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In this paper, the authors report the structure of the TBLR1 gene …

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Discussions and conclusion
- TBLR1 regulates the expression of nuclear hormone receptor co-repressors

TBLR1 gene is expressed widely and Northern analysis, using sequences from the ORF as a probe, revealed size heterogeneity of the expressed mRNAs (3.9, 4.7 kb and ~7.3 kb). These are due to both the presence of alternatively spliced messages and the presence of multiple polyadenylation sites. The 4.7 kb form is more abundant in hematopoietic tissues. In addition to the heterogeneity in message size, the protein also has several isoforms. The isoforms are produced by the use of alternative splice donor sites. TBLR1β, differs from the original sequence at the carboxyl-terminus (3' end of the ORF). The mouse homologue shows mRNA size heterogeneity that is similar to the human RNA. Both the human and mouse genes have a complex and somewhat unusual genomic structure. The human gene contains 18 highly conserved exons spread over 230 kb of genomic DNA. The first and second exons are ~140 kb upstream of the third, which in turn, is separated from the fourth exon by 33 kb of intronic DNA. The translational start site is located an additional 11 kb downstream in the middle of the fifth exon. The gene also has an extremely long 3'UTR that includes a sequence deposited in GenBank as DC42. This is described as an intronless gene for which there is no independent evidence of expression and may not actually encode a functional message. Human TBLR1 maps to chromosome 3q26 while mouse TBLR1 is located between 3A2 and 3A3.

The broad evolutionary conservation of this gene family is of note. Homologs are present in yeast, plants, fish and flies as well as in mammals and the insect proteins are > 80% similar to the mammalian ones. No homology was detected to prokaryotic genomes.

The cDNAs for TBLR1 encode members of the β-transducin or WD-repeat family [22]. Eight WD-repeats are present in both the human and mouse proteins. The splice site that distinguishes between TBLR1α and TBLR1β is immediately 3' of the last WD-repeat sequence signature. At the nucleotide level, the sequence of TBLR1α has 79% homology to Homo sapiens mRNA for transducin (β)-1 like protein (TBL1) [GenBank: Y12781]. The only homology of TBLR1 and TBL1 to the prototypic signal transducing guanine nucleotide binding regulatory (G) protein β sub- unit is in the WD-repeat domains. TBL1 has been mapped to the X chromosome and deletions in the region containing TBL1 are associated with adult onset sensorineural deafness[23]. Both TBLR1 and TBL1 are homologous to a Drosophila protein called ebi The regions of maximal nucleotide conservation between TBL1 and ebi are at the N-terminal end of the molecule and in the WD40 repeats. The C-terminal ends of these proteins are not homologous. The carboxyl terminal exon of TBLR1α has some homology to several members of the Arp2/3 complex of proteins that control actin polymerization [24]. The equivalent region of TBLR1β has no homology to any known protein. The N-terminal 100 amino acids of TBLR1 co-precipitates with both HDAC3 and SMRT. The data indicate that TBLR1 binds directly to SMRT, which in turn binds HDAC3. SMRT binding of HDAC3 plays an important role in regulating chromatin structure and gene expression[5,25,26] The homologous region of TBL1 also co-precipitates with these co-repressors [9,10] and has been reported to form an effective transcriptional repressor. This portion of the molecule contains a LisH motif. LisH is a β-propeller-binding motif and is thought to mediate the dimerization of WD-40 proteins [27-29] Sif2p, the yeast homolog of TBLR1, is a tetramer and contains an unusual eight-bladed β-propeller structure, that apparently mediates tetramerization [30]. Mutations in conserved LisH amino acids significantly reduced the half-life of TBL1 and altered its intracellular localization. TBL1 mutated in the LisH domain was not imported into the nucleus [31]. Our data indicates that elimination of the LisH domain prevents interaction with SMRT.

The actual role that the TBLR1 family plays in cell physiology remains unclear. Like the proverbial blind man trying to describe an elephant, each laboratory has found a role for family members in their own system. Presumably, TBL1 and TBLR1 are multi-functional proteins. We found TBLR1 in a screen for messages over-expressed in early hematopoietic cells [16]. TBL1 deletions are associated with deafness [23]. The drosophila homolog, ebi has been reported to regulate epidermal growth factor receptor signaling [32] promote the degradation of a repressor of neuronal differentiation (Ttk88), and to limit S phase entry. A cDNA, described as the "human homologue of ebi" was reported to encode a protein that plays a role in the ubiquitin pathway [33]. More recently, both TBL1 and TBLR1 were reported to be required for transcriptional activators of nuclear hormone receptors and other regulated transcription factors [34]. Cofactor exchange through ubiquitin-dependent protein degradation was proposed to account for the transcriptional activation dependent on TBL1 and/or TBLR1. Yoon et al. [35] were unable to find any evidence for transcriptional activation in their studies, which postulated a role for TBL1/TBLR1 in histone code reading. Knock-down experiments using small interference RNA (siRNA) techniques indicate that TBL1 and TBLR1 are functionally redundant but essential for repression by unliganded thyroid hormone receptors [9]. Tomita et al. used the frog oocyte system to demonstrate that unliganded TBLR1 interacts with T3R and recruits TBLR1 to its chromatinized target promoter in vivo, accompanied by histone deacetylation and gene repression and showed that the recruitment of TBLR1 or related proteins is important for repression by unliganded T3R [11]. Yoon et al. have demonstrated that there is an additional site of interaction between a site near the most N-terminal of the WD-40 repeats of TBL1 and the RD4 region of N-Cor [9]. These interaction sites on SMRT flank the SANT motifs and it has been suggested that they play a role in interpreting the histone code and promoting histone deacetylation [36]. Expression of a TBLR1 fragment that contains this interaction site, but lacks the C-terminal two-thirds of the molecule including the WD-40 repeats, leads to a partial proliferation arrest, suggesting again, the multiplicity of roles that these family member play [18-21].

The proteins of the TBLR1 family also contain variant F-box near the N terminus of the molecule. F-box proteins, in combination with Skp1 and Cullin, form part of an E3 ubiquitin ligase that plays a critical role in the targeting of phosphorylated proteins for ubiquitination and subsequent proteasomal degradation. Because proteolysis is irreversible, proteasomal degradation provides a unidirectional regulatory switch. The initiation of DNA replication, chromosome segregation, and exit from mitosis are all triggered by the destruction of key regulatory proteins [37] [38-39] The F-boxes in TBLR1, TBL1 and ebi all lack a tryptophan near the NH2-end of the motif that is associated with Skp1 binding to F-box proteins that recognize phosphorylated protein [40]. A cDNA, described only as the "human homologue of ebi" encodes a protein that the authors believe plays a role in the ubiquitin mediated destruction of β-catenin [33] and they suggest that this ebi can bind Skp1 and is a key component in a pathway that targets unphosphorylated proteins for ubiquitination and subsequent proteasomal degradation [33] [41].

Ectopic expression of full-length TBL1 potentiates repression by unliganded T3R. TBL1 does not bind directly to T3R; it binds to SMRT and/or N-CoR and this interaction was reported to contribute an autonomous repression function to the complex. The methods used in these experiments could not distinguish among the possibilities; 1) that TBL1 changed the structure of N-CoR, perhaps by altering the accessibility of the SANT motifs, producing increased repression, 2) that it increased the quantity of N-CoR present in the target cells or 3) that it facilitated the recruitment of an additional co-repressor. We have shown that over expression of TBLR1 or either the C- or N-terminal truncates increases the expression of NHR co-repressors. Co-transfection of TBLR1 with SMRT(1-300) leads to greatly enhanced expression of a truncated SMRT in a transient expression model and cells in which TBLR1 is stably over expressed have elevated levels of endogenous N-Cor. Co-transfection with the wild type TBLR1 as well as both the N- and C-terminal truncates leads to a significant increase in both SMRT(1-300) and endogenous N-CoR expression. Thus, both ends of the molecule can independently contribute to co-repressor stabilization. Alternatively, the region between a.a. 80 and 175, and shard in all of our constructs) may play an autonomous role in increasing co-repressor expression. Since SMRT(1-300) is degraded via a ubiquitin-mediated mechanism and inhibition of ubiquitination leads to elevation of SMRT, our data suggest the possibility that TBLR1 may act by preventing the degradation of nuclear co-repressors. This stabilization appears to be part of a complex regulatory network that governs the level of unliganded nuclear receptor. Although this result is the opposite of what might have been predicted on the basis of the results described above for ebi-mediated targeting of β-catenin [33]both sets of data suggest that the TBLR1 family interacts with proteins subject to proteasomal degradation and the actual outcome, protection or targeting, may depend on either the nature of the target protein or the identity of the other proteins that interact with TBLR1. It may also be controlled by which of the TBLR1 family members does the interacting.

The mechanism through which the steady state level of SMRT and N-CoR expression is regulated is not obvious. The truncated construct of TBLR1 [TBLR1[δN] containing the C-terminal half of the molecule lacks the sequence involved in binding to the RD1 region of the co-repressors but has the greatest effect on both SMRT and N-CoR expression. Although this fragment contains most of the sequence that Yoon et al. suggested is responsible for binding the RD4 region of the co-repressor, the SMRT(1-300) fragment that we used lacks this RD4 region. Thus can not identify the binding partner responsible for the increased co-repressor levels found in cells over-expressing TBLR1. It may be that oligimerization of TBLR1 is required co-repressor stabilization and that a heteromeric multimer of a truncated form with the wild type may form a complex with SMRT that is an unsuitable substrate for proteasomal degradation. Pulse-chase experiments with co-transfected SMRT(1-300) suggest that ectopically expressed TBLR1 [δN] stabilizes SMRT (data not shown) but the pulse chase experiments do not provide an explanation for the increased level of SMRT expression associated with co-transfection of either the N-terminal fragment of TBLR1 or the whole molecule.

The fluorescence studies show that the intracellular distribution of TBLR1 is influenced by the metabolic state of the cells. The increase in nuclear staining may either represent translocation to the nucleus or nuclear trapping. In either case, it is likely that the TBLR1 that accumulates in the nucleus is degraded with different kinetics than the cytoplasmic material and that differential localization as well as alterations in proteasomal targeting, are the explanation for the effect of increased TBLR1 expression on co-repressor levels.

These results suggest that the TBL1-TBLR1 family plays a role in the regulation of N-Cor and/or SMRT expression; the amino terminal end docks the molecule to the co-repressor, perhaps altering its intracellular localization and targeting it for degradation. The carboxyl end appears to contribute to this activity but also appears to be the source of tissue specificity.

In summary, we have identified TBLR1 as a transcriptional regulator interacting with the co-repressors of nuclear hormone receptor activity. We have cloned the gene encoding this protein and identified it as a member of a small family of proteins that include at least two isoforms encoded by the same gene and closely related, X- and Y linked proteins, called TBL1X and TBL1Y. The evidence suggests that these proteins act by altering the stability of the co-repressor complex and that they may also contribute, or recruit other proteins that provide, an autonomous repressor function.

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