Polyglutamine expansion reduces the association of TATA-binding protein with DNA and induces DNA binding-independent neurotoxicity

Abstract

TATA-binding protein (TBP) is essential for eukaryotic gene transcription. Human TBP contains a polymorphic polyglutamine (polyQ) domain in its N terminus and a DNA-binding domain in its highly conserved C terminus. Expansion of the polyQ domain to >42 glutamines typically results in spinocerebellar ataxia type 17 (SCA17), a neurodegenerative disorder that resembles Huntington disease. Our recent studies have demonstrated that polyQ expansion causes abnormal interaction of TBP with the general transcription factor TFIIB and induces neurodegeneration in transgenic SCA17 mice (Friedman, M. J., Shah, A. G., Fang, Z. H., Ward, E. G., Warren, S. T., Li, S., and Li, X. J. (2007) Nat. Neurosci. 10, 1519-1528). However, it remains unknown how polyQ expansion influences DNA binding by TBP. Here we report that polyQ expansion reduces in vitro binding of TBP to DNA and that mutant TBP fragments lacking an intact C-terminal DNA-binding domain are present in transgenic SCA17 mouse brains. polyQ-expanded TBP with a deletion spanning part of the DNA-binding domain does not bind DNA in vitro but forms nuclear aggregates and inhibits TATA-dependent transcription activity in cultured cells. When this TBP double mutant is expressed in transgenic mice, it forms nuclear inclusions in neurons and causes early death. These findings suggest that the polyQ tract affects the binding of TBP to promoter DNA and that polyQ-expanded TBP can induce neuronal toxicity independent of its interaction with DNA.

FIGURE 1.

Polyglutamine expansion inhibits the intrinsic binding of TBP to DNA. A, Coomassie staining of purified His₆-tagged TBPs (31Q and 71Q, arrows) used in EMSAs. B, representative EMSA showing that polyQ-expanded TBP binds less TATA box DNA than normal TBP. Recombinant TBPs (130–220 ng) were incubated with a radiolabeled 45-mer probe containing the adenoviral E1b TATA box, and protein-DNA complexes were resolved on a 4% polyacrylamide gel. C, a Western blot probed with an N-terminal TBP antibody (N-12) demonstrating that equivalent amounts of soluble TBP were used in EMSAs. Note the absence of aggregated TBP-71Q protein in the stacking gel (bracket) after incubation under gel shift conditions.

FIGURE 2.

DNA binding capability of polyQ-expanded TBP determines its effect on TATA-dependent transcription activity. A, schematic structure of full-length (71Q-F) and truncated (71Q-T) His₆-tagged TBPs with an expanded polyQ domain (71Q). hTBP, human TBP; mTBP, mouse TBP. B, representative EMSA showing that TBP-71Q-T, unlike TBP-71Q-F, was unable to interact with TATA box DNA. Binding reactions contained an increasing amount of purified recombinant proteins (150, 225, and 340 ng), and Western blotting with anti-His confirmed that a similar amount of TBP-F and TBP-T was used (lower panel). Control, no TBP added. C, schematic layout of the EGFP reporter used to assess activated, TATA-dependent transcription (top panel). Reporter assays showing the effect of full-length TBP with 31Q or 71Q (middle panel) and truncated TBP with 13Q or 71Q (bottom panel) on activated transcription are presented below. The data are expressed as the means + S.E. (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 3.

polyQ-expanded TBP fragments that lack an intact C terminus are present in SCA17 mouse brains. A, schematic representation of mouse TBP showing the epitopes of different antibodies used to evaluate expression. B, Western blot analysis of lysates from cerebral cortex of a symptomatic TBP-71Q-F mouse (line 16, 7 months of age; Ref. 24) and a wild-type (WT) littermate. Note that the polyglutamine (1C2, left panel) and N-terminal TBP (1TBP18, middle panel) antibodies recognized transgenic TBP (arrowheads) and lower molecular weight TBP bands (asterisks) that were specific to TBP-71Q-F mouse brain samples. The lower bands were not detected by an antibody against C-terminal TBP (EM192, right panel). C, Western blot analysis of lysates from cerebella of TBP-71Q-F mice and wild-type littermates (line 16, 7 months of age; Ref. 24). Arrowhead, full-length transgenic TBP; asterisk, transgenic TBP fragments. Note that the expanded polyQ tract (71Q) retarded the migration of transgenic proteins in the gel and that high molecular weight TBP-71Q-F oligomers could be detected with an N-terminal antibody (1TBP18, bracket) in A and C. The arrows indicate endogenous TBP in all panels.

FIGURE 4.

Nuclear inclusions in SCA17 cell models contain N-terminal TBP. A, double immunofluorescent staining of HEK293 cells transfected with TBP-105Q-F. Most TBP aggregates (arrows) could be labeled with an N-terminal (1TBP18) but not a C-terminal (EM192) TBP antibody. B, some TBP-105Q-F-transfected cells contained aggregates (arrows) that could be labeled by both N-terminal and C-terminal antibodies. C, representative double immunofluorescent staining of cultured cerebellar granule neurons (15 days in vitro) from TBP-105Q-F mice with antibodies against N-terminal (1TBP18) or C-terminal TBP (EM192). Note that only the antibody against N-terminal TBP labeled nuclear TBP inclusions (arrows). Scale bars, 10 μm.

FIGURE 5.

Truncated, polyQ-expanded TBP that is unable to bind DNA aggregates extensively in transfected cells. A, double immunofluorescent staining of HEK293 cells transfected with truncated TBPs containing polyQ tracts of different length using antibodies to TBP (N-12) and polyglutamine (1C2). Note that anti-TBP detected nuclear TBP aggregates, whereas 1C2 showed only diffuse staining. B, immunofluorescent expression analysis of full-length (TBP-F, upper panel) and truncated (TBP-T, lower panel) TBPs in HEK293 cells. Transfected cells were labeled with an N-terminal TBP antibody (N-12). Note that TBP-T forms more aggregates than TBP-F. Scale bars, 10 μm.

FIGURE 6.

Nuclear aggregates formed by truncated, polyQ-expanded TBP sequester TFIIB. Double immunofluorescent staining of HEK293 cells expressing TBP-71Q-T (upper panel) and TBP-105Q-T (lower panel) with anti-TBP (1TBP18) and anti-TFIIB. Note that endogenous TFIIB localized to nuclear TBP aggregates (arrows). Scale bar, 10 μm.

FIGURE 7.

Truncated, polyQ-expanded TBP accumulates in nuclei and forms nuclear inclusions in the brains of transgenic mice. Striatal sections (top panel) from wild-type, transgenic SCA17 (TBP-105Q-F; Ref. 24), and truncated TBP (TBP-105Q-T) transgenic mice were labeled with 1C2. Representative 1C2 staining of cerebral cortex, hippocampus, and cerebellum from a TBP-105Q-T transgenic mouse is included (middle panel). The arrows indicate nuclear inclusions, which are conspicuous in the high magnification micrographs (bottom panel). Immunohistochemical analysis was performed on mice at 7 weeks of age. Scale bars, 10 μm.

FIGURE 8.

Model for mutant TBP-mediated transcriptional dysregulation in SCA17. Increased interaction of soluble polyQ-expanded TBP with TFIIB and/or other transcription factors may allow for recruitment of the former to certain TATA-containing promoters. Because this soluble form of TBP is not inherently defective, its recruitment can stimulate TATA-dependent transcriptional activity (upper panel). However, mutant TBP can no longer productively interact with promoter DNA after proteolytic processing and/or misfolding. TBP aggregates can sequester particular transcription factors, such as TFIIB, and thereby reduce their availability at certain promoters. Decreased transcriptional activity is the likely consequence of aberrant interaction of misfolded TBP with basal transcription factors or activator proteins (lower panel).