Title

Identification and Characterization of Transcriptional Control Elements Regulating a Novel Post-Receptor Signaling Mediator, TNIP1

Date of Completion

January 2012

Keywords

Biology, Molecular|Philosophy of Science

Degree

Ph.D.

Abstract

TNFα interacting protein 3 interacting protein 1 (TNIP1) is a novel nuclear receptor interacting protein, isolated and characterized as a corepressor of RARs and PPARs. TNIP1 has also been shown to regulate a variety of other receptor-mediated events such as programmed cell death and cell cycling stemming from TNF and EGF signaling, respectively. Changes in TNIP1 expression levels are likely to impact the biological endpoints of these different pathways. As the importance of TNIP1 becomes more apparent, it is crucial to determine what controls its expression levels, as can be determined by a study of its promoter. We isolated ∼6kb of the human TNIP1 promoter and examined it both in silico and experimentally for transcriptional control elements with an eye directed at constitutive and inducible elements. Sequence analysis predicted two specificity protein (Sp) sites in the proximal region of the promoter and multiple NF-κB sites throughout the promoter. We predict the Sp family of transcription factors is responsible for much of TNIP1's constitutive activity and NF-κB for its inducible expression. Transcriptional activation studies revealed NF-κB, Sp1 and Sp3 positively regulate TNIP1. Furthermore, EMSA and ChIP demonstrated the physical association between NF-κB, Sp1, Sp3 and specific regions of TNIP1 promoter. Decreased Sp1 protein via siRNA or Sp binding to cognate sites by mithramycin decreased TNIP1 mRNA while the potent NF-κB activator TNFα increased TNIP1 expression. In summary, Sp1 and Sp3 contribute to the constitutive regulation of TNIP1 promoter through two proximal sites and NF-κB contributes to the inducible regulation of TNIP1 via two distal sites. Changes in endogenous Sp or NF-κB levels or pharmacological control of their activity would be expected to affect TNIP1 expression, which, in turn, could ultimately regulate TNIP1-related biological endpoints such as cell death, proliferation, and inflammation, and more globally, diseases such as psoriasis and rheumatoid arthritis. ^