Review
Progesterone receptors, their isoforms and progesterone regulated transcription

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Abstract

This review discusses mechanisms by which progesterone receptors (PR) regulate transcription. We examine available data in different species and tissues regarding: (1) regulation of PR levels; and (2) expression profiling of progestin-regulated genes by total PRs, or their PRA and PRB isoforms. (3) We address current views about the composition of progesterone response elements, and postulate that PR monomers acting through “half-site” elements are common, entailing cooperativity with neighboring DNA-bound transcription factors. (4) We summarize transcription data for multiple progestin-regulated promoters as directed by total PR, or PRA vs. PRB. We conclude that current models and methods used to study PR function are problematical, and recommend that future work employ cells and receptors appropriate to the species, focusing on analyses of the effects of endogenous receptors targeting endogenous genes in native chromatin.

Highlights

► Progesterone receptor (PR) levels are regulated by estrogen-dependent and independent pathways. ► PR mediated gene regulation as studied by expression profiling. ► Prevalence of PRE half-sites suggests that PR monomers and dimers can regulate transcription. ► PR, transcription factor cooperativity, and role of co-response elements. ► The PR isoforms, PR-A and PR-B, regulate overlapping and distinct gene subsets.

Introduction

Like all nuclear receptors, progesterone receptors (PR) are transcription factors that consist of a DNA binding domain (DBD), sandwiched between an upstream N-terminal region that contains activation (AF) and inhibitory (IF) functions, and a downstream hinge region and C-terminal ligand binding domain (LBD) (Hovland et al., 1998). There are two PR isoforms, PRA and PRB, which differ only in that human PRB contain an additional 164 amino acid far N-terminal region called the “B-upstream segment” (BUS) that confers AF3 activity. BUS is missing in PRA (Sartorius et al., 1994b). Site-specific mutations of amino acids in BUS that are responsible for its AF3 activity destroy PRB-specific gene regulation without however, switching PRB to PRA (Tung et al., 2006). This suggests that global structural differences between PRB and PRA apart from BUS control their unique properties. We review below the distinctive transcriptional activities of the two PR isoforms.

In normal human tissues including the breast, PRA and PRB are generally expressed at similar levels but at least in some breast cancers, their ratio is dysregulated (Graham et al., 1995b, Hopp et al., 2004). Classically, transcription mediated by PR is viewed as follows: (1) the unliganded receptors are cytoplasmic and bound to heat shock proteins in the absence of progestins (P). (2) Liganded receptors are released from heat shock proteins, dimerize and translocate to the nucleus. (3) There they locate specific palindromic DNA binding sites called progesterone response elements (PREs) in promoters of PR regulated genes, after which, (4) transcription is initiated through recruitment of a transcription complex. While this may be the case for some tissues, in some contexts, on some promoters, this simple model cannot explain data showing that: (1) PR are largely localized to the nucleus even in the absence of P; (2) dimerization may not be required for PR mediated transcription; (3) the endogenous structures of PREs have not been defined by unbiased in vivo analyses but are unlikely to be “classical”; and (4) PR also signal through cell membrane and cytoplasmic pathways.

The functions of steroid receptors generally, and of PR specifically, have been studied in a variety of systems. For PR, the most widely used are artificial reporters consisting of tandem palindromic PREs separated by random intervening sequences, linked to luciferase. These double PRE (PRE2) reporters are then transiently or stably transfected into cells that may or may not be relevant to the question under study; conditions and cells are optimized to yield maximum luciferase activity; and conclusions are drawn regarding PR structure, PR function, PR isoform specificity, and coregulators of PR-dependent transcription. In an attempt to address more physiologically relevant conditions, other studies choose bona fide genes regulated by PR, map their proximal promoters (usually less than 5 Kb) by deletion analysis, and search for classical PREs within regions considered to be functionally important. To confirm the validity of any conclusions, candidate sequence(s) are subjected to protein:DNA interaction studies by electrophoretic mobility shift assays, DNA footprinting, or methylation interference; all of which use “naked” DNA. More recently, nuclear receptor binding sites and kinetics of protein:DNA interactions have been analyzed by chromatin immunoprecipitation (ChIP), which has the advantage of searching for PR binding sites in the context of chromatin. However, even ChIP assays are biased by being limited to a search of specified DNA regions or sequences. The least biased methods are ChIP-on-chip, which combines ChIP with microarray technology (chip) and identifies DNA-binding sites on a genome-wide basis; or ChIP-seq, which combines ChIP with massively parallel DNA sequencing. Studies examining PR-regulated promoters have, as of this writing, been limited to proximal promoter regions using ChIP assays. Neither ChIP-on-chip nor ChIP-seq has been reported. Thus many important PR regulatory regions including ones in introns, 3′ untranslated regions (UTR), or tens of thousands of Kb removed from transcriptional start sites remain unexamined, and true endogenous DNA sequences to which PR bind either directly or indirectly remain largely unknown.

In this review we examine available data in a variety of tissues and species addressing: (1) regulation of PR levels by estrogenic and non-estrogenic signaling; (2) analysis of genes regulated by total PR, or by PRA vs. PRB using expression profiling; (3) our current understanding about the composition of PREs, and transcriptional regulation via PR monomers, PRE half-sites and cooperativity with other transcription factors. (4) We review in detail transcriptional regulation of multiple P-regulated promoters by total PR and the two PR isoforms. Although many signaling pathways converge on PR and influence their transcriptional activity (reviewed in Daniel et al. (2009)), even in the absence of ligands (Jacobsen et al., 2005), and non-genomic effects of PR have also been described (Boonyaratanakornkit et al., 2001), this review is limited to the genomic transcriptional effects of liganded PR.

Section snippets

Regulation of PR expression

To understand PR function, it is important to review factors that regulate PR levels. In T47D human breast cancer cells, which are the major models for human PR, synthesis of the PRA and PRB isoforms is driven by transcription from two promoters (Kastner et al., 1990) located at −711 to +31 and +464 to +1105 of the transcription start-site, respectively. At least six transcripts encode PR (Kastner et al., 1990, Wei et al., 1988) containing translational start sites not only for PRA and PRB, but

Gene regulation: expression profiling

In the last few years, expression profiling studies have greatly expanded our knowledge of human P-regulated genes and the role, if any, of PR homo- and heterodimers. These studies indicate that isoform-specific gene regulation by PR is largely tissue, cell type and promoter specific. As a result, since most studies use cancer cells, conclusions may not apply to normal, physiological states. PR gene regulation has been studied mainly in ER+ T47D human breast cancer cells because, typical of

Composition of PREs

The activity of PR has been extensively studied on two exogenous models: the mouse mammary tumor virus-long terminal repeat (MMTV-LTR) and tandem PREs (PRE2) derived from the second PRE of the rat tyrosine amino transferase (TAT) promoter. These sequences, which usually respond to both PR and GR (and often to AR and mineralocorticoid receptors as well), are often referred to as GRE/PREs. It is not surprising that GR and PR recognize the same DNA elements since their core DBDs exhibit 90% amino

Detailed promoter analyses and PR isoforms

The majority of studies addressing transcriptional mechanisms of PR focus on the proximal promoter (usually 5 Kb or less from the 5′-UTR) of the regulated gene, cloned upstream of a reporter, and transfected into cells that contain endogenous or transfected PR. Deletion and/or mutation analysis of the targeted promoter mark PR functional sites, which are then analyzed for presence of “consensus” PRE palindromes, and/or for DNA:PR protein interactions. Often these analyses point to non-consensus

Summary

Progesterone is a key physiologic hormone of women and its receptors play a major role in the diagnosis and treatment of breast cancers. Nevertheless its fundamental mechanisms of action remain in doubt. The receptor proteins have not been purified, crystallographic structural data are unavailable, virtually nothing is known about authentic DNA binding sites on promoters of PR regulated genes, and very little about the true nature of PR interactions with native chromatin. As we review here, in

Acknowledgments

The authors are grateful for the support of the NIH (R01 CA026869-32), the Avon Foundation for Women, the Breast Cancer Research Foundation, and the National Foundation for Cancer Research. We regret that space limitations prevented discussion of the important work of many colleagues.

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