TRATAMIENTO DE LOS DATOS

The primary mechanism of TH action is the modulation of gene expression via binding of T3 to specific TH nuclear receptors (TR; reviewed in Hollenberg 1998, Zhang & Lazar 2000). TR are encoded by two genes, c-erbAa and c-erbAP, located on human chromosomes 17 and 3 respectively. Alternative splicing of 3' exons of c- erbAa results in mRNAs encoding three proteins, including the T3-binding T R al, as well as non T3-binding variants c-erbAa2 and c-erbAa3. The non-coding strand of c- erbAa gives rise to Rev-erbAa, which lacks TH binding activity. Alternative 5'-exon usage of c-erbAP gives rise to mRNAs encoding TR^l, TRp2, as well as the newly identified protein, TR|33 (Williams 2000), all of which bind T3. Truncated protein products of the c-erbAa 2ind c-erbAP genes (termed c-erbAAal, c-erbAAa2 and

TRAp3) also exist and may function as transcriptional repressors (Chassande et al. 1997, Williams 2000).

TR-dependant regulation of transcription is mediated by interaction of the receptor with a TH response element (TRE) located within the promoter of target genes (reviewed in Zhang & Lazar 2000). TREs consist of two hexamer half sites with the consensus sequence AGGTCA. The two half sites may be arrange as palindromes (PAL; AGGTCA - TGACCT), inverted palindromes (IPL; TGACCT - AGGTCA) or direct repeats (DR; AGGTCA-AGGTCA). TR bind to a TRE either as homodimers or as heterodimers, usually with a retinoid X receptor (RXR) isoform. Homodimers form mostly in the absence of T3, when they tend to bind DR and IPL and act to repress basal transcription. Binding of T3 causes dissociation of homodimers and promotes heterodimer formation, and results in T3-dependent regulation of target genes; either stimulation of transcription of positively regulated genes or repression of transcription of negatively regulated genes. In addition to TR and basal transcription factors such as RNA polymerase II, TATA binding protein (TBP) and TBP-associated proteins (TAF^s), efficient activation of T3-dependent transcription also requires nuclear co-activator proteins.

1.5.1.1 Nuclear co-regulators

Current findings suggest the existence of co-regulator complexes involved in the regulation of transcription by nuclear receptors (McKenna et al. 1999). Most co­ regulators are ubiquitously expressed, albeit at differing concentrations in each tissue

(O’Malley et al. 2001). Consequently, TH responsiveness of a particular cell type may be partly dependent on its complement of co-regulators.

Several co-activator proteins capable of interacting with TR in the presence of T3 have been identified (Hollenberg 1998, Zhang & Lazar 2000). Thyroid hormone receptor interacting protein-1 (TRIP-1), the first TR coactivator to be described, has been implicated in stabilisation of the TR, possibly through interaction with TBP (Lee et at. 1995, Fraser et al. 1997). CREE (cAMP response element-binding protein) binding protein (GBP) promotes transcription via its histone acetyltransferase activity, which disrupts chromatin structure, allowing recruitment of transcription machinery to the target gene (Ogryzko et al. 1996, Bannister & Kouzarides 1996). GBP interacts with TR either directly or via association with members of the steroid receptor co-activator (SRG) family of co-activators {e.g. AGTR, SRG-1), some of which also posses histone acetyltransferase activity (Zhang & Lazar 2000). The importance of SRG-1 in T3 action is demonstrated by the observation that mice lacking functional SRG-1 exhibit T3 resistance (Weiss et al. 1999). SRG-1 is expressed in fetal rat brain before the onset of fetal thyroid function, possibly in order to mediate maternal TH action via TR in this tissue (lannacone et al. 2002)

The binding of TR to TRE in the absence of ligand results in silencing of gene expression on genes that are positively regulated by T3 (reviewed in Hollenberg 1998, Zhang & Lazar 2000). Silencing activity of the unliganded TR has been attributed to co-repressor proteins that bind to TR in the absence of ligand. Two such proteins, nuclear receptor corepressor (NGoR) and silencing mediator of retinoic acid and thyroid hormone receptors (SMRT), interact with TR in vitro in the absence of ligand (Zamir et al. 1997a, Gohen et al. 1998). Both co-repressors form a ternary complex with other co-repressors, mSin3A and mSin3B and the histone deacetylases HD AGI and mRPD3 (Alland et al. 1997, Nagy et al. 1997). Histone deacetylation gives rise to a more compact chromatin structure, resulting in the repression of transcription. Small unique corepressor (SUN-GoR) also potentiates repression by TR, possibly through interaction with the orphan receptor Rev-erbAa (Zamir et al.

1997b). Of the various co-repressors, NcoR is expressed in fetal brain and thus may be involved in regulating the action of unliganded TR during early brain development (lannacone et al. 2002).

1.5.1.2 Orphan receptors

The exact function of the non-T3 binding variants is unknown. It has been suggested that c-erbAa2may serve as a negative regulator of T R a l, TR|3l and TR^2by competing for binding to TRE (Koenig et al. 1989). However, whilst c-erbAa2 is present in brain throughout development and in TH-responsive cultures (Section 1.5.2),

it is not certain whether its cellular distribution in vivo is similar to that of the T3- binding isoforms. The orphan receptor, chicken ovalbumin upstream promoter transcription factor (COUP-TF) represses TR mediated transcription by competing for TRE (Cooney et al. 1993). COUP-TF binds to the promoter region of the gene encoding Purkinje cell protein-2 (PCP-2) in vitro and represses T3-dependent activation of this gene (Anderson et al. 1998). Furthermore, COUP-TF is specifically expressed in the immature fetal and neonatal Purkinje cell and its expression diminishes as PCP-2 expression becomes TH-sensitive (Anderson et al. 1998). COUP-TF also competes with TR for RXR, thus limiting TR-RXR heterodimer formation (Cooney et al. 1993). Nevertheless, certain genes are TH-responsive from fetal to adult stages irrespective of c-erbAa2 or COUP-TF expression (Section 1.6.2).