terminal maturation on the basis of their expression of specific surface antigens (c-kit and TER119) and we analysed their profile of transcription by DNA microarrays analysis.
By comparing the expression profiles of these populations we identified a set of genes whose expression correlates with the globin switch and the establishment of definitive hemato- and erythro-poiesis. Among them, we wish to study the most promising genes (selected on the basis of their pattern of expression, on the extent of the variation of their expression and on their function), using both molecular and functional assays.
In particular, we will overexpress the corresponding cDNA in primary mouse fetal liver hematopoietic cells grown in vitro and in immortalized cell lines that can be induced to differentiate in vitro. We will also inhibit the expression of the genes of interest by RNA interference. We will then evaluate the consequences of the perturbation of the level of these transcripts on the ability to complete the erythroid differentiation and maturation programs as well as on the expression of globin transcripts.
References
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