Cis-acting DNA sequence at a replication origin promotes repeat expansion to fragile X full mutation.
Bottom Line: This origin is absent in FXS human embryonic stem cells (hESCs), which have the SNP variant C, but present in the nonaffected hESCs, which have a T variant.Interestingly, premutation hESCs have a replication origin and the T variant similar to nonaffected hESCs.These results suggest that a T/C SNP located at a replication origin could contribute to the inactivation of this replication origin in FXS hESCs, leading to altered replication fork progression through the repeats, which could result in repeat expansion to the FXS full mutation.
Affiliation: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461 firstname.lastname@example.org email@example.com.Show MeSH
Related in: MedlinePlus
License 1 - License 2
Mentions: A T/C SNP (ss71651738) previously identified 53 kb upstream of the FMR1 CGG repeats was linked to FXS patients in chromosome haplogroup D, a haplogroup at high risk of expansion (Table S1; Ennis et al., 2007). To determine whether the replication initiation site overlaps with this SNP in the MRE1b element, we mapped the replication origin upstream of the repeats in more detail using the nascent strand abundance assay (using the protocol of Liu et al., 2010). As a quality control of the isolated nascent strand DNA, we measured the enrichment of a known replication origin, Or6 (Fig. 1, B and D; Gerhardt et al., 2006). We designed six primer pairs at and surrounding the SNP ∼50 kb upstream of the repeats (Fig. S1, A–C). In the nonaffected control hESC H14 and a fetal fibroblast line GM00011 (Fig. 1 A), we detected an enrichment of nascent strands at the SNP site in comparison to the surrounding DNA segments. In addition, we detected lower levels of nascent strands at the site where the C primer pair binds, probably as a result of a rarely used replication origin at this site. In FXS hESCs SI-214 and WCMC37, we detected no significant enrichment of nascent strand DNA at the SNP site in comparison to control primer C, indicating that an active replication initiation site was missing in FXS hESCs (Fig. 1 C). Thus, the nascent strand abundance analysis revealed that the replication initiation sites ∼50 kb upstream of the CGG repeats overlaps with the SNP in nonaffected cells.
Affiliation: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461 firstname.lastname@example.org email@example.com.