Mechanism of Repression of the Inhibin α-Subunit Gene by Inducible 3′,5′-Cyclic Adenosine Monophosphate Early Repressor

Anna D Burkart,Kelly E Mayo,Abir Mukherjee

doi:10.1210/me.2005-0204

Abstract

The rodent ovary is regulated throughout the reproductive cycle to maintain normal cyclicity. Ovarian follicular development is controlled by changes in gene expression in response to the gonadotropins FSH and LH. The inhibin alpha-subunit gene belongs to a group of genes that is positively regulated by FSH and negatively regulated by LH. Previous studies established an important role for inducible cAMP early repressor (ICER) in repression of alpha-inhibin. These current studies investigate the mechanisms of repression by ICER. It is not clear whether all four ICER isoforms expressed in the ovary can act as repressors of the inhibin alpha-subunit gene. EMSAs demonstrate binding of all isoforms to the inhibin alpha-subunit CRE (cAMP response element), and transfection studies demonstrate that all isoforms can repress the inhibin alpha-subunit gene. Repression by ICER is dependent on its binding to DNA as demonstrated by mutations to ICER's DNA-binding domain. These mutational studies also demonstrate that repression by ICER is not dependent on heterodimerization with CREB (CRE-binding protein). Competitive EMSAs show that ICER effectively competes with CREB for binding to the inhibin alpha CRE in vitro. Chromatin immunoprecipitation assays demonstrate a replacement of CREB dimers bound to the inhibin alpha CRE by ICER dimers in ovarian granulosa cells in response to LH signaling. Thus, there is a temporal association of transcription factors bound to the inhibin alpha-CRE controlling inhibin alpha-subunit gene expression.

Highlights

THE INTRACELLULAR SIGNALING molecule cAMP functions in all cells to regulate gene expression
To determine whether deletion of the ␥-exon or differences between inducible cAMP early repressor (ICER) DBD I and II would have an effect on isoform binding to the noncanonical inhibin ␣ CRE, recombinant HA-ICER protein was made in HeLaT4 cells and used in subsequent EMSAs
Amounts of crude nuclear extracts that contained equivalent levels of ICER protein were ascertained by Western blotting, as shown in Fig. 1B, and identical amounts were used in EMSAs

Summary

Introduction

THE INTRACELLULAR SIGNALING molecule cAMP functions in all cells to regulate gene expression. These proteins contain leucine zipper motifs at their carboxy terminus along with a stretch of basic residues, which allows for DNA binding and dimerization [1] Two members of this family are the CRE-binding protein (CREB) and CRE-modulatory protein (CREM). There are proteins that inhibit either the recruitment of CBP to the promoter, such as the human T cell leukemia virus type 1 oncoprotein Tax [12], or the interaction of CBP with p300/ CBP-associated factor, such as the adenoviral oncoprotein E1A [9, 13] In addition to these proteins, there are bZIP repressors, including a class of truncated isoforms, which can bind to CRE and CRE-like elements [14].

Methods

Results

Conclusion