Molecular mechanisms of transcriptional repression by the orphan receptor SHP

Abstract: SHP (Small Heterodimer Partner) is an atypical orphan member of the mammalian nuclear receptor family that consists of only a putative ligand-binding domain and thus cannot bind DNA. The aim of this thesis was to investigate the molecular mechanisms of the transcriptionally inhibitory effect of SHP. This was achieved by analysis of structural motifs within SHP, by studying nuclear receptor interactions, by isolation of upstream target co-factors and by characterization of SHP mutations. In the first study, we provide evidence that SHP binds directly to the estrogen receptors a and beta via LXXLL-related motifs. Similar motifs, referred to as nuclear receptor (NR) boxes, are usually critical for the binding of co-activators to the ligand-regulated activation domain AF-2 within nuclear receptors. We demonstrate that SHP variants, carrying either interaction-defective NR-box mutations or a deletion of the repressor domain, have lost the capacity to inhibit agonist-dependent estrogen receptor activation. Our study suggests that SHP acts as a transcriptional co-regulator by inhibiting the activity of nuclear receptors via occupation of the co-activator binding surface and via active repression. However, active repression mechanisms have remained elusive and may involve factors distinct from known nuclear receptor co-repressors. In the second study, we describe the isolation of mouse EID1 (E1A-like inhibitor of differentiation 1) as the first co-inhibitor for SHP. We characterize the interactions between SHP and EID1 and identify two repression-defective SHP mutations that have lost the ability to bind EID1. We suggest histone acetyltransferases and histones as targets for EID1 action, and we propose that SHP inhibition of transcription involves EID1 antagonism of p300/CBP-dependent co-activator functions. Recent evidence suggests the existence of a larger family of EID1-related proteins. In the third study, we describe a third family member designated EID3 that is highly expressed in testis and shows homology to a region of EID1 implicated in binding to p300/CBP. We demonstrate that EID3 acts as a potent inhibitor of nuclear receptor transcriptional activity by a mechanism that is independent of direct interactions with nuclear receptors, including SHP. However, EID3 directly binds to the C-terminus of CBP, which has been implicated to act as the interaction surface for nuclear receptor co-activators. Consistent with this idea, EID3 prevents recruitment of CBP to a natural nuclear receptor-regulated promoter. Our study suggests that EID-family members act as inhibitors of p300/CBP-dependent transcription in a tissue-specific manner. In the fourth study, we characterize GPS2 (G-protein pathway suppressor 2), a subunit of the N-CoR/HDAC3 co-repressor complex, as an interaction partner for SHP. Specific interactions between GPS2 and SHP have been verified in vitro and in vivo and appear physiologically relevant as GPS2 mRNA is expressed in SHP target tissues. It is shown that GPS2 increases SHP-mediated repression of NR target genes by enhancing intrinsic repression of the SHP ligand-binding domain. Taken together, these results suggest that SHP recruits a conserved co-repressor complex to nuclear receptor target genes via direct interactions with the GPS2 subunit. SHP fulfils specific roles as inducible co-repressors in the feedback regulation of LRH-1 (liver receptor homologue 1) target genes involved in bile acid synthesis. In the fifth study, we have identified and characterized the homeodomain protein Prox-1 (Prospero-related homeobox 1) as an additional co-repressor for LRH-1. We show that Prox-1 binds to LRH-1 via two LXXLL motifs and represses the LRH-1 dependent expression of SHP. Our hypothesis is that Prox-1 may modulate LRH-1 target gene expression during development and under normal physiological conditions, whereas SHP may primarily serve as a metabolic sensor under conditions of increased metabolic activity such as a high-cholesterol diet.