Identification and characterisation of a steroid response element-binding protein

• Main Author: Crawford, Louise
• Published: University of Edinburgh 1991
• Subjects: 612.6
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.645119

The steroid hormone receptors (SR) are nuclear transcription factors which, upon activation by hormone binding, bind specifically to short DNA sequences called steroid response elements (SRE) in steroid regulated genes, and alter the transcription rates of those genes. The consensus oestrogen (ERE) or glucocorticoid (GRE) response element can work alone as a hormone-dependent transcriptional enhancer <i>in vivo</i>, when linked to a heterologous promoter. However, highly specific binding of purified SR to a SRE <i>in vitro</i>, has not been demonstrated; in many cases, purified oestrogen (ER) and glucocorticoid (GR) receptor discern between their specific SRE and non-specific DBA with less than 10-fold discrimination. Several studies have implicated the involvement of accessory proteins which increase the affinity of purified SR for its SRE <i>in vitro</i>. <i>In vivo</i>, such accessory proteins may be involved in high affinity binding of SR to a SRE to confer transcriptional enhancement. This thesis describes the identification and characterisation of a steroid response element-binding protein (SRE-BP) and argues that by modulating the interaction of different SRs with their target SREs, the SRE-BP plays a role in steroid hormone action. The SRE-BP present in HeLa, GH<SUB>3</SUB> and CV-1 cells, and in liver tissue, binds specifically to two classes of functionally distinct SRE. In gel retardation experiments the SRE-BP binds preferentially to oligonucleotides containing a consensus ERE or a symmetrical GRE; it binds less well to a mutant GRE and binds neither to a symmetrical thyroid response element nor to other unrelated transcription factor binding sites. Using gel filtration chromatography, the SRE-BP has been partially purified and shown to have a relative molecular weight under non-denaturing conditions of im205kD. The same species that exists in solution is also the DNA binding form as determined by pore gradient gel electrophoresis. Crosslinking experiments show that the SRE-BP is a multisubunit protein with a tentatively deduced DNA-binding sununit of im40kD. The SRE-BP is, therefore, a sequence specific DNA binding protein. It is neither ER nor GR as demonstrated by its cell type distribution, DBA sequence specificity, and its relative molecular weight. Preliminary evidence is presented which suggests that HeLa WCE containing SRE-BP activity increase the binding of <i>in vitro</i> translated ER to a consensus ERE in a gel retardation assay. A role for accessory proteins in steroid receptor/DNA binding is further substantiated by the finding that <i>in vitro</i> translated ER binds to an ERE as part of a 360kD complex and not simply as a 130kD homodimer.