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. Author manuscript; available in PMC: 2009 Apr 30.
Published in final edited form as: Mol Endocrinol. 2007 May 22;21(8):1924–1939. doi: 10.1210/me.2007-0035

Fig. 1. Different Corepressor Splice Variants Differ in Their Ability to Interact with TRα.

Fig. 1

A, Schematic representations of the SMRT and N-CoR alternative splice variants are shown. The locations of the repression domains (RD) and the CoRNR box motifs (S3, S2, S1, N3, N2, N1) are indicated. Overall length of each variant is expressed in codons. The receptor interaction domains of these corepressors, indicated by an arrow, were fused to GAL4DBD for use in the two-hybrid studies (see also D). B, SMRTτ and TRα interact strongly in a mammalian two-hybrid assay. A GAL4DBD-SMRTτ construct and a GAL4AD-TRα construct were cotransfected into CV-1 cells together with a GAL-17mer luciferase reporter. Relative luciferase activity was calculated as the absolute luciferase activity normalized to the expression of a constitutive β-galactosidase construct used as an internal transfection control. Negative controls included the use of empty GAL4DBD and/or empty GAL4AD constructs, as indicated, or performing the transfection in the presence of T3, which releases corepressor from TRα. C, Different corepressor splice variants differ in their interaction with TRα. The receptor interaction domains of the various corepressor splice variants in A were introduced into the GAL4DBD construct, as noted below the panel, and tested for their ability to interact with the GAL4AD-TRα construct in the mammalian two-hybrid assay. D, Schematic representations of the SMRT and N-CoR mutants used and their expression levels are shown. Mutations were introduced into individual CoRNR boxes in the GAL4DBD-SMRTsp18 (codons 1675–2508) and GAL4DBD-N-CoR (codons 1634–2453) constructs as indicated by the × symbols and by the mS2, mS3, etc. nomenclature. The relative levels of expression of each of these constructs, determined by Western blotting, are depicted to the right. Alternatively, specific CoRNR boxes were exchanged between SMRT and N-CoR (e.g. N-CoR N3:S2:S1 contains the N3 motif of N-CoR linked to the S2 and S1 motifs of SMRT). E, Both the iteration and the identity of the CoRNR motifs in SMRT and N-CoR determine the ability of the corepressors to interact with TRα. The mammalian two-hybrid assay in C was repeated using the GAL4DBD-SMRTsp18 and GAL4DBD-N-CoR constructs bearing disruptions in specific CoRNR boxes. Single mutants are as depicted in D; double mutants (e.g. mN1/mN2) bear disruptive mutations in two CoRNR boxes. The mammalian two-hybrid interaction of TRα with the N-CoR construct was defined as 1.