Hepatocyte Nuclear Factor-4 Controls Transcription from a TATA-less Human Sex Hormone-binding Globulin Gene Promoter

Marja Jänne,Geoffrey L Hammond

doi:10.1074/jbc.273.51.34105

Abstract

Hepatocytes are the major source of sex hormone-binding globulin (SHBG), a glycoprotein that transports sex steroids in the blood and regulates their access to target tissues. The human SHBG proximal promoter was analyzed by DNase I footprinting, and the functional significance of 6 footprinted regions (FP1-FP6) within the proximal promoter was studied in human HepG2 hepatoblastoma cells. Two footprinted regions (FP1 and FP3) contain binding sites for the chicken ovalbumin upstream promoter-transcription factor (COUP-TF) and hepatocyte nuclear factor-4 (HNF-4). In experiments where SHBG promoter-luciferase reporter gene constructs were co-transfected into HepG2 cells with COUP-TF and/or HNF-4 expression vectors, HNF-4 markedly increased transcription, whereas COUP-TF suppressed this probably by displacing HNF-4 from their common FP1-binding site. This COUP-TF/HNF-4-binding site within FP1 includes a TTTAA sequence, located at nucleotides -30/-26 upstream of the transcription start site, which fails to interact with human TFIID, TATA-binding protein in vitro. When this sequence was replaced with an idealized HNF-4-binding site, the transcriptional activity of the promoter increased in HepG2 cells. Taken together, these data imply that an interplay between COUP-TF and HNF-4 at a site within FP1 regulates human SHBG expression and that HNF-4 controls transcription from this TATA-less promoter by somehow substituting for TATA-binding protein in the recruitment of a transcription preinitiation complex.

Highlights

Sex hormone-binding globulin (SHBG)1 is a plasma glycoprotein that transports sex steroid hormones and regulates their access to target cells (1)
The alteration of the TTTAAC sequence to either TCCCAC or TATAAA within FP-1 reduced the transcriptional activity of the Ϫ137/ϩ7SHBG promoter in HepG2 cells (Fig. 4A), and the same alterations within an footprint 1 (FP1) oligonucleotide abolished any protein binding in an Electrophoretic Mobility Shift Assay (EMSA)
The activity of the SHBG promoter, in which both chicken ovalbumin upstream promoter-transcription factor (COUP-TF)/HNF4-binding sites were disrupted, was reduced by 73%. These results indicate that both COUP-TF/hepatocyte nuclear factor-4 (HNF-4)-binding sites in the human SHBG promoter are functional in hepatoblastoma cells and provide additional evidence that they act independently of each other

Summary

Introduction

Sex hormone-binding globulin (SHBG)1 is a plasma glycoprotein that transports sex steroid hormones and regulates their access to target cells (1). Each human SHBG promoter deletion fragment was analyzed in the context of a luciferase reporter gene in HeLa cells to assess the cellular specificity of its transcriptional activity (not shown).

Results

Conclusion