1. Volume 53, Issue 1, Pages 19-31 (January 2014)
The DNA Replication Program Is Altered at the FMR1 Locus in Fragile X Embryonic Stem Cells 
Jeannine Gerhardt, Mark J. Tomishima, Nikica Zaninovic, Dilek Colak, Zi Yan, Qiansheng Zhan, Zev Rosenwaks, Samie R. Jaffrey, Carl L. Schildkraut 
Molecular Cell 
Volume 53, Issue 1, Pages (January 2014)
DOI: /j.molcel
Copyright © 2014 Elsevier Inc. Terms and Conditions

2. Figure 1
The DNA Replication Profile Differs at the Endogenous FMR1 Locus in FXS hESCs and Nonaffected hESCs
(A) To determine repeat length, genomic DNA was digested with EcoRI and methylation-sensitive enzyme EagI and analyzed by Southern blot.
(B) The table summarizes the results from the Southern blot, the Asuragen PCR assay, and capillary electrophoresis (Figure S2), which were used to accurately determine the repeat lengths (PCR was applicable for less than 280 repeats). Very few cells contain the premutation repeat length (indicated by asterisks).
(C) Schematic of the steps of SMARD that enable the visualization of single replicating DNA molecules. First, cells are pulsed with IdU (red) and CldU (green). Cells are then embedded in agarose and lysed. After digestion with SfiI enzyme, DNA molecules are separated by PFGE, and the target molecules are identified by Southern blot. The target sequences are then stretched on silanized glass slides, and the labeled DNA molecules are detected by immunostaining (red and green). Two FISH probes surrounding the repeats were used to identify the FMR1 locus.
(D–G) (Top) Map of the SfiI segment containing the FMR1 gene and CGG repeats. The positions of the FISH probes are marked in blue, and the CGG repeats are indicated by a red vertical bar. (Middle) Photomicrographs of labeled DNA molecules from nonaffected hESC H14 (D), H9 (E), FXS hESC SI-214 (F), and WCMC37 (G) are ordered according to replication fork (yellow arrows) progression in the 5′→3′ and 3′→5′ directions; replication initiation; and termination. (Bottom) The percentage of molecules with IdU incorporation (first pulse) is calculated from the DNA molecules shown above. See also Figures S1 and S2.
Molecular Cell  , 19-31DOI: ( /j.molcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 2
The DNA Replication Profile Is Similar in Nonaffected, FXS Fetal Fibroblast, and Differentiated FXS Embryoid Bodies
(A–D) (Top) Map of the FMR1 gene locus with SfiI restriction enzyme cutting sites and FISH probes indicated (blue). A vertical red bar marks the CGG repeats. (Bottom) Photomicrographs of labeled DNA molecules from nonaffected (MEC) cortex microvascular endothelial cells (A), nonaffected fetal fibroblasts GM00011 (B), FXS fetal fibroblasts GM07072 (C), and FXS fetal fibroblasts GM05252 (D) are arranged by replication fork progression in 5′→3′ and 3′→5′ directions, replication initiation, and termination sites. Yellow arrows mark the location of the replication forks in each individual DNA molecule. The percentage of molecules with IdU incorporation (first pulse) is calculated from DNA molecules shown above.
(E) FXS hESC SI-214 and WCMC37 were differentiated into embryoid bodies (EBs). After 12 days, EBs were analyzed by flow cytometry to confirm that differentiation marker SSEA1 is expressed and the pluripotency marker SSEA4 is not expressed (table).
(F and G) (Top) Map of the SfiI segment containing the FMR1 gene and CGG repeats. The positions of the FISH probes are marked in blue, and CGG repeats are indicated by a red vertical bar. (Middle) Photomicrographs of labeled DNA molecules from FXS SI-214 EB (F) and WCMC37 EB (G) are ordered according to replication fork (yellow arrows) progression in the 5′→3′ and 3′→5′ directions, replication initiation, and termination.
(H) The percentage of molecules with IdU incorporation (first pulse) is calculated from the DNA molecules shown above and from Figure 1. See also Figure S3.
Molecular Cell  , 19-31DOI: ( /j.molcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

4. Figure 3
Differences in the Replication Program Upstream of the FMR1 Locus Cause the Altered Replication Fork Progression at the FMR1 Locus in FXS ESCs
(A) Map of the FMR1 locus with the position of the FMR1 gene, CGG repeats, FISH probes (blue), and DNA segments analyzed by SMARD; 119 kb SfiI fragments (brown) and 152 kb SbfI segment (green).
(B–E) (Top) Map of the restriction segment with the location of the FISH probes. (Bottom) Photomicrographs of labeled DNA molecules from nonaffected hESC H14 (B and C) and FXS hESCs SI-214 (D and E) are ordered by replication fork progression (yellow arrows) in 3′→5′, 5′→3′, replication initiation, and termination sites.
(F and G) The percentage of replication forks for each 5 kb DNA segment was determined upstream of the FMR1 gene (F) and downstream of the FMR1 gene (G) in nonaffected human ESC H14 (blue) and FXS hESC SI-214 (red).
Molecular Cell  , 19-31DOI: ( /j.molcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

5. Figure 4
Replication Initiation Sites Located Approximately 40–50 kb Upstream of the FMR1 Gene Are Absent in FXS hESCs
(A–C) Summary of the results displayed schematically with the map of the FMR1 locus on the top (A) and the replication profiles of H14 (B) and SI-214 (C) on the bottom. Replication fork progression is bidirectional from the origin (near to the SfII restriction site) (D) upstream of the FMR1 gene. This potential replication origin is absent in FXS hESCs (C and E).
(D–G) (Top) Map of the PmeI segment containing the SfiI site in the middle of the fragment. The positions of the FISH probes are marked in blue, and CGG repeats are indicated by red vertical bar. (Middle) Photomicrographs of labeled DNA molecules from nonaffected H14 (D), H9 (F), and FXS hESC SI-214 (E), WCMC37 (G) are ordered according to replication fork (yellow arrows) progression in the 5′→3′ and 3′→5′ directions, replication initiation, and termination.
(H) The percentage of molecules with IdU incorporation (first pulse) is calculated from the DNA molecules shown above for each cell line. See also Figure S4.
Molecular Cell  , 19-31DOI: ( /j.molcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

6. Figure 5
DNA replication Forks Stall at CGG/CCG Repeats in the FMR1 Locus
(A and B) The percentage of molecules with replication forks for each 5 kb DNA interval was counted at the FMR1 locus in (A) nonaffected hESC H14, H9, and (B) FXS hESC SI-214, WCMC37. The replication fork in 3′→5′ direction is indicated by > and 5′-3′ direction by <.
(C and D) The percentage of molecules with replication forks per 5 kb interval downstream of the FMR1 gene is shown for (C) hESC H14 and (D) FXS hESC SI-214. See also Figure S5.
Molecular Cell  , 19-31DOI: ( /j.molcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

7. Figure 6
Model for Repeat Instability at the Endogenous FMR1 Locus in FXS hESCs
(A) The replication forks stall at the CGG/CCG repeats for a short period of time (black rectangle) in human fibroblasts and hESCs. The replication fork stalling is caused by single-stranded CCG/CCG repeats, which form secondary hairpin-like structures. The stability of the hairpin-like secondary structures decreases according to length and the sequence of the repeats in following order: CGG repeats form a more stable hairpin then CCG repeats (Paiva and Sheardy, 2004). Repeat contractions occur when the replication machinery skips the repetitive hairpin on the template strand. Repeat expansion can occur after replication fork stalling during replication fork reversal and restart, leading to the formation of a repetitive hairpin on the nascent strand.
(B) In nonaffected hESCs the DNA replication forks progress through the CGG (<50) repeats from either upstream or downstream of the FMR1 gene promoting repeat stability.
(C) In FXS hESCs, the absence of the replication origin(s) 50 kb upstream (H9 50 and 40 kb upstream) of the repeats leads to DNA replication fork progression through the CGG (>200) repeats from primarily one direction, thereby changing the direction of replication through the repeats from using the CGG strand as the template for lagging-strand synthesis to predominantly using the CCG strand. The replication forks initiate from a replication origin downstream of the FMR1 gene. These results support the origin switch model. In FXS hESCs the replication fork direction through the repeats is probably more prone to form hairpin-like secondary structures during lagging-strand synthesis, which results in TNR instability. The replication forks stall at the secondary CGG/CCG structures multiple times, causing DNA polymerase to slip and add repeats (expansion) or skip repeats (contraction).
Molecular Cell  , 19-31DOI: ( /j.molcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions