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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.

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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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A replication initiation site upstream of the repeats overlaps with an SNP in nonaffected cells and is absent in FXS hESCs. (A and C) The previously identified replication initiation sites ∼50 kb upstream of the repeats (Gerhardt et al., 2014) were mapped more in detail by nascent strand abundance. (top) Map of the FMR1 locus upstream of the repeats. The SfiI restriction enzyme site and position of primers used in the nascent strand abundance assay are indicated. SNP primer pair binds at the SNP T/C (ss71651738 or WEX70) on the X chromosome (Chr. X; Fig. S1, A and B), which cosegregates with a chromosome haplotype at the highest risk for repeat expansion (Table S1; Ennis et al., 2007). Primer pairs A, B1, B2, B3, and C amplify surrounding genomic regions. (A, bottom) Nascent strand DNA was isolated from nonaffected hESCs (H14) and fetal fibroblast (GM00011). Nascent strand enrichment at the SNP was determined by real-time PCR using specific primer pairs. (B) As a control, the nascent strand enrichment was determined at the well-characterized Or6 replication origin (Gerhardt et al., 2006) on chromosome 12 (with Or6 primer pair) and at the adjacent control region (N primer pair). (C and D) Nascent strand DNA was isolated from FXS hESCs SI-214 and WCMC37 upstream of the CGG repeats (C) and at a control region (D). Enrichment of the nascent strand at the SNP and neighboring genomic regions was determined by real-time PCR with primer pairs shown above the map. No nascent strand enrichment was obtained for primer B. There was no significant enrichment of nascent strand DNA in FXS hESCs at the SNP in comparisons to control region C in contrast to nonaffected cells in three separate experiments (SI-214: **, P = 0.075; WCMC37: **, P = 0.81; H14: *, P < 0.0005; GM00011: *, P = 0.0047; also see Fig. S1). Error bars indicate the standard error of the mean of three experiments.
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fig1: A replication initiation site upstream of the repeats overlaps with an SNP in nonaffected cells and is absent in FXS hESCs. (A and C) The previously identified replication initiation sites ∼50 kb upstream of the repeats (Gerhardt et al., 2014) were mapped more in detail by nascent strand abundance. (top) Map of the FMR1 locus upstream of the repeats. The SfiI restriction enzyme site and position of primers used in the nascent strand abundance assay are indicated. SNP primer pair binds at the SNP T/C (ss71651738 or WEX70) on the X chromosome (Chr. X; Fig. S1, A and B), which cosegregates with a chromosome haplotype at the highest risk for repeat expansion (Table S1; Ennis et al., 2007). Primer pairs A, B1, B2, B3, and C amplify surrounding genomic regions. (A, bottom) Nascent strand DNA was isolated from nonaffected hESCs (H14) and fetal fibroblast (GM00011). Nascent strand enrichment at the SNP was determined by real-time PCR using specific primer pairs. (B) As a control, the nascent strand enrichment was determined at the well-characterized Or6 replication origin (Gerhardt et al., 2006) on chromosome 12 (with Or6 primer pair) and at the adjacent control region (N primer pair). (C and D) Nascent strand DNA was isolated from FXS hESCs SI-214 and WCMC37 upstream of the CGG repeats (C) and at a control region (D). Enrichment of the nascent strand at the SNP and neighboring genomic regions was determined by real-time PCR with primer pairs shown above the map. No nascent strand enrichment was obtained for primer B. There was no significant enrichment of nascent strand DNA in FXS hESCs at the SNP in comparisons to control region C in contrast to nonaffected cells in three separate experiments (SI-214: **, P = 0.075; WCMC37: **, P = 0.81; H14: *, P < 0.0005; GM00011: *, P = 0.0047; also see Fig. S1). Error bars indicate the standard error of the mean of three experiments.

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.


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)

A replication initiation site upstream of the repeats overlaps with an SNP in nonaffected cells and is absent in FXS hESCs. (A and C) The previously identified replication initiation sites ∼50 kb upstream of the repeats (Gerhardt et al., 2014) were mapped more in detail by nascent strand abundance. (top) Map of the FMR1 locus upstream of the repeats. The SfiI restriction enzyme site and position of primers used in the nascent strand abundance assay are indicated. SNP primer pair binds at the SNP T/C (ss71651738 or WEX70) on the X chromosome (Chr. X; Fig. S1, A and B), which cosegregates with a chromosome haplotype at the highest risk for repeat expansion (Table S1; Ennis et al., 2007). Primer pairs A, B1, B2, B3, and C amplify surrounding genomic regions. (A, bottom) Nascent strand DNA was isolated from nonaffected hESCs (H14) and fetal fibroblast (GM00011). Nascent strand enrichment at the SNP was determined by real-time PCR using specific primer pairs. (B) As a control, the nascent strand enrichment was determined at the well-characterized Or6 replication origin (Gerhardt et al., 2006) on chromosome 12 (with Or6 primer pair) and at the adjacent control region (N primer pair). (C and D) Nascent strand DNA was isolated from FXS hESCs SI-214 and WCMC37 upstream of the CGG repeats (C) and at a control region (D). Enrichment of the nascent strand at the SNP and neighboring genomic regions was determined by real-time PCR with primer pairs shown above the map. No nascent strand enrichment was obtained for primer B. There was no significant enrichment of nascent strand DNA in FXS hESCs at the SNP in comparisons to control region C in contrast to nonaffected cells in three separate experiments (SI-214: **, P = 0.075; WCMC37: **, P = 0.81; H14: *, P < 0.0005; GM00011: *, P = 0.0047; also see Fig. S1). Error bars indicate the standard error of the mean of three experiments.
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fig1: A replication initiation site upstream of the repeats overlaps with an SNP in nonaffected cells and is absent in FXS hESCs. (A and C) The previously identified replication initiation sites ∼50 kb upstream of the repeats (Gerhardt et al., 2014) were mapped more in detail by nascent strand abundance. (top) Map of the FMR1 locus upstream of the repeats. The SfiI restriction enzyme site and position of primers used in the nascent strand abundance assay are indicated. SNP primer pair binds at the SNP T/C (ss71651738 or WEX70) on the X chromosome (Chr. X; Fig. S1, A and B), which cosegregates with a chromosome haplotype at the highest risk for repeat expansion (Table S1; Ennis et al., 2007). Primer pairs A, B1, B2, B3, and C amplify surrounding genomic regions. (A, bottom) Nascent strand DNA was isolated from nonaffected hESCs (H14) and fetal fibroblast (GM00011). Nascent strand enrichment at the SNP was determined by real-time PCR using specific primer pairs. (B) As a control, the nascent strand enrichment was determined at the well-characterized Or6 replication origin (Gerhardt et al., 2006) on chromosome 12 (with Or6 primer pair) and at the adjacent control region (N primer pair). (C and D) Nascent strand DNA was isolated from FXS hESCs SI-214 and WCMC37 upstream of the CGG repeats (C) and at a control region (D). Enrichment of the nascent strand at the SNP and neighboring genomic regions was determined by real-time PCR with primer pairs shown above the map. No nascent strand enrichment was obtained for primer B. There was no significant enrichment of nascent strand DNA in FXS hESCs at the SNP in comparisons to control region C in contrast to nonaffected cells in three separate experiments (SI-214: **, P = 0.075; WCMC37: **, P = 0.81; H14: *, P < 0.0005; GM00011: *, P = 0.0047; also see Fig. S1). Error bars indicate the standard error of the mean of three experiments.
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.

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