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

Abstract

An origin of bidirectional DNA replication was mapped to the promoter of the FMR1 gene in human chromosome Xq27.3, which has been linked to the fragile X syndrome. This origin is adjacent to a CpG island and overlaps the site of expansion of the triplet repeat (CGG) at the fragile X instability site, FRAXA. The promoter region of FMR2 in the FRAXE site (approximately 600 kb away, in chromosome band Xq28) also includes an origin of replication, as previously described [Chastain II, P.D., Cohen, S.M., Brylawski, B.P., Cordeiro-Stone, M., Kaufman, D.G., 2006. A late origin of DNA replication in the trinucleotide repeat region of the human FMR2 gene. Cell Cycle 5, 869-872]. FMR1 transcripts were detected in foreskin and male fetal lung fibroblasts, while FMR2 transcripts were not. However, both FMR1 and FMR2 were found to replicate late in S phase (approximately 6 h into the S phase of normal human fibroblasts). The position of the origin of replication relative to the CGG repeat, and perhaps the late replication of these genes, might be important factors in the susceptibility to triplet repeat amplification at the FRAXA and FRAXE sites.

Figure 1

Nascent strand abundance analysis in the human FMR1 promoter region of NHF1-hTERT cells. Each marker shown in this histogram was tested at least three times using nascent DNA prepared from normal human fibroblasts (NHF1-hTERT cells). Error bars show the standard deviation. A second preparation of nascent DNA from these cells was also tested (each marker tested at least twice) with the same results. 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.

Figure 2

Nascent strand abundance analysis in the human FMR1 promoter region of GM1604-hTERT cells. The relative abundance of markers in a preparation of short nascent DNA from GM1604-hTERT, a fetal lung fibroblast cell line, was determined as described for NHF1-hTERT cells. Each primer set was tested at least twice and error bars show the standard deviation. These results indicate that the origin of replication associated with the FMR1 promoter is found in the same location in both the neonatal and fetal fibroblast cell lines that were tested.

Figure 3

Reverse Transcription PCR. RT-PCR of FMR1 and FMR2 messenger RNA in total RNA isolated from log phase NHF1-hTERT (lane 1), 1604-hTERT (lane 2), and CRL-1502 (lane 3) cells. Products were visualized on a 2% agarose gel stained with ethidium bromide (shown in reverse contrast). A negative control (no template DNA) was also included (lane 4). Results show that FMR1 is expressed in both cell types analyzed for replication origin activity while FMR2 is expressed in neither. We also analyzed cDNA from CRL-1502 cells (female fetal lung fibroblast cells) as a positive control. The resulting PCR product had the expected size, indicating that the FMR2 primer set was working correctly.

Figure 4

Determination of the timing of replication of the FMR1 gene in NHF1-hTERT cells. Newly synthesized DNA from seven different 1-h S phase intervals was tested by quantitative PCR to determine when the FMR1 gene region replicates. Panel A: Image of PCR products separated on an ethidium bromide-stained agarose gel (shown in reverse contrast). S-phase fractions were each tested in duplicate. Images were scanned and bands quantified as described in the Materials and Methods section. Panel B: Bar graph illustrating the abundance of the marker in each of the seven 1-h S-phase samples. Relative abundance was calculated from the linear regression equation of the standard curve and expressed as a percentage of the highest value. Results are the average of two tests and error bars indicate the standard deviation. A second synchronization was tested with similar results. These data indicate that FMR1 replicates very late in S phase, approximately at the same time as the FMR2 gene region (Chastain et al., 2006).

Fig. 5

Schematic representation of the relative position of initiation region and triplet repeats for the FMR1 and FMR2 origins. The boxed areas in A and C represent the “initiation region” for the found origins spanning from the midpoint of the sequence between markers D and E to the midpoint of the sequence between marker E and F (illustrated in Fig. 1). The position and type of triplet repeats are indicated for FMR1 (A, B) and FMR2 (C, D) (Chastain et al., 2006). The schematics in A and C illustrate the position of leading and lagging replicating strands with respect to the active origin and the triplet repeats. The schematics in B and D illustrate a hypothetical origin active on the 3′ site of the respective origin, and the consequent possible formation of misprimed fragments during replication. The asterisk indicates the triplet repeat configuration in the template that is more likely to cause mispriming in nascent fragments.