Sp1 and TAFII130 Transcriptional Activity Disrupted in Early Huntington’s Disease Anthone W. Dunah, Hyunkyung Jeong, April Griffin, Yong-Man Kim, David G. Standaert, Steven M. Hersch, M. Maral Mouradian, Anne B. Young, Naoko Tanese, Dimitri Krainc

Yeast Two-Hybrid System
Bait and prey constructs were generated from PCR amplification of the respective cDNA's with incorporation of an EcoR1 site in the 5' end and BamH1 site in the 3' end to allow simple unidirectional cloning into the two-hybrid vector (S1). TAFII130 constructs were made as described (S2). These insertions were in-frame with the LexA DNA binding domain and a hemagglutinin tag of the pJG4-5 vector. All the constructs were verified by DNA sequencing for correct reading frame and orientation. All assays were performed in triplicates and protein expression verified by Western blotting. The ability of the LexA fusion to bind operator was confirmed by a repression assay. The two-hybrid screen assays were performed as described (S1).

Co-precipitation Assays
Human HEK293 cells were transfected with expression vectors using FuGENE6 Transfection Reagent (Roche) according to the manufacturer's instructions. Total amount of plasmid DNA in individual plates was adjusted using empty vector plasmid. Thirty six hours after transfection, cells were sonicated for 5 sec in lysis buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% sodium deoxycholate, 0.5 mM DTT, 0.2 mM PMSF, 10 mM NaF, 10 mM sodium pyrophosphate, 10 g/ml leupeptin and 10 g/ml aprotinin). Immunoprecipitations were performed with anti-huntingtin antibody (Chemicon, MAB2166) and protein G Agarose (Roche) followed by washing four times with RIPA (75mM NaCl, 25mM Na₂S0₄1mM EDTA, 0.5% sodium deoxycholate, 0.5% TritonX-100) or ice-cold phosphate buffered saline (PBS). Immunoprecipitates or total lysates were separated on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to immunoblotting with anti-huntingtin and anti-Flag (Sigma) or anti-HA antibodies (TAFII130). For coimmunoprecipitation experiments in human brain, tissues were homogenized using a polytron (Brinkmann Instruments) in ice-cold TE buffer (10 mM Tris-HCl, 5 mM EDTA, pH 7.4) containing 320 mM sucrose and centrifuged at 15,000 rpm at 4°C for 20 min. The pellet was solubilized by adding 1% sodium deoxycholate in 50 mM Tris-HCl, pH 9.0 followed by incubation at 36°C for 30 min. A 1/10 volume of a buffer containing 1% Triton X-100, 50 mM Tris-HCl, pH 9.0 was then added and the preparation was dialyzed against binding buffer (50mM Tris-HCl, pH 7.4, 0.1% Triton X-100) overnight at 4°C. The sample was centrifuged at 37,000 x g at 4°C for 40 min. The supernatant was used for immunoprecipitation. Precoupling of antibodies to Protein A Sepharose beads and immunoprecipitation procedures were performed as previously described (S3). SDS-PAGE and the transfer of proteins to nitrocellulose were performed according to standard protocols. Samples were analyzed by SDS-PAGE , immunoblotted and probed with anti-Sp1 antibody (Santa Cruz, sc-59) and anti-huntingtin antibody (Chemicon, MAB2166). The same blot was probed with anti-cadherin (cell adhesion molecule) antibody as a control for nonspecific interaction. The concentration of antibodies used for the immunoblots was 1-2 g/ml. Experiments were performed with postmortem HD brains from presymptomatic patients (shown here), as well as affected HD patients. Age-matched control brains were used in each experiment.

Eletrophoretic Mobility Shift Assays
Human brain tissue Grades 1- 4 according to Vonsattel classification (S4) from HD patients and age-matched controls originated from the Brain Bank at McLean Hospital in Boston, from the Vonsattel lab and from our labs. Two of the three donors of Grade 1 HD brains were presymptomatic and one of them is shown here. EMSA for cultured cells and frozen human tissue was performed as described (S1, S5), except for the modifications as below. Nuclear extracts were prepared from the caudate nucleus and hippocampus. These regions were selected because of their differential vulnerability in HD (S4). Frozen postmortem human brain tissue (0.2 g) was rinsed in PBS buffer and spun at 1,500 x g for 5 min. NP-40 (0.5% final) was added and homogenization performed with pestle, followed by incubation for 15 min to lyse the cells, and microcentrifuged for 2 min. The nuclear pellet was resuspended in buffer C (20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM each of EDTA, DTT, EGTA and PMSF) and placed on a rotary shaker for 20 min, followed by centrifugation at 11,000 x g for 5 min, and removal of the supernatant that contained soluble nuclear proteins. We obtained about 4g of nuclear protein per mg of frozen tissue. EMSA was performed with labeled D2 receptor promoter fragment containing Sp1 site (underlined) (5'-CAGGGTGACCCCGCCCCCTCCTCC-3') (S6). The protein-DNA complexes were resolved on a 4% native polyacrylamide gel in 0.25 X TBE (44.5 mM Tris-HCl, 44.5 mM boric acid, 1 mM EDTA) and visualized by autoradiography. The slowest migrating complex was supershifted with Sp1 antibody. To determine the molecular weight of retarded Sp1 band, we UV cross-linked the proteins to the labeled DNA probe, separated the DNA-protein complexes on a native gel, cut out each shifted complex and the crosslinked proteins were run on a SDS-denaturing polyacrylamide gel. For reactions analyzing the binding of purified Sp1 (Promega; 1.5 footprinting unit per reaction) to labeled consensus Sp1 site (underlined) (Promega; 5'-ATTCGATCGGGGCGGGGCGAGC- 3', 50000 cpm) EMSA was performed as in (S1) except that 5mM DTT and 1 g/ml poly(dI-dC) were used. GST-huntingtin fusions (0.2 g/l) were a gift of E. Wanker and were purified as described (S7). An approximate 1:1 ratio of purified proteins was used. Coomassie blue staining and Western blotting were used to examine the purified proteins.

Neuronal Transfections and Immunocytochemistry
Striatal neurons were prepared from embryonic (E17-18) rats and transfected as previously described (S1, S8). Plates were coated overnight with 0.1 mg/ml poly-L- ornithine solution. The cells were incubated in 5% CO₂ for 1 hr with 3 ml of fresh DMEM (w/out added glutamine). DNA/calcium phosphate precipitate was prepared by mixing one volume of DNA in 250 mM CaCl₂ with an equal volume of 2 X HeBS (274 mM NaCl, 10 mM KCl, 2.4 mM Na₂HPO₄, 15 mM D-glucose, 42 mM HEPES, pH 7.05). The precipitate was allowed to form for 25-30 min in the dark at room temperature before addition to the cultures. Six to eight g of plasmid DNA were used for each 60-mm-diameter plate. 120 l of the DNA/calcium phosphate precipitate were added drop-wise to each 60-mm-diameter dish and mixed gently. Plates were then returned to the 5% CO₂ incubator. The incubation was stopped after 15-25 min by aspirating the media. Cells were then washed three times with 3 ml of DMEM (w/out added glutamine). The saved conditioned medium was added back to each plate, and the cells were returned to the 5% CO₂ incubator at 37 °C. CAT assays were carried out using the CAT/enzyme-linked immunosorbent kit (Roche), and the results were normalized to -galactosidase activity. Transfected neurons were scored for cell death as described (S8). Cells were stained with the DNA dye Hoechst 33258. Neurons were scored as dead if they stained positively with TUNEL and had pyknotic and fragmented nuclei. Equal expression of transfected DNA's was confirmed by Western blot analysis. For immunocytochemistry, cultured striatal neurons were transfected at DIV 8 (8 days after plating) with either huntingtin constructs alone or a combination of huntingtin, Sp1 and HA-tagged TAFII130, and double-labeled at DIV 10. Endogenous dopamine D2 receptor was stained with polyclonal D2 antibody (Santa Cruz, sc-9113). Other antibodies used included mouse monoclonal anti-huntingtin (Chemicon, MAB2166), anti-flag (against flag-huntingtin), and anti-HA. Neurons were fixed with ice-cold methanol for 5 min at -20°C, washed in PBS three times for 5 min at room temperature, and incubated with primary antibodies in staining buffer (GDB, 30 mM phosphate buffer, pH 7.4, containing 0.2% gelatin, 0.5% Triton X-100, and 0.8 M NaCl) overnight at 4°C. The cells were then washed three times in wash buffer (WB, 20 mM phosphate buffer, pH 7.4, 0.5 M NaCl) for 10 min at room temperature. After incubating the cells with secondary antibody in GDB for 1 hr at room temperature, they were washed sequentially in WB and PBS for 10 min at room temperature. Experiments were performed in triplicate. Confocal microscopy was used to visualize the cells.

Real-time PCR
Individual huntingtin-, TAFII130- and Sp1-transfected cells identified by immunostaining, as well as untransfected neurons were aspirated into glass microelectrodes by applying negative pressure under visual control (S9). After aspiration, the electrode was broken and the contents ejected into a thin-walled PCR tube. At least 10 cells were pooled together in each group for analysis of D2 receptor expression. Experiments were performed in triplicate. D2 receptor RNA was isolated with Picopure RNA Isolation Kit (Arcturus), and levels analyzed by cDNA synthesis and PCR with SYBR Green dye (PE Biosystems). The thermal cycling program for all primer sets was 94°C for 1 min, 58°C for 1 min, and 72°C for 1.5 min for 45 cycles. All primer sets had PCR efficiency above 90% at 58°C. Primers for the D2 cDNA were D2R1- 5'-GCA GTC GAG CTT TCA GAG CC-3' and 5'-TCT GCG GCT CAT CGT CTT AAG-3' (404 and 317 bp; GenBank accession M36831) (S9). The accumulation of fluorescent products was monitored by iCycler (Bio-Rad). PCR baseline subtracted threshold values were calculated for each amplicon. PCR products were verified by melting temperature profiles of final products and by sequencing of PCR reactions. All RNA values were normalized to -actin whose mRNA expression was not changed in transfected neurons, as determined by real-time RT-PCR. Negative controls for contamination from extraneous and genomic DNA were run for every batch of neurons. To ensure that genomic DNA did not contribute to the PCR products, neurons were aspirated and processed in the normal manner, except that the reverse transcriptase was omitted. Contamination from extraneous sources was checked by replacing the cellular template with water. Both controls were consistently negative in these experiments.

Immunohistochemistry and Western Blots
Series of 50µm sections were processed for immunohistochemistry. To reduce endogenous peroxidase activity and nonspecific antibody binding, sections were incubated in 3% hydrogen peroxide and then PBS containing 4% normal goat serum (NGS) followed by PBS rinses. Sections were incubated in 2% NGS/PBS containing the primary antibody and 50µg/ml of biotin for 48-72 hr. Sections were incubated overnight in biotinylated goat anti-mouse secondary antibody (Vector) in PBS containing 2% NGS and in avidin-biotin complex (Vector ABC Elite) for 4 hr. Final development was done by incubation in 0.05% 3,3'-diaminobenzidine tetrahydrochloride (DAB, Sigma) and 0.01% hydrogen peroxide in 50 mM Tris buffer (pH 7.5) for 5-15 min. Specific controls included omission of the primary antibody to determine the amount of background generated from the detection assay and preabsorption with excess target proteins when available to demonstrate specificity. We used Sp1 antibody (Santa Cruz, sc-59) and EM48 antibody which recognizes the N-terminal portion of huntingtin (amino acids 1-256, excluding polyglutamine and polyproline repeat stretches) that selectively immunoreacts with aggregates in human tissue (S10). Western blots were performed as described (S11). Tissues were homogenized in 10 volumes of 50 mM Tris (pH 7.5) and 1% Triton X-100. The homogenate was spun for 30 min at 150,000xg. The pellet was resuspended in 50mM Tris (pH 7.5) with 2% SDS and boiled for 4 min. Samples were spun at 1,000 x g for 2 min, and the supernatant assayed for protein concentration, and equal amounts of protein analyzed by SDS-PAGE and immunoblotting.

References and Notes
S1. D. Krainc et al., J. Biol. Chem. 273, 26218 (1998).
S2. D. Saluja et al., Mol. Cell. Biol. 18, 5734 (1998).
S3. A. W. Dunah et al., Mol. Pharmacol. 53, 429 (1998).
S4. J. P. Vonsattel, R. H. Myers, T. J. Stevens, R. J. Ferrante, E. D. Bird, J. Neuropathol. Exp. Neurol. 44, 559 (1985).
S5. D. K. Lahiri, Y. Ge, Brain Res. Protoc. 5, 257 (2000).
S6. S. Yajima, S-H. Lee, T. Minowa, M. M. Mouradian, DNA Cell Biol. 17, 471 (1998).
S7. J. S. Steffan et al., Nature 413, 739 (2001).
S8. F. Saudou et al., Cell 95, 55 (1998).
S9. D. J. Surmeier, W-J.Song, Z. Yan, J. Neurosci. 16, 6579 (1996).
S10. C. A. Gutekunst et al., J. Neurosci. 19, 2522 (1999).
S11. F. C. Nucifora et al., Science 291, 2423 (2001).
S12. D. Krainc et al., unpublished data.
S13. We thank H. Zoghbi, R. Tjian, R. Kingston, R. Luthi-Carter, M. DiFiglia, M. J. Kim, J.P. Vonsattel, A. McCampbell, X-J. Li, K. Kegel, R. Ratan, R. Ferrante, and J-H Cha for helpful discussion and suggestions; Harvard Brain Tissue Resource Center, J. P. Vonsattel and K. Newell for human postmortem brain tissues; L. Orlando, J-H Li and Z. Qiu for help with preparation of striatal cultures; F. Norflus for help with transgenic animals; V. Chen, D. Trotti and W. Bi for help with real-time RT-PCR; J. Cohen for help with immunohistochemistry; F. Saudou for huntingtin expression plasmids; and C. M. Chiang for TAFII55. This study was supported by Hereditary Disease Foundation and NIH (NS02174) to D.K; Parkinson's Disease Foundation Grant to A.W.D.; NIH (5R37AG13617) to A.B.Y.; NIH (NS35255 and AT00613), the HDF, and the HDSA Coalition for the Cure to S. M. H.; NIH (NS34361) to D.G. S.; N.T. was supported in part by The Irma T. Hirschl Trust.

Supplemental Figure 1. Binding of MEF2 transcription factor to DNA is not altered in caudate of HD brain. Nuclear extracts were prepared from the caudate of human control (lane 1) or HD brain (lane 3) and EMSA was performed as in Fig. 2C. Competition with unlabeled probe is shown in lanes 2 and 4. The probe was a double-stranded oligonucleotide containing the brain creatine kinase MEF2 binding site (5'-ATGGGCTATAAATAGCCGCCA-3'). To determine the specificity of retarded bands we performed supershift experiments with antibodies against MEF2C and MEF2A proteins (S12).

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Supplemental Figure 2. Levels of Sp1 and TAFII130 proteins are not altered in the caudate nucleus of PSP postmortem brain. Triton X-insoluble protein fractions were collected from caudate nucleus of human control brain (lane 1) and PSP brain (lane 2) and Western blot analysis was performed as in Figure 4B. Densitometric analysis was performed to correct for differences in the expression of tubulin. Expression of TAFII130 and Sp1 was not significantly different in control and PSP brain (S12).

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Supplemental Figure 3. A model depicting transcriptional downregulation of Sp1-dependent genes in HD brain. We propose that soluble mutant huntingtin (Htt) contributes to loss of Sp1 binding to DNA and disruption of Sp1/TAFII130 activation function leading to transcriptional dysregulation in early stages of Huntington's Disease.

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