A KNOCKDOWN MUTATION CREATED BY CIS-ELEMENT GENE TARGETING REVEALS THE DEPENDENCE OF ERYTHROID CELL MATURATION ON THE LEVEL OF TRANSCRIPTION FACTOR GATA-1

The hematopoietic-restricted transcription factor GATA-1 is required f or both mammalian erythroid cell and megakaryocyte differentiation. To define the mechanisms governing its transcriptional regulation, we re placed upstream sequences including a DNase I hypersensitive (HS) regi on with a neomycin-resistance cassette by homologous recombination in mouse embryonic stem cells and generated mice either harboring this mu tation (neo Delta HS) or lacking the selection cassette (Delta neo Del ta HS). Studies of the consequences of these targeted mutations provid e novel insights into GATA-1 function in erythroid cells, First, the n eo Delta HS mutation leads to a marked impairment in the rate or effic iency of erythroid cell maturation due to a modest (4- to 5-fold) decr ease in GATA-1 expression. Hence, erythroid differentiation is dose-de pendent with respect to GATA-1, Second, since expression of GATA-1 fro m the Delta neo Delta HS allele in erythroid cells is largely restored , transcription interference imposed by the introduced cassette must a ccount for the ''knockdown'' effect of the mutation, Finally, despite the potency of the upstream sequences in conferring high-level, develo pmentally appropriate expression of transgenes in mice, other cis-regu latory elements within the GATA-I compensate for its absence in erythr oid cells, Our work illustrates the usefulness of targeted mutations t o create knockdown mutations that may uncover important quantitative c ontributions of gene function not revealed by conventional knockouts.