THAP1 is an atypical zinc finger protein with a highly conserved, N-terminal C2CH module (CX2-4CX35-53CX2H), termed THAP, which defines a family of over 100 proteins in diverse species, including 12 human THAPs (Roussigne et al. 2003a,b; Clouaire et al. 2005). This metal-coordinating signature encodes a DNA-binding domain that recognizes an 11-nucleotide sequence within the promoters of target genes, the best characterized of which are cell cycle-related genes such as RRM1 (ribonucleotide reductase M1; Clouaire et al. 2005; Cayrol et al. 2007; Bessière et al. 2008; Campagne et al. 2010). Although DNA binding by a recombinant, isolated THAP domain has been demonstrated in cell-free systems (Clouaire et al. 2005; Bessière et al. 2008), it is hypothesized that, in vivo, full-length THAP1 may form oligomeric complexes, either with itself or other proteins, to achieve full transcriptional activity (Campagne et al. 2010).
In the present study, we addressed this question by examining the ability of THAP1 variants bearing different epitope tags to associate with each other when expressed in cultured cells. A number of different DYT6-related mutants were evaluated, including five which target residues within the C-terminal coiled-coil domain and two which affect the N-terminal DNA-binding module. This analysis identified a DYT6 mutant which loses the ability to bind wild-type THAP1, while also revealing a 13-amino acid (aa) sequence which appears necessary for THAP1 dimerization. This region overlaps with a predicted nuclear localization signal (NLS) and, as a result, proved equally critical for nuclear translocation by THAP1. A potential structural model of this domain was therefore generated to predict the spatial segregation between residues likely to mediate dimerization versus ones believed to interact with the cellular nuclear import machinery. Collectively these data provide further insight into the role of the C-terminal domain in THAP1, which may assist in future efforts to predict functional consequences of different DYT6-related mutations in this region.
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The function of a wide range of cellular proteins may be regulated, at least in part, by dimerization, which may involve binding of identical subunits to each other (homodimers) and/or the interaction between members of the same (homotypic) or different (heterotypic) gene families (Klemm et al. 1998; Marianayagam et al. 2004; Amoutzias et al. 2008). Among the different categories of proteins known to dimerize are multiple transcription factors, including the basic-region leucine zipper, homeodomain leucine zipper, basic-region helix-loop-helix, nuclear receptors, transcription factor family named for four members (MCM1, Agamous, Deficiens, SRF) (MADS-box), signal transducers and activators of transcription, and nuclear factor-κB families (for review, see Amoutzias et al. 2008). In the case of transcription factors, dimerization can exert both qualitative and quantitative effects on gene expression. The types of homo- and heterotypic dimers formed by a given factor can greatly expand its repertoire of compatible DNA elements. In addition, the signals which trigger dimer formation and/or dissociation by a given factor may influence its DNA-binding activity and thereby regulate transcription.
Dimerization has been postulated as a mechanism regulating activity of THAP proteins (Sabogal et al. 2010), although direct demonstrations of dimer formation by specific THAP family members are relatively few. Two examples of THAP proteins confirmed to form homodimers are the Drosophila melanogaster P-element transposase KP repressor protein (Lee et al. 1996) and the Caenorhabditis elegans protein, CTPB-1 (Nicholas et al. 2008), both of which display activity dependent on oligomeric status. Aside from THAP1, none of the other human THAPs have yet been shown to homodimerize, although THAP11 and THAP7 may bind each other within a transcriptional complex (Dejosez et al. 2008). The likelihood that THAP1 homodimerizes has been suggested indirectly by reports that: (i) the promoters of at least some target genes contain two copies of its binding element (Cayrol et al. 2007; Kaiser et al. 2010); and (ii) the affinity of monomeric THAP1 for DNA is relatively weak, indicating that it may need to form a complex with itself or other proteins to effectively bind its target element (Campagne et al. 2010). More direct evidence of THAP1 dimerization has been provided by yeast double hybrid screens, demonstrating interactions between different THAP1 constructs (Rual et al. 2005; Lanati et al. 2010). The present study, demonstrating an interaction between tagged variants in cultured cells, confirms that THAP1 associates with itself, while further identifying specific residues that modulate the interaction.
Our analysis of the Q154fs180X and Δ167–213 mutants indicates that the ability of THAP1 to bind itself is dependent on residues within a 13-aa region (154–166) of the coiled-coil domain. This sequence, together with upstream residues within the coiled-coil domain, contains elements consistent with leucine zippers in other dimerizing transcription factors. Within a leucine zipper, dimer formation is driven by interhelical interactions between hydrophobic residues at the a and d positions (aa′ and dd′ where ′ denotes the position on the opposite monomer), as well as interhelical electrostatic interactions between differently charged residues at g and e positions (ge′ where e′ is five residues towards the C-terminal on the opposite monomer) (Alber 1992; Ellenberger et al. 1992; Thompson et al. 1993; Vinson et al. 2002). Thermodynamic studies have shown that the most stable dimers contain: (i) isoleucine (Acharya et al. 2002) or valine (Tripet et al. 2000) at a positions; (ii) leucine at d positions (Harbury et al. 1993; Moitra et al. 1997); and (iii) one of four polar amino acids (glutamate, glutamine, arginine, or lysine) at g and e positions (Vinson et al. 2002), with the strongest dimers produced by interactions between a positively charged arginine and a negatively charged glutamate (Krylov et al. 1994). Consistent with these rules, THAP1 contains isoleucine and valine at a2 and a3, respectively, and leucines at d2 and d3. This scheme also accurately describes all of the ge′ pairs that would exist in the predicted THAP1 dimer, specifically: arginineglutamate (g1e2′); glutaminearginine (g2e3′); and lysineglutamine (g3e4′).
Leucine zippers in transcription factors such as the basic region leucine zipper transcription factors are typically comprised of four or five heptads (Vinson et al. 2002), but stable oligomerization has been achieved by even a two-heptad domain provided a sufficient interface of consecutive hydrophobic residues is present (Burkhard et al. 2000; Lu and Hodges 2004). The Q154fs180X mutant most likely fails to meet these criteria, given that: (i) the helical wheel analysis demonstrated no more than 2–3 continuous hydrophobic residues at any point in this region, suggesting that an amphipathic helix is unlikely to form; and (ii) secondary structure analysis indicated that the novel sequence encoded by the frameshift lacks helical structure and may be disordered.
In addition to the Q154fs180X frameshift, the I149T mutation also targets a critical residue within this domain, specifically the isoleucine at a2. However, the threonine substitution did not disrupt dimerization; rather, it displayed an apparent tendency across experiments to immunoprecipitate more wtTHAP1-FLAG than was pulled out by V5-wtTHAP1. Leucine zipper proteins which normally contain threonine at a2 have been characterized (Porte et al. 1997), and a dimer involving a threonineisoleucine aa′ interaction (as would occur between I149T and wtTHAP1) is reportedly stable (Acharya et al. 2006). However, substitutions at a positions may also change the packing geometry between coils to produce aberrant trimers and even tetramers in place of dimers (Harbury et al. 1993). The behavior of the I149T mutant in the coimmunoprecipitation assay could suggest a similar effect on oligomerization by THAP1; if so, then the interaction with its target DNA element might be altered because of formation of aberrant oligomeric complexes. Nevertheless, further biophysical analyses are required to test these possibilities.
Bonetti et al. (2009) identified a DYT6-related missense mutation at position 170 (C170R) and hypothesized that it might negatively affect dimerization based on a proposed structural model of the coiled-coil domain. Our results confirm the importance of the coiled-coil domain for THAP1 self-association but suggest that residues beyond position 166 are not required. The C-terminal portion of the coiled-coil domain may instead mediate binding of THAP1 to other proteins, although this hypothesis remains to be tested. Lanati et al. (2010) also proposed a hypothetical model of THAP1 dimerization, predicting that individual monomers may associate via the zipper-like motif in the coiled-coil domain. The observed dimerization defect in the Q154fs180X mutant, which loses most of the zipper-like motif, provides direct evidence to support this prediction. In addition, we extended the previous in silico analyses of the THAP1 coiled-coil domain to predict how critical sequences within the bipartite NLS might be spatially oriented within a potential dimer structure. In cargo proteins with bipartite NLS sequences similar to THAP1, the upstream and downstream clusters of basic residues interact with a minor and major binding pocket, respectively, on carrier proteins such as importin-α that mediate nuclear entry (Conti et al. 1998; Fontes et al. 2000). We hypothesize that the translocation defect in the Q154fs180X mutant is most likely caused by the loss of the downstream basic cluster, 158KLRKKLK164. Given that the fractionation/co-immunoprecipitation analysis suggested that THAP1 binds itself within the cytoplasm, it seems likely that the basic residues of the NLS must be displayed on exposed surfaces of the resulting dimer to allow interaction with the nuclear import machinery. The helical wheel and Richardson plots predict such an arrangement.
Although the I149T mutation also falls within the NLS, it did not prevent nuclear localization. The linker sequence in a bipartite NLS is believed to orient the upstream and downstream basic clusters in the proper conformation to allow binding to the two pockets on an importin protein (Lange et al. 2010). Although some mutations within NLS linkers have been shown to disrupt nuclear trafficking, others have been reported with no apparent effect (Moore et al. 1998; Lange et al. 2010). Our results demonstrate that the I149T substitution in THAP1 is an example of the latter. Among the DYT6 mutants tested, the only other variant to exhibit altered intracellular localization was C54Y, which frequently accumulated within large perinuclear inclusions that did not colocalize with two markers of the nuclear envelope (NE), lamin A/C and lamin B receptor. This finding bears some similarity to previous descriptions of the DYT1 dystonia protein, torsinA, a AAA+ chaperone protein localized to the lumen of the NE and endoplasmic reticulum (Granata and Warner 2010; Bragg et al. 2011). The DYT1 mutant form, torsinAΔE, accumulates within membrane inclusions derived from the NE and endoplasmic reticulum (Hewett et al. 2000; Kustedjo et al. 2000; Bragg et al. 2004b; Gonzalez-Alegre and Paulson 2004; Goodchild and Dauer 2004; Naismith et al. 2004), yet only when expressed at high levels in cultured cells (Bragg et al. 2004c) with only limited and controversial evidence that it may do so in brains of DYT1 individuals (Standaert 2011). It is unclear whether the immunoreactive inclusions in cells expressing the C54Y mutant are relevant to DYT6 pathogenesis, or if they bear any relationship to the aberrant membrane structures induced in cultured cells by torsinAΔE. Yet, given that NE dysfunction has been postulated as a mechanism underlying DYT1 dystonia (for review, see Granata et al. 2009), this observation may suggest that there could be interactions between THAP1 and the NE that warrant further investigation.
The DYT6 mutants tested here are associated with a range of clinical phenotypes, both in terms of disease onset (early vs. late) and somatic distribution of symptoms (generalized vs. focal). The clinical spectrum of DYT6 dystonia has grown more heterogeneous as additional THAP1 mutations have been identified, and it has been difficult to account for the phenotypic variability at the level of genotype. Among the descriptions of DYT6 mutations reported to date, there does not appear to be a consistent genotype : phenotype relationship in terms of symptom distribution, but there is a potential trend related to disease onset. Early onset dystonia has typically been documented for THAP1 mutations within the N-terminal DNA binding module and in C-terminal mutations which disrupt the NLS; whereas the mutations associated with late onset disease have generally been C-terminal variants not predicted to alter nuclear localization (Bonetti et al. 2009; Bressman et al. 2009; Djarmati et al. 2009; Fuchs et al. 2009; Paisán-Ruiz et al. 2009; De Carvalho Aguiar et al. 2010; Groen et al. 2010; Houlden et al. 2010; Söhn et al. 2010; Xiao et al. 2010; Zittel et al. 2010; Cheng et al. 2011; Clot et al. 2011; Jech et al. 2011; Schneider et al. 2011). In that regard, our observation of normal nuclear localization by the I149T mutation in the NLS appears consistent with this trend, as this mutation has been linked to late onset dystonia. A potential exception is the ΔF132 mutation which falls outside the N-terminal binding domain and NLS yet is associated with early onset generalized dystonia. However, unlike most of the known DYT6 mutations, the ΔF132 deletion was detected on both alleles, which may account for the severity of the phenotype.
In summary, this study performed a phenotypic analysis of wtTHAP1 and a collection of DYT6 mutant forms in cultured cells, with an emphasis on probing the effects of different mutations on the protein’s ability to bind itself. The demonstration that the Q154fs180X mutant is defective in both dimerization and nuclear translocation confirmed the importance of specific residues within the coiled-coil domain for both of these functions. Given that other DYT6 mutations have been identified within this region, these data may ultimately guide further structure : function analyses of this domain, which could prove useful in understanding the heterogeneous clinical phenotypes associated with different DYT6 disease variants.
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- Materials and methods
- Supporting Information
Figure S1. Confirmation of THAP1 dimerization via pull-down assay. Purified recombinant wtTHAP1-FLAG was captured on anti-FLAG agarose beads and used as bait to pull down V5-THAP1 variants in lysates from transfected HEK-293T cells. Consistent with the coimmunoprecipitation analysis, all V5-THAP1 variants were pulled down by wtTHAP1-FLAG, except for the Q154fs180X mutant. No V5-wtTHAP1 was pulled down by anti-FLAG beads in the absence of immobilized wtTHAP1-FLAG (control).
Figure S2. Intracellular localization of DYT6 mutants. Immunofluorescence of U2OS cells expressing V5-tagged THAP1 variants, stained with anti-V5 monoclonal antibody (red), wheatgerm agglutinin-Alexa488 (membranes, green), and TO-PRO®-3-iodide (nuclei; blue). Several of the DYT6 mutants tested produced staining patterns which could not be distinguished from that of wtTHAP1. (A-D) V5-wtTHAP1 was detected primarily in the nucleus and, in many cells, also in the cytosol. Similar distributions were observed for the F81L (E-H), ΔF132 (I-L), T142A (M-P), and A166T (Q-T) mutants. Images captured via laser confocal microscopy at final magnification of 100× under oil immersion. Scale bar = 10 μM.
Figure S3. Colocalization of wtTHAP1 and C54Y variants with lamin A/C. Double label immunofluorescence in transfected U20S cells demonstrating localizations of V5-wtTHAP1 (A-D) or V5-C54Y mutant (E-H), the nuclear envelope marker, lamin A/C, and TO-PRO®-3-iodide (nuclei). Lamin A/C immunoreactivity (green) consisted of prominent labeling in and around the nucleus which overlapped to some extent with intranuclear V5 staining (red), particularly for wtTHAP1 (D). However, the perinuclear inclusions produced by the C54Y mutant were not immunoreactive for lamin A/C (H). Images shown were captured by laser confocal microscopy at 100× final magnification under oil immersion. Scale bars = 10 μM.
Figure S4. Colocalization of wtTHAP1 and C54Y with lamin B receptor. Double label immunofluorescence in transfected U20S cells demonstrating localizations of V5-wtTHAP1 (A-D) or V5-C54Y mutant (E-H), the nuclear envelope marker, lamin B receptor, and TO-PRO®-3-iodide (nuclei). Similar to Lamin A/C, staining for lamin B receptor (green) showed strong labeling around the nucleus with diffuse immunoreactivity also within the nucleoplasm. Although some colocalization with V5 (red) was detected in the nucleus, the C54Y+ inclusions did not colocalize with lamin B receptor (E). Images shown were captured by laser confocal microscopy at 100× final magnification under oil immersion. Scale bars = 10 μM.
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