Switching to IgE can be activated by a combination of IL4, which induces transcription in that 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 concentrate on the human B cell line CL-01, which has been reported to undergo switch recombination. We have shown that IL4 causes a 100X increase in transcript, both unspliced and spliced, over the IgE switch region. 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 are now extending these observations to studying the distribution of the histone variants H3.3 and H2A.Z. In addition we are studying DNA methylation in the same region. 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. Further work is in progress to determine potential sites of AID binding in this region, as well as the distribution of RNA polymerase II. Efforts are also underway to find conditions that stimulate switching to IgE.

Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2009
Total Cost
$215,095
Indirect Cost
City
State
Country
Zip Code
Gupta, Shikha; Gellert, Martin; Yang, Wei (2012) Mechanism of mismatch recognition revealed by human MutSβ 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