Nuclear extracts from globin-synthesizing cells enhance globin transcription in vitro

Abstract

In vitro transcription studies of cloned messenger RNA-coding genes have yielded considerable information regarding the sequence elements and protein factors involved in transcription initiation and RNA processing. Fractionation of whole-cell, S-100 protein and nuclear extracts reveals the existence of both general class II and gene-specific transcription initiation factors. Because the soluble in vitro transcription systems prepared from cells in culture are largely nonspecific for the origin of the template DNA, they are highly suited to searching for tissue-specific and gene-specific transcription regulatory factors. In the experiments reported here, we have added a nuclear extract prepared from human erythroleukaemia-like cells (K562, which can be induced to synthesize epsilon- and gamma-globin mRNA and protein) to several deproteinized DNA templates, and monitored transcription levels in a HeLa cell-free transcription system. The K562 nuclear extract enhanced transcription of beta-, epsilon- and gamma-globin genes by as much as 30-fold compared with control non-globin templates. These results suggest the presence of a globin gene regulatory factor in erythroleukaemia cell nuclei.