Structural and functional characterization of the human FMR1 promoter reveals similarities with the hnRNP-A2 promoter region
Structural and functional characterization of the human FMR1 promoter reveals similarities with the hnRNP-A2 promoter regionRégen Drouin1,2, Martin Angers1,2, Nancy Dallaire1,2, Timothy M. Rose3, Edouard W. Khandjian1,2 and François Rousseau1,2,*
1Unité de Recherche en Génétique Humaine et Moléculaire,Centre de Recherche, Pavillon Saint-François d'Assise, Centre Hospitalier Universitaire de Québec, 10 rue de l'Espinay, Québec, Québec G1L 3L5, Canada, 2Département de Biologie Médicale, Faculté de Médecine, Université Laval, Québec, Canada and 3Department of Pathobiology, School of Public Health and Community Medicine, University of Washington, Seattle, WA 98195, USA
Received May 28, 1997;Revised and Accepted August 8, 1997
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.
The fragile X syndrome, a leading cause of inherited mental retardation (1 ) is associated with an unstable expansion of a polymorphic CGG trinucleotide repeat array localised in the 5' untranslated region (UTR) of the first exon of the FMR1 gene (2 -4 ). This gene spans 38 kb and comprises 17 exons (5 ) and a 5' CpG island of ~1 kb which encompasses the triplet repeats (2 ). The expansion of the CGG repeats is associated with abnormal DNA methylation of the CpG island and correlates with the transcriptional silencing of FMR1 expression in fragile X patients (2 ,3 ,6 -11 ). Although it has been shown that FMR1 is widely expressed in different tissues during embryogenesis (12 ,13 ), as well as in young and adult tissues (9 ,11 ,14 ,15 ), little is known about the control elements in the promoter region. In a transgenic experiment with a reporter gene, a 2.8 kb fragment spanning the region 5' of exon 1 appeared sufficient to produce a tissue distribution of expression of the reporter gene similar to the endogenous murine Fmr1 gene (16 ). Primer extension studies have identified a transcription initiation site upstream of the CGG repeat array and 26 bp downstream of a TATA-like sequence (TTACA) (17 ). In this same study, deletion analysis detected high CAT promoter activity associated with a 466 bp PstI-XhoI fragment containing part of the CpG island with 272 bp upstream and 193 bp downstream of the transcription initiation site. Deletion of the transcription initiation site and the 193 bp downstream sequences from this gene fragment inhibited all promoter activity. Promoter activity was also inhibited after in vitro DNA methylation of cytosines in all CpG dinucleotides in the 466 bp promoter fragment.
Clues to understanding both the regulation of FMR1 gene expression and the functions of its encoded protein (FMRP) can be obtained from the study of the factors that modulate FMR1 transcription. We have identified cis-controlling elements in the FMR1 promoter by in vivo DMS (dimethylsulfate) footprinting. Protein binding to these elements was detected in normal cells (lymphocytes, fibroblasts and lymphoblastoid cells) but not in those with fragile X mutations. Computer-aided analysis identified sequences in the promoter region of the gene coding for heterogenous ribonucleoprotein (hnRNP)-A2 which had striking similarity to the transcriptional regulatory elements showing protein-DNA interactions in the FMR1 promoter.
Principle of in vivo DMS footprinting by ligation-mediated polymerase chain reaction (LM-PCR). When applied to living cells, DMS diffuses across the plasma and nuclear membranes into the nucleus where it preferentially methylates guanine residues through the major groove at the N-7 position. Guanine residues in contact with sequence-specific DNA-binding proteins display a different degree of reactivity with DMS compared with guanine residues not in contact with binding proteins. Proteins in contact with DNA either decrease accessibility of specific guanines to DMS (protection) or, often at the edges of a footprint, increase reactivity (hyperreactivity) (18 ). Hyperreactivity can also indicate a greater DMS accessibility allowed by special in vivo DNA structures. The glycosylic bond of methylated guanines, as well as the DNA phosphate backbone at the apurinic sites, can be cleaved by hot piperidine leaving a 5'-phosphate (19 ). These 5'-phosphate single-strand breaks can be quantitatively mapped at the resolution of a single base by the LM-PCR technique coupled with analysis on a DNA sequencing gel. The unique aspect of LM-PCR is the blunt-end ligation of an asymmetric double-stranded linker onto the 5'-end of each nicked DNA molecule (20 ). The blunt end is created by the extension of a gene-specific primer #1 until a strand break is reached. This linker provides a common sequence at all 5'-ends. A PCR amplification is then performed using the linker primer and the gene specific primer #2 which is nested 3' of the first gene specific primer (#1). The size of amplification products then corresponds to the position of strand breaks in relation to the position of gene specific primer #2 in the promoter region. The PCR products are size-fractionated on a sequencing gel. LM-PCR amplification with 20 thermal cycles preserves the quantitative representation of each DMS-induced strand break in the original DNA sample. From our experiments and those of others, LM-PCR gives a reproducible quantification of the DMS-induced strand breaks within a given DNA sample (21 -24 ). In vivo footprinting is considered to be the most accurate predictor of the state of transcriptional activity of genes because it more accurately represents the DNA-protein interactions encountered in living cells. In vitro assays using purified naked DNA are compromised because many of the specific DNA structures present in vivo have been lost during the DNA purification steps (18 ,25 ,26 ).
In vivo DMS protein footprints of the FMR1 promoter. Transcription initiation of the FMR1 gene has been detected at position +1 (Fig. 3 ) within a 466 bp fragment which has been shown to have promoter activity (17 ). We have analyzed both strands of a 1.1 kb sequence in the 5' region of the FMR1 gene for in vivo protein footprints. The region studied corresponds to ~620 bp upstream of the major transcription start site (located at position 13719 in the GenBank sequence #L29074), the first exon including the 5'UTR (273 bp in GenBank) and 51 bp of the initial coding sequence, and ~170 bp of the first intron. As indicated above, the 5'UTR containing the CGG repeats is expanded in DNA samples from patients with fragile X syndrome. We were unable to obtain interpretable results across the G-C rich CGG repeat region probably due to secondary structure problems in the amplification and gel analysis steps.
In primary fibroblasts, lymphoblasts and peripheral lymphocytes from healthy male donors which contain a transcriptionally active FMR1 gene, only four DMS footprints were observed (Figs 1 , 2 and 3 ). These footprints were present within a 91 bp region 5' of the major transcription start site (Fig. 3 ). They were localised at nucleotides -131 to -123 (5'-GCGCATGCG-3'), nt -95 to -90 (5'-GGGCGG-3'), nt -68 to -60 (5'-GGGGGAGGG-3') and nt -50 to -40 (5'-GATCACGTGAC-3'). The 5' and 3' boundaries of these four sequences involved in DNA-protein interactions were easily identified (Figs 1 and 2 ). The sequences covering nt -620 to -135 and the proximal 130 bp of the first intron did not show any evidence for protein-DNA interactions (data not shown). None of these four footprints was detected in any of the fibroblasts or lymphoblasts from male patients with fragile X syndrome who have a transcriptionally inactive FMR1 gene (Fig. 1 ). In addition, the studied promoter region in the fragile X cells showed no other indication of protein binding or special DNA structures. For these patients, the FMR1 gene has a large expansion of >230 CGG repeats in the 5'UTR. This expansion is associated with an abnormal methylation of the CpG residues as indicated by the inability to cleave the classical EagI site in this region (27 ). The presence of this EagI restriction site for each sample was confirmed by genomic sequencing (provided by the LM-PCR procedure). Furthermore, methylation-sensitive LM-PCR analysis (18 )of CpGs within the 1.1 kb region studied confirmed that all CpGs were methylated in these cells (unpublished results).
We used TESS (Transcription Element Search Software) to search for potential transcription factor binding sites within the proximal FMR1 promoter sequence. The sequence of the FMR1 promoter is shown in Figure 3 with the positions corresponding to some of the potential transcription factor binding sites indicated. In general, the FMR1 promoter is very GC rich and multiple copies of the consensus binding site for the transcription activator Sp1 (28 ) are present. Sequences matching other transcription factor binding sites were also identified including AP2, UBP1, AGP/EBP, Myc and Zeste as indicated in Figure 3 . Moreover, several transcription factor binding sites corresponded to the sequences within the four footprints identified above. As shown in Table 2 and Figure 3 , the sequence across the most 5' footprint in the 1.1 kb fragment at position -131 to -122 (CGCGCATGCGCG) is identical to the palindromic consensus binding site (YGCGCAYGCGCR) for [alpha]-PAL/NRF1. [alpha]-PAL was initially detected as a transcription factor involved in the regulation of the expression of the eukaryotic Initiation Factor 2 alpha (eIF-2[alpha]), a translation initiation factor (29 ). Cloning studies have shown this factor to be identical to nuclear respiratory factor (NRF)-1 which stimulates the transcription of nuclear genes whose products function in the mitochondria (30 ). All of the protected guanines in the -131 to -122 footprint were present in the [alpha]-PAL/NRF1 consensus binding site (see Fig. 3 ) indicating a close correlation between the experimentally determined footprint and the predicted [alpha]-PAL/NRF1 binding site.
The sequence across the second in vivo footprint (-98, GAGGGCGGGGC, -88), as shown in Figure 3 , has 10/11 identical nucleotides with the consensus binding site (GGGGGCGGGGY) for Sp1, a ubiquitous transcription activator involved in cell development and differentiation (Table 1 ) (28 ). The sequence across the third in vivo footprint (-68, GGGGGAGGG, -60) is completely contained within the consensus binding site for the histone H4 gene-specific transcription activator (H4TF1; GGGGGAGGG) and Sp1-like transcription factors (GGGGGCGGGGY; 31 ), and is partially contained within the consensus binding site for the transactivator AP-2 (MKCCCSCNGGCG; 32 ) (see Fig. 3 and Table 1 ). The sequence across the fourth in vivo footprint (-50, GATCACGTGACG, -38) is identical to the core binding site (CACGTG) for c-MYC and MYC-like transcription factors and matches well with an extended consensus binding site for c-MYC (RACCACGTGCTC; 33 ).
Consensus sequences recognized by transcriptional factors
M = A,C; N = A,C,G,T; K = G,T; R = A,G; S = G,C; Y = C,T.
Analysis of the sequence (+12, GGCGCTCAGCTCC, +24) across the singular DNA structure which was identified at nt +14 in the LM-PCR study above, revealed the presence of a palindromic structure, centered at nt +18. TESS analysis also revealed similarities between this sequence and the binding site (CGCTCA) for the Zeste transcription factor in Drosophila. As indicated above, however, no protein footprint was associated with this DNA region in either actively transcribed or silent FMR1 genes.
In order to determine whether sequences similar to those of the FMR1 promoter exist in the promoter regions of previously characterized genes, the 1100 bp fragment of the FMR1 gene was used as a probe in a search of the non-redundant nucleotide sequence database using the BLAST search algorithm. This type of search is constrained by the defined word length of 12 used in the BLAST search which only allows sequences containing an initial match of 12 identical nucleotides in a row to be further considered for similarity scores. Most identified promoter elements have sequence lengths <12 nt. Unexpectedly, a 36 bp sequence within the promoter region of the hnRNP-A2 gene (from -896 to -854; GenBank accession no. U09120) was identified as having an 83% match with a sequence between -96 and -60 of the FMR1 promoter region (Fig. 5 b). This corresponds to the region in FMR1 containing the footprints for the Sp1, AP2 and H4TF1/Sp1-like binding sites, and is herein denoted as region II (Figs 3 and 4 ). While the BLAST score for the nucleotide match (0.94) would not normally be considered significant for gene to gene comparisons, such a match between small promoter elements could. Therefore, a comparison of the two DNA sequences was undertaken. This analysis demonstrated complete conservation across the region of the footprints in the FMR1 promoter region with the corresponding sequence in the hnRNP-A2 promoter region (Fig. 5 b). In addition, the sequence of the hnRNP-A2 gene also corresponded to the consensus binding sites for the Sp1, AP2 and H4TF1/Sp1-like transcription factors, as seen in FMR1 (see Table 1 ). Subsequent analysis of this region in both genes revealed a second region of nucleotide similarity upstream of region II. This region, called region I, contained [alpha]-PAL/NRF1 and AGP/EBP consensus binding sites in both promoters, although the hnRNP-A2 sequence was inverted in relationship to the FMR1 sequence (Figs 3 and 5 a). Both promoter regions contained comparable palindromic sequences within the predicted transcription factor binding sites, and the guanines protected in the FMR1 footprint were conserved in the hnRNP-A2 sequence (Fig. 5 a). Recently, a chicken homolog of [alpha]-PAL/NRF-1 was identified (34 ). This factor which represses transcription of histone H5 has high affinity binding to the consensus site `RCGCRYGCGY' which is identical to the potential [alpha]-PAL/NRF-1 binding site in hnRNP-A2 (Table 1 ).
The hnRNP-A2 promoter sequence was also analyzed using the TESS software and a number of Sp1 and H4TF1/Sp1-like binding sites were identified (Fig. 4 ).
Human skin fibroblasts from healthy male donors and the fibroblasts from a male with the fragile X syndrome were grown in DMEM supplemented with 10% calf serum (FCS). For LM-PCR (see below) analysis, the culture medium was replaced with fresh unsupplemented medium containing 0.2% dimethylsulfate (DMS, Aldrich) (22 ). After incubation for 6 min at room temperature, the cell monolayer was washed with Ca-Mg-free HBSS (Hanks' Balanced Salt Solution) medium and detached by trypsinization. After cell lysis, nuclei were isolated and the DNA purified as described (20 ,22 ). Peripheral blood lymphocytes were isolated from heparinized blood by centrifugation on Ficoll-Hypaque (Pharmacia) gradients. Epstein-Barr-virus-transformed lymphoblastoid cell lines originating from healthy female and male donors as well as from fragile X patients were maintained in RPMI medium supplemented with 15% FCS. Lymphocytes and lymphoblastoid cell lines were treated with 0.2% DMS for 6 min prior to isolation of the nuclei. DNA concentration was measured by fluorometry after staining with 4'-6-diamidino-2-phenylindole (DAPI) (20 ). For comparison, DNA purified from lymphocytes was treated with 0.5% DMS (in vitro treatment) as described (23 ). In the present study, six different types of cells were studied: fragile X mutated primary fibroblasts NIGMS #GM04024B, fragile X mutated lymphoblastoid cell line RMGA#Q.691, normal lymphoblastoid cells from a male RMGA#Q.001 and a female RMGA#Q.772 as well as normal low-passage foreskin fibroblasts cultures and fresh lymphocytes from two different normal male donors.
DMS-exposed DNA was treated with hot piperidine (Fluka) to convert methylated bases to DNA strand breaks (23 ). The single-strand break frequency in the total genomic DNA was determined by alkaline agarose (1.5%) gel electrophoresis (58 ).
Details of the LM-PCR protocol used for this work have already been published (20 ). The procedure can be divided into six steps: (i) primer extension of an annealed gene-specific oligonucleotide (primer 1) to generate blunt ends from nicked genomic DNA; (ii) ligation of a universal asymmetric double-strand linker; (iii) PCR amplification using a second gene-specific oligonucleotide (primer 2); (iv) separation of the DNA fragments on a sequencing polyacrylamide gel; (v) transfer of the DNA to a nylon membrane by electroblotting; (vi) hybridization of a radiolabeled probe prepared by repeated primer extension using a third gene-specific oligonucleotide (primer 3).
Approximately 1100 base pairs of the human FMR1 gene (GenBank accession no. L29074: from bp 13080 to 14180), including the promoter region, part of the first exon and proximal part of the first intron, were studied (see Results) on both strands using the primer sets described in Table 2 . Primer extension was initiated with 0.5-1.0 [mu]g of DNA in duplicate. Initially, one half of each duplicate sample was analyzed as described (20 ). If there was no significant variation between duplicate samples, the remainder of the two samples were pooled and a combined gel was produced. Thus, each analysis represents the sum of both duplicate samples. The nylon membranes were exposed to a phosphor-sensitive imaging plate (Type III-s) and the band intensities were quantified by Phosphoimager, Fuji BAS 1000 (Fuji Medical Systems USA Inc., Stanford, CT).
DNA analysis by Maxam-Gilbert cleavage reactions was carried out as described (23 ), except DNA for the `adenine' reactions was treated with K2PdCl4 at pH 2.0 followed by piperidine treatment (59 ). Chemically cleaved G, A, T+C and C samples were included along with the other samples in the LM-PCR assays in order to provide base markers in the sequencing gels.
Total protein extracts were prepared by lysing the cells in SDS sample buffer (68 mM Tris-HCl, 2%SDS, 2% [beta]-mercaptoethanol, 6% glycerol) followed by sonication and heat denaturation in a boiling bath for 3 min. Immunoblot analyses were performed using the ECL (DuPont) system as described (15 ). FMRP was detected using 1C3 monoclonal antibody (9 ) and hsp90 was detected with affinity-purified rabbit polyclonal antibodies (60 ).
To determine whether similarities exist between the FMR1 promoter and other characterized nucleotide sequences, we performed a BLASTN search against the non-redundant DNA database, as implemented in the Baylor College of Medicine Search Launcher (http://dot.imgen.bcm.tmc.edu:9331/seq-search/gene-search.html). To identify potential transcription regulatory sites in the FMR1 and hnRNP-A2 promoters, we used TESS (Transcription Element Search Software) as implemented on the Baylor College of Medicine Search Launcher (as above). This software accesses the TRANSFAC MATRIX database Release 3.1 which contains the consensus binding sites for a variety of transcription factors.
We thank S. Tremblay for cell culture, J.-P. Therrien for assistance with the Phosphorimager and M. Vaillancourt for his help in the initial steps of this work. We thank D. Devys and J.-L. Mandel for 1C3 antibody and R. Tanguay for the affinity-purified hsp90 antibody. This project was supported by the Canadian Genetic Diseases Network (MRC/NSERC NCE Program) and by the Medical Research Council (MRC) of Canada. Lymphoblastoid cell lines of fragile X patients were established by the `Banque Lymphoblastique du Réseau de Médecine Génétique Appliquée du FRSQ'. R.D. is a research scholar of the Cancer Research Society Inc./MRC program. M.A. holds a studentship from the Department of Medical Biology, Faculty of Medicine, Université Laval. F.R. is an MRC Scientist.
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*To whom correspondence should be addressed. Tel: +1 418 525 4470; Fax: +1 418 525 4481; Email: francois.rousseau@crsfa.ulaval.ca
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Table 1.
