A large and growing number of co-factors have been shown to interact with liganded nuclear hormone receptors and enhance their transcriptional activation. These include: steroid receptor coactivator 1 (SRC1); SRC2/transcriptional intermediary factor 2 (TIF2) / glucocorticoid receptor interacting protein 1 (GRIP1); SRC3/ amplified in breast cancer 1 (AIB1)/ receptor associated coactivator 3 (RAC3)/ p300/CBP cointegrator associated protein (p/CIP)/ nuclear receptor coactivator (ACTR)/ thyroid receptor activator molecule 1 (TRAM 1); peroxisome proliferator activated protein binding protein (PBP); TR accesory proteins (TRAPs) /vitamin D receptor interacting proteins (DRIPs); p300/CBP associated factor (p/CAF), and cAMP response element binding protein (CREB) binding protein (CBP)/ p300. among others (reviewed elsewhere (2,16,31,32,99,100).

At present, the precise roles of all these putative coactivators are not known; however, it appears that there are at least two major complexes involved in ligand-dependent transcriptional activation: the steroid receptor co-activator (SRC) complex and the vitamin D receptor interacting protein/TR associated protein (DRIP/TRAP) complex Fig. 3d-6). SRCs (SRC-1,SRC-2, and SRC-3) are 160 kD proteins that associate with nuclear hormone receptors, including TRs, and enhance their ligand-dependent transcription (100,110-112). SRCs also interact with the CREB-binding protein (CBP), the co-activator for cAMP-stimulated transcription as well as the related protein, p300, which interacts with the viral co-activator E1A (113-116). Recent studies also have shown that CBP/p300 can interact with PCAF (p300/CBP-associated factor), the mammalian homolog of a yeast transcriptional activator, general control nonrepressed protein 5, GCN5. Like GCN5, PCAF has intrinisic histone acetyltransferase activity (HAT) activity. Both PCAF and CBP interact with TBP associated factors (TAFs) and RNA pol II. Thus, PCAF and CBP possess dual functional roles both as adaptors of nuclear receptors to the basal transcriptional machinery as well as enzymes that can alter chromatin structure by histone acetyl transferase (HAT) activity. SRC‑1 and CBP may coordinate with TRs to synergize further the actions of TH, and also allow for the convergence of plasma membrane and nuclear hormone receptor signaling pathways in the cell.

The DRIP/TRAP complex also interacts with liganded VDRs and TRs (117-120). However, none of the subunits are members of the SRC family or their associated proteins. Instead, several DRIP/TRAP components are mammalian homologs of the yeast Mediator complex, which associates with RNA Pol II. Thus, TR recruits DRIP/TRAP complex which, in turn, may recruit or stabilize RNA Pol II holoenzyme via their shared subunits. It is noteworthy that DRIP/TRAP complex does not appear to have intrinisic HAT activity. Recent chromatin immunoprecipitation assays of proteins bound to hormone response elements (HREs), suggest that there may be a sequential, possibly cyclical recruitment, of co-activator complexes to hormone response elements by liganded nuclear hormone receptors (121-123). Studies of co-activator recruitment to TH-regulated genes showed distinct temporal patterns of recruitment . Last, other co-factors such as SW1/Snf and BRG-1 may be involved in early chromatin remodeling before the co-activator complexes are recruited to the TREs .

In contrast to positively-regulated target genes, negatively-regulated genes can be stimulated in the absence of TH and repressed by TH (Figure 3d-5, above). Regulation of TRH and the TSH α and β -subunit genes have been studied most extensively as models of negatively-regulated genes. From a physiological perspective, negative-regulation of these genes represents a critical aspect of feedback control of the TH axis. The T3-responsive regions of these negatively- regulated genes have been localized to the proximal promoter regions (127-129). However, TR binding to putative TREs in these promoters is relatively weak in comparison to the binding sites in positively-regulated genes.

There are several different potential mechanisms for negative regulation by TH. Negative regulation may involve receptor interference with the actions of other transcription factors or with the basal transcription apparatus (130, 131). For instance, TR can inhibit the activity of AP-1, a heterodimeric transcription factor composed of Jun and Fos. T3-mediated repression of the prolactin promoter has been proposed to occur by preventing AP-1 binding (132). The TR also interacts with other classes of transcription factors, including NF-1, Oct-1, Sp-1, p53, Pit-1, and CTCF (133-138). By binding to these, or other positive transcription factors, the TH receptor may be able to inhibit gene expression by protein-protein interactions. Negative regulation may also occur by TR directly binding to DNA. A negative TRE from the TSHβ gene resides in an exon downstream of the start site of transcription (129) raising the possibility that it occludes the formation of a transcription complex. (Figure 3d-6, above) Additionally, liganded TRs may potentially recruit positive cofactors off DNA (squelching), which in turn, could lead to decreased transcription of target genes.

Transcriptional CoRs and CoAs, or even novel co-factors, may be involved in the control of negatively regulated genes. In contrast to the basal repression by unliganded TR in the case of positively regulated genes, CoRs cause basal activation of the TSH and TRH genes (127-129). CoAs also play an apparently paradoxical role in T3-dependent repression of negatively regulated genes (139, 140). Moreover, both SRC-1 knockout mice and knockin mice which express a TRβ mutant with a mutation in the helix 12 region (that interacts with CoAs) have defective negative regulation of TSH (141,142). On the other hand, HDACs may be recruited by TRs during ligand-dependent negative regulation in some instances (143). Cofactor-associated changes in histone acetylation, and alterations in chromatin structure, may therefore be involved in negative regulation by TR, although the precise effects and particular co-factors involved remained to be elucidated. Last, there appears to be some TR isoform-specificity in the negative regulation of certain target genes (72,144).

Phenotypic EFFECTS OF TRα AND TRα KNOCKOUTS

Recently, targeted gene inactivation or knockout (KO) of TR isoforms, and “knockin” of mutant TRs to their native TR genomic locii have provided new information on the mechanisms of TH action (17, 18). The ability to disrupt TR genes by targeted mutagenesis has been particularly challenging given there is more than one gene encoding TRs, multiple splicing variants (TRα 1, α 2, TRβ 1, TRβ 2), and an additional transcript (Rev-erbA) derived from the opposite strand of the TRα gene (2, 16, 18, 99). Two TRα knockout mouse lines have been generated that display different phenotypes (145, 146). It is likely this difference is due to the different sites in the TRα gene locus used for homologous recombination to generate the knockout mice. The TRα gene is complex as it encodes TRα 1, α 2 (which cannot bind T3), and rev-erbA (generated from the opposite strand encoding TRα ) (2, 16, 99). KO mice in which both TRα 1 and α 2 were deleted (TRα -/-) had a more severe phenotype with hypothyroidism, intestinal malformation, growth retardation, and early death shortly after weaning (145). T3 injection prevented the early death of pups. KO mice which lacked only TRα 1 (TRα 1-/-) had a milder phenotype with decreased body temperature and prolonged QT intervals on electrocardiograms (146). The phenotypic effects of the loss of TRα 1 are relatively mild (146-148). Unexpectedly, there is no evidence of resistance to TH, as occurs with the TRβ knockout. Disruption of the TRα 1 causes lower heart rates (19% reduced) and prolonged QRS and QT durations. These cardiac effects persist after hormone replacement. No changes were found in the levels of known TH-responsive genes in the heart (e.g., sarcoplasmic Ca2+ ATPase, Na+-K+ ATPase, β -adrenergic receptors). The bradycardic effect of the TRα 1 knockout may result from alterations in the sympathetic or parasympathetic nervous systems or it could result from an intrinsic defect in cardiac myocytes. The TRα 1-deficient mice also have a 0.5oC reduction in body temperature that is independent of TH levels. The mice have normal amounts of brown adipose tissue.

Samarut and co-workers have reported generation of short TRα isoforms from intronic transcriptional start sites which have dominant negative activity on TR function (73), and it is likely these short TRα isoforms are responsible for the more severe phenotype of the TRα -/- mice. In this connection, TRα KO mice which did not express either TRα 1 and α 2 (TRα o/o), had a milder phenotype than TRα -/- mice which expressed only the short TRα isoforms (17,18). Interestingly, TH stimulation of some target genes was increased, perhaps due to the absence of α 2 which inhibits normal TR-mediated transcription (149).

Targeted disruption of the TRβ locus created a mouse deficient in both TRβ 1 and TRβ 2 (17, 18). These mice had elevated circulating TSH and T4 levels, thyroid hyperplasia, as well as hearing defects (150, 151). These findings are similar to the index patients with resistance to TH who were later shown to have homozygous deletion of TRβ (4, 152). Thus, the mouse model appears to faithfully reproduce some of the features seen in humans with resistance to TH who are lack TRβ or express a dominant negative mutant TRβ (4). TRβ 2-selective knockout mice also have been generated and exhibited elevated levels of TH and TSH suggesting TRβ 2 plays the major role in regulating TSH (144). TRβ 2-selective knockout mice also have abnormal color discrimination and suggest TRβ 2 may play a role in cone development of the retina (153).

The relatively mild phenotypes of the TRα 1 and TRβ KO mice suggest the two isoforms have redundant roles in the transcriptional regulation of many target genes. In this connection, microarray studies of TRα and TRβ KO mice showed similar gene regulation profiles in the absence and presence of T3 (82). Surprisingly, when both TR isoforms were abolished, the resultant double knockout mice (TRα 1-/- TRβ -/-) were viable (108, 109). Thus, the absence of TRs is compatible with life. These mice had markedly elevated T4, T3, and TSH as well as large goiters. They also showed decreased growth, fertility, heart rate as well as bone density and development. Interestingly comparison of cDNA microarrays of double KO and hypothyroid mice showed only partial overlap of their gene regulation profiles, confirming the observation that the absence of receptor can give a different phenotype than lack of hormone. It is likely that basal transcription occurs even in the absence of receptor whereas basal repression of target genes occurs in the absence of hormone.

Cheng and colleagues have generated a “knockin” mouse model in which a mutant TRβ from a patient with RTH was introduced into the endogenous TRβ gene locus (154). These mice have a phenotype similar to patients with RTH, as the heterozygous mice showed elevated serum T4 and TSH, mild goiter, hypercholesterolemia, impaired weight gain, and abnormal bone development. Homozygous mice had markedly elevated serum T4 and TSH, and a much more severe phenotype than heterozygous mice. Wondisford and colleagues also have generated a “knockin” mouse expressing mutant TRβ (155). These mice had abnormal cerebellar development and function, and learning deficits. These latter studies suggest that expression of mutant TRβ under the control of endogenous TRβ promoter produces many of the clinical features of RTH in mice. This same group also recently developed a knock-in of a mutant TRβ that cannot bind DNA. This model should be useful in distinguishing signaling and developmental patterns due to protein-protein interactions of TRs (as well as non-genomic pathways) from those that require TR binding to TREs of target genes (156).