title: Histone H3.3 promotes IgV gene diversification by enhancing formation of AID-accessible single-stranded DNA.
abstract: Immunoglobulin diversification is driven by activation-induced deaminase (AID), which converts cytidine to uracil within the Ig variable (IgV) regions. Central to the recruitment of AID to the IgV genes are factors that regulate the generation of single-stranded DNA (ssDNA), the enzymatic substrate of AID Here, we report that chicken DT40 cells lacking variant histone H3.3 exhibit reduced IgV sequence diversification. We show that this results from impairment of the ability of AID to access the IgV genes due to reduced formation of ssDNA during IgV transcription. Loss of H3.3 also diminishes IgV R-loop formation. However, reducing IgV R-loops by RNase HI overexpression in wild-type cells does not affect IgV diversification, showing that these structures are not necessary intermediates for AID access. Importantly, the reduction in the formation of AID-accessible ssDNA in cells lacking H3.3 is independent of any effect on the level of transcription or the kinetics of RNAPII elongation, suggesting the presence of H3.3 in the nucleosomes of the IgV genes increases the chances of the IgV DNA becoming single-stranded, thereby creating an effective AID substrate.
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.
authors.forename: Marina
authors.initials: M
authors.lastname: Romanello
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.
authors.forename: Davide
authors.initials: D
authors.lastname: Schiavone
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.
authors.forename: Alexander
authors.initials: A
authors.lastname: Frey
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge, UK jes@mrc-lmb.cam.ac.uk.
authors.forename: Julian E
authors.identifier: 0000-0002-5031-3780
authors.initials: JE
authors.lastname: Sale
mesh_terms: D000818:Animals; D002460:Cell Line; D002645:Chickens; D003564:Cytidine Deaminase / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism*; D006657:Histones / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000378:metabolism*; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
chemical_list: D004277:DNA, Single-Stranded; D006657:Histones; D007135:Immunoglobulin Variable Region; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase
keywords: activation‐induced deaminase (AID); histone H3.3; immunoglobulin diversification; single‐stranded DNA; transcription