Histone 3 lysine 4 methylation during the pre-B to immature B-cell transition

Abstract

The relationship between chromatin modification and lymphocyte development is still poorly understood. Here we show a correlation between methylation of lysine 4 on histone 3 (H3-K4) and activation of several loci required for the pre-B cell to immature B-cell developmental transition. A critical step in this transition is the induction of V(D)J recombination at the Igkappa locus. Upon activation of Igkappa recombination, a >10-fold enrichment of both di- and trimethylated H3-K4 is observed at Jkappa targeting signals, but not at an analogous targeting signal in the T-cell receptor alpha locus or, surprisingly, at several Vkappa signals. However, H3-K4 methylation is restricted to the actively recombining fraction of Jkappa recombination targeting signals, consistent with a direct relationship between H3-K4 methylation and signal activity. Correlations between increased H3-K4 methylation and induction of transcription are also observed at some, but not all, loci where transcription is induced. H3-K4 methylation may therefore be a widely used but not universal means for controlling chromatin activity in this developmental transition.

Figure 1

Levels of tri-Me-H3-K4 detected at selected sites. (A) ChIP analysis of extracts from ts-Ab-MuLV cells. L, extracts from ts-Ab-MuLV cells grown at permissive temperature; H, extracts from ts-Ab-MuLV cells grown at non-permissive temperature for 24 h. Additional 5- and 2-fold dilutions of H input DNA are shown to confirm PCR is in the linear range. Input DNA template (left of figure) was compared to DNA template immunoprecipitated with a non-specific control antibody (IgG) or using the tri-Me-H3-K4 antibody, and template detected using 5′ of Jκ1 or TCR primer pairs as noted. (B) Levels of tri-Me-H3-K4 at selected sites. Approximate locations of primer pairs noted on the locus diagram (details in Materials and Methods). The amount of immunoprecipitated DNA, averaged from two independent inductions, for a given primer pair is expressed as a fraction of input DNA for both L and H samples. Error bars represent standard deviation. (C) Levels of di-Me-H3-K4 at selected sites from one representative experiment of two. Primer pair locations as in (B).

Figure 2

Ordering of recombination and H3-K4 methylation near Jκ1. (A) Approximate location of PCR primers used in (B) and (C); numbers identify each PCR product used in subsequent panels. (B) Relative levels of coding junctions from input DNA are plotted against levels of tri-Me-H3-K4 detected at the indicated sites before (L) or after shift of cells to the non-permissive temperature (H) for 12, 16, 20 or 24 h. Initial levels of coding junctions at L were defined as 1. Data are from a representative experiment. (C) ChIP analysis of extracts from ts-Ab-MuLV cells grown at non-permissive temperature for 24 h. Additional 5- and 2-fold dilutions of input DNA are shown to confirm LM-PCR is in the linear range; see (A) for cartoon representation of LM-PCR. Input DNA template (left of panel) was compared to DNA template immunoprecipitated with a non-specific control antibody (IgG) or using the tri-Me-H3-K4 antibody.

Figure 3

Tri-Me-H3-K4 and activation of transcription. (A) Relative levels of RNA before (L) and after (H) shifting to the non-permissive temperature using RT–PCR and ORF primer pairs for each gene, as indicated in the Materials and Methods. A GAPDH RT–PCR was used to correct for differences in RNA between samples. (B) ChIP data showing levels of tri-Me-H3-K4 in Spi-B, RAG1 and IRF-4 as assessed by ORF primer pairs for each gene.

Figure 4

Levels of tri-Me-H3-K4 in primary pre-B cells. T0, immediately before IL-7 withdrawal; T15, 15 h after IL-7 withdrawal.