Several regions of the Saccharomyces cerevisiae genome are subject to position-dependent transcriptional repression mediated by a multi-component nucleosome-binding complex of silent information regulator proteins (Sir2p, Sir3p and Sir4p). These proteins are present in limiting amounts in the nucleus and are targeted to specific chromosomal regions by interaction with sequence-specific DNA-binding factors. Different sites of repression compete for Sir complexes, although it is not known how Sir distribution is regulated. In a screen for factors that interact with Sir4p amino terminus, SIF2 has been cloned; this protein encodes a WD40-repeat-containing factor that disrupts telomeric silencing when overexpressed. In contrast to deletion of SIR4, SIF2 deletion improves telomeric repression, suggesting that under normal conditions Sif2p antagonizes Sir4p function at telomeres. Sif2p overexpression alters the subnuclear localization of Sir4p, but not its protein expression level, suggesting that Sif2p may recruit Sir4p to nontelomeric sites or repression. The sif2 mutant strains are hypersensitive to a range of stress conditions, but do not have decreased viability and do not alter repression in the rDNA. In conclusion, Sif2p resembles the Sir4p regulatory proteins Sir1p and Uth4p in that it competes for the functional assembly of Sir4p at telomeres, yet unlike Sir1p or Uth4p, it does not target Sir4p to either mating-type or rDNA loci (Cockell, 1998).
A novel gene, transducin (beta)-like 1 (TBL1, Drosophila homolog: Ebi), has been identified in the Xp22.3 genomic region, that shows high homology with members of the WD-40-repeat protein family. The gene contains 18 exons spanning approximately 150 kb of the genomic region adjacent to the ocular albinism gene (OA1) on the telomeric side. However, unlike OA1, TBL1 is transcribed from telomere to centromere. Northern analysis indicates that TBL1 is ubiquitously expressed, with two transcripts of approximately 2.1 kb and 6.0 kb. The open reading frame encodes a 526-amino acid protein, which shows the presence of six beta-transducin repeats (WD-40 motif) in the C-terminal domain. The homology with known beta-subunits of G proteins and other WD-40-repeat containing proteins is restricted to the WD-40 motif. Genomic analysis has revealed that the gene is either partly or entirely deleted in patients carrying Xp22.3 terminal deletions. The complexity of the contiguous gene-syndrome phenotype shared by these patients depends on the number of known disease genes involved in the deletions. Interestingly, one patient carrying a microinterstitial deletion involving the 3' portion of both TBL1 and OA1 shows the OA1 phenotype associated with X-linked late-onset sensorineural deafness. An involvement of TBL1 in the pathogenesis of the ocular albinism with late-onset sensorineural deafness phenotype is postulated (Bassi, 1999).
The corepressor SMRT mediates repression by thyroid hormone receptor (TR) as well as other nuclear hormone receptors and transcription factors. A novel SMRT-containing complex has been isolated from HeLa cells. This complex contains transducin beta-like protein 1 (TBL1), whose gene is mutated in human sensorineural deafness. It also contains HDAC3, a histone deacetylase not previously thought to interact with SMRT. TBL1 displays structural and functional similarities to Tup1 and Groucho corepressors, sharing their ability to interact with histone H3. In vivo, TBL1 is bridged to HDAC3 through SMRT and can potentiate repression by TR. Intriguingly, loss-of-function TRbeta mutations cause deafness in mice and humans. These results define a new TR corepressor complex with a physical link to histone structure and a potential biological link to deafness (Guether, 2000).
Evidence is presented that both corepressors SMRT and N-CoR exist in large protein complexes with estimated sizes of 1.5-2 MDa in HeLa nuclear extracts. Using a combination of conventional and immunoaffinity chromatography, a SMRT complex has been isolated and histone deacetylase 3 (HDAC3) and transducin (beta)-like I (TBL1), a WD-40 repeat-containing protein, have been identified as the subunits of the purified SMRT complex. The HDAC3-containing SMRT and N-CoR complexes can bind to unliganded thyroid hormone receptors (TRs) in vitro. In Xenopus oocytes, both SMRT and N-CoR also associate with HDAC3 in large protein complexes and injection of antibodies against HDAC3 or SMRT/N-CoR leads to a partial relief of repression by unliganded TR/RXR. These findings thus establish both SMRT and N-CoR complexes as bona fide HDAC-containing complexes and shed new light on the molecular pathways by which N-CoR and SMRT function in transcriptional repression (Li, 2000).
Destruction of ß-catenin is regulated through phosphorylation-dependent interactions with the F box protein ß-TrCP. A novel pathway for ß-catenin degradation was discovered involving mammalian homologs of Drosophila Sina (Siah), which bind ubiquitin ß-conjugating enzymes, and Ebi, an F box protein that binds ß-catenin independent of the phosphorylation sites recognized by ß-TrCP. A series of protein interactions were identified in which Siah is physically linked to Ebi by association with a novel Sgt1 homolog SIP that binds Skp1, a central component of Skp1-Cullin-F box complexes. Expression of Siah is induced by p53, revealing a way of linking genotoxic injury to destruction of ß-catenin, thus reducing activity of Tcf/LEF transcription factors and contributing to cell cycle arrest (Matsuzawa, 2001).
A pathway linking the RING protein Siah-1 to the F box protein Ebi has been mapped and it has been shown that Ebi can bind ß-catenin. Unlike ß-TrCP, however, which requires GSK3ß-mediated phosphorylation of ß-catenin on serine 33 and serine 37, Ebi interacts with ß-catenin independently of these phosphorylation sites. Also, the Siah binding protein SIP associates with complexes containing Ebi but not ß-TrCP, suggesting differences compared to previously characterized E3 ubiquitin ligase complexes, where E2 enzymes are supplied via Cullin-mediated interactions with RING-containing proteins such as Rbx-1/Roc-1. Recent identification of interactions between Siah-1 and the ß-catenin binding protein APC suggest that this scaffold protein represents a point of common intersection of the Wnt and Siah-1 pathways for ß-catenin degradation (Matsuzawa, 2001).
Two alternative pathways for regulation of ß-catenin levels are presented, involving different F box proteins (Ebi versus ß-TrCP). One pathway is initiated by increases in the expression of Siah-family proteins, which can be induced, for example, by p53 in response to DNA damage, and involves sequential protein interactions with SIP, Skp1, and Ebi. Ebi binds ß-catenin, thus recruiting it to the Siah-1-SIP-Skp1 complex for polyubiquitination and subsequent proteosome-mediated degradation. Siah-1 binds the E2 UbcH5. The other pathway is regulated by Wnt signals (Dsh) and possibly PI3K/Akt. This pathway is phosphorylation dependent and involves GSK3ß-induced phosphorylation of Ser-33 and Ser-37 on ß-catenin, allowing ß-TrCP binding, resulting in recruitment of ß-catenin to Skp1-Cullin-1- ß-TrCP complexes (SCF). Cullin-1, in collaboration with other proteins, supplies this SCF complex with E2s, such as UbcH3. APC is required for both pathways as a scaffold protein, binding ß-catenin via one domain and also binding Siah-1 and GSK3ß (Matsuzawa, 2001).
In the fly, Sina recruits E2s to Phyllopod/Tramtrack complexes, targeting Tramtrack for ubiquitination. The ebi-gene product also binds Tramtrack and promotes its degradation in vitro and when expressed in insect cells in culture. Loss-of-function mutations of ebi cause Tramtrack accumulation and prevent R7 cell differentiation. Similar to ß-TrCP, the ebi gene of Drosophila encodes an F box/WD-40-repeat protein with sequence homology to Cdc4 (yeast), Sel-10 (C. elegans), and Slimb (Drosophila), suggesting that it provides a functional connection between a Sina-regulated pathway and SCF complexes. How this linkage between Sina and SCF complexes is achieved, however, has been unclear (Matsuzawa, 2001).
The finding that SIP functions as a molecular bridge between the human homologs of Sina and the SCF-component Skp1 provides evidence of a physical linkage between components of these two ubiquitin ligase systems, thus corroborating the genetic evidence from Drosophila that these two pathways for targeted protein degradation interact. The Drosophila ortholog of SIP is also capable of bridging the fly Skp1 and Sina proteins in three-hybrid experiments. Thus, an evolutionarily conserved network of protein interactions exists in which Siah-1 (Sina) binds to SIP, which in turn binds to Skp1, which binds Ebi (Matsuzawa, 2001).
p53 can induce expression of Siah-family genes in mammals, establishing p53 as one factor capable of invoking Siah-dependent pathways for protein degradation. Siah-family proteins are normally maintained at a relatively low level through ubiquitination-dependent protein turnover, where human Siah-1 and Siah-2 promote their own degradation through interactions of their RING domains with E2s. This therefore suggests that activation of p53 leads to a burst of Siah-1 mRNA and protein production, triggering the Siah/SIP/Skp1/Ebi pathway for ß-catenin degradation. In contrast to Siah-family proteins, it seems unlikely that SIP, Skp1, or Ebi are limiting components of this pathway, since overexpression of them has little effect on ß-catenin levels (Matsuzawa, 2001).
Though p53-mediated degradation of ß-catenin correlates with cell cycle arrest, it remains to be established whether these events are functionally linked. Activation of Tcf/LEF-family transcription factors by ß-catenin is known to induce expression of cyclin D1, c-myc, and other genes important for cell proliferation, making it plausible that ß-catenin degradation is linked to p53-mediated cell cycle arrest. However, given the role established for the cyclin-dependent kinase inhibitor p21Waf1 in mediating G1 arrest induced by p53, it is unclear whether a parallel pathway for ß-catenin degradation would be required. Circumstances have been described where p53 fails to induce cell cycle arrest despite inducing p21Waf1 expression, raising the question of whether p21Waf1 is necessary but insufficient for p53-mediated G1 arrest. Recently, a genetic interaction between ebi and p21Waf1 has been identified using an assay in Drosophila where flies are engineered to ectopically express human p21Waf1 in the developing eye disc (Boulton, 2000). Specifically, mutant alleles of ebi abrogated inhibition of S phase entry by p21Waf1, implying a need for Ebi in p21-mediated cell cycle arrest. Flies with mutant ebi also display ectopic S phases and overproliferation phenotypes (Boulton, 2000), further implying a role for ebi in growth suppression. Defects in cell cycle arrest in ebi mutants, however, do not necessarily implicate ß-catenin/Armadillo. For example, p53 can induce degradation of c-Myb through a proteosome-dependent mechanism partly mediated by Siah (Tanikawa, 2000). Thus, Ebi may have other targets in addition to ß-catenin that are relevant to mechanisms of p53-mediated cell cycle arrest. Future experiments should explore whether the fly homolog of p53 is linked to an ebi-dependent pathway for cell cycle arrest entailing degradation of Armadillo. In the M1 cell model, p53 induces both G1 arrest and apoptosis. Though Ebi(DeltaF)-expressing M1 cells may exhibit some delay in p53-induced apoptosis, this could result indirectly because of failed G1 arrest. Moreover, Siah-1 often fails to induce apoptosis when overexpressed in cells. However, links of Siah to apoptosis can occur under some circumstances, as demonstrated by the observation that coexpression of Siah-1 with a Siah binding protein Pw1/Peg3 causes apoptosis, whereas neither Siah-1 nor Pw1/Peg3 alone are sufficient. Mutations affecting components of the Wnt-signaling pathway are commonly observed in human cancers, resulting in aberrant accumulation of ß-catenin and activation of Tcf/LEF-target genes. Wnt-family ligands, frizzled-family receptors, and the signaling proteins downstream of these define one mechanism for regulating ß-catenin levels. However, additional inputs into pathways controlling ß-catenin turnover have recently been identified, including a mitogen-activated protein kinase pathway involving a Tak1 homolog and Nemo-like kinases in C. elegans and a cell adhesion-dependent pathway involving integrin-linked kinase. The findings reported here reveal yet another pathway for regulating ß-catenin levels that is linked at least in part to p53-dependent responses to genotoxic injury. It is speculated that loss of p53 or components of the Siah/SIP/Skp/Ebi pathway for ß-catenin destruction may contribute to aberrant ß-catenin accumulation in cancers (Matsuzawa, 2001).
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