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Cis-acting DNA sequence at a replication origin promotes repeat expansion to fragile X full mutation.

Gerhardt J, Zaninovic N, Zhan Q, Madireddy A, Nolin SL, Ersalesi N, Yan Z, Rosenwaks Z, Schildkraut CL - J. Cell Biol. (2014)

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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461 jeannine.gerhardt@gmail.com carl.schildkraut@einstein.yu.edu.

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Premutation hESCs, which do not expand to full mutation, have a different replication program than FXS hESCs and a similar replication program to nonaffected hESCs. (A) The stability of the CGG repeats in premutation hESCs was analyzed by EcoRI and EagI Southern blotting. (B) Schematic of the steps of SMARD: First, cells are pulsed with IdU and CldU. Cells are then embedded in agarose and lysed. After digestion with a restriction enzyme, DNA molecules are separated by PFGE. Target segments were identified by Southern blotting and stretched on silanized glass slides. The replicated DNA molecules are detected by immunostaining and FISH. (C and D) The replication program was analyzed by SMARD in premutation and FXS hESCs as previously described (Gerhardt et al., 2014). (top) Map of the 102-kb PmeI segment containing the SNP variant T/C and the CGG repeats. The positions of the FISH probes are marked in blue and through the DNA molecules by yellow lines. (bottom) Photomicrographs of labeled DNA molecules from WCMC37 FXS hESCs (C) and WCMC5 premutation hESCs (D) are ordered according to replication fork (yellow arrowheads) progression; replication initiation, 5′→3′ and 3′→5′ directions, and termination are depicted. In contrast to FXS hESCs, the premutation hESCs WCMC5 contains a replication origin 53 kb upstream of the repeats. (E) The replication program was analyzed by SMARD at the FMR1 locus containing the CGG repeats in premutation hESCs (120-kb SfiI segment). The CGG repeats are indicated by a red vertical line.
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fig4: Premutation hESCs, which do not expand to full mutation, have a different replication program than FXS hESCs and a similar replication program to nonaffected hESCs. (A) The stability of the CGG repeats in premutation hESCs was analyzed by EcoRI and EagI Southern blotting. (B) Schematic of the steps of SMARD: First, cells are pulsed with IdU and CldU. Cells are then embedded in agarose and lysed. After digestion with a restriction enzyme, DNA molecules are separated by PFGE. Target segments were identified by Southern blotting and stretched on silanized glass slides. The replicated DNA molecules are detected by immunostaining and FISH. (C and D) The replication program was analyzed by SMARD in premutation and FXS hESCs as previously described (Gerhardt et al., 2014). (top) Map of the 102-kb PmeI segment containing the SNP variant T/C and the CGG repeats. The positions of the FISH probes are marked in blue and through the DNA molecules by yellow lines. (bottom) Photomicrographs of labeled DNA molecules from WCMC37 FXS hESCs (C) and WCMC5 premutation hESCs (D) are ordered according to replication fork (yellow arrowheads) progression; replication initiation, 5′→3′ and 3′→5′ directions, and termination are depicted. In contrast to FXS hESCs, the premutation hESCs WCMC5 contains a replication origin 53 kb upstream of the repeats. (E) The replication program was analyzed by SMARD at the FMR1 locus containing the CGG repeats in premutation hESCs (120-kb SfiI segment). The CGG repeats are indicated by a red vertical line.

Mentions: Next, we asked whether the premutation hESCs, which contain a T, have a similar replication program to nonaffected hESCs. Nonaffected hESCs contain the SNP variant T and an active replication origin 53 kb upstream of the repeats (Gerhardt et al., 2014). This is in contrast to FXS hESCs, which contain a C and the replication origin is missing. First, we tested whether the CGG repeats in the premutation hESCs containing the SNP variant T expand further in cell culture. Therefore, we analyzed the repeat length in multiple passages of the premutation hESC WCMC5 by Southern blotting over the course of several months (Fig. 4 A). We observed no large changes in the repeat length in these cell passages. We also analyzed cell passages by Asuragen PCR analysis. All passages contained the same repeat length (73 repeats). We concluded that the repeats in the premutation hESCs WCMC5 do not expand further in cell culture.


Cis-acting DNA sequence at a replication origin promotes repeat expansion to fragile X full mutation.

Gerhardt J, Zaninovic N, Zhan Q, Madireddy A, Nolin SL, Ersalesi N, Yan Z, Rosenwaks Z, Schildkraut CL - J. Cell Biol. (2014)

Premutation hESCs, which do not expand to full mutation, have a different replication program than FXS hESCs and a similar replication program to nonaffected hESCs. (A) The stability of the CGG repeats in premutation hESCs was analyzed by EcoRI and EagI Southern blotting. (B) Schematic of the steps of SMARD: First, cells are pulsed with IdU and CldU. Cells are then embedded in agarose and lysed. After digestion with a restriction enzyme, DNA molecules are separated by PFGE. Target segments were identified by Southern blotting and stretched on silanized glass slides. The replicated DNA molecules are detected by immunostaining and FISH. (C and D) The replication program was analyzed by SMARD in premutation and FXS hESCs as previously described (Gerhardt et al., 2014). (top) Map of the 102-kb PmeI segment containing the SNP variant T/C and the CGG repeats. The positions of the FISH probes are marked in blue and through the DNA molecules by yellow lines. (bottom) Photomicrographs of labeled DNA molecules from WCMC37 FXS hESCs (C) and WCMC5 premutation hESCs (D) are ordered according to replication fork (yellow arrowheads) progression; replication initiation, 5′→3′ and 3′→5′ directions, and termination are depicted. In contrast to FXS hESCs, the premutation hESCs WCMC5 contains a replication origin 53 kb upstream of the repeats. (E) The replication program was analyzed by SMARD at the FMR1 locus containing the CGG repeats in premutation hESCs (120-kb SfiI segment). The CGG repeats are indicated by a red vertical line.
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fig4: Premutation hESCs, which do not expand to full mutation, have a different replication program than FXS hESCs and a similar replication program to nonaffected hESCs. (A) The stability of the CGG repeats in premutation hESCs was analyzed by EcoRI and EagI Southern blotting. (B) Schematic of the steps of SMARD: First, cells are pulsed with IdU and CldU. Cells are then embedded in agarose and lysed. After digestion with a restriction enzyme, DNA molecules are separated by PFGE. Target segments were identified by Southern blotting and stretched on silanized glass slides. The replicated DNA molecules are detected by immunostaining and FISH. (C and D) The replication program was analyzed by SMARD in premutation and FXS hESCs as previously described (Gerhardt et al., 2014). (top) Map of the 102-kb PmeI segment containing the SNP variant T/C and the CGG repeats. The positions of the FISH probes are marked in blue and through the DNA molecules by yellow lines. (bottom) Photomicrographs of labeled DNA molecules from WCMC37 FXS hESCs (C) and WCMC5 premutation hESCs (D) are ordered according to replication fork (yellow arrowheads) progression; replication initiation, 5′→3′ and 3′→5′ directions, and termination are depicted. In contrast to FXS hESCs, the premutation hESCs WCMC5 contains a replication origin 53 kb upstream of the repeats. (E) The replication program was analyzed by SMARD at the FMR1 locus containing the CGG repeats in premutation hESCs (120-kb SfiI segment). The CGG repeats are indicated by a red vertical line.
Mentions: Next, we asked whether the premutation hESCs, which contain a T, have a similar replication program to nonaffected hESCs. Nonaffected hESCs contain the SNP variant T and an active replication origin 53 kb upstream of the repeats (Gerhardt et al., 2014). This is in contrast to FXS hESCs, which contain a C and the replication origin is missing. First, we tested whether the CGG repeats in the premutation hESCs containing the SNP variant T expand further in cell culture. Therefore, we analyzed the repeat length in multiple passages of the premutation hESC WCMC5 by Southern blotting over the course of several months (Fig. 4 A). We observed no large changes in the repeat length in these cell passages. We also analyzed cell passages by Asuragen PCR analysis. All passages contained the same repeat length (73 repeats). We concluded that the repeats in the premutation hESCs WCMC5 do not expand further in cell culture.

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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461 jeannine.gerhardt@gmail.com carl.schildkraut@einstein.yu.edu.

Show MeSH
Related in: MedlinePlus