RESCUE OF GATA-1-DEFICIENT EMBRYONIC STEM-CELLS BY HETEROLOGOUS GATA-BINDING PROTEINS

Totipotent murine embryonic stem (ES) cells can be differentiated in v itro to form embryoid bodies (EBs) containing hematopoietic cells of m ultiple lineages, including erythroid cells. In vitro erythroid develo pment parallels that which is observed in vivo. ES cells in which the gene for the erythroid transcription factor GATA-1 has been disrupted fail to produce mature erythroid cells either in vivo or in vitro. Wit h the EB in vitro differentiation assay, constructs expressing heterol ogous GATA-binding proteins were tested for their abilities to correct the developmental defect of GATA-1-deficient ES cells. The results pr esented here show that the highly divergent chicken GATA-1 can rescue GATA-1 deficiency to an extent similar to that of murine GATA-1 (mGATA -1), as determined by size and morphology of EBs, presence of red cell s, and globin gene expression. Furthermore, GATA-3 and GATA-4, which a re normally expressed in different tissues, and a protein consisting o f the zinc fingers of GATA-1 fused to the herpes simplex virus VP16 tr anscription activation domain were able to compensate for the GATA-1 d efect. Chimeric molecules in which both zinc fingers of mGATA-1 were r eplaced with the zinc fingers of human GATA-3 or with the single finge r of the fungal GATA factor areA, as well as a construct bearing the z inc finger region alone, displayed rescue activity. These results sugg est that neither the transcription activation domains of mGATA-1 nor i ts zinc fingers impart erythroid cell specificity for its action in vi vo. Rather, it appears that specificity is mediated through the cis-ac ting control regions which determine spatial and temporal expression o f the GATA-1 gene. Furthermore, our results demonstrate that the zinc finger region may have a biological function in addition to mediating DNA binding.