Alternative mRNA Splicing of SMRT Creates Functional Diversity by Generating Corepressor Isoforms with Different Affinities for Different Nuclear Receptors

Abstract

Many eukaryotic transcription factors are bimodal in their regulatory properties and can both repress and activate expression of their target genes. These divergent transcriptional properties are conferred through recruitment of auxiliary proteins, denoted coactivators and corepressors. Repression plays a particularly critical role in the functions of the nuclear receptors, a large family of ligand-regulated transcription factors involved in metazoan development, differentiation, reproduction, and homeostasis. The SMRT corepressor interacts directly with nuclear receptors and serves, in turn, as a platform for the assembly of a larger corepressor complex. We report here that SMRT is expressed in cells by alternative mRNA splicing to yield two distinct variants or isoforms. We designate these isoforms SMRT␣ and SMRT and demonstrate that these isoforms have significantly different affinities for different nuclear receptors. These isoforms are evolutionarily conserved and are expressed in a tissue-specific manner. Our results suggest that differential mRNA splicing serves to customize corepressor function in different cells, allowing the transcriptional properties of nuclear receptors to be adapted to different contexts.Nuclear receptors are transcription factors that play multiple roles in metazoan development and physiology (1-6). Nuclear receptors operate by binding to specific promoter elements on DNA and by modulating transcription of adjacent target genes in response to hormone ligand (3, 7-9). The nuclear receptors include, among others, the thyroid hormone receptors (TRs), 1 the retinoic acid receptors (RARs), and the retinoid X receptors (RXRs) (3-5, 7, 10, 11). Each of these receptors localizes to the nucleus and binds to DNA in both the absence and presence of hormone ligand. These receptors can repress transcription of their target genes in the absence of hormone, but activate target gene transcription upon binding to hormone agonist (3, 7-9, 12, 13). This bimodal transcriptional regulation is accomplished through a hormone-regulated exchange of a corepressor complex, found on the nuclear receptor in the absence of hormone, for a coactivator complex recruited in the presence of hormone agonist (14). Corepressor and coactivator protein complexes regulate transcription through direct interaction with the basal transcription machinery and through modification of chromatin structure (15).Both activation and repression are essential for correct receptor function. For example, RAR-mediated repression is required for appropriate anterior/posterior segregation in vertebrates, and disruption leads to aberrant head formation during murine development (16). TR-mediated repression is required for correct Xenopus larval development, and abrogation of repression leads to premature metamorphosis (17). Aberrations in the regulation of repression can result in human disease. For example, resistance to thyroid hormone syndrome, an inherited endocrine disorder, has been mapped to mutations in TRs that disrupt the hormone-...

Thyroid hormone receptors mutated in liver cancer function as distorted antimorphs

“…We determined if the HCC mutations altered the relative affinity of TRb1 for SMRT versus N-CoR. As shown in prior studies (Zamir et al, 1997;Goodson et al, 2005), wild-type TRb1 interacted more weakly with GST-SMRT than with GST-N-CoR in the absence of hormone, but released from both corepressors at comparable T3 concentrations (Figure 2b, solid line). Interestingly, the affinities of the TRb1 HCC mutants for SMRT versus N-CoR differed.…”

“…Wild-type and mutant TRs were also cloned into pFastBac1 (Invitrogen, Carlsbad, CA, USA) using EcoRI restriction sites for the TRb1 clones and EcoRI (5 0 ) and HindIII (3 0 ) for the TRa1 clones, and wild-type TRa1 and TRa1-I were cloned into pCI-neo using EcoRI (5 0 ) and NotI (3 0 ). The pSG5-wild-type TRb1, pSG5.2 expression vector, DR4-thymidine kinase promoter (TK)-luciferase reporter, AP-1 collagenase luciferase reporter, pRSV-c-jun, pGEX-MPa-SRC1, pGEX-MPc-SMRT and pGEX-N-CoR constructs were previously described (Weinberger et al, 1986;Sharif and Privalsky, 1992;Wong and Privalsky, 1998;Jonas and Privalsky, 2004;Goodson et al, 2005).…”

EBV nuclear antigen EBNALP dismisses transcription repressors NCoR and RBPJ from enhancers and EBNA2 increases NCoR-deficient RBPJ DNA binding

“…RBPJ is in repressive complexes containing NCoR1 and -2, SKIP, and SHARP (17)(18)(19)(20)(21)(22)(23). NCoR recruits class I and II histone deacetylases (HDAC), including HDAC3, which repress transcription and associate with matrix-associated deacetylase (MAD) bodies, subnuclear sites of repressed transcription (24,25).…”

Multiple mutations contribute to repression by the v-Erb A oncoprotein

“…Whereas c-Erb A binds N-CoR more strongly than SMRT (Cohen et al, 1998(Cohen et al, , 2000(Cohen et al, , 2001Webb et al, 2000;Makowski et al, 2003;Goodson et al, 2005;Zamir et al, 1997), we found that v-Erb A displayed a nearequal interaction with SMRT as with N-CoR. Restoring the c-Erb A helix 12 to v-Erb A restored the c-Erb A preference for N-CoR.…”

“…As anticipated, both v-Erb A SMRT corepressor is closely related to N-CoR but differs in its affinity for different nuclear receptors (Privalsky, 2004). Although alternative mRNA splicing can produce corepressors with differing receptor affinities, c-Erb A generally interacts more strongly with NCoR than with SMRT (Zamir et al, 1997;Cohen et al, 1998Cohen et al, , 2000Cohen et al, , 2001Webb et al, 2000;Makowski et al, 2003;Goodson et al, 2005). To examine this question for v-Erb A, we tested SMRT constructs in place of NCoR in our EMSA supershifts.…”