Fig. 5.
Mutant htt decreases the association of Sp1 with the GLT-1 promoter. (A and B) EM48 immunostaining of cultured wild-type mouse astrocytes that were transfected with NLS-exon1 htt (green) containing 150Q or 20Q. The high-magnification graphs in (B) show that mutant htt (150Q) forms nuclear aggregates, whereas the normal htt fragment (20Q) is diffuse in the nucleus (blue). (C) Immunoprecipitation of Sp1 in transfected astrocytes shows that more mutant htt (150Q) than normal htt (20Q) is coprecipitated with Sp1. (D) Coexpression of the GLT-1 promoter reporter with N-terminal htt fragments containing different numbers of amino acids (67, 208, 508) plus either 20Q, 150Q, or 120Q. Note that mutant htt fragments (67–150Q and 208–120Q) significantly inhibit the reporter activity. ***, P < 0.001. (E) Chromatin immunoprecipitation (ChIP) assay showing decreased association of Sp1 with the GLT-1 promoter in the frontal brain tissue of htt-160Q transgenic mice compared with wild-type littermate controls. Rabbit anti-Sp1 and IgG, which served as a control, were used for immunoprecipitation. C, no template. (F) Quantification of the ratios of PCR products from immunoprecipitated (IP) to input. **, P < 0.01 compared to wild-type (WT) control (n = 4).