RICERCA ITALIANA     

Molecular analysis of erythropoiesis: Post-genomic and functional approach

Università degli Studi di Napoli "Federico II"

Abstract

Erythropoiesis is a finely modulated differentiation process which allows the production of mature red cells starting from bone marrow undifferentiated staminal cells. Although several genes, involved in the process, have been identified, cloned and functionally characterized, the large part of the precise molecular mechanisms of erythropoiesis and their detailed sequence are still unknown. The general aim of our project is the elucidation of the major phenomena which are at the basis of a efficacious erythopoiesis, in view, at the same time, of analysing some diseases whose causes and/or mechanisms are unclear or completely unknown.
Our program includes the bioinformatic evaluation of expression profiles of three cellular populations (CD34+, BFU-E and CFU-E); profiles already available at the coordinator unit. These findings will be employed in order to identify, at transcriptional level, the main molecular events observable during the erythropoiesis.
These data will be confirmed, at protein level, by bidimensional SDS-PAGE analyses. The main proteins identified will characterized by mass spectrometry, while their level variations by DIGE. In addition, by means of biochemical investigation and approaches of cellular and molecular biology , we will try to identify the interactors of stomatin, a protein involved in some forms of stomatocytosis. An additional theme which will be investigated in details is the modulation of cell division cycle during >>>

Principal Investigator

Achille IOLASCON Università degli Studi di NAPOLI "Federico II"

Research Objectives

The final goal of the proposed research project is to shed new light on the complex molecular interactions which occur during normal and pathological erythropoiesis. In order to reach this aim, we joined a number of research units, each having a great experience in its specific field, which cover a wide spectrum of red cell functions. A general theme of the program is the analysis of the process from a mechanistic point of view (namely studying protein-protein interaction) which represents the natural evolution of the genomic era and of a simple description (although important) of expression profiles (mRNA level) or the bidimensional SDS PAGE (protein level).
The identification of protein interaction, in our view, will give dynamic information, which represent sound bases in order to construct a functional map of red cell maturation.

Several specific aims, described below, will be pursued but it is important to stress that they do not represent single goals but pieces of a general puzzle:
1) Some groups will define complete expression maps of the three major populations occurring along the erythrocytic differentiation route, namely CD34+, BFU-E and CFU-E cells. The expression profiles will be obtained either at mRNA or protein level. The transcriptional maps, already available, at least in part, in the laboratory of the program coordinator, will initially identify genes whose expression is strongly modulated (both activated or reduced) during >>>

First Results

Summary of the partial results of the first part:
- map of the expression profiling (mRNA) during normal erythropoiesis
- proteomic 2D map during normal erythropoiesis
- modulation of cell cycle proteins during erythroid differentiation
- mappa proteomica bidimensionale dell'eritropoiesi fisiologica
- Functional characterization of RPS19
- Phisiology of band 3 domainsWill be realized the following points:
-expression profiling during erythroid maturation in CDA-II
- Functional chacarcterizaztion of K-Cl cotransportater
- proteomic mapping of stomatocytes
- Analysis of RPS19 partener in BD
- band 3 folding during spehrocytosisIdentification of candidate genes for stomatocytosis and cloning

Analysis of K-Cl transport in thalassemic patients as well as of protein stabiliring AHSP and
FECH

Identification of "D map in stomatocytosis

Pharmacolocig manipulation (via HDAC) of erythropoiesis

Studies on the interaction between RPS19 and ACP and S100A10

Timescale

24 months

National and international background

The hematopoietic system can be envisioned as a continuum of functional compartments. In general, the more primitive stem cell compartment is made up of pluripotent stem cells with high self-renewal capacity, the capacity to give rise at all lineages, and a low incidence of mitotically active cells.
The first cell phenotype, that can be easily identified and prepared is represented by the CD34+ cells. CD34+ are grown in specific media for long term culture. The cultures are then halted at specific recognizable points as BFU-E or CFU-E clls. With a long standing of these culture it is possible to observe the appearance of the more mature cells, until the reticulocytes.
Several transcriptional and growth factors are known to play a role during different steps of erythropoiesis. For example, it is firmly established that GATA-1 and FOG are transcription factors up-regulated during the process of red cell maturation. Similarly, it has been demonstrated that the up-regulation of PU.1 (another important hemopoietic transcription factor) causes in the erythroid precursors the arrest of differentiation and the induction of apoptosis. With regard to growth factors specific for erythropoiesis, it is obvious underline the role of erythropoietin.
Although the majority of the erythropoietic steps is almost delineated, a large part of the molecular picture is still lacking. These data offer the unique possibility of developing mice ablated of specific genes in >>>