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. 2021 Oct 29;297(6):101372. doi: 10.1016/j.jbc.2021.101372

Figure 4.

Figure 4

Two RE1 consensus sites are critical for EAAT2 promoter activities and REST activation.A, two putative RE1 consensus sites (−663 and −131) in the EAAT2 promoter were identified. After transfection of REST, REST binding to its consensus sites in the EAAT2 promoter was determined by ChIP assay. Quantification of REST binding activities to these two RE1 consensus sites was measured by quantitative PCR from H4 astrocytes. B, DAPA was performed to determine REST binding to the oligonucleotide sequence of its consensus sites in astrocyte nuclear extracts after REST overexpression. C, EMSA was performed in nuclear extracts prepared from H4 astrocytes as described in the Experimental procedures section. The red arrow indicates the protein–DNA complex form for both RE1 consensus sites, and the blue arrow indicates unbound oligos. D, site-directed mutagenesis of two RE1 conserved motifs on EAAT2 promoter, followed by luciferase assay. E, after astrocytes were cotransfected with WT or mutant EAAT2 promoter and REST expression vectors, promoter activity was determined by luciferase assay. F, after H4 astrocytes were cotransfected with WT or deletion constructs of EAAT2 promoter and REST expression vectors, luciferase assay was performed. G, after transfection of REST in human primary astrocytes, REST binding to its consensus sites in the EAAT2 promoter was determined by ChIP assay, followed by quantitative PCR to quantify REST binding activities to these two RE1 consensus sites of the EAAT2 promoter. ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; #p < 0.05; ##p < 0.01; ###p < 0.001; ####p < 0.0001 compared with the controls; @p < 0.05; @@@@p < 0.0001 compared with each other (one-way ANOVA followed by Tukey's post hoc test; n = 3). Data are expressed as mean ± SD. The data shown are representative of three independent experiments. ChIP, chromatin immunoprecipitation; DAPA, DNA affinity precipitation assay; EAAT2, excitatory amino acid transporter 2; RE1, repressor element 1; REST, repressor element 1-silencing transcription factor.