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.
Institute for Clinical and Molecular Virology, Erlangen University Medical School, D-91054 Erlangen, Germany.
Anja
A
Naumann
Norbert
N
Hochstein
Stefanie
S
Weber
Ellen
E
Fanning
Walter
W
Doerfler
D020121:5' Untranslated Regions / Q000235:genetics; D000328:Adult; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D018899:CpG Islands; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D019175:DNA Methylation; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics*; D005600:Fragile X Syndrome / Q000235:genetics*; D016678:Genome; D015894:Genome, Human; D045325:HCT116 Cells; D006801:Humans; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017422:Sequence Analysis, DNA; D013447:Sulfites / Q000494:pharmacology
D020121:5' Untranslated Regions; D004247:DNA; D051860:Fragile X Messenger Ribonucleoprotein 1; D009687:Nuclear Proteins; D013447:Sulfites; C497683:FMR1 protein, human