Interaction of the Transcription Factors USF1, USF2, and α-Pal/Nrf-1 with the FMR1 Promoter

Abstract

Hypermethylation of the FMR1 promoter reduces its transcriptional activity, resulting in the mental retardation and macroorchidism characteristic of Fragile X syndrome. How exactly methylation causes transcriptional silencing is not known but is relevant if current attempts to reactivate the gene are to be successful. Understanding the effect of methylation requires a better understanding of the factors responsible for FMR1 gene expression. To this end we have identified five evolutionarily conserved transcription factor binding sites in this promoter and shown that four of them are important for transcriptional activity in neuronally derived cells. We have also shown that USF1, USF2, and ␣؊Pal/Nrf-1 are the major transcription factors that bind the promoter in brain and testis extracts and suggest that elevated levels of these factors account in part for elevated FMR1 expression in these organs. We also show that methylation abolishes ␣؊Pal/Nrf-1 binding to the promoter and affects binding of USF1 and USF2 to a lesser degree. Methylation may therefore inhibit FMR1 transcription not only by recruiting histone deacetylases but also by blocking transcription factor binding. This suggests that for efficient reactivation of the FMR1 promoter, significant demethylation must occur and that current approaches to gene reactivation using histone deacetylase inhibitors alone may therefore have limited effect.Fragile X syndrome is caused by the expansion of a CGG repeat in the 5Ј-untranslated region of the fragile X mental retardation (FMR1) gene (1, 2). This results in hypermethylation of the promoter and transcriptional silencing (3). The major symptoms of fragile X syndrome, mental retardation and macroorchidism, are consistent with the observation that high levels of FMR1 expression occurs in specific cells in brain and testis (4). The GC-rich human FMR1 promoter lacks a typical TATA-box and contains several potential Sp1 binding sites as well as an E-box and putative binding sites for the transcription factors ␣-Pal/Nrf-1, 1 AP2, AGP/EBP, and Zeste (5). Four regions of protein binding in the unmethylated promoter of the FMR1 gene have been described by in vivo dimethyl sulfate footprinting analysis in human fibroblasts, peripheral lymphocytes, and lymphoblastoid cell lines (5, 6). These footprints correspond to the ␣-Pal/Nrf-1 site, 2 GC-boxes, and the E-box. However, the transcription factors that interact with these sites have not yet been identified. Moreover, whereas these footprints do reflect in vivo interactions, their relevance to the regulation of FMR1 transcription in brain and testis, the two major affected organs, is unknown.Because FMR1 knockout mice demonstrate learning deficits and macroorchidism similar to those seen in fragile X patients (7), and mice show patterns of temporal and tissue-specific FMR1 expression similar to humans (8), many of the control elements important for FMR1 gene regulation are also likely to be evolutionarily conserved. To identify conserved promoter elements, we compared the ...

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Fragile X syndrome: causes, diagnosis, mechanisms, and therapeutics

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