Introduction

　You may have heard the topic of "stem cell" or "stem cell research" over the past several years. There have been reports of successful treatments of myocardial infaction or damaged blood vessles by stem cell injection and subsequent cell regneration. Recent research has found that stem cells exist in various organs and ittsues, providing us with the hope of cell-based therpaies and cure for life-threatening diseases.

The existence of hematopoietic stem cells (HSCs) has been known for over five decades. HSCs possess the amazing ability of differentiate into more than 0 different teypes of blood and immune cells, such as erythrocytes, leucocytes, B-cells, and T-cells. However, the mechanisms and molecules invovled in the HSC-differentiation are largely unknown. We have been focusing on this precise topoic from the perspective of "transcription factors." Our ultimate purpose is for the research information to benefit patients with leukemia, thalassemia, and other blood-related disease and other hematological disorders. (Figure.1)

Our Research

　Blood cells differentiate from HSCs into erythrocytes, leucocytes (neutrophils, eosinophils, mast cells, monocytes/macrophage, B-, T-lymphocytes), and platelets. It is thought that multiple transcription factors create time- and lineage-specific signaling cascades to tightly regulate the intricate process.

　GATA-family of transcription factors consists of six members, who are divided into two groups depending on their expression patterns. While GATA-4, 5, and 6 are invovled in endoderm development (heart, gut, and liver), GATA-1, 2, and 3 are mainly expressed in hematopoietic cells and T-lymphocytes. Of the three factors, GATA-1is expressed in erythrocytes, megakaryocytes, eosinophils, and mast cells. Its gene expression gradually increases during differentiation thereby tightly regulating the down-stream erythorid-specific gene expression.

In order to sutdy the relationship between erythropoiesis and GATA-1 gene expression, we created a transgenic reporter mouse model using the GATA-1 hematopoietic regulatory domain (G1-HRD) to drive the expression of GFP (G1-HRD-GFP). GFP expression pattern of bone marrow cells in G1-HRD-GFP mice was same as the expression pattern of endogenous GATA-1 gene. We successfullly identified the two bone marrow erythroid progenitor cells, which were equivalent to BFU-E and CFU-E, by FACS analysis using GFP-positive cells with CD71 transferrin-receptor expression as the cell-sorting marker. BFU-E and CFU-E equivalent erythroid progenitors were named EEP (early erythroid progenitor) and LEP (late erythroid progenitor), respectively. Furthermore, mRNA extractd from EEP and LEP allowed us to determine that the highest GATA-1 expression occurs in LEP fraction, while high GATA-2 expression is observed in EEP fraction.¹ These findings are of particular interest when considering the ability of GATA-1 to repress GATA-2 gene expression during erythropoiesis, otherwise known as the "GATA Switch." (Figure 2)

　Recently, there have been several reports of GATA-1 N-terminus mutation found in Down's Syndrome associated acute megakaryocytic leukemia (DS-AMKL). Additionally, similar N-terminus mutation has been also observed in transient myeloproliferative disorder (TMD) that occurs specifically in newborns with Down's Syndrome. These clinical findings suggest the important function of GATA-1 N-terminus in megakaryocytic differentiation.

Using MGS cell line derived from DS-AMKL, we investigated the GATA-1 N-terminal function and erythroid differentiation. MGS cells only express GATA-1 lacking the N-terminus (GATA-1ΔNT). Over expression of wildtype GATA-1 in MGS cells using retroviral transfection was sufficient to drive erythropoiesis, which was confirmed by the increase in Glycophorin A-positive cells by flow cytometry. Whereas over expression of GATA-1ΔNT could not induce Glycophorin A-positive cells (Figure 3). ²

In order to examine the role GATA-1ΔNT plays in erythropoiesis in vivo, we utilized the Complimentation Rescue Method to generate GATA-1.05::GATA-1ΔNT transgenic mice. These mice can only express 5% of normal GATA-1 protein level in addition to having GATA-1ΔNT as a transgene. Erythroid differentiation was observed in high expressing GATA-1ΔNT line which was absent in low expressing GATA-1ΔNT line. We are currently investigating the functional importance of GATA-1 N-terminus in erythroid differentiation.

We have yet to understand why AMKL occurs with high prevelance in children affected by Down's Syndrome. In addition to the expression of GATA-1 lacking the N-terminus, we can surmise that the thrid copy of Chromosome 21 could be a factor in the development of DS-AMKL. Thus, further research is necessary looking into other blood-cell related transcription factors, such as RUNX1 or Bach1, which are both located on Chromosome 21.

Our laboratory is utilizing both molecular biology techniques and transgenic mouse technology to evaluate the role of GATA-1 N-terminus in blood cell differentiation and proliferation using in vitro and in vivo approach.