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 (reviewed in ref. 1). Fractionation of whole-cell², S-100 protein³ and nuclear extracts⁴ reveals the existence of both general class II^5–9 and gene-specific^8,10,11 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^1,12, 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 ε- and γ-globin mRNA and protein^13,14) to several deproteinized DNA templates, and monitored transcription levels in a HeLa cell-free transcription system². The K562 nuclear extract enhanced transcription of β-, ε- and γ-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.