Two breakpoint clusters at fragile site FRA3B form phased nucleosomes

Abstract

Fragile sites are gaps and breaks in metaphase chromosomes generated by specific culture conditions. Fragile site FRA3B is the most unstable site and is directly involved in the breakpoints of deletion and translocation in a wide spectrum of cancers. To learn about the general characteristics of common fragile sites, we investigated the chromatin structure of the FRA3B site. Because FRA3B spans several hundred kilobases, we focused our study on two breakpoint clusters found in FRA3B. Using various nucleases, we demonstrated that these two regions contain phased nucleosomes, regardless of treatment with aphidicolin. Because these regions are located in intron 4 of the FHIT gene, it is very interesting to observe phased nucleosomes over these regions, which are several hundred kilobases downstream from the promoter. Further, by using nucleosome assembly assays, we demonstrate that these two regions do not contain strong nucleosome positioning elements. These results suggest that other factors appear to cooperate with the DNA sequence of these regions to impart nucleosome phasing. This study provides the first information on the chromatin structure of breakpoint regions in a common fragile site. The observation of phased nucleosomes over these breakpoint regions could offer a foundation to understand the expression of fragile sites.

Figure 1

Genomic organization of the FHIT/FRA3B region. The FHIT sequence is shown from intron 3 through intron 6. The position of the unstable FRA3B region is indicated by a hatched bar. The major breakpoint clusters are marked by black blast symbols within the 2150-bp EcoRI fragment of the proximal region and the 1899-bp AflIII fragment of the distal region. Solid circles represent the locations of Alu elements. (E) EcoRI; (A) AflIII.

Figure 2

The FRA3B distal breakpoint region does not have a condensed chromatin structure. Nuclei isolated from aphidicolin-treated (lanes 3,4,13,14), untreated GM 13069 cells (lanes 5,6,15,16) or from FRAXA cells (lanes 7,8,17,18) were incubated with 25 or 100 U of AflIII (indicated by triangles), and purified DNA was completely digested with EcoRI. Genomic DNA from the GM 13069 and FRAXA cells was completely digested with EcoRI (lanes 1,9,11,19) or EcoRI and AflIII (lanes 2,10,12,20). Southern blot analysis was carried out first with the FRA3B distal region specific probe (the C/I probe, top), then reprobing with the W/X probe, specific for the FRAXA site (bottom). The EcoRI fragment of the FRAXA site is 5.2 kb in GM13069 cells, whereas due to CGG expansion, it is 6.8 kb in FRAXA expressing cells. The positions of the distal breakpoint cluster of the FRA3B site and the breakpoint region (the CGG repeating sequence) of the FRAXA site are indicated by blasts. (E) EcoRI; (A) AflIII.

Figure 3

No DNase I hypersensitive sites in either breakpoint region of FRA3B. Nuclei were isolated from aphidicolin-treated and untreated cells and incubated with DNase I at 10, 25, 50, and 100 U/mL at 37°C for 5 min. Reactions were stopped by a solution containing EDTA and EGTA and DNA was extracted. (A) For probing the FRA3B distal breakpoint region, DNA was further digested with EcoRI and AflIII and hybridized with the D8/D9 probe to visualize a 1899-bp AflIII fragment of the distal breakpoint region (the 1899-bp AflIII fragment of FRA3B does not contain an internal EcoRI site). The blot was also hybridized with the C/I probe (see Fig. 1) to examine the same fragment from the other end (data not shown). As a control to insure the condition of DNase I digestion, the same blot was hybridized with the DHFR probe to visualize a 1777-bp EcoRI fragment of the DHFR promoter region (the 1777-bp EcoRI fragment of the DHFR gene does not contain an internal AflIII site). (B) For probing the proximal breakpoint region, DNase I-treated DNA was further digested with EcoRI and hybridized with the 17/18 probe to visualize a 2150-bp EcoRI fragment of the FRA3B proximal breakpoint region. The same blot was hybridized with the DHFR probe to visualize a 1777-bp EcoRI fragment of the DHFR promoter region (data not shown). The major breakpoint clusters are marked by blasts within the 1899-bp AflIII fragment of the distal region and the 2150-bp EcoRI fragment of the proximal region. The transcription initiation site of the DHFR gene is indicated by a right angle arrow.

Figure 4

Both major breakpoint regions of FRA3B contain contiguously phased nucleosomes. Nuclei were isolated from aphidicolin-treated and untreated cells and incubated with MNase at 0.00375, 0.0125, 0.0375, and 0.125 U/mL at 37°C for 5 min. Reactions were stopped and DNA was extracted. (A) The Southern blot probing strategy was the same as described in Fig. 3. For probing the FRA3B distal breakpoint region, MNase-treated DNA was further digested with EcoRI and AflIII and hybridized with the D8/D9 probe (left) and the C/I probe (right). The same blot was hybridized with the DHFR probe (data not shown) (B) For probing the proximal breakpoint region, MNase-treated DNA was further digested with EcoRI and hybridized with the 17/18 probe. The same blot was hybridized with the DHFR probe to examine the DHFR promoter region. Symbols are the same as in Figure 3.

Figure 5

The formation of nucleosome arrays on a plasmid containing a 1899-bp FRA3B distal breakpoint sequence. A diagram of the plasmid with a 1899-bp FRA3B distal breakpoint sequence inserted at the SmaI site of the polylinker region (white bar) of the pGEM3zf(+) vector is shown. The single arrow indicates the direction of the FHIT gene. The position of the distal breakpoint cluster of the FRA3B site is indicated by a blast. The position of the high-flexibility DNA sequence is indicated by a hatched bar. (E) EcoRI; (A) AflIII. RSF-assembled plasmids (lanes 1–4, the plasmid containing the FRA3B sequence; lanes 5–8, the parental plasmid) were partially digested by two different concentrations of MNase. The MNase-treated DNA was purified, and half of the DNA was used directly in lanes 1, 2, 5, and 6. The other half of the DNA samples was further digested by HindIII (lanes 3,4,7,8), and the MNase digestion patterns were analyzed by Southern blots. The 26-nt hybridization probe is located between the HindIII and SmaI sites. The multiple arrows to the left indicate the nucleosome ladder.