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. 2016 Oct 20;64(2):388-404.
doi: 10.1016/j.molcel.2016.09.017.

FANCD2 Facilitates Replication Through Common Fragile Sites

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Free PMC article

FANCD2 Facilitates Replication Through Common Fragile Sites

Advaitha Madireddy et al. Mol Cell. .
Free PMC article

Abstract

Common fragile sites (CFSs) are genomic regions that are unstable under conditions of replicative stress. Although the characteristics of CFSs that render them vulnerable to stress are associated mainly with replication, the cellular pathways that protect CFSs during replication remain unclear. Here, we identify and describe a role for FANCD2 as a trans-acting facilitator of CFS replication, in the absence of exogenous replicative stress. In the absence of FANCD2, replication forks stall within the AT-rich fragility core of CFS, leading to dormant origin activation. Furthermore, FANCD2 deficiency is associated with DNA:RNA hybrid formation at CFS-FRA16D, and inhibition of DNA:RNA hybrid formation suppresses replication perturbation. In addition, we also found that FANCD2 reduces the number of potential sites of replication initiation. Our data demonstrate that FANCD2 protein is required to ensure efficient CFS replication and provide mechanistic insight into how FANCD2 regulates CFS stability.

Keywords: DNA replication; DNA:RNA hybrids; Fanconi anemia; cancer; common fragile sites; genomic instability.

Conflict of interest statement

The authors have NO commercial affiliations or conflicts of interest.

Figures

Figure 1
Figure 1. The replication profile is altered at the endogenous CFS-FRA16D locus in the absence of FANCD2
(A) Common fragile site FRA16D locus map Locus map of the CFS-FRA16D (brown line-1.5 Mb) that contains the AT-rich fragility core (pink line–280 kb) and overlaps the WWOX tumor suppressor gene (dark blue line–1.1 Mb). The locus was divided into 4 segments based on restriction enzyme availability. The coordinates of the different regions are summarized in Table S1, providing additional information about fosmids and primers used to identify the regions. (B) Locus map of RR1-PmeI segment containing a portion of the AT-rich fragility core. The segments are aligned according to the positions of the FISH probes (blue) on the map. (C–F) Top; Locus map of PmeI digested RR1 segment. Middle; Aligned photomicrograph images of labeled DNA molecules from (C) Non-affected 1 (GM02184), (D) FANCD2−/−-L-1 (PD20), (E) FANCD2−/−-L-2 (2742) and (F) FANCD2−/−-L-1 + FANCD2 (corrected) lymphoblast. The yellow arrows indicate the sites along the molecules where the IdU transitioned to CldU. The molecules are arranged in the following order: molecules with initiation events, molecules with 3’ to 5’ progressing forks, molecules with 5’ to 3’ progressing forks and molecules with termination events. White ovals indicate regions of replication fork pausing and correspond to the pausing peaks listed in Table S2. Bottom; The percentage of molecules incorporating IdU (red) is calculated from the replication program (middle) and is represented as a histogram.
Figure 2
Figure 2. DNA replication forks stall within the fragility core of CFS-FRA16D in FANCD2−/− lymphoblasts
(A) Locus map of RR2-SbfI segment containing a portion of the AT-rich fragility core. The FISH probes that identify the segment are labeled in blue. (See also Figure S2) (B–D) Top; Locus map of the SbfI digested RR2 segment. Middle; Aligned photomicrograph images of labeled DNA molecules from (B) Non-affected I (GM02184), (C) FANCD2−/−-L-1 (PD20) patient derived lymphoblast, and (D) FANCD2−/−-L-2 (2742) lymphoblasts. White ovals indicate regions of replication fork (yellow arrow) pausing and correspond to the pause peaks listed in Table S2. (E) Top; Locus map of the RR1 + RR2 regions. The RR1 quantification was included here to enable the visualization of replication pausing along the complete length of the fragility core. Bottom; The percentage of molecules with replication forks at each 10 kb interval of RR2 (left, quantification of molecules shown in Fig. 3B–D) and RR1 (right, quantification of molecules shown in Fig. 2B–D) in the non-affected I (GM02184) line, FANCD2−/−-L-1 (PD20) and the FANCD2−/−-L-2 (2742) lymphoblasts. The replication forks moving in the 3’ to 5’ direction and the forks moving in the 5’ to 3’ direction are denoted by purple < and orange > colors respectively. A high percentage of molecules with replication forks in a particular 10 kb interval is indicative of fork pausing in that interval. Black arrows denote the most prominent pause peaks and correspond to the white ovals in the SMARD profile. Refer Table S2 for the coordinates of the 10 kb region corresponding to the pause peaks.
Figure 3
Figure 3. In the absence of FANCD2, cells activate dormant origins associated with replication stalling at the AT-rich fragility core of CFS. (See also Figure S2, S4)
(A) Locus map of R3-SbfI segment that lies outside the AT-rich fragility core to the right. The FISH probes that identify the segment are labeled in blue. Combinations of two-three probes were used to identify the R3 segment. (B–E) Top; Locus map of the SbfI digested Region 3 (R3) segment. Middle; Aligned photomicrograph images of labeled DNA molecules from (B) non-affected I (GM02184), (C) FANCD2−/−-L-1 (PD20), (D) FANCD2−/−-L-2 (2742), (E) FANCD2−/−-L-1 + FANCD2 (corrected) patient derived lymphoblast. The molecules are arranged as in Fig. 2. Bottom; The percentage of molecules incorporating IdU (red) is calculated from the replication program (middle) and is represented as a histogram. (F) Schematic representation of dormant origin activation when replication forks pause in the 5’ to 3’ direction
Figure 4
Figure 4. The absence of FANCD2 protein affects the replication program of CFS-FRA16D in lymphoblasts but not in fibroblasts
(A) Locus map of the RR1-PmeI and R3-SbfI segments. The FISH probes that identify the segment are labeled in blue. (B–C) Top; Locus map of PmeI digested RR1 segment. Middle; Aligned photomicrograph images of labeled DNA molecules from (B) Non-affected-F (IMR90 fibroblast), (C) FANCD2−/−-F-1 (PD20) patient fibroblasts. The yellow arrows indicate the sites along the molecules where the IdU transitioned to CldU. The molecules are arranged as in Fig. 2. Bottom; The percentage of molecules incorporating IdU (red) is represented as a histogram. (D–E) Top; Locus map of SbfI digested R3 segment. Middle; Aligned photomicrograph images of labeled DNA molecules from (D) Non-affected-F (IMR90 fibroblast), (E) FANCD2−/−-F-1 (PD20) patient fibroblasts. (F–G) Top; Locus map of the RR1 region. Bottom; The percentage of molecules with replication forks at each 10 kb interval of RR1 (quantification of molecules shown in Fig. 4B–C) in (F) Non-affected-F (IMR90 fibroblast), (G) FANCD2−/−-F-1 (PD20) patient fibroblasts. Black arrows denote the most prominent pause peaks and correspond to the white ovals in the SMARD profile.
Figure 5
Figure 5. Replication pausing at CFS-FRA16D, observed in the absence of FANCD2 is associated with the accumulation of DNA:RNA hydrids
(A) DNA:RNA hybrid accumulation in FANCD2-deficient lymphoblasts. DRIP-qPCR using the anti-DNA:RNA hybrid S9.6 monoclonal antibody, in non-affected I (GM02184-grey bar), FANCD2−/−-L (red bars) lymphoblasts. The samples obtained by immunoprecipitation with either treated (+) or not treated (−), with RNase H1, as indicated. Signal values of DNA:DNA hybrids, immunoprecipitated in each region are normalized to input values. Data represent mean ± SEM from three independent experiments. (B) Immunoblot analysis to detect the relative levels of the RNaseH1 protein expression in the non-affected 1 (GM02184) cells transfected with the control GFP vector (Lane 1-CONTROL + GFP), non-affected 1 (GM02184) cells transfected with the RNaseH1 overexpression vector (Lane 2-CONTROL+RNH1), FANCD2−/−-L lymphoblast cells transfected with the control GFP vector (Lane 3-FANCD2−/−-L-1+GFP) and FANCD2−/−-L lymphoblast cells transfected with the RNaseH1 overexpression vector (Lane 4-FANCD2−/−-L-1+RNH1). Proteins from whole cell extracts were separated, immunoblotted and detected with RNaseH1 antibody. (C–D) Top; Locus map of PmeI digested RR1 segment. Middle; Aligned photomicrograph images of labeled DNA molecules from (C) non-affected I or control + RNH1, (D) FANCD2−/−-L-1+RNH1 lymphoblast. The molecules are arranged as in Fig. 2. Bottom; The percentage of molecules incorporating IdU (red) is represented as a histogram. (E–F) Top; Locus map of the SbfI digested Region 3 (R3) segment. Middle; Aligned photomicrograph images of labeled DNA molecules from (E) Non-affected I or Control + RNH1, (F) FANCD2−/−-L-1+RNH1 lymphoblast. (See also Figure S7)
Figure 6
Figure 6. The absence of other key FA proteins or the monoubiquitination of FANCI or FANCD2 only moderately alters CFS-FRA16D replication. (See also Figure S5)
(A) Schematic representation of results from Fig. 4 (SbfI segment), for comparison of replication program at the R3 segment of CFS-FRA16D, Top; the locus map, Bottom Left; replication profile of the non-affected-L cell line Bottom Right; replication profile of the FANCD2−/−-L lymphoblast. (B–E) Top; Locus maps of SbfI digested R3 segment. Middle; Aligned photomicrograph images of labeled DNA molecules from (B) FANCA−/−-L, (C) FANCImonoub−/−-L, (D) FANCD2monoub−/−-L and (E) FANCD1−/−-L patient derived lymphoblast. The molecules are arranged as in Fig. 2. Bottom; The percentage of molecules incorporating IdU (red) is represented as a histogram. (F) Percentage of molecules with initiation sites in Region 3 of non-affected I (GM02184 - grey bar), FANCD2−/−-L (red bars), FANCA−/−-L (green bar), FANCImonoub−/−-L (blue bar), FANCD2monoub−/−-L (purple bar) and FANCD1−/−-L (orange bar) patient derived lymphoblast. Error bars represent mean ± s.d. from two independent experiments (*P<0.05).
Figure 7
Figure 7. FANCD2 deficiency is associated with altered replication initiation and instability at CFS loci
(A) Cytogenetic FISH analysis using the IGH/MAF probe set, to detect and compare breaks at CFS-FRA16D in lymphoblasts and fibroblasts, in the presence and absence of Aphidicolin. The MAF probe set consist of two probes that flank the FRA16D locus such that a split signal (two red dots within a chromatid) represents a FRA16D break (modified from (Bergsagel and Kuehl, 2001)). Top; Table representing the percentage of FRA16D breaks in the presence and absence of 0.2 µM aphidicolin in Non-affected-L, FANCD2−/−-L (PD20), FANCD2−/−+FANCD2-L-1, FANCD2monoub−/−-L-1, FANCD2−/−-F-1 (PD20), FANCD2−/−+FANCD2-F-1. Bottom; Representative images of FRA16D breaks. White arrows indicate FRA16D breaks. (B) Top; Locus map of CFS-FRA16D spanning the ~600 kb, RR1+R3 segments. Bottom; Observed sites of initiation in non-affected 1 (GM02184 – grey arrow), non-affected 2 (GM03798 - grey arrow), FANCD2−/−-L-1 (PD20) (red arrow), FANCD2−/−-L-2 (2742) lymphoblasts (red arrows), FANCD2−/−-L-3 (2717) (red arrow), FANCA−/−-L (green arrows), FANCImonoub−/−-L (blue arrows), FANCD2monoub−/−-L (purple arrows) and FANCD1−/−-L (orange arrows). (See also Figure S6) (C) Model depicting the consequence of FANCD2 deficiency on CFS replication. Under unperturbed replicative conditions (+FA/BRCA), replication forks progressing from distantly fired origins replicate the CFS locus (black line). Upon reaching the AT-rich fragility core (pink line), replication forks efficiently replicate through WWOX transcription associated DNA:RNA hybrids (green) and AT associated secondary structures (black hairpins) likely aided by the FANCD2 protein, in association with the FA/BRCA pathway. This ensures CFS replication completion genomic stability. In the absence of FANCD2, replication forks pause (overlapping yellow arrows) within the fragility core and this is accompanied by the activation of a dormant origin (red circle). Replication pausing likely persists due to the absence of a functional FA/BRCA pathway to resolve DNA:RNA hybrids (green arrow) and other AT associated secondary structures (black hairpins). Furthermore, the absence of FANCD2 further affects CFS replication by restraining the number of sites at which dormant origins fire, collectively leading to genomic instability.

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