Characterization of FMR1 Promoter Elements by In Vivo-Footprinting Analysis

Abstract

Fragile X syndrome is associated with silencing of the FMR1 gene. We studied the transcriptional regulation, by analysis of the FMR1 promoter region for the presence of in vivo protein/DNA interactions and for cytosine methylation at the single-nucleotide level. Four protein-binding sites were present in the unmethylated promoter of the active FMR1 gene. In the methylated promoter of inactive genes no footprints were detected, and no evidence of active repression was found in the region investigated. We propose that the silencing of FMR1 gene transcription results from a lack of transcription-factor binding.

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

“…2). The four described previously in vivo protein binding sites, ␣ϪPal/Nrf-1, the 2 Sp1 or GC boxes, and the E-box (sometimes referred to as the c-Myc binding site) (5,6) are conserved in all five species (shown in the dark gray boxes in Fig. 2).…”

“…Sp3 Binds a FMR1 Promoter Subfragment-Factor binding to the 2 GC boxes in the human promoter has been reported in fibroblasts and lymphoid cells (5,6) and in vitro on a 71-bp promoter-containing fragment in extracts of the neuronally derived cell line SK-N-SH (15). However, GC-box-specific factors did not bind the full-length promoter in any of our extracts under a variety of reaction conditions; unlabeled GC-box oligonucleotides did not alter the EMSA profile (Fig.…”

“…2). These include the four in vivo protein binding sites found in fibroblasts and lymphoid cells (5,6), namely the E-box, two GC-boxes, and a ␣ϪPal/Nrf-1 binding site. The conservation of these sites among species as evolutionarily divergent as primates, canids, and rodents, which last shared a common ancestor 80 -120 million years ago, indicates that they are important for regulation of this gene.…”

“…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.…”

“…We also demonstrate that binding of one of these factors is abolished by methylation, and binding of the other two factors is also affected. This may have implications for therapeutic strategies aimed at reactivating the gene, since it indicates that methylation of the FMR1 promoter in Fragile X patients does not simply inhibit transcription via the formation of transcriptionally inactive chromatin, as suggested from in vivo dimethyl sulfate footprinting in lymphoblasts (6) and from the presence of deacetylated histones on the FMR1 promoter in individuals with Fragile X syndrome (9).…”