MULTIPLE REGIONS OF P45 NF-E2 ARE REQUIRED FOR BETA-GLOBIN GENE-EXPRESSION IN ERYTHROID-CELLS

Regulated expression of genes in the beta-globin cluster depends upon sequences located between 5 and 20 kb upstream of the epsilon gene, kn own as the locus control region (LCR). beta-Globin expression in murin e erythroleukemia (MEL) cells depends on NF-E2, a transcription factor which binds to enhancer sequences in the LCR. To gain insight into th e mechanism of globin gene activation by NF-E2, an NF-E2 null MEL cell line was used to map regions of NF-E2 required for beta-globin expres sion. Within the transactivation domain, two discrete proline-rich reg ions were required for rescue of beta-globin expression. The first was located at the N-terminus of NF-E2, while the second was located N-te rminal of the cap 'n collar (CNC) domain. Other proline-rich sequences were dispensable, indicating that proline content per se does not det ermine NF-E2 activity. Mutations within the conserved CNC domain marke dly diminished rescue of beta-globin expression. This domain was requi red, in addition to the basic leucine zipper domain, for DNA binding a ctivity. The requirement for discrete proline-rich sequences within th e transactivation domain suggests that globin gene expression in MEL c ells depends on specific interactions between NF-E2 and downstream eff ector molecules.