The DSIF subunits Spt4 and Spt5 have distinct roles at various phases of immunoglobulin class switch recombination

PLoS Genet. 2012;8(4):e1002675. doi: 10.1371/journal.pgen.1002675. Epub 2012 Apr 26.

Abstract

Class-switch recombination (CSR), induced by activation-induced cytidine deaminase (AID), can be divided into two phases: DNA cleavage of the switch (S) regions and the joining of the cleaved ends of the different S regions. Here, we show that the DSIF complex (Spt4 and Spt5), a transcription elongation factor, is required for CSR in a switch-proficient B cell line CH12F3-2A cells, and Spt4 and Spt5 carry out independent functions in CSR. While neither Spt4 nor Spt5 is required for transcription of S regions and AID, expression array analysis suggests that Spt4 and Spt5 regulate a distinct subset of transcripts in CH12F3-2A cells. Curiously, Spt4 is critically important in suppressing cryptic transcription initiating from the intronic Sμ region. Depletion of Spt5 reduced the H3K4me3 level and DNA cleavage at the Sα region, whereas Spt4 knockdown did not perturb the H3K4me3 status and S region cleavage. H3K4me3 modification level thus correlated well with the DNA breakage efficiency. Therefore we conclude that Spt5 plays a role similar to the histone chaperone FACT complex that regulates H3K4me3 modification and DNA cleavage in CSR. Since Spt4 is not involved in the DNA cleavage step, we suspected that Spt4 might be required for DNA repair in CSR. We examined whether Spt4 or Spt5 is essential in non-homologous end joining (NHEJ) and homologous recombination (HR) as CSR utilizes general repair pathways. Both Spt4 and Spt5 are required for NHEJ and HR as determined by assay systems using synthetic repair substrates that are actively transcribed even in the absence of Spt4 and Spt5. Taken together, Spt4 and Spt5 can function independently in multiple transcription-coupled steps of CSR.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antigens, Nuclear / genetics
  • Antigens, Nuclear / metabolism
  • B-Lymphocytes / immunology
  • B-Lymphocytes / metabolism
  • Cell Culture Techniques
  • Chromatin* / genetics
  • Chromatin* / metabolism
  • Chromosomal Proteins, Non-Histone* / genetics
  • Chromosomal Proteins, Non-Histone* / immunology
  • Cytidine Deaminase / genetics
  • DNA Cleavage
  • DNA End-Joining Repair / genetics
  • DNA Repair* / genetics
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Gene Rearrangement / immunology
  • Histone-Lysine N-Methyltransferase / genetics
  • Histone-Lysine N-Methyltransferase / metabolism
  • Histones / genetics
  • Histones / metabolism
  • Homologous Recombination* / genetics
  • Homologous Recombination* / immunology
  • Immunoglobulin Class Switching* / genetics
  • Immunoglobulin Class Switching* / immunology
  • Immunoglobulins* / genetics
  • Immunoglobulins* / metabolism
  • Ku Autoantigen
  • Mice
  • Protein Processing, Post-Translational
  • Signal Transduction
  • Transcriptional Elongation Factors* / genetics
  • Transcriptional Elongation Factors* / immunology

Substances

  • Antigens, Nuclear
  • Chromatin
  • Chromosomal Proteins, Non-Histone
  • DNA-Binding Proteins
  • Histones
  • Immunoglobulins
  • Transcriptional Elongation Factors
  • SPT5 transcriptional elongation factor
  • Histone-Lysine N-Methyltransferase
  • Cytidine Deaminase
  • Xrcc6 protein, mouse
  • Ku Autoantigen