title: Topoisomerase I deficiency causes RNA polymerase II accumulation and increases AID abundance in immunoglobulin variable genes.
abstract: Activation-induced deaminase (AID) is a DNA cytosine deaminase that diversifies immunoglobulin genes in B cells. Recent work has shown that RNA polymerase II (Pol II) accumulation correlates with AID recruitment. However, a direct link between Pol II and AID abundance has not been tested. We used the DT40 B-cell line to manipulate levels of Pol II by decreasing topoisomerase I (Top1), which relaxes DNA supercoiling in front of the transcription complex. Top1 was decreased by stable transfection of a short hairpin RNA against Top1, which produced an accumulation of Pol II in transcribed genes, compared to cells transfected with sh-control RNA. The increased Pol II density enhanced AID recruitment to variable genes in the λ light chain locus, and resulted in higher levels of somatic hypermutation and gene conversion. It has been proposed by another lab that AID itself might directly suppress Top1 to increase somatic hypermutation. However, we found that in both AID(+/+) and AID(-/-) B cells from DT40 and mice, Top1 protein levels were identical, indicating that the presence or absence of AID did not decrease Top1 expression. Rather, our results suggest that the mechanism for increased diversity when Top1 is reduced is that Pol II accumulates and recruits AID to variable genes.
authors: Laboratory of Molecular Biology and Immunology, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, United States.
authors: Robert W
authors: RW
authors: Maul
authors: Laboratory of Molecular Biology and Immunology, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, United States.
authors: Huseyin
authors: H
authors: Saribasak
authors: Laboratory of Molecular Biology and Immunology, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, United States.
authors: Zheng
authors: Z
authors: Cao
authors: Laboratory of Molecular Biology and Immunology, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, United States. Electronic address: gearhartp@mail.nih.gov.
authors: Patricia J
authors: PJ
authors: Gearhart
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology*; D002460:Cell Line; D002645:Chickens / Q000235:genetics / Q000276:immunology; D003564:Cytidine Deaminase / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D055786:Gene Knockout Techniques; D005803:Genes, Immunoglobulin; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D051379:Mice; D012319:RNA Polymerase II / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D027041:Somatic Hypermutation, Immunoglobulin; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
chemical_list: D007135:Immunoglobulin Variable Region; D004247:DNA; D012319:RNA Polymerase II; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase; D004264:DNA Topoisomerases, Type I
keywords: Activation-induced deaminase; DT40; Gene conversion; RNA polymerase II; Somatic hypermutation; Topoisomerase I