Switching to IgE can be activated by a combination of IL4, which induces transcription through the IgE switch region, and activation of the cell surface CD40 receptor, which induces cell proliferation. Activation can be produced by anti-CD40 antibody or by a trimeric form of CD40 ligand. Our studies have concentrated on the human B cell line CL-01, which has been reported to undergo switch recombination, and have more recently included primary human B cells. We have shown that IL4 causes a 100X increase in transcript, both unspliced and spliced, over the IgE switch region in CL=01 cells. We also observed a sizable (5X) increase in transcription of AID, the cytidine deaminase that is responsible for initiating CSR. A high resolution study of histone modifications in uninduced and induced CL-01 cells has been carried out, focusing on the region between the I-epsilon promoter and the 3 end of the IgE switch region. This is the most important region associated with this CSR event. The largest increases were found for the dual modification acetyl-H3K9/14 (or acetyl-H3K9 alone) and for trimethyl-H3 K4, while other modifications including histone H4 tetra-acetylation (K5/8/12/16) and H3 K27 trimethylation were not greatly altered following IL4 induction. This pattern is consistent with broad activation of the region. Furthermore, most of the modifications were unevenly distributed: for example, dimethyl-H3K4, tetraacetyl-H4, and acetyl-H3 K9/14 all were concentrated over the I-epsilon promoter. To aid in interpreting these results, the abundance of nucleosomes was mapped across the same region, and a relative depletion over I-epsilon was observed, without any further change on induction. We have shown that treatment of the cells with 5-aza-deoxycytidine, a known demethylating agent, leads to an additional increase in IL4-stimulated transcription. We have also mapped the distribution of RNA polymerase II, as well as potential sites of AID binding in this region. Working together with our collaborators in Great Britain, we have also studied histone modifications in primary human B cells from several subjects. The patterns are on average similar to that in CL-01 cells, but with notable variations between individuals, which will be the subject of future work. After the completion of this set of experiments, production of a manuscript required considerable effort and coordination among the several groups of authors. The work has now been published.
|Gupta, Shikha; Gellert, Martin; Yang, Wei (2012) Mechanism of mismatch recognition revealed by human MutSÎ² bound to unpaired DNA loops. Nat Struct Mol Biol 19:72-8|
|Dayal, Sandeep; Nedbal, Jakub; Hobson, Philip et al. (2011) High resolution analysis of the chromatin landscape of the IgE switch region in human B cells. PLoS One 6:e24571|
|Grundy, Gabrielle J; Yang, Wei; Gellert, Martin (2010) Autoinhibition of DNA cleavage mediated by RAG1 and RAG2 is overcome by an epigenetic signal in V(D)J recombination. Proc Natl Acad Sci U S A 107:22487-92|