Characterization of an Inverted Repeat with a Zero Spacer (IR0)-Type Retinoic Acid Response Element from the Mouse Nuclear Orphan Receptor TR2-11 Gene

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Abstract
An inverted repeat with zero nucleotides in the spacer (IR0, 5‘- GGGTCA CGAACT -3‘) element was localized in the proximal promoter region of the mouse TR2-11 gene, and characterized as a functional retinoic acid response element (RARE). In gel mobility shift assays, heterodimers of retinoic acid receptor α (RARα) and retinoid X receptor β (RXRβ) bound specifically to this element. Neither receptor alone was able to bind to this element efficiently. The dissociation constant (Kd) with respect to RAR−RXR binding was estimated to be 8 nM. The biological activity of this IR0 element was assessed in a heterologous reporter system. The IR0-containing reporter was induced by RA in COS-1 cells in the presence of exogenously provided RARα and RXRβ. In addition, the IR0 oligomers could be bound by nuclear extracts isolated from COS-1 cells harboring the expression vectors for RARα and RXRβ, but not by extracts isolated from control COS-1 cells. RA responsiveness of this IR0 was further confirmed in P19 cells that expressed endogenous RARs and RXRs. Collectively, these data demonstrated, for the first time, the presence of a natural RARE of the IR0 type, and suggested a potential cross-talk between nuclear orphan receptor TR2-11 and RAR−RXR families.