Identification of Nuclear Factors that Enhance Binding of the Thyroid Hormone Receptor to a Thyroid Hormone Response Element

Abstract

Using a gel shift assay, we analyzed the binding of in vitro translated alpha- and beta-thyroid hormone (T3) receptors to a T3-response element (TRE) derived from the rat GH gene. No receptor-TRE complexes were observed when translated receptor alone was incubated with the TRE. However, addition of a nuclear extract from liver to the translational products resulted in the formation of two receptor-DNA complexes for both the alpha- and beta-receptors. These complexes were shown to contain translated receptor by comigration of 32P-labeled TRE and 35S-labeled receptor in the gel shift assay. A competition experiment demonstrated that formation of the complexes was sequence specific. Preincubation of the liver nuclear extract at 60 C abolished formation of both complexes indicating that receptor-TRE binding required a heat-labile nuclear factor. Phosphocellulose chromatography of the nuclear extract resulted in separation of the activities required for formation of the two complexes. Analysis of nuclear extracts from different tissues revealed that one complex formed in the presence of all extracts, whereas the second complex appeared predominantly with a nuclear extract from liver. Addition of T3 to the binding reaction had no effect on receptor-TRE complex formation. We suggest that nuclear factors interact with the T3 receptor to enhance hormone-independent binding to a TRE.

Variable RXR requirements for thyroid hormone responsiveness of endogenous genes

“…RXRs originally were described as nuclear proteins that enhance the binding of TRs to DNA in EMSAs (Murray and Towle, 1989), and were subsequently shown to have similar effects on a broader subset of nuclear receptors, such as retinoic acid and vitamin D receptors (Kliewer et al, 1992;Leid et al, 1992;Yu et al, 1991). However, the precise role of RXRs in T3 action has been difficult to define in part because there are no RXR-null mammalian cells, as noted above.…”

Endogenous retinoid X receptors can function as hormone receptors in pituitary cells.

“…While retinoid-dependent transactivation by RXR is mediated in part by RXR homodimers (66), RXR is also able to form heterodimers with TR and VDR (7,18,36,37,46,48,64,65). In each case, target gene binding by the nuclear hormone receptor is greatly enhanced by its partnership with RXR.…”

Nuclear factors specifically favor thyroid hormone binding to c‐ErbAα1 protein (thyroid hormone receptor α) over‐expressed in E. coli

“…1C and D). The applied heating conditions were known to be able to totally destroy, in nuclear extracts, any T3 binding activity [22] or any amplifying activity on TR binding to DNA [11]. Our present finding suggests that the nuclear factors that enhance T3 binding to rec-TR~ may be heterogeneous.…”

“…As evoked above, nuclear extracts are known to play an amplifying role in TR binding to DNA whether TR were produced by in vitro translation in reticulocyte lysate [11] or in bacteria [21]. Working with 32p-labelled oligonucleotides that contained TRE sequences, different authors described both an increase in the amount of TR bound to TRE and qualitative changes with the formation of high Mr complexes with heatlabile nuclear TRAP (heterodimers and oligomers in EMSA) [12,13].…”

“…Fax: (33) 91 79 65 11. transcriptional activation [10]. A series of recent reports shows that TR interaction with TRE, and T3-mediated transcriptional activity of TR, are amplified by other nuclear auxiliary proteins (TRAP) [11,12], including the retinoid receptors RXR [13][14][15].…”