# GENE TRANSCRIPTION IN ESTROGEN-DEPENDENT DIFFERENTIATION

> **NIH NIH R01** · UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN · 1994 · $205,273

## Abstract

A single exposure of male Xenopus laevis to estradiol-17-beta induces
hepatic transcription of the genes coding for the egg yolk precursor
protein, vitellogenin (VIT), and elicits the long-term induction of Xenopus
estrogen receptor (XER) mRNA and serum retinol binding protein gene
transcription. To study estrogen regulation of gene expression we developed
stable Xenopus liver and fibroblast cell lines, cloned and expressed the
Xenopus estrogen receptor (XER), and used mutagenesis and cotransfection to
analyze the domain structure of the XER, and to identify regulatory
sequences in the vitellogenin promoter.

We have identified a far upstream palindrome related to the consensus
estrogen response element (ERE) which may mediate autoregulation of XER
gene expression. The putative XER ERE, and the native ERE, will be compared
in transfections, and in binding and competition studies using our purified
mutant XER.

A specific transcription activation domain has not been identified in the
XER or human ER. We shall test the ability of acidic amphipathic helices
located in the hormone binding domain of the XER, and synthetic
transcription activators, to increase the activity of wild type and mutant
XER.

We have overexpressed and purified a 111 amino acid XER mutant, which binds
to the ERE and retains considerable ability to activate transcription. In a
collaborative study, this mutant, and other XER mutants, will be bound to
EREs separated by a few hundred nucleotides. We will examine the ability of
the ERE-bound XER mutants to establish direct protein-protein contact by
looping out the DNA.

Electrophoresis, protein cross-linking using bifunctional reagents, and
electron microscopy will be used to identify and characterize the
complexes.

Using purified XER mutants and our high efficiency HeLa cell transcription
extract we will:  (A) Correlate in vivo activity of mutant XERs with their
ability to activate transcription in extracts. (B) Use our powerful
synthetic ERE-TATA promoters as immobilized templates to begin to
characterize the components of a simplified XER regulated transcription
complex. (C) Use an immobilized native VIT promoter to determine whether
the XER remains bound to the promoter during transcription, or dissociates
after facilitating formation of an initiation complex involving an
essential activator element we have identified. (D) The activator element
linked to a TATA box is a powerful transcription activator, yet it is
inactive in the VIT promoter unless XER is bound.  We shall determine
whether its activity is suppressed by unliganded XER, or by nucleosome
phasing.

## Key facts

- **NIH application ID:** 2197350
- **Project number:** 5R01HD016720-13
- **Recipient organization:** UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
- **SAM.gov UEI:** Y8CWNJRCNN91
- **Principal Investigator:** DAVID J SHAPIRO
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 1994
- **Award amount:** $205,273
- **Award type:** 5
- **Project period:** 1982-07-01 → 1995-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/2197350

## Citation

> US National Institutes of Health, RePORTER application 2197350, GENE TRANSCRIPTION IN ESTROGEN-DEPENDENT DIFFERENTIATION (5R01HD016720-13). Retrieved via MORVS 2026-10-25 from https://api.morvs.ai/grant/nih/2197350. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*