TY - JOUR
T1 - Immunoglobulin switch μ sequence causes RNA polymerase II accumulation and reduces dA hypermutation
AU - Rajagopal, Deepa
AU - Maul, Robert W.
AU - Ghosh, Amalendu
AU - Chakraborty, Tirtha
AU - Khamlichi, Ahmed Amine
AU - Sen, Ranjan
AU - Gearhart, Patricia J.
PY - 2009/6/8
Y1 - 2009/6/8
N2 - Repetitive DNA sequences in the immunoglobulin switch μ region form RNA-containing secondary structures and undergo hypermutation by activation-induced deaminase (AID). To examine how DNA structure affects transcription and hypermutation, we mapped the position of RNA polymerase II molecules and mutations across a 5-kb region spanning the intronic enhancer to the constant μ gene. For RNA polymerase II, the distribution was determined by nuclear run-on and chromatin immunoprecipitation assays in B cells from uracil-DNA glycosylase (UNG)-deficient mice stimulated ex vivo. RNA polymerases were found at a high density in DNA flanking both sides of a 1-kb repetitive sequence that forms the core of the switch region. The pileup of polymerases was similar in unstimulated and stimulated cells from Ung- /- and Aid- /- Ung- /- mice but was absent in cells from mice with a deletion of the switch region. For mutations, DNA was sequenced from Ung- /- B cells stimulated in vivo. Surprisingly, mutations of A nucleotides, which are incorporated by DNA polymerase η, decreased 10-fold before the repetitive sequence, suggesting that the polymerase was less active in this region. We propose that altered DNA structure in the switch region pauses RNA polymerase II and limits access of DNA polymerase η during hypermutation.
AB - Repetitive DNA sequences in the immunoglobulin switch μ region form RNA-containing secondary structures and undergo hypermutation by activation-induced deaminase (AID). To examine how DNA structure affects transcription and hypermutation, we mapped the position of RNA polymerase II molecules and mutations across a 5-kb region spanning the intronic enhancer to the constant μ gene. For RNA polymerase II, the distribution was determined by nuclear run-on and chromatin immunoprecipitation assays in B cells from uracil-DNA glycosylase (UNG)-deficient mice stimulated ex vivo. RNA polymerases were found at a high density in DNA flanking both sides of a 1-kb repetitive sequence that forms the core of the switch region. The pileup of polymerases was similar in unstimulated and stimulated cells from Ung- /- and Aid- /- Ung- /- mice but was absent in cells from mice with a deletion of the switch region. For mutations, DNA was sequenced from Ung- /- B cells stimulated in vivo. Surprisingly, mutations of A nucleotides, which are incorporated by DNA polymerase η, decreased 10-fold before the repetitive sequence, suggesting that the polymerase was less active in this region. We propose that altered DNA structure in the switch region pauses RNA polymerase II and limits access of DNA polymerase η during hypermutation.
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U2 - 10.1084/jem.20082514
DO - 10.1084/jem.20082514
M3 - Article
C2 - 19433618
AN - SCOPUS:67449161835
SN - 0022-1007
VL - 206
SP - 1237
EP - 1244
JO - Journal of Experimental Medicine
JF - Journal of Experimental Medicine
IS - 6
ER -