Changes in E2F DNA-binding activity during induced erythroid differentiation.

Abstract

The cytodifferentiation agent hexamethylene bisacetamide (HMBA) is an inducer of differentiation of a variety of transformed cells, including the murine erythroleukemia (MEL) cell line. Induction of differentiation of MEL cells is a multistep process resulting in cessation of cell division and phenotypic maturation (including hemoglobin production). To investigate HMBA-induced MEL cell differentiation, we have analyzed the regulation of the E2F transcription factor. E2F regulates the transcription of several genes whose products are involved in both cell cycle regulation and differentiation. In nuclear extracts from uninduced MEL cells, three complexes were detected using gel mobility assays with the E2F/E2 oligonucleotide. The complex with the fastest mobility is the free form of E2F binding to DNA, and the more slowly migrating complexes contain E2F, p107, and cdk2. By 8 h of HMBA induction and for the remainder of the differentiation process, the free E2F complex is not detected, and only complexes of slower mobility, which contain p107 and cdk2, are found. The level of p107 protein increases during induction of differentiation; there is no change in the level of cdk2 protein and E2F-4 and DP-1 proteins during the first 4 days. The level of E2F-1 mRNA does not change, but a new form of E2F is detected during induction of differentiation. Thus, HMBA causes a selective loss in the free E2F DNA-binding complex, an increase in p107 protein, and an increase in a form of E2F protein during MEL cell differentiation.