Spt5 accumulation at variable genes distinguishes somatic hypermutation in germinal center B cells from ex vivo-activated cells

J Exp Med. 2014 Oct 20;211(11):2297-306. doi: 10.1084/jem.20131512. Epub 2014 Oct 6.

Abstract

Variable (V) genes of immunoglobulins undergo somatic hypermutation by activation-induced deaminase (AID) to generate amino acid substitutions that encode antibodies with increased affinity for antigen. Hypermutation is restricted to germinal center B cells and cannot be recapitulated in ex vivo-activated splenic cells, even though the latter express high levels of AID. This suggests that there is a specific feature of antigen activation in germinal centers that recruits AID to V genes which is absent in mitogen-activated cultured cells. Using two Igh knock-in mouse models, we found that RNA polymerase II accumulates in V regions in B cells after both types of stimulation for an extended distance of 1.2 kb from the TATA box. The paused polymerases generate abundant single-strand DNA targets for AID. However, there is a distinct accumulation of the initiating form of polymerase, along with the transcription cofactor Spt5 and AID, in the V region from germinal center cells, which is totally absent in cultured cells. These data support a model where mutations are prevalent in germinal center cells, but not in ex vivo cells, because the initiating form of polymerase is retained, which affects Spt5 and AID recruitment.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3' Untranslated Regions
  • Animals
  • B-Lymphocytes / immunology*
  • B-Lymphocytes / metabolism*
  • Chromosomal Proteins, Non-Histone / metabolism*
  • Cytidine Deaminase / genetics
  • DNA Polymerase II / metabolism
  • DNA, Single-Stranded / metabolism
  • Female
  • Gene Order
  • Genetic Loci
  • Germinal Center / immunology*
  • Germinal Center / metabolism*
  • Immunoglobulin Variable Region*
  • Lymphocyte Activation / genetics
  • Lymphocyte Activation / immunology
  • Male
  • Mice
  • Models, Biological
  • Sequence Deletion
  • Somatic Hypermutation, Immunoglobulin*
  • Transcriptional Elongation Factors / metabolism*

Substances

  • 3' Untranslated Regions
  • Chromosomal Proteins, Non-Histone
  • DNA, Single-Stranded
  • Immunoglobulin Variable Region
  • Transcriptional Elongation Factors
  • SPT5 transcriptional elongation factor
  • DNA Polymerase II
  • AICDA (activation-induced cytidine deaminase)
  • Cytidine Deaminase