Structural and Functional Characterization of the Human FMR1 Promoter Reveals Similarities with the hnRNP-A2 Promoter Region

Abstract

Fragile X mental retardation syndrome is associated with an expansion of a CGG repeat within the 5'UTR of the first exon of the FMR1 gene, abnormal methylation of the CpG island in the promoter region, and a transcriptional silencing of this gene. We studied transcriptional regulation of the FMR1 gene using protein footprint analysis of the active and inactive gene in vivo . We identified four footprints within the FMR1 promoter region which correspond to consensus binding sites of known transcription factors, alpha-PAL/NRF1, Sp1, H4TF1/Sp1-like and c-myc. These footprints were present in normal cells with a transcriptionally active FMR1 gene. The same footprints were present in different cell types: primary fibroblasts, lymphoblastoid cells and peripheral lymphocytes. However, for the 1.1 kb region analyzed, no footprints were detected in a variety of cell types derived from patients with fragile X syndrome which have a transcriptionally inactive FMR1 gene. A BLAST nucleotide search identified sequence similarities between the region of the FMR1 gene containing the footprints and an analogous region within the promoter region of the gene for the heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a member of a family of ribonucleoproteins implicated in mRNA processing and nuclear-cytoplasm transport. The nucleotide sequences identified in the hnRNP-A2 promoter region correspond to the same consensus binding sites showing DNA-protein interactions in the FMR1 gene. Our previous functional studies and the studies of others demonstrate that FMR proteins, like hnRNP-A2, are also ribonucleoproteins which appear to be involved in mRNA transport. The results from our footprint studies suggest that the expression of the FMR1 gene is regulated by the binding of specific transcription factors to sequence elements in the 5' region of the gene and that this expression may be regulated by elements in common with the hnRNP-A2 gene. Common regulation of these two genes might play an important role in the cooperative processing and transport of mRNA from the nucleus to the translation machinery.

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

“…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).…”

“…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.…”

“…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.…”

Mapping of an origin of DNA replication in the promoter of fragile X gene FMR1

“…The width of each bar in the histogram is proportional to the size of the PCR product and is plotted based on sequence data available at genome.ucsc.edu. Below the histogram are indicated the percent GC in 5-base windows (dark areas: high GC content, light areas: low GC content), the position of the transcriptional promoter (Drouin et al, 1997) and FMR1 exon 1, the associated CpG island and trinucleotide repeat (based on data also available at genome.ucsc.edu). The shaded box drawn around the peak of abundance delimits the boundary of the "initiation region" as defined in the text.…”