An origin of DNA replication in the promoter region of the human fragile X mental retardation (FMR1) gene

Fragile X syndrome, the most common form of inherited mental retardation in males, arises when the normally stable 5 to 50 CGG repeats in the 5' untranslated region of the fragile X mental retardation protein 1 (FMR1) gene expand to over 200, leading to DNA methylation and silencing of the FMR1 promoter. Although the events that trigger local CGG expansion remain unknown, the stability of trinucleotide repeat tracts is affected by their position relative to an origin of DNA replication in model systems. Origins of DNA replication in the FMR1 locus have not yet been described. Here, we report an origin of replication adjacent to the FMR1 promoter and CGG repeats that was identified by scanning a 35-kb region. Prereplication proteins Orc3p and Mcm4p bind to chromatin in the FMR1 initiation region in vivo. The position of the FMR1 origin relative to the CGG repeats is consistent with a role in repeat maintenance. The FMR1 origin is active in transformed cell lines, fibroblasts from healthy individuals, fibroblasts from patients with fragile X syndrome, and fetal cells as early as 8 weeks old. The potential role of the FMR1 origin in CGG tract instability is discussed.     

Instability of the CGG repeat and expression of the FMR1 protein in a male fragile X patient with a lung tumor.

The molecular mechanism of the fragile X syndrome is based on the expansion of an CGG repeat in the 5' UTR of the FMR1 gene in the majority of fragile X patients. This repeat displays instability both between individuals and within an individual. We studied the instability of the CGG repeat and the expression of the FMR1 protein (FMRP) in several different tissues derived from a male fragile X patient. Using Southern blot analysis, only a full mutation is detected in 9 of the 11 tissues tested. The lung tumor contains a methylated premutation of 160 repeats, whereas in the testis, besides the full mutation, a premutation of 60 CGG repeats is detected. Immunohistochemistry of the testis revealed expression of FMR1 in the spermatogonia only, confirming the previous finding that, in the sperm cells of fragile X patients with a full mutation in their blood cells, only a premutation is present. Immunohistochemistry of brain and lung tissue revealed that 1% of the cells are expressing the FMRP. PCR analysis demonstrated the presence of a premutation of 160 repeats in these FMR1-expressing cells. This indicates that the tumor was derived from a lung cell containing a premutation. Remarkably, despite the methylation of the EagI and BssHII sites, FMRP expression is detected in the tumor. Methylation of both restriction sites has thus far resulted in a 100% correlation with the lack of FMR1 expression, but the results found in the tumor suggest that the CpGs in these restriction sites are not essential for regulation of FMR1 expression. This indicates a need for a more accurate study of the exact promoter of FMR1.     

Stable DNA Methylation Boundaries and Expanded Trinucleotide Repeats: Role of DNA Insertions

The human genome segment upstream of the FMR1 (fragile X mental retardation 1) gene (Xq27.3) contains several genetic signals, among them is a DNA methylation boundary that is located 65–70 CpGs upstream of the CGG repeat. In fragile X syndrome (FXS), the boundary is lost, and the promoter is inactivated by methylation spreading. Here we document boundary stability in spite of critical expansions of the CGG trinucleotide repeat in male or female premutation carriers and in high functioning males (HFMs). HFMs carry a full CGG repeat expansion but exhibit an unmethylated promoter and lack the FXS phenotype. The boundary is also stable in Turner (45, X) females. A CTCF-binding site is located slightly upstream of the methylation boundary and carries a unique G-to-A polymorphism (single nucleotide polymorphism), which occurs 3.6 times more frequently in genomes with CGG expansions. The increased frequency of this single nucleotide polymorphism might have functional significance. In CGG expansions, the CTCF region does not harbor additional mutations. In FXS individuals and often in cells transgenomic for EBV (Epstein Barr Virus) DNA or for the telomerase gene, the large number of normally methylated CpGs in the far-upstream region of the boundary is decreased about 4-fold. A methylation boundary is also present in the human genome segment upstream of the HTT (huntingtin) promoter (4p16.3) and is stable both in normal and Huntington disease chromosomes. Hence, the vicinity of an expanded repeat does not per se compromise methylation boundaries. Methylation boundaries exert an important function as promoter safeguards.     

A Distinct DNA-Methylation Boundary in the 5′- Upstream Sequence of the FMR1 Promoter Binds Nuclear Proteins and Is Lost in Fragile X Syndrome

We have discovered a distinct DNA-methylation boundary at a site between 650 and 800 nucleotides upstream of the CGG repeat in the first exon of the human FMR1 gene. This boundary, identified by bisulfite sequencing, is present in all human cell lines and cell types, irrespective of age, gender, and developmental stage. The same boundary is found also in different mouse tissues, although sequence homology between human and mouse in this region is only 46.7%. This boundary sequence, in both the unmethylated and the CpG-methylated modes, binds specifically to nuclear proteins from human cells. We interpret this boundary as carrying a specific chromatin structure that delineates a hypermethylated area in the genome from the unmethylated FMR1 promoter and protecting it from the spreading of DNA methylation. In individuals with the fragile X syndrome (FRAXA), the methylation boundary is lost; methylation has penetrated into the FMR1 promoter and inactivated the FMR1 gene. In one FRAXA genome, the upstream terminus of the methylation boundary region exhibits decreased methylation as compared to that of healthy individuals. This finding suggests changes in nucleotide sequence and chromatin structure in the boundary region of this FRAXA individual. In the completely de novo methylated FMR1 promoter, there are isolated unmethylated CpG dinucleotides that are, however, not found when the FMR1 promoter and upstream sequences are methylated in vitro with the bacterial M-SssI DNA methyltransferase. They may arise during de novo methylation only in DNA that is organized in chromatin and be due to the binding of specific proteins.     

Gene structure and expression of the 5'-(CGG)(n)-3'-binding protein (CGGBP1)

The human nuclear 5'-(CGG)(n)-3'-binding protein (CGGBP1) influences the expression of the fragile X mental retardation (FMR1) gene by specifically interacting with the 5'-(CGG)(n>5)-3' repeat in its 5' untranslated region. Here, we show that CGGBP1 binds to 5'-(CGG)(n)-3' repeats with n>or=5 and to interrupted repeats. The genomic and mRNA organization of the human and murine CGGBP1 genes was studied and the human gene was mapped to chromosome 3p. Due to alternative polyadenylation, mRNAs of 1.2 and 4.5 kb are transcribed at varying ratios in human and murine cells and in embryonic, fetal, and adult tissues. The human and the murine genes, including promoters and large parts of the untranslated regions, are highly conserved. A sequence of 235 nucleotides 5' upstream of CGGBP1 is essential for promoter activity in transfection experiments. Complete in vitro methylation inactivates the promoter, which is unmethylated in human cells as shown by bisulfite genomic sequencing.     

甲基化特异性PCR检测FMR1 和XIST基因甲基化实验方法的建立

建立一种快速、灵敏的检测脆性X智障基因（Fragile X mental retardation, FMR1）和X染色体失活基因（X chromosome inactivation,XIST）甲基化的方法,用亚硫酸氢钠和对苯二酚对基因组DNA进行脱氨基修饰。以修饰后的DNA为模板,用两套不同的引物对：1对甲基化特异性引物和1对非甲基化特异性引物扩增FMR1基因（CGG）n重复序列区、FMR1 和XIST 基因的启动子区。PCR产物进一步克隆、测序。以亚硫酸氢钠和对苯二酚脱氨基修饰后的DNA为模板,进行PCR扩增后的产物与预期基因目的基因片段大小相符合,无非特异性扩增产物。测序结果表明,FMR1、XIST基因中的非甲基化的C碱基转变为U碱基,而CpG岛被甲基化的C碱基不改变。成功地建立了检测FMR1、XIST甲基化的方法,为实验室诊断脆性X综合征提供了新的方法。     

A fragile X case with an amplification/deletion mosaic pattern

Fragile X syndrome is the most common cause of hereditary mental retardation. The FMR1 gene, which is involved in fragile X syndrome, contains a polymorphic CGG repeat, which expands in affected patients. Expanding triplet repeats have been shown to be a new type of mutation, termed "dynamic mutation", responsible for more than 12 genetic diseases. These mutations occur as multiple steps rather than as a single event. The first step leads to an unstable allele that then becomes increasingly unstable generally achieving further increases in copy or occasionally contraction. In this report, we describe a fragile X boy with both a hypermethylated full mutation and a deletion of 905 bp encompassing the CGG repeat. The upstream breakpoint is 438 bp 5' to the CGG repeat and the downstream breakpoint is 420 bp 3' of the triplet repeats. The deletion includes the ATG starting codon for translation of the FMR1 gene. This was confirmed by using FMRP immunocytochemistry both on blood smears and hair roots. The deleted region is flanked by a ccgg direct repeat next to the breakpoints; this may have had a critical role in the formation of a secondary DNA structure leading to the deletion.     

Elevated Fmr1 mRNA levels and reduced protein expression in a mouse model with an unmethylated Fragile X full mutation

The human FMR1 gene contains a CGG repeat in its 5' untranslated region. The repeat length in the normal population is polymorphic (5-55 CGG repeats). Lengths beyond 200 CGGs (full mutation) result in the absence of the FMR1 gene product, FMRP, through abnormal methylation and gene silencing. This causes Fragile X syndrome, the most common inherited form of mental retardation. Elderly carriers of the premutation, defined as a repeat length between 55 and 200 CGGs, can develop a progressive neurodegenerative syndrome: Fragile X-associated tremor/ataxia syndrome (FXTAS). In FXTAS, FMR1 mRNA levels are elevated and it has been hypothesised that FXTAS is caused by a pathogenic RNA gain-of-function mechanism. We have developed a knock in mouse model carrying an expanded CGG repeat (98 repeats), which shows repeat instability and displays biochemical, phenotypic and neuropathological characteristics of FXTAS. Here, we report further repeat instability, up to 230 CGGs. An expansion bias was observed, with the largest expansion being 43 CGG units and the largest contraction 80 CGG repeats. In humans, this length would be considered a full mutation and would be expected to result in gene silencing. Mice carrying long repeats ( approximately 230 CGGs) display elevated mRNA levels and decreased FMRP levels, but absence of abnormal methylation, suggesting that modelling the Fragile X full mutation in mice requires additional repeats or other genetic manipulation.