Title

Functional analysis of interaction between nuclear retinoid receptors and a novel keratinocyte derived coregulator TNIP1

Date of Completion

January 2010

Keywords

Health Sciences, Pharmacy

Degree

Ph.D.

Abstract

Retinoids are natural and synthetic derivatives of vitamin A with wide ranging physiological functions. Nearly all of these are mediated through retinoid binding to two distinct classes of retinoid receptors: RAR and RXR which belong to the nuclear receptor (NR) superfamily. Transcriptional control by NRs is mediated not only through their ligands, but also through coregulator proteins which act either as coactivators or corepressors of NR activity. In a yeast two-hybrid screen seeking novel coregulators, we isolated TNIP1, a protein previously reported to interact with HIV proteins and also to repress NF-κB activity. TNIP1 contains two NR box motifs suggesting it may be a coregulator. TNIP1 does not interact with RXRα but associates with RARα and RARγ in a ligand dependent fashion and exhibits other interaction hallmarks of a coactivator. However, TNIP1 represses RAR activity in the presence of ligand. This makes TNIP1 an unusual coregulator—a corepressor of agonist bound NRs. The repression is partially relieved by coactivator SRC1, suggesting interference with coactivator recruitment as a mechanism of TNIP1 action. TNIP1 does not associate with HDAC enzymes, suggesting the TNIP1 repression is HDAC independent. ^ With this atypical function, we sought to determine what might contribute to control of TNIP1 expression. We carried out in silico analysis of its promoter which predicted several retinoic acid response elements. Transcriptional activation studies revealed that TNIP1 promoter is positively regulated by RARs and RXRγ. EMSA showed RAR binding at distinct sites in the distal portion of the TNIP1 promoter, which were confirmed as response elements. ^ Additionally, we generated and validated a polyclonal antibody against TNIP1. We used it to confirm wide distribution of the protein, demonstrate its nuclear and cytoplasmic localization, and establish its colocalization with RARα in the epidermis. Our findings reveal a potential regulatory feedback loop where TNIP1 expression is increased by RARs which, in turn, attenuates their activity. Such regulatory feedback loops between coregulators and their target NRs may serve to buffer cells against extremes of hormone-regulated signaling, such as the presence of toxic ligand levels, or cells being exposed to ligand at inappropriate times. ^