The Pit-1β Domain Dictates Active Repression and Alteration of Histone Acetylation of the Proximal Prolactin Promoter

Scott E Diamond,Arthur Gutierrez-Hartmann

doi:10.1074/jbc.m006048200

Scott E Diamond, Arthur Gutierrez-Hartmann

Open Access

https://doi.org/10.1074/jbc.m006048200

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Abstract

A critical problem in current molecular biology is to gain a detailed understanding of the molecular mechanisms by which related transcription factor isoforms with identical DNA sequence specificity mediate distinct transcription responses. Pit-1 and Pit-1beta constitute such a pair of transcription factor isoforms. Pit-1 enhances the Ras signaling pathway to the prolactin promoter, and Pit-1beta represses basal prolactin promoter activity as well as Ras signaling to the prolactin promoter in pituitary cells. We have previously demonstrated that the beta-domain amino acid sequence dictates the transcriptional properties of Pit-1beta. Here, we show that five hydrophobic beta-domain residues are required for Pit-1 isoform-specific repression of Ras signaling, and we demonstrate that sodium butyrate and trichostatin A, pharmacological inhibitors of histone deacetylation, as well as viral Ski protein, a dominant-negative inhibitor of recruitment of N-CoR/mSin3 histone deacetylase complexes, specifically reverse beta isoform-specific repression of Ras signaling. Moreover, we directly demonstrate, with a chromatin immunoprecipitation assay, that the Pit-1beta isoform alters the histone acetylation state of the proximal prolactin promoter. This differential analysis of Pit-1/Pit-1beta isoform function provides significant insights into the structural determinants that govern how different transcription factors with identical DNA sequence specificity can display opposite effects on target gene activity.

Highlights

Most transcription factors are members of extended families defined by conserved structural motifs, typically in the DNAbinding domain, yet differing in other domains, especially the transactivation domain (TAD).1 A number of transcription factors are expressed as a set of proteins derived from a single gene via alternative promoter usage or splicing events that result in virtually identical transcription factor isoforms that, can mediate distinct responses (e.g. PR-A versus PR-B; TR␤1 versus TR␤2; Oct 2.1 versus Oct 2.5; Ets-1 versus Ets-1⌬VII; and Pit-1 versus Pit-1␤) [1,2,3,4,5,6]
We focus on identifying key ␤-domain residues that are responsible for the ␤ isoform-specific repression of Ras signaling to the rPRL promoter, as well as identifying a mechanism for this repression
Replacement of each of these residues with alanine identified five hydrophobic residues that are required for the ␤-domain to act as a transcriptional repressor of Ras signaling to the rPRL promoter

Summary

Introduction

Most transcription factors are members of extended families defined by conserved structural motifs, typically in the DNAbinding domain, yet differing in other domains, especially the transactivation domain (TAD).1 A number of transcription factors are expressed as a set of proteins derived from a single gene via alternative promoter usage or splicing events that result in virtually identical transcription factor isoforms that, can mediate distinct responses (e.g. PR-A versus PR-B; TR␤1 versus TR␤2; Oct 2.1 versus Oct 2.5; Ets-1 versus Ets-1⌬VII; and Pit-1 versus Pit-1␤) [1,2,3,4,5,6]. In GH4 pituitary cells, the two isoforms have opposite transcriptional effects with regard to Ras signaling to the rPRL promoter; Pit-1 enhances and Pit-1␤ represses the oncogenic RasVal-12 response [9, 22].

Results

Conclusion