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* Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal;
Program in Neurogenetics, Department of Neurology, David Geffen School of Medicine-UCLA, Los Angeles, USA;
UnIGENe, Institute for Molecular and Cell Biology, Porto, Portugal; and
Department of Biology, University of Utah, Salt Lake City, Utah, USA
1Correspondence: Life and Health Sciences Research Institute, Health Sciences School, University of Minho, Campus de Gualtar, 4710–057 Braga, Portugal. E-mail: pmaciel{at}ecsaude.uminho.pt
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Key Words: polyglutamine disorders • ubiquitin-proteasome pathway • microarray • ataxia • knockout
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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, 2)
. MJD is caused by the expansion of a CAG tract in the ATXN3 gene, which encodes for ataxin-3, a 42 kDa protein that resides in both the nucleus and cytoplasm, and has also been associated with the nuclear matrix (3
4
5)
.
MJD belongs to a group of human disorders known as polyglutamine (polyQ) diseases, which includes Huntington’s disease (HD), spinal and bulbar muscular atrophy (SBMA), dentatorubropallidoluysian atrophy (DRPLA), and other spinocerebellar ataxias such as SCA1, 2, 6, 7, and 17 (6)
. All these disorders are caused by a CAG trinucleotide expansion in the coding sequence leading to an expanded polyQ tract within the protein. The polyQ expansion increases protein misfolding and makes them prone to aggregate in nuclear and cytoplasmic ubiquitinated inclusions (7)
. A toxic gain of function due to the polyQ expansion has been proposed as a common pathogenic mechanism underlying the polyQ diseases (8)
. However, despite the ubiquitous expression of both normal and disease proteins, only a particular subset of neurons is selectively affected in each disease, emphasizing the importance of studying native protein function to help elucidate the basis of neurodegeneration (9)
. For example, studies of 
1A voltage-dependent calcium channel involvement in SCA6 and the androgen receptor (AR) in SBMA suggested that the pathology and selective neuronal vulnerability are partially related to the normal function of the proteins and not just a general effect of polyQ expansion (10
11
12)
. Moreover, although MJD is considered a pure dominant disorder, homozygous patients with two mutant alleles have been reported to show a more severe and earlier onset disease phenotype (13
14
15)
, raising the possibility that the normal function of ataxin-3 may modulate disease.
Ataxin-3 interacts with VCP/p97, a conserved AAA ATPase that appears to be responsible for shuttling a large number of polyubiquitinated proteins to the proteasome (16
17
18)
. Human ataxin-3 binds polyubiquitinated proteins, associates with the proteasome, and exhibits deubiquitinating (DUB) activity against K-48 polyubiquitin chains, suggesting a role in the ubiquitin-proteasome pathway (UPP) (17
, 19
20
21)
. DUB enzymes are a heterogeneous group of proteins capable of removing ubiquitin from ubiquitinated substrates, hence regulating various biological processes (22)
.
It has also been suggested that ataxin-3 might be involved in transcription repression (23)
. Ataxin-3 binds CBP, p300, and PCAF and can inhibit transcription by these coactivators. Ataxin-3 can bind histones through its josephin domain and inhibit their acetylation, thus repressing transcription (23)
.
The analysis of evolutionarily conserved orthologous genes in simple genetic models can play an important role in assessing the function of human genes. In this study, we identified the Caenorhabditis elegans ATXN3 orthologue and characterized its pattern of expression, developmental regulation, and subcellular localization. We demonstrate that C. elegans ataxin-3 has DUB activity, dependent on the conserved putatively catalytic cysteine. We also extensively analyzed the first known animal model for ataxin-3 deficiency: two C. elegans strains with deletions in the atx-3 gene. We show that despite no overt phenotype, both strains share deregulation of genes involved in the UPP, structure, motility, and signal transduction.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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25
26)
.
Animal culture and observation of phenotypes
Animals were maintained at 20°C in NGM or peptone plates (27)
. Both atx-3 knockout strains were outcrossed five times. Gene deletion of atx-3(gk193) was confirmed by single worm polymerase chain reaction (PCR) using nested primers atx3_EL, atx3_ER, atx3_IL, and atx3_IR. Deletion of strain atx-3(tm1689) was confirmed using primers atx3_EL2, atx3_ER2, atx3_IL2, and atx3_IR2 (supplemental Table 1). Lack of protein was confirmed by Western blot using anti-303JSP (described below). Synchronous cultures were obtained by bleaching (27)
.
Animals were observed with a Zeiss SV11 stereoscope or a Zeiss Axio Imager D1 microscope to detect anatomic, behavior or other visible alterations.
Cloning and sequence analysis of C. elegans atx-3 cDNA
The expressed sequence tag (EST) clone yk77c4 corresponding to atx-3 cDNA was sequenced and the missing 5'end was obtained using the RACE system (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s instructions. Using random primers and the enzyme SuperScript II RT (Invitrogen), cDNA was obtained from total RNA. The PCR reaction was performed using the reverse primer ceNde and SL1 primer with an additional PstI restriction site (supplemental Table 1). The amplified fragment was subcloned into NdeI and PstI restriction sites of the initial yk77c4 construct, and the full-length cDNA plasmid obtained was confirmed by sequencing.
GFP reporter strains and colocalization studies
We analyzed strain BC14364, an integrated transgenic strain with GFP under the control of the putative atx-3 promoter (2 kb upstream of atx-3 start codon).
In addition, we created and analyzed a strain named ATX-3::GFP that has ATX-3 fused with the GFP under the control of 5 kb of the putative ATX-3 promoter. To obtain this reporter strain, genomic DNA was amplified using Expand Long Template PCR System (Roche, Nutley, NJ, USA). Primers were designed to amplify the full-length atx-3 gene and its 4.5 kb upstream region using PROMF and PROMR (supplemental Table 1), including an additional BamHI restriction site. The resulting fragment was digested by HindIII/BamHI and cloned into the pPD95–77 vector (Fire vectors) in-frame with the GFP gene. The construct was confirmed by sequencing and coinjected with the lin-15 (pbLH98) marker into the lin-15(n765) strain (28)
; a stable extrachromosomal transgenic line was obtained, which we named ATX-3::GFP.
ATX-3::GFP animals were washed several times with M9 buffer and incubated with 4',6'-diamidino-2-phenylidole (DAPI) diluted in ethanol (150 nmol/ml) for 30 min. After this incubation, worms were rehydrated with M9, placed in agarose pads, and visualized under a microscope Olympus BX61. Images were analyzed and merged using the microscope software.
Antibody production and immunoblotting analysis
The anti-ataxin-3 rabbit polyclonal antibody, anti-303JSP (supplemental Fig. 1), was generated against the peptide containing the last 14 amino acids (ERFEKKKEERNDEK) of the worm protein and affinity purified (Davids Biotechnologies).
For regular immunoblotting, worms were collected from plates with M9 buffer, resuspended in lysis buffer (100 mM Tris, pH=6.8, 2% SDS, 15% glycerol), and boiled for 5 min. The lysate was centrifuged and the supernatant was saved. Protein concentration was determined by the Bradford method and 75 µg of total protein extracts were separated in 10% SDS-PAGE. Blots were probed with anti-303JSP (1:1000) and monoclonal anti-actin (1:2500; Sigma) as primary antibodies and anti-rabbit (1:10,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-mouse (1:5000; Santa Cruz) as secondary antibodies. Detection was performed by chemiluminescence (Pierce, Rockford, IL, USA).
Nuclear and cytosolic (S100) extracts were prepared as described previously (29)
. Cytosolic extracts (75 µg) and 200 µg of the nuclear fraction were separated in a 10% SDS-PAGE gel. Blots were probed with anti-303JSP (1:200), monoclonal anti--tubulin (1:5000; Santa Cruz), or anti-histone H3 (1:500, Upstate Biotechnology, Lake Placid, NY, USA) as primary antibodies and anti-rabbit (1:10,000; Santa Cruz) or anti-mouse (1:5000; Santa Cruz) as secondary antibodies. Detection was performed by chemiluminescence (Pierce).
Expression and purification of recombinant His-tagged ATX-3
The atx-3 full cDNA was cloned into the pDEST17 vector containing a His6-tag (Invitrogen). Mutagenic clones were obtained using the Quick-Change Site Direct Mutagenesis kit (Stratagene, San Diego, CA, USA) following described protocol (30)
. Primer sequences are described in supplemental Table 1. Cysteine C20 replacement by alanine was obtained using primer C20A_hisATX-3F. The clone of the protein corresponding to the josephin domain was obtained using primer J1_hisATX-3F. Serine to alanine mutations in UIM1 and in UIM2 were obtained using primers S232A_hisATX-3F and S260A_hisATX-3F, respectively. Recombinant protein was obtained by heterologous expression in Escherichia coli BL21SI strains, as described (31)
. Proteins were then purified using the His-Trap Kit according to the manufacturer’s conditions (Amersham, Arlington Heights, IL, USA).
Deubiquitination assays
Five hundred nanograms of recombinant ATX-3 were incubated with 500 ng of K48 polyubiquitin chains (Affinity, Neshanic Station, NJ, USA; Biomol, Plymouth Meeting, PA, USA) in 20 µl of assay buffer (50 mM HEPES, 0.5 mM EDTA, 1 mM DTT, 0.1 mg/ml BSA, and protease inhibitor cocktail (Roche)) for 16 h at 30°C. Reactions were stopped by adding SDS buffer and proteins were separated in a 4–20% SDS-PAGE gel. After transfer, the membrane was probed with monoclonal antiubiquitin FK2 (1:2000; Affinity, Biomol) and secondary anti-mouse (1:10,000; Santa Cruz).
Microarray experimental design and data analysis
Synchronous cultures of N2, atx-3(gk193), and atx-3(tm1689) were grown at 20°C; when animals reached adulthood, they were washed off the plates with M9, pelleted, and frozen. Total RNA was isolated with Trizol (Invitrogen); quality was assessed with the Agilent Bioanalyzer (Santa Clara, CA, USA), and 500 ng of total RNA was amplified and labeled using the Agilent Low RNA Input Linear Amplification Kit. Each of three independent paired knockout and wild-type replicate comparisons were hybridized onto the C. elegans oligo microarray from Agilent; dye swap was performed to eliminate the influence of dye bias for a total of 12 arrays. Arrays were scanned with the Agilent scanner and images were analyzed using Agilent Feature Extraction (V.8.0) software. Data were read into R/Bioconductor and normalized (Lowess) between arrays. A linear model (Limma package in Bioconductor) to the data and a contrast analysis were performed. Differentially expressed (DE) genes were selected using a Bayesian approach with a false discovery rate of 0.1%. The union of DE genes between the two ko strains was 290. Genes were categorized combining gene description, Gene Ontology, and Interpro description since Gene Ontology alone was insufficient (poorly annotated). This information was obtained using Biomart (http://www.biomart.org/). Human gene orthologues of worm DE genes were obtained in R/Bioconductor using Wormbase best blastp hits file (ftp://ftp.wormbase.org/pub/wormbase/acedb/current_release/best_blastp_hits.WS158.gz). Of 290 initial genes, we obtained 140 putative human protein orthologues. Enrichment analysis was done using David software (http://david.niaid.nih.gov/david/ease.htm) and performing 
2 statistical analysis.
Quantitative and semiquantitative real-time PCR
Primers were designed using Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) (supplemental Table 2). Two micrograms of three independent samples (distinct from those used for hybridizations) of each strain were DNase treated (Promega, Madison, WI, USA) and converted into cDNA using iScript (Bio-Rad, Hercules, CA, USA) following standard protocols from the manufacturers.
PCR reactions included Platinum Sybr Green qPCR SuperMix-UDG with ROX (Invitrogen), 3 µl of 10-fold diluted cDNA, and 0.6 µM of each primer. Each reaction was run in triplicate in an ABI PRISM 7900 HT system. Fold change was calculated using the delta-delta method (32)
with act-1 and hprt as reference genes (supplemental Table 2).
For semiquantitative PCR, atx-3 and act-1 were amplified in the same reaction following described protocol (33)
. The PCR product was separated in a 2% agarose gel, visualized with AlphaImager (Alpha Innotech Corporation, San Leandro, CA, USA), and analyzed densitometrically with the corresponding AlphaEase software. Atx-3 expression levels were normalized using actin.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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). The corresponding gene, atx-3, is located on chromosome V; it consists of 1.3 kb and comprises four exons. The C. elegans atx-3 gene does not contain the repetitive (CAG)n tract present in humans. ATX-3 consists of a conserved josephin domain (7–193 residues) and, near the C-terminal, has two ubiquitin-interacting motifs (UIMs) (Fig. 1A
, B). Using PSORTII, we found a putative bipartite nuclear localization signal (NLS) that is also found in the homologous proteins of other species (Fig. 1A
, B). Based on PSORTII analysis of subcellular localization, ATX-3 has a 43% probability of having a nuclear localization and a 35% probability of being in the cytoplasm.
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Caenorhabditis elegans ATX-3 has deubiquitinating activity
Domain similarity strongly suggests a conserved functional role (or roles) between worm and human ataxin-3. The catalytic amino acid triad (C, H, and N), characteristic of human ataxin-3 and several ubiquitin proteases, was also present in worm ATX-3 (Fig. 1B
). To test its function directly, we expressed recombinant worm ATX-3 and assessed its ability to cleave K48 polyubiquitin chains, a common substrate for DUB assays. Like human ataxin-3, ATX-3 was able to cleave K48 polyubiquitin chains in vitro and had higher activity, with larger chains containing four or more ubiquitin molecules (Fig. 1C
). Mutations in the predicted catalytic cysteine C20, corresponding to C14 in humans, inhibited this activity (Fig. 1C
). Moreover, we noted that the josephin domain itself was capable of cleaving polyubiquitin chains and that mutations in the UIMs did not seem to interfere with the DUB function of ATX-3 (Fig. 1C
). However, total ubiquitination levels in atx-3 mutant strains did not appear to be different from wild-type, which suggests compensatory mechanisms or substrate specificity (data not shown).
Atx-3 is expressed widely throughout development
We next investigated where and at which life stages atx-3 was expressed in worms. The C. elegans atx-3 expression pattern was determined using strain BC14364, which has an integrated transgene containing the GFP gene under the control of 2 kb upstream of the atx-3 start codon; and strain ATX-3::GFP, which has the worm ATX-3 protein fused to a GFP-tag. Young embryos did not present a GFP signal, but expression was observed in late embryogenesis and at all stages of postnatal development: eggs, L1, L2, L3, L4, and adults (Fig. 2
A, data not shown). Adult animals showed stronger expression than did larval stages and eggs; this was also proved by RT-polymerase chain reaction (RT-PCR) (Fig. 2B
), suggesting potential developmental dynamics in atx-3 function. Both transgenic strains had a generalized expression pattern, with a strong signal in the spermatheca and vulval muscle (Fig. 2A
; data not shown). We observed high fluorescence in neuronal dorsal and ventral cord and neurons of the head and tail. Expression was also observed in the hypoderm, body muscles, and coelomocytes.
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Colocalization studies using DAPI staining of the ATX-3::GFP transgenic strain showed that ATX-3 is present in both the nucleus and cytoplasm (Fig. 2C
); however, our subcellular fractioning and Western blot analysis of wild-type animals shows that ATX-3 is more abundant in the cytoplasm (Fig. 2D
).
Atx-3 loss of function mutants have no overt phenotype
In this work, we characterized two atx-3 knockout strains: atx-3(gk193) and strain atx-3(tm1698). Atx-3(gk193) has a 287 bp deletion of the putative promoter region and 79 bp of the first exon. Atx-3(tm1698) has a 660 bp deletion covering most of the gene (Fig. 3
A). Both strains were outcrossed five times and the deletions were confirmed by nested PCR (Fig. 3B
). Absence of the protein was determined by Western blot using our anti-303JSP antibody (Fig. 3C
, Supplemental Fig. 1).
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These two strains are, to our knowledge, the first known animals lacking the protein ataxin-3. The animals were viable and showed no obvious morphological abnormalities. The life span of the mutants, as well as brood size, did not differ from wild-type (Supplemental Fig. 2), which suggests that the atx-3 gene is not essential for embryonic development or adult life.
Detailed microscopic analysis revealed no defects in cell number and position. Locomotion and other behavioral aspects such as feeding, defecation, touch sensitivity, heat stress, avoidance, and chemotaxis assays appear to be normal (data not shown; Supplemental Figs. 3, 4).
Transcriptomic profile of atx-3 knockout strains
Considering the transcriptional repressor role proposed for ataxin-3 and the lack of an evident phenotype, we performed a large-scale microarray analysis to test the hypothesis that we would observe a molecular phenotype for the mutant animals that might provide insight into ATX-3 function.
The Agilent platform used in this study comprises >20,000 predicted genes, which correspond to the whole C. elegans genome. Adult animals were selected since they showed higher expression of ATX-3. We selected three independent RNA samples of each knockout strain to be hybridized with three matched wild-type samples and performed dye swap. Applying conservative criteria to identify only those genes with the clearest differential expression (Bayesian approach, false discovery rate=0.1%), we identified 290 differential expressed (DE) genes shared by the two knockout strains compared with wild-type (Fig. 4
, supplemental Table 3). The array data were consistent and variance was extremely low. Although the two knockout strains can be considered independent replicates of each other, we performed real-time PCR to provide additional confirmation evidence. Not surprisingly, 8/8 genes were further confirmed using independent samples (Fig. 5
B).
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These 290 DE genes, which correspond to 1.4% of the worm genome, are likely to be biologically relevant because they were deregulated similarly in both knockout strains. Of these, 253 were up-regulated and 37 were down-regulated. To understand the biological significance of the overall changes in gene expression, DE genes were categorized based on their concise description (Wormbase), Gene Ontology, and Interpro description. Half of the DE genes (143) had unknown function. For some of the other 147 genes, the precise biological role is not known yet. However, we were able to annotate their putative function based on indirect evidence such as expression pattern, RNAi screens, protein structural domains, and orthology/homology with other species. Analysis of the annotated DE genes shows a clear deregulation of several biological processes common to both atx-3 mutants: cell structural/motility components (50%), signal transduction (20%), and the UPP (8%) (Fig. 5A
). Other genes (22%) were involved in diverse metabolic pathways and were not specifically categorized.
Ubiquitin-proteasome pathway
Genes encoding components of the SCF complex such as skr-8, 10, and skr-13 (skr: skp1 related) were down-regulated in the knockout strains. The SCF complex is a multisubunit ubiquitin ligase (E3) that facilitates the recognition of substrates by ubiquitin-conjugating enzymes (E2, Ubc); it is formed by four components: Skp1, a cullin, Rbx/Roc/Hrt1, and an F-box protein (34)
. In addition, we observed a down-regulation of a large number of genes encoding F-box proteins in the knockout strains (Supplemental Table 3). F-box proteins act as SCF adapters and can bind multiple specific substrates, often in a phosphorylation-dependent manner (35)
. An example is pes-2, which encodes for an F-box protein probably involved in the UPP, which may also play a role in life span determination (36)
.
Expression of the ubxn-5 gene was enriched 3-fold in the absence of ATX-3. Ubxn-5 encodes a protein with a UBX domain usually found in ubiquitin-regulatory proteins, members of the ubiquitination pathway (37)
. The putative ATPase encoding gene C10G11.8 was significantly overexpressed in knockout strains. C10G11.8 protein shows similarity with human 26S proteasome regulatory subunit 4.
Structural and motility-related genes
Analysis of the mouse ataxin-3 expression pattern linked ataxin-3 to structural and/or motility proteins (38)
. Ten collagens that are essential proteins involved in the formation of the worm cuticle were enriched in the absence of atx-3 (Supplemental Table 3). In addition, myo-3, which encodes the minor isoform of myosin heavy chain, and nmy-1, which encodes a class II nonmuscle myosin heavy chain, were significantly up-regulated. MYO-3 is highly expressed in body and vulval muscles, as is ATX-3. Nmy-1 has been involved in embryonic morphogenesis (39)
. Although both strains share these alterations in motility-related genes, the animals apparently have normal movement and muscle organization.
Thirty-seven major sperm proteins (MSP) comprising central components underlying worm sperm motility were highly enriched in both knockout strains (Supplemental Table 3). MSPs are involved in extracellular signaling to promote oocyte maturation and cytoskeleton function (40)
. Other cytoskeleton-related genes such as ssq-4 (similar to human cytoskeletal 9 keratin), ifp-1 (intermediate filament), and ssq-2 (elastin precursor) were also up-regulated in the absence of ATX-3.
Other pathways
Some genes involved in signal transduction were altered in atx-3 knockout strains (supplemental Table 3). Par-4 encodes a serine-threonine kinase involved in development (41)
, which was down-regulated in knockout strains. Some other genes abundant during spermatogenesis (42)
were up-regulated in mutant strains, such as R13H9.5, which encodes a protein kinase, and ZK354.8, which encodes a protein tyrosine phosphatase.
In addition, bath-31 and egl-38 were down-regulated in the absence of atx-3. The egl-38 encodes a Pax5 transcription factor homologue, and its mutations result in a discrete set of defects in developmental pattern formation (43)
. Bath-31 encodes a protein that has a BTB/POZ domain. Proteins with a BTB/POZ domain have been shown to mediate transcriptional repression and to interact with components of histone deacetylase corepressor complexes, including N-CoR and SMRT (44
, 45)
.
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DISCUSSION |
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The fact that the two proteins share the same molecular partners also provided further evidence of their functional identity. Caenorhabditis elegans ATX-3 has been shown to interact with CDC-48.2, the worm homologue of VCP/p97, a known molecular interactor of human ataxin-3 (46)
.
Our findings showed an overlap between mouse and worm ataxin-3 temporal expression and cell type localization. Our previous work showed that mouse ataxin-3 is expressed throughout development and is abundant in muscle, neurons, and sperm (38)
, similar to worm ATX-3 expression.
The preserved protein motifs and expression pattern, conserved molecular partners, and in vitro deubiquitinating activity suggest that ATX-3 is the worm functional orthologue of human ataxin-3.
Transcriptional signature of ATX-3 knockout models
The ATX-3 knockout animals were viable and with no overt phenotype. This can be explained by functional redundancy and extensive adaptative modifications due to the absence of a protein that participates in a crucial cellular pathway. Nonetheless, the transcriptomic profile of the two ATX-3 knockout strains revealed strong overlapping changes in gene expression, providing evidence of a clear molecular phenotype. The ATX-3 knockout strains share the deregulation of genes related to several key biological processes, specifically the UPP, cell structure and motility, and signal transduction. Gene ontology analysis was also performed on the human orthologues of the DE genes and revealed a clear enrichment of similar pathways found in worms (data not shown).
ATX-3 and the ubiquitin-proteasome pathway (UPP)
UPP has a central role in the degradation of key regulators and is involved in the pathogenesis of several human diseases such as Parkinson’s disease (47)
. Considering the close relationship between ataxin-3 and the UPP, it was not surprising that several genes involved in this pathway were altered in the absence of ATX-3. Ubxn-5 was enriched in both knockout strains. In yeast, some UBX proteins have been found to act as adaptors for CDC-48 (orthologue of VCP/p97) -dependent protein degradation through the UPP (37)
. Another C. elegans UBX protein, UBXN-1, has been shown to interact with one of the worm VCP/p97 orthologues (46)
, raising the possibility of UBXN-5 being another adaptor for VCP/p97-dependent protein degradation pathway (Fig. 6
A). C10G11.8, a proteasome 26S regulatory subunit 4, was up-regulated in knockout strains. This subunit also interacts with VCP/p97 (48)
and participates in the initial recognition and binding of substrates by the proteasome. Enrichment of these two genes may suggest a cellular attempt to compensate for the absence of a protein involved in the protein degradation pathway and strengthens the biological significance of ataxin-3 and VCP/p97 interaction.
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Another important player in the UPP is the SCF [skp-1, Cdc53 (cullin), F-box] complex, an E3 ubiquitin ligase that targets several proteins for proteasomal degradation such as transcription factors, signal transducers, and cell cycle regulators. Substrates are recruited to the core of the SCF complex through specific adapter subunits called F-box proteins, each of which binds multiple specific substrates. Our microarray data showed that several skp1-related (skr) genes (skr-8, 10, and 13), as well as some F-box protein encoding genes, were down-regulated in atx-3 knockout strains. A genomewide computational analysis of functional interactions in C. elegans was recently described in which several SCF-associated genes were identified as putative genetic interactors of atx-3 (49)
.
Addition or removal of Nedd8 from the cullin subunit of the SCF regulates E3 activity (50
, 51)
. Our unpublished observations that Nedd8 interacts with human and worm proteins and that ataxin-3 has deneddylase activity in vitro (A. Ferro et al., personal communication) emphasize the possibility of a functional role in the SCF complex. We propose a model whereby ATX-3 interacts directly with Nedd8, which can regulate SCF complex activity and interfere with the degradation of several proteins, including transcription factors and developmental regulators (Fig. 6A
). This can potentially explain the large number of spermatogenesis and developmental-related genes altered in the absence of ATX-3. Another C. elegans DUB enzyme, UCH-1, has also been implicated in spermatogenesis and embryonic development (52)
.
ATX-3 in transcriptional regulation
ATX-3 can have several modi operandi in transcriptional regulation. First, through its DUB activity, ATX-3 can modulate transcription factor and repressor degradation by removing the polyubiquitin degradation signal. Second, it can control transcription factor degradation through the SCF complex (Fig. 6A
). In addition, ATX-3 may have a more direct effect over transcription regulation. Human ataxin-3 has a NLS and is transported into the nucleus (53)
, where it regulates transcription through at least two independent mechanisms (Fig. 6B
; see 23
). Worm ATX-3 also has the conserved NLS; it is present in the nucleus and its absence leads to a preponderance of up-regulated genes, supporting a role for the transcriptional repressor.
Reinforcing this hypothesis, our gene expression analysis of ATX-3 mutants revealed a strong up-regulation of collagens, which are constituents of the extracellular matrix (ECM), and a microarray study of cell lines expressing ataxin-3 by Evert et al. suggested that normal ataxin-3 seems to be involved in the transcriptional repression of ECM-associated components (54)
. Other ATX-3 repressor activity direct targets can be identified by ATX-3 overexpression (to observe enhanced repressor activity on some genes), yeast two hybrid screening (to detect potential corepressor activity partners), and chromatin immunoprecipitation (if ATX-3 binds directly to histones/DNA).
Based on these observations, we support a model where ataxin-3 functions in the UP/SCF degradation pathway and in transcriptional regulation. Moreover, these functions may be cell-specific, potentially explaining why only a subset of neurons is affected in the human disease despite widespread expression of mutant and normal ataxin-3.
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CONCLUSIONS |
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. Our microarray data provided some likely connections between ATX-3, the UP/SCF degradation pathway, structural and motility components, signal transduction and transcription regulation. It also showed a novel and unexpected linkage of ATX-3 with C. elegans development, given that the absence of ATX-3 strongly modifies expression of several development-related genes.
Additional studies are needed to clarify the specific physiological roles of ataxin-3, and the two knockout strains characterized here will be useful. Furthermore, the hypothetical networks and models developed can serve as a critical platform for our understanding of normal ataxin-3 function(s) and its role in modulating the disease course.
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ACKNOWLEDGMENTS |
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Received for publication July 27, 2006. Accepted for publication November 14, 2006.
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42 kDa that was present in wild-type animals and absent in both mutant strains.
