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(The FASEB Journal. 2007;21:488-496.)
© 2007 FASEB

A mechanism for mutational inactivation of the homeodomain protein TGIF in holoprosencephaly

Nathalie Ferrand*, Christine Demange*, Céline Prunier{dagger}, Su Ryeon Seo* and Azeddine Atfi*,1

* INSERM U 673, Hôpital St-Antoine, Paris, France; and

{dagger} Department of Cell Biology, Lerner Research Institute, Cleveland, Ohio, USA

1Correspondence: INSERM U 673, Hôpital St-Antoine, 184 Rue du Faubourg St-Antoine, 75571, Paris Cedex 12, France. E-mail: atfi{at}st-antoine.inserm.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The homeodomain protein TGIF functions as a negative modulator for multiple classes of transcription factors. Loss of function mutations in a single copy of TGIF result in holoprosencephaly, a developmental anomaly leading to severe forebrain and craniofacial malformations. However, the mechanisms by which these mutations disrupt the functions of TGIF remain to be elucidated. Here we show that a holoprosencephaly mutation (P63R) interferes with the ability of TGIF to act as a corepressor for c-Jun and Smad2, suggesting that this holoprosencephaly mutation may lead to a general defect in the TGIF protein. In fact, we observed that the P63R mutation affects folding of the TGIF protein, resulting in the disruption of the diffuse nuclear staining pattern characteristic of wild-type (WT) TGIF and the accumulation of TGIF in nuclear aggregates. We also show that the mutant TGIF.P63R is degraded more rapidly when compared with WT TGIF and that this degradation occurs through the ubiquitin-proteasome pathway. Furthermore, we observed that TGIF.P63R homodimerizes with WT TGIF to sequester it into nuclear aggregates and to enhance its ubiquitin-dependent degradation. These results reveal an important mechanism for the degradation of TGIF through the ubiquitin-proteasome pathway, whose deregulation might contribute to the development of human holoprosencephaly.—Ferrand, N., Demange, C., Prunier, C., Seo, S. R., Atfi, A. A mechanism for mutational inactivation of the homeodomain protein TGIF in holoprosencephaly.


Key Words: nuclear aggregates • severe forebrain and craniofacial malformations • transcriptional repression • ubiquitin-dependent degradation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HOLOPROSENCEPHALY (HPE) IS THE most common congenital structural forebrain anomaly in humans during development. HPE is associated with mental retardation and craniofacial malformations. The etiology of HPE is very heterogeneous and comprises environmental factors and genetic causes. At least 12 different loci have been associated with HPE, and several distinct human genes for HPE have been identified (1) . One of the identified genes codes for the TG-interacting factor (TGIF), a member of the TALE superfamily of homeodomain proteins. TGIF originally identified by its ability to bind a specific retinoid receptor (RXR) response element; it has been suggested to repress transcription by competing with retinoid receptors for binding to DNA (2) . Subsequently, TGIF was shown to function as an important negative regulator in the transforming growth factor ß (TGF-ß) signaling pathway (3) . This pathway is initiated when the type I receptor, activated by the type II receptor, phosphorylates Smad2 and Smad3. Phosphorylated Smad2 and Smad3 associate with Smad4 and the complexes translocate to the nucleus, where they act as transcriptional activators for TGF-ß-responsive genes (4 5 6) . The mechanism of TGIF-mediated inhibition of TGF-ß signaling has been attributed primarily to the ability of TGIF to recruit to Smad2, a transcriptional repressor complex containing histone deacetylases (3) . Recently we reported that TGIF can also associate with the E3 ubiquitin ligase Tiul1 (for TGIF-interacting ubiquitin ligase 1) to target Smad2 for ubiquitin-dependent degradation, suggesting that TGIF may repress TGF-ß signaling through distinct mechanisms (7) . In addition to inhibiting TGF-ß signaling and RXR, it has been shown that TGIF binds directly to c-Jun to repress its transcriptional activity (8) . These observations suggest that TGIF acts to repress transcription in multiple regulatory pathways either as a DNA binding repressor or as a corepressor in association with other DNA binding proteins.

TGIF defects associated with HPE are deletions of a single copy of the TGIF gene with no loss of heterozygosity or missense mutations, which might result in a loss of function (1) . However, the physiological relevance of alterations of TGIF in promoting HPE is still unknown. Here we show that a mutant form of TGIF (TGIF.P63R) identified in human HPE (9) is constitutively degraded, and this decrease in stability occurs as a result of TGIF ubiquitination with subsequent degradation through the proteasome. We found that TGIF.P63R associates with wild-type (WT) TGIF to enhance its ubiquitin-dependent degradation. These studies reveal a mechanism for HPE whereby genetic defects in TGIF induce its degradation through the ubiquitin-mediated pathway.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Expression vectors
Expression vectors for Myc-Smad2, Gal4-Smad2, hemagglutinin (HA)-c-Jun, His-Ub, RasV12, HA-TßRI, HA-TßRI.act, G5E1B-Lux, activating protein (AP)1-Lux, ARE3-Lux, FAST1, HA-TGIF, GFP-TGIF, Myc-TGIF were previously described (8 , 10 , 11) . The dominant-negative mutants of E2 ubiquitin ligases—His-UbcH5a.DN, His-UbcH5b.DN, His-UbcH5c.DN, His-UbcE20K.DN—were a gift from Dr. K. Iwai. Expression vectors for GFP-TGIF.P63R or Flag-TGIF.P63R were constructed using the pEGFPC2 vector (Clontech, Palo Alto, CA, USA) or pcDNA3-Flag (kindly provided by Dr. K. Miyazono) and the TGIF cDNA from HA-TGIF.P63R, a gift from Dr. M. Meunke. A polymerase chain reaction (PCR) -based procedure with HA-TGIF as a template was used to generate Myc-TGIF.N(1–164), Myc-TGIF.C(108–272), Flag-TGIF.N(1–164), and Flag-TGIF.C(108–272).

Cell lines and gene expression analysis
COS-7, HepG2, and 293 cells were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS) and 5 mM glutamine. For luciferase reporter assays, cells were plated to semiconfluency and 24 h later transfected with expression vectors by the LipofectAMINE method. To induce the Gal4-Smad2 or ARE3-Lux reporters, cells were either left untreated or treated with human TGF-ß1 (Sigma, St. Louis, MO, USA) at 2 ng/ml for 16 h. Luciferase activity was measured using the luciferase assay system described by the manufacturer (Promega, Madison, WI, USA) and normalized for transfection efficiency using an expression vector coding for Renilla luciferase.

Immunoprecipitation and immunoblotting
Cells were transfected with expression vectors by the LipofectAMINE method; 48 h post-transfection they were lysed at 4°C in TNMG buffer (20 mM Tris-HCl [pH 8], 150 mM NaCl, 5 mM MgCl2, 10% glycerol, 0.5% Nonidet P-40, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, and 20 µg/ml leupeptin). Lysates were subjected to immunoprecipitation with the indicated antibody for 2 h, followed by adsorption to Sepharose-coupled protein G for 1 h. The beads were washed five times in lysis buffer, and samples were analyzed by SDS-PAGE and immunoblotting. To determine total protein levels, aliquots of cell lysates were subjected to direct immunoblotting.

Pulse-chase analysis
For experiments examining the metabolic stability of TGIF or TGIF.P63R, cells were preincubated for 1 h in Met/Cys-free DMEM and pulsed for 30 min with 100 µCi/ml [35S]-Met/Cys. Cells were then washed extensively with DMEM and chased for the indicated periods in DMEM/0.2% FCS. Samples were subjected to immunoprecipitation with anti-hemagglutinin antibody and the immunoprecipitates were resolved by SDS-PAGE, visualized by PhosphorImager, and quantified.

Immunofluorescence
COS-7 cells were transfected with the indicated expression vectors by the LipofectAMINE method. After 48 h, the dishes were fixed in 4% paraformaldehyde for 30 min at room temperature and permeabilized in 0.1% triton X-100. Cells were incubated overnight at 4°C with the primary antibody, washed, and incubated with Texas Red-conjugated secondary antibody. The slides were mounted and examined on a Leica fluorescence microscope.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The HPE mutation P63R affects the folding of TGIF
To begin to understand the mechanisms underlying the loss of TGIF function in HPE, we focused our analyses on a missense mutation P63R found in human HPE (9) . Initially, we tested the potential effects of this HPE mutation on repression of c-Jun-activated transcription using an AP1-Lux reporter (12) . In contrast to WT TGIF (TGIF.WT), expression of TGIF.P63R had little or no ability to repress c-Jun-induced transcription (Fig. 1 A). In the course of these analyses, we also investigated the effect of TGIF.P63R on Smad2-mediated transcriptional activation by using Gal4-Smad2 and the reporter G5E1B-Lux. As shown in Fig. 1B , the presence of the P63R mutation in TGIF suppressed the ability of TGIF to repress TGF-ß-induced Smad2 transcriptional activity. Similar results were obtained when the effect of TGIF.P63R on the reporter ARE3-Lux, which is sensitive to TGF-ß-activated Smad2 (13 , 14) , was examined (Fig. 1C ).


Figure 1
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  		Figure 1. The HPE mutation P63R affects the repressive function of TGIF. A) HepG2 cells were cotransfected with AP1-Lux alone or together with c-Jun in either the absence or presence of TGIF.WT or TGIF.P63R. B) HepG2 cells were cotransfected with Gal4-Smad2 together with G5E1B-Lux in either the absence or presence of TGIF.WT or TGIF.P63R. Cells were treated with (black bars) or without (open bars) TGF-ß for 16 h prior to lysis. C) HepG2 cells were cotransfected with ARE3-Lux together with FAST1 in either the absence or presence of TGIF.WT or TGIF.P63R. Cells were treated with (black bars) or without (open bars) TGF-ß for 16 h prior to lysis. In all cases (A–C), cell lysates were analyzed for luciferase activity. Luciferase activity was normalized and expressed as the mean ± SD of triplicates from a representative experiment performed at least three times.

To determine the basis for the inactivating effects of the P63R mutation, we investigated whether it could disrupt the colocalization of TGIF with c-Jun or Smad2. Analysis of cells transfected with TGIF.WT and c-Jun revealed that both TGIF and c-Jun localized in the nucleus, and that the distribution of TGIF was indistinguishable from cells transfected with TGIF alone (Fig. 2 A). In cells transfected with TGIF.P63R, the localization of c-Jun was normal, whereas TGIF.P63R accumulates in intranuclear aggregates (Fig. 2A ). A similar localization of TGIF.P63R was observed in cells transfected with Smad2 and a constitutively activated form of the TGF-ß type I receptor (TßRI) (15) , which induces accumulation of Smad2 in the nucleus (Fig. 2B ). These findings suggest that the mutation may lead to disruption of the diffuse nuclear pattern of the TGIF protein.


Figure 2
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  		Figure 2. The HPE mutation P63R affects the folding of the TGIF protein. A) COS-7 cells were transfected with the indicated combinations of GFP-TGIF.WT, GFP-TGIF.P63R, and HA-c-Jun. 48 h after transfection, cells were fixed and stained with anti-hemagglutinin antibody. The localization of HA-c-Jun (red) or GFP-TGIF mutants (green) was visualized by a fluorescence microscope. B) COS-7 cells were transfected with Myc-Smad2 together with a constitutively activated form of the TGF-ß type I receptor in either the presence or absence GFP-TGIF.WT or GFP-TGIF.P63R. Cells were stained with anti-Myc antibody as in panel A; localization of Smad2 (red) or TGIF (green) was visualized by a fluorescence microscope. C) COS-7 cells were transfected with HA-TGIF.WT or HA-TGIF.P63R and cell lysates were immunoblotted with anti-hemagglutinin antibody. D) COS-7 cells were transfected with GFP-TGIF.WT or GFP-TGIF.P63R in the presence or absence of the constitutively active Ras (Ras.V12) and cell lysates were immunoblotted with anti-GFP antibody.

To more carefully assess the inactivating effects of P63R, we transfected TGIF mutants into COS7 cells and investigated their expression by Western blot. Similar to previous observations (3) , we noted that TGIF.WT migrated as a doublet of 30–35 kDa. Introduction of P63R into TGIF resulted in a significant decrease in the steady-state levels of the protein (Fig. 2C ), which is consistent with the hypothesis that this mutation may affect the integrity of the TGIF protein.

Previous studies have shown that activation of the Ras-ERK (extracellular-regulated kinase) signaling pathway causes phosphorylation of TGIF, leading to stabilization of the TGIF protein (16) . Thus, we investigated whether the mutation P63R might interfere with the ability of the Ras-ERK pathway to stabilize TGIF. Figure 2D showed that expression of a constitutively activated form of Ras, Ras.V12, resulted in the accumulation of the upper form of TGIF.WT, the stable form of the protein (16) . In contrast, we were unable to detect any alterations in the levels of TGIF.P63R in cells coexpressing Ras.V12 (Fig. 2D ). These results suggest that the mutation P63R may disrupt the regulation of TGIF stabilization by the Ras-ERK pathway.

The P63R mutant of TGIF causes an increased rate of protein degradation
To determine whether the decreased level of TGIF.P63R was due to an enhanced turnover of the TGIF mutant protein, we monitored the rate of degradation of TGIF.WT or TGIF.P63R by pulse-chase analysis. As shown in Fig. 3 , the level of newly synthesized TGIF.P63R was indistinguishable from that of TGIF.WT, suggesting that TGIF.P63R and TGIF.WT are translated at similar rates. Steady-state levels of WT TGIF remained constant throughout the time course of the experiment (Fig. 3) . In contrast, the newly synthesized TGIF.P63R protein disappeared more rapidly when compared with TGIF.WT (Fig. 3) . Thus, it is likely that the mutation P63R accelerates the turnover of the TGIF protein.


Figure 3
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  		Figure 3. The mutant P63R increased turnover of the TGIF protein. 293 cells transfected with HA-TGIF.WT or HA-TGIF.P63R were pulse labeled with [35S]-Met/Cys and chased for the times indicated. Then cell lysates were immunoprecipitated with anti-hemagglutinin antibody, and [35S]-labeled proteins were visualized by PhosphorImager and quantified. Labeled proteins are plotted at each time point as the percentage of the amount present at time 0.

The P63R mutant of TGIF is degraded through the ubiquitin-proteasome pathway
To approach the question of how P63R enhances the turnover of TGIF, we evaluated the involvement of the ubiquitin-proteasome pathway in this process by investigating the effect of the proteasome inhibitor MG132 on protein stability. As shown in Fig. 4 A, treatment of cells with MG132 caused an increase in the steady-state level of TGIF.P63R. To provide further evidence that TGIF.P63R is targeted for degradation through the ubiquitin-proteasome pathway, various dominant-negative E2 ubiquitin-conjugating enzymes were tested for their ability to block the ubiquitin-dependent degradation of TGIF.P63R. These mutant enzymes contain catalytic site mutations and have been shown to behave in a dominant-negative manner (17) . We noted that expression of dominant-negative mutants of UbcH5a, Ubc5Hb, and Ubc5Hc caused an increase in the steady-state levels of TGIF.WT or TGIF.P63R (Fig. 4B ). This effect was specific to the UbcH5 family because transfection of the dominant-negative mutant UbcE20K was accompanied by a decrease in the expression level of both TGIF.WT and TGIF.P63R (Fig. 4B ). We also tested whether the mutation P63R enhances ubiquitination of TGIF. As shown in Fig. 4C , little or no ubiquitinated forms of TGIF.WT were detected in control cells. However, exposure of cells to MG132, which prevents the degradation of ubiquitinated proteins, led to a slight accumulation of ubiquitin-TGIF.WT conjugates. Under these experimental conditions, TGIF.P63R yielded highly ubiquitinated forms of the mutant protein, reminiscent of its rapid degradation (Fig. 4C ).


Figure 4
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  		Figure 4. The mutant TGIF.P63R is degraded through the ubiquitin-proteasome pathway. A) 293 cells were transfected with HA-TGIF.WT or HA-TGIF.P63R. Before lysis, cells were either left untreated or were treated with MG132 (10 µM) for 6 h. Cell lysates were immunoblotted with anti-hemagglutinin antibody. B) 293 cells were transfected with HA-TGIF.WT or HA-TGIF.P63R, and the indicated dominant-negative mutants of E2 ligases were tagged with His. Cell lysates were immunoblotted with anti-hemagglutinin or anti-His antibodies. C) 293 cells were transfected with HA-TGIF.WT or HA-TGIF.P63R in the presence or absence of His-Ub. Before lysis, cells were either left untreated or treated with MG132 (10 µM) for 6 h. Cell lysates were immunoprecipitated with anti-His antibody and immunoblotted with anti-hemagglutinin antibody. D) COS-7 cells were transfected with GFP-TGIF.WT or GFP-TGIF.P63R in the presence or absence of Myc-Ub. Localization of TGIF mutants (green) or Ub (red) was visualized by a fluorescence microscope. Colocalization of TGIF or TGIF.P63R with Ub appears as yellow.

Protein ubiquitination is a feature of nuclear aggregates found in several human diseases, including Huntington and Parkinson (18) . To provide further evidence that TGIF.P63R is constitutively ubiquitinated, we examined the colocalization of TGIF.P63R with ubiquitin by immunofluorescence analysis. In cells coexpressing TGIF.WT, we observed that TGIF was localized exclusively in the nucleus whereas ubiquitin was distributed throughout the cell, with cytoplasmic staining being stronger than nuclear staining (Fig. 4D ). However, when TGIF.P63R was expressed, ubiquitin was found predominantly in the nucleus and localized extensively with TGIF.P63R in nuclear aggregates (Fig. 4D ). Thus, it is likely that the nuclear aggregates of TGIF.P63R are ubiquitinated.

Homodimerization of TGIF
As both TGIF.P63R and TGIF.WT transcripts were expressed in the HPE sample (9) , we speculated that TGIF.P63R might interfere with the function of the WT allele. At first we looked for possible homodimerization of TGIF. Immunoprecipitation of cell lysates from transfected 293 cells with an antibody directed against Myc-TGIF.WT revealed the presence of HA-TGIF.WT, which was absent in a control transfection in which only HA-TGIF.WT was expressed (Fig. 5 A), indicating that TGIF can undergo homodimerization. As the P63R mutation localizes to the N domain of TGIF, we analyzed in more detail the homodimerization of TGIF by transfecting 293 cells with the N domain or C domain of TGIF.WT tagged with Flag or Myc. As shown in Fig. 5B , the isolated C domain interacted with itself but not with the N domain. Furthermore, the N domain of TGIF failed to interact with itself, suggesting that the homodimerization of TGIF is mediated primarily by the C domain, which is not affected by the P63R mutation. Next, we examined whether TGIF.P63R could form a complex with TGIF.WT. To minimize the difference in expression levels between TGIF.WT and TGIF.P63R, we transfected various amounts of expression vectors to obtain steady-state levels of the mutant proteins closer to that of endogenous TGIF (data not shown). Under these conditions, we observed that TGIF.P63R retained its capacity to undergo homodimerization with TGIF.WT (Fig. 5C ).


Figure 5
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  		Figure 5. Homodimerization of TGIF. A) 293 cells were transfected with the indicated combinations of HA-TGIF.WT and Myc-TGIF.WT. Cell lysates were immunoprecipitated with anti-Myc antibody and immunoblotted with anti-hemagglutinin antibody. In all the following experiments, the total expression levels of proteins were determined by immunoblotting with the appropriate antibody. B) 293 cells were transfected with the indicated combinations of Myc-TGIF.N, Myc-TGIF.C, Flag-TGIF.N, and Flag-TGIF.C. Cell lysates were immunoprecipitated with anti-Myc antibody and immunoblotted with anti-Flag antibody. C) 293 cells were transfected with Myc-TGIF.WT together with HA-TGIF.WT or HA-TGIF.P63R. Cell lysates were immunoprecipitated with anti-Myc antibody and immunoblotted with anti-hemagglutinin antibody.

The P63R mutant of TGIF induces constitutive degradation of the WT allele
Our previous analysis indicated that TGIF.P63R is constitutively degraded through the ubiquitin-proteasome pathway. This finding prompted us to consider whether TGIF.P63R might increase the accessibility of TGIF.WT to the ubiquitin-proteasome machinery, facilitating its degradation. In fact, coexpression of TGIF.P63R resulted in an ~ 3-fold increase in the ubiquitination of TGIF.WT (Fig. 6 A). Confirmation of these results was obtained by experiments showing that expression of TGIF.P63R can induce an ~ 3-fold decrease in the expression levels of WT TGIF (Fig. 6B ). This increased ubiquitin-dependent degradation of the WT allele does not occur as a result of homodimerization between two molecules of TGIF because expression of two different tagged TGIF.WT failed to increase the ubiquitination of each other (data not shown). These results suggest that TGIF.P63R may engage TGIF.WT in a complex that is constitutively targeted by the ubiquitin-proteasome pathway.


Figure 6
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  		Figure 6. TGIF.P63R engages WT TGIF in a complex that is constitutively targeted by the ubiquitin-proteasome pathway. A) 293 cells were transfected with the indicated combinations of HA-TGIF.WT, GFP-TGIF.P63R, and His-Ub. Cells were either left untreated or treated with MG132 (10 µM) for 6 h and cell lysates were immunoprecipitated with anti-His antibody and immunoblotted with anti-hemagglutinin antibody. B) 293 cells were transfected with the indicated combinations of Myc-TGIF.WT, HA-TGIF.P63R, and Myc-Smad2 (used as an internal control). Cell lysates were immunoblotted with anti-Myc or anti-hemagglutinin antibodies. Analysis of the Myc-Smad2 expression showed that equal amounts of proteins were loaded in the gel. C) COS-7 cells were transfected with HA-TGIF.WT alone or together with GFP-TGIF.P63R. 48 h after transfection, the cells were fixed and stained with anti-hemagglutinin antibody. Localization of HA-TGIF.WT (red) or GFP-TGIF.P63R (green) was visualized by a fluorescence microscope. Colocalization of TGIF.WT with TGIF.P63R appears as yellow.

The second approach we employed to investigate whether the expression of TGIF.P63R might have an effect on the function of the WT allele took advantage of the finding that the TGIF.P63R mutant forms nuclear aggregates. Thus, we examined the localization of TGIF.WT in the presence or absence of TGIF.P63R. In cells expressing TGIF.WT alone, TGIF immunoreactivity exhibits diffuse nuclear staining (Fig. 6C ). However, when TGIF.P63R was coexpressed, TGIF.WT was found predominately in nuclear aggregates and colocalized extensively with TGIF.P63R (Fig. 6C ). The ability of TGIF.P63R to sequester TGIF.WT in nuclear aggregates was observed in > 90% of the detectable coexpressing levels of TGIF.P63R. Together with our observation that TGIF.P63R can form a stable complex with TGIF.WT, these results suggest that TGIF.P63R may function to recruit TGIF.WT in nuclear aggregates, perhaps away from some of TGIF’s targets.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In humans, deletion or mutation of a single copy of the TGIF gene causes holoprosencephaly, a developmental anomaly leading to severe forebrain and craniofacial malformations (1) . One missense mutation (P63R) of TGIF identified in human holoprosencephaly results in the alteration of proline 63 to arginine. An indication that this mutation leads to the loss of TGIF function came from gene analysis using the TGF-ß-responsive reporter 3TP-Lux, which contains elements from the plasminogen activator inhibitor I (PAI-1) (9 , 19) . However, it is not clear whether this mutation results in holoprosencephaly due to defects in the TGF-ß pathway or in other TGF-ß-independent responses. In this study we show that P63R interferes with the ability of TGIF to act as a transcriptional corepressor for both c-Jun and Smad2, suggesting that this HPE mutation may lead to a general defect in the TGIF protein. Consistent with this, we observed that P63R causes an accumulation of TGIF in nuclear aggregates and facilitates its targeting for degradation through the ubiquitin-proteasome pathway (Fig. 7 ). These results suggest a new mechanism for the loss of function of TGIF in human holoprosencephaly.


Figure 7
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  		Figure 7. Schematic diagram illustrating inactivation of TGIF by the holoprosencephaly mutation P63R. The homeodomain protein TGIF functions as an important negative modulator for the TGF-ß signaling pathway through association with the transcription factor Smad2, which is directly activated by the TGF-ß receptor at the cell membrane. In addition to inhibition of TGF-ß signaling, TGIF can inhibit other classes of transcription factors, the most characterized of which is c-Jun, which functions in a variety of signaling pathways. In our proposed model, P63R interferes with the ability of TGIF to act as a transcriptional corepressor for both c-Jun and Smad2, suggesting that this holoprosencephaly mutation may lead to a general defect in the TGIF protein. In support of this notion, P63R mutation affects the folding of the TGIF protein, resulting in the accumulation of TGIF in nuclear aggregates, perhaps away from some TGIF’s target genes. The general defect of the TGIF protein generated by the P63R mutation is also manifested by findings that the mutant TGIF.P63R is constitutively degraded through the ubiquitin-proteasome pathway.

Several lines of evidence support the conclusion that the mutation P63R causes constitutive degradation of TGIF protein through the ubiquitin-proteasome pathway. First, examination of the half-life of TGIF demonstrates that the mutant TGIF.P63R is degraded more rapidly compared with the WT counterpart. Second, this decrease in protein stability was accompanied by a similar decrease in steady-state levels of the mutant TGIF protein. Third, treatment of cells with MG132 or transfection of dominant-negative mutants of E2 ligases yielded an increase in the steady-state levels of TGIF.P63R. Finally, the mutant TGIF.P63R is highly ubiquitinated when compared with TGIF.WT. Thus, enhancing ubiquitination of TGIF could contribute to the loss of the ability of TGIF to repress transcription in response to TGF-ß signaling or other signal inputs. On the other hand, the severe defects in brain and craniofacial development caused by TGIF.P63R suggest that the mutant of TGIF may not be regulated by modifiers that normally bring the levels over the threshold needed for proper development. In these respects, it should be noted that epidermal growth factor, via the Ras-Erk pathway, causes phosphorylation of TGIF, leading to its stabilization (16) . We observed here that expression of a constitutively activated Ras, RasV12, had little or no effect on the steady-state levels of TGIF.P63R while enhancing the stabilization of TGIF.WT. Based on these findings, it is reasonable to suggest that the mutation P63R may escape from regulation by environmental cues that normally ensure the stabilization of TGIF.

The ubiquitin conjugation system plays an important role in several biological processes, and alterations in the activity of the ubiquitin system have been implicated in several human disorders (20 21 22) . Ubiquitin conjugation to a protein substrate involves a cascade of enzymatic reactions including E1, E2, and E3. Among these, the E3 ubiquitin ligases play a crucial role in determining substrate specificity and subsequent degradation by the 26S proteasome (20 21 22) . Recently, we identified the E3 ubiquitin ligase Tiul1 as a partner of TGIF that targets active components of the TGF-ß signaling pathway for degradation (7) . TGIF does not seem to be targeted for degradation by Tiul1, but rather functions to form with Smad2 and Tiul1 a stable complex from which Tiul1 then induces ubiquitin-dependent degradation of Smad2. In several attempts we were unable to see an effect of Tiul1 on the ubiquitin-dependent degradation of TGIF or TGIF.P63R, ruling out the possibility that Tiul1 functions as an ubiquitin ligase that mediates constitutive degradation of the mutant TGIF.P63R found in human holoprosencephaly. The E3 ubiquitin enzymes involved in the degradation of TGIF and/or TGIF.P63R remain to be defined. In any case, our data with the proteasome inhibitor MG132 and the dominant-negative mutants E2 strongly suggest that the constitutive degradation of TGIF.P63R occurs through ubiquitin-mediated degradation.

The paradigm for the role of certain genes in human diseases is that they are trans-acting and recessive at the cellular level (i.e., loss of one allele has no effect on cell function) and homozygous inactivation is required for the development of diseases. However, there is increasing evidence that several mutated genes are not recessive at the level of the cell. For example, humans who are heterozygous for the WT allele of p53 developed cancer with very high frequency and often at an early age (23) . In the heterozygous state, mutations in the APC gene may promote excessive proliferation of the colon epithelium (24) . In human colon cancer, only one of the two Smad2 alleles is mutated; such a mutant allele encodes a Smad2 protein with a point mutation (Smad2.P445H) that interacts with WT Smad2, resulting in its retention in the cytoplasm (11) . The findings outlined in the present study provide new insights into how a mutant allele can act in a dominant fashion through its WT counterpart in human diseases. In fact, we demonstrate that TGIF.P63R can homodimerize with TGIF.WT to increase its accessibility to the ubiquitin-proteasome machinery. We also demonstrate that the homodimerization of TGIF.P63R with TGIF.WT leads to sequestration of the WT allele in nuclear aggregates. Thus, by functioning to induce the constitutive degradation and mislocalization of the WT allele, TGIF.P63R could generate devastating defects in human development, leading to holoprosencephaly. However, it remains unclear whether the P63R mutation causes human holoprosencephaly due to loss of TGIF functions or produces novel abnormal functions, because recent works have shown that target disruption of the TGIF gene in mice did not result in holoprosencephaly (25 26 27) . Tgif–/– mice were viable, fertile, and indistinguishable from WT littermates, suggesting a possible functional redundancy of TGIF potentially provided by the closely related TGIF2. Based on these observations, it is tempting to speculate that expression of the P63R mutant may have a dominant-negative effect that will not only eliminate normal TGIF function, but also interfere with the function of TGIF2 or other proteins. Therefore, it will be interesting to determine whether having mutated forms of the TGIF protein may be more detrimental than a complete loss of the protein.


   ACKNOWLEDGMENTS
 
This work was supported by INSERM, Centre National de la Recherche Scientifique (CNRS), la Ligue contre le Cancer Comité de Paris, and ARC (Association pour la Recherche sur le Cancer).

Received for publication August 23, 2006. Accepted for publication August 29, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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