Abstract
The thyroid hormone receptor (TR) regulates the expression of target genes upon binding to triiodothyronine (T3) response elements. In the presence of T3, the TR recruits coactivating proteins that both modulate and integrate the ligand response. We report here the cloning of a novel protein using the TR ligand-binding domain as bait in the yeast two-hybrid system. Analysis of a putative full-length clone demonstrates a cDNA sequence that encodes a protein of 920 amino acids with a size of 120 kDa (p120). Alignment with known sequences shows homology to a previously identified protein of unknown function, termed skeletal muscle abundant protein. Interaction studies demonstrate that p120 interacts with the TR AF-2 domain in the presence of ligand through a 111-amino acid region. Northern analysis demonstrates widespread expression in human tissues. Cotransfection assays in CV-1 cells demonstrate that p120 enhances TR-mediated transactivation on multiple T3 response elements in the presence of T3. In addition, CREB-binding protein synergizes with p120 to enhance this effect. When linked to the GAL4 DNA-binding domain, p120 is an activator of transcription alone. Thus, p120 satisfies a number of important criteria as a nuclear receptor coactivator.
Highlights
The thyroid hormone receptor (TR),1 which is a member of the steroid/thyroid hormone receptor superfamily, regulates the expression of many target genes positively [1, 2] or negatively [3,4,5,6] through its ability to bind to thyroid hormone response elements (TREs) [1, 2]
The recently identified coactivators SRC-1, TIF2, and RIP140 all appear to bind the nuclear receptors (NRs) AF-2 domain, one report suggests that SRC-1 may interact with other regions of the TR [12, 14, 27]
We identified a number of positive clones, two of which showed a significant enhancement of interaction in the presence of T3 (Fig. 1). One of these clones was identified as TRIP1, which has been previously demonstrated to interact with the TR in yeast and mammalian cells [17]
Summary
The thyroid hormone receptor (TR), which is a member of the steroid/thyroid hormone receptor superfamily, regulates the expression of many target genes positively [1, 2] or negatively [3,4,5,6] through its ability to bind to thyroid hormone response elements (TREs) [1, 2]. On positive TREs, the TR acts as a repressor in the absence of thyroid hormone (T3) through its ability to interact with the nuclear corepressor family of proteins, which bind to the. In the presence of ligand, these corepressors are released from the TR, and proteins termed coactivators are recruited to mediate the ligand-dependent response [11]. The recently identified coactivators SRC-1, TIF2, and RIP140 all appear to bind the NR AF-2 domain, one report suggests that SRC-1 may interact with other regions of the TR [12, 14, 27]. It has been reported that activation by NRs can be enhanced by the CREB-binding protein-binding protein (CBP/p300) through a direct ligand-dependent interaction [13]. CBP/p300 interacts with SRC-1, suggesting that CBP/p300 may play a role as an integrator of NR-mediated activation of gene expression (28 –30) by signaling RNA polymerase II or by acting to modify histone structure through an intrinsic histone acetyltransferase activity [31]
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