Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2006 Mar 28;103(13):5030-5.
doi: 10.1073/pnas.0506548103. Epub 2006 Mar 17.

Downstream boundary of chromosomal R-loops at murine switch regions: implications for the mechanism of class switch recombination

Feng-Ting Huang  1 , Kefei Yu, Chih-Lin Hsieh, Michael R Lieber
Affiliations

Downstream boundary of chromosomal R-loops at murine switch regions: implications for the mechanism of class switch recombination

Feng-Ting Huang et al. Proc Natl Acad Sci U S A. .

Abstract

R-loops form at Sgamma3 and Sgamma2b Ig class switch regions in the chromosomes of stimulated murine primary B cells and are suspected to be a general feature of mammalian class switch regions. The in vivo upstream boundary of the R-loops is known to begin within the switch repeats. To determine how precisely the R-loop structure conforms to the repetitive zone of the murine Sgamma3 and Sgamma2b switch regions, a chemical probing method was used to obtain structural information on the downstream boundary. We find that only 61-67% of the R-loops terminate within the Sgamma3 and the Sgamma2b repetitive zones, and the remainder terminate downstream, usually within the first 600 bp immediately downstream of the core switch repeats. Interestingly, the nontemplate strand G density falls to the random level gradually through this same region. Hence, the R-loops terminate as the G-richness of the nascent RNA strand falls. This finding is consistent with thermodynamic predictions for RNA:DNA duplex strength relative to that of DNA:DNA duplexes. This result contrasts with the location of known recombination breakpoints, which correlate not with G-richness and R-loop location but rather with AGCT density. The implications of these findings are discussed in the context of models for the targeting of class switch recombination.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Single-strandedness on the top strand of the murine Sγ3 region in stimulated B cells. (A) Top strand sequence without the RNase H treatment before the bisulfite treatment. Primary splenic B cells were stimulated in culture for 2 days with LPS. Genomic DNA was prepared and then treated with sodium bisulfite as described in Materials and Methods. A single round of PCR (30 cycles) was done by using one regular (native sequence) primer and one converted primer whose sequence is complementary to the top strand where the C's have been converted to U's. FTH13, the converted primer containing seven C's converted to seven T's, is located 400 bp downstream of the beginning of the murine Ig Sγ3 region. KY320, the native primer, is located 674 bp downstream of the end of the Sγ3 region. Each long line represents an independent molecular clone. The small vertical bar on each line indicates a C on the sequence converted to a T. The asterisk indicates that the region of conversion extends to the KY320 site. The word “gap” indicates that the clone has gaps in the long stretches of C to T conversion. (The gaps have more than two consecutive unconverted C's). In the diagram at the top, the long ellipse represents the Sγ3 region, which is 1,801 bp long. The region between the Sγ3 and the Cγ3 (constant region of the Igγ3) is 2,080 bp. (B) Top strand sequence with RNase H treatment before bisulfite treatment. The experiment was done as in A, except that an RNase H incubation was done before bisulfite treatment. The bottom line displays every C residue on the top strand of the PCR product.
Fig. 2.
Fig. 2.
Single-strandedness on the top strand immediately downstream of murine Sγ3 in stimulated B cells. Two primers were used in an initial round of PCR (30 cycles) and yielded a faint band. To generate more DNA for cloning, this band was cut out and amplified with the same primers with an additional round of 30 cycles. FTH5, the converted primer containing seven C's converted to seven T's, is located immediately within the downstream edge of the murine Ig Sγ3 region. FTH7, a native primer, is located 130 bp downstream of the beginning of the first Cγ3 exon. Each long line represents an independent molecular clone. The small vertical bar on each line indicates a C on the sequence converted to T. The most bottom line displays every C residue on the top strand of the PCR product.
Fig. 3.
Fig. 3.
Single-strandedness on the top strand within and downstream of the Sγ2b switch region in stimulated B cells. (A) The experiment was done as in Fig. 1A. In the diagram at the top, the half ellipse represents the downstream part of Sγ2b region. The region between the Sγ2b and the Cγ2b (constant region of the Igγ2b) is 1,837 bp. FTH71, a converted primer, is located 400 bp upstream of the end of Sg2b. FTH73 is a native primer that is located 100 bp downstream of Sγ2b. (B) The same DNA as in A was analyzed by using a different downstream native primer. FTH88, a native primer, is located 800 bp downstream of Sγ2b. The bottom line displays every C residue on the top strand of the PCR product. RNase H destroyed most of the R-loops.
Fig. 4.
Fig. 4.
Plots of the downstream edge of the R-loops, the G density, and the AGCT locations for murine Sγ3 through the initial portion of the first constant exon. (A) The relative position of the 482-bp Iγ3 exon, the 805-bp Iγ3–Sg3 intervening region, the 1,801-bp Sγ3, the 2,080-bp Sγ3–Cγ3 intervening region, and the first 1,801 bp of the Cγ3 region (exon/intron boundaries not specified) are shown. R-loop downstream boundaries based on Figs. 1 and 2 are shown as dots above or below the line, respectively. (B) The AGCT plot of the sequence in A. The short vertical bars on the plot indicate the AGCT sites. (C) The G density plot of the sequence in A.
Fig. 5.
Fig. 5.
Plots of the downstream edge of the R-loops, the G density, and the AGCT locations for murine Sγ2b through the initial portion of the first constant exon. (A) The relative position of the 3,807-bp Sγ2b, the 1,837-bp Sγ2b–Cγ2b intervening region, the 356-bp Ig2b–Sγ2b intervening region, the 400-bp Iγ2b exon, and 3,807 bp of the Cγ2b constant exons/introns (boundaries not shown). R-loop downstream boundaries based on Fig. 3 are shown as dots above (from Fig. 3A) or below (from Fig. 3B) the line, respectively. (B) The AGCT plot of the sequence in A. The short vertical bars on the plot indicate the AGCT sites. (C) The G density plot of the sequence in A.
See this image and copyright information in PMC

References

    1. Chaudhuri J., Alt F. W. Nat. Rev. Immunol. 2004;4:541–552. - PubMed
    1. Yu K., Lieber M. R. DNA Repair. 2003;2:1163–1174. - PubMed
    1. Janeway C. A., Travers P., Walport M., Shlomchik M. Immunobiology. New York: Garland; 2001.
    1. Etzioni A., Ochs H. D. Pediatr. Res. 2004;56:519–525. - PubMed
    1. Luby T. M., Schrader C. E., Stavnezer J., Selsing E. J. Exp. Med. 2001;193:159–168. - PMC - PubMed

Publication types

MeSH terms