Occupancy and synergistic activation of the FMR1 promoter by Nrf-1 and Sp1 in vivo.
Fragile X syndrome is due to mutation of the FMR1 gene. The most common mutation is an expansion of a CGG repeat in the 5' UTR that triggers dense DNA methylation and formation of a heterochromatin-like structure which lead to transcriptional silencing. In vitro experiments have identified several transcription factors, including Sp1, Nrf-1 and USF1/2, as potential regulators of normal FMR1 promoter activity. Using CpG methylation-deficient Drosophila cells, we demonstrate in vivo that Nrf-1 and Sp1 are strong, synergistic activators of an unmethylated human FMR1-driven reporter, while USF1/2 and Max repress this activation. In addition, analyses of transcription factor activity upon DNA methylation of the reporter show that Sp1 activity was largely intact when the promoter was densely methylated, but Nrf-1 transactivation was very sensitive to dense methylation. Notably, Nrf-1 transactivation was relatively insensitive to methylation of cytosines only at its binding site. FMR1 reporter activity is also reduced in HeLa cells after expression of a short interfering RNA directed against endogenous Nrf-1. Using chromatin immunoprecipitation, we demonstrate directly that Sp1 and Nrf-1 occupy the human FMR1 promoter in vivo and these interactions are disrupted in fragile X patient cells. In addition, we discover that Max resides at the FMR1 promoter and show that USF1/2 but not c-Myc are present at endogenous FMR1. These findings provide the first direct in vivo evidence identifying the specific transcription factors that regulate FMR1.
Smith
Karen T
KT
Department of Biochemistry and Graduate Program in Genetics and Molecular Biology, Emory University School of Medicine, Atlanta, GA 30322, USA.
Coffee
Bradford
B
Reines
Daniel
D
D000818:Animals; D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004330:Drosophila / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins / Q000378:metabolism; D051860:Fragile X Messenger Ribonucleoprotein 1; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics*; D051266:Nuclear Respiratory Factor 1; D011401:Promoter Regions, Genetic; D016601:RNA-Binding Proteins / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D051720:Upstream Stimulatory Factors
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D051860:Fragile X Messenger Ribonucleoprotein 1; D009419:Nerve Tissue Proteins; D051266:Nuclear Respiratory Factor 1; D016601:RNA-Binding Proteins; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D051720:Upstream Stimulatory Factors; C497683:FMR1 protein, human; C102283:Max protein, Drosophila; C497181:USF1 protein, human; C088388:alpha-Pal transcription factor