Restricting somatic mutation to the Ig V region by chromatin modification

Abstract

Somatic hypermutation (SHM) of the immunoglobulin genes is a centroblast B lymphocyte-specific mutational process that is targeted and restricted to the rearranged variable (V) region. Activation-induced cytidine deaminase (AID) initiates the mutational process by directly deaminating cytidines along transcribed DNA. Still, little is known about how SHM is restricted to the V region. We hypothesize that a microenvironment of chromatin alterations is induced at the V region to restrict the mutational machinery.;The BL2 Burkitts lymphoma cell line can be induced to undergo SHM at the Ig heavy chain V region. After 72 hours of stimulation with anti-IgM and T cells, there is an approximately eight-fold increase in the frequency of V region mutation and not in the C region. We believe that the normal process of SHM has been triggered in the BL2 cells based on the characteristics of V region mutations observed.;Since the unstimulated BL2 cells do not have detectable levels of AID mRNA, the onset of AID expression may explain the onset of SHM. Furthermore, when AID is overexpressed in the BL2 cells, SHM is constitutive showing decreased hot spot focusing and elevated targeting to GC base pairs. Interestingly, C region mutations arise. In BL2 cells, one mechanism of regulating SHM may be to induce AID expression at limited amounts such that its activity on transcribed DNA is restricted to the V region.;The regulation of AID expression, however, does not explain how SHM is restricted to the V region and much of this work is focused upon whether histone acetylation facilitates targeting SHM to the V region. The chromatin immunoprecipitation assay demonstrated that histone H4 hyperacetylation occurs at the V region, but not the C region, after 72 hours only under the mutation-inducing conditions. Changes in histone acetylation at the V region are independent of AID expression and are observed in BL2 cells expressing anti-sense for AID. Trichostatin A (TSA), a histone deacetylase inhibitor caused an increase in histone H4 hyperacetylation at the Ig gene. When BL2 cells are co-stimulated in the presence of TSA, the induced levels of AID extended mutations to the C region. HDAC2, a histone deacetylase, may be a target for TSA inhibition. HDAC2 is associated with the Ig gene and TSA causes their dissociation. HDAC2 may contribute to restricting mutations to the V region by preventing histone hyperacetylation from occurring downstream at the C region.;Stimulation with anti-IgM and T cells initiates two independent events: the expression of AID and increased histone acetylation in the V region microdomain. The observed epigenetic alterations in the V region microdomain are independent of AID expression and function. We propose that histone hyperacetylation at the V region plays an important role in restricting SHM in a V region-specific manner at physiological AID levels.