Chromatin modifications in immunoglobulin switch recombination

白介素4通过IgE切换区诱导转录，细胞表面CD40受体激活诱导细胞增殖，两者结合可激活向IgE的转换。激活可由抗CD40抗体或CD40配体的三聚体形式产生。我们的研究集中在人类B细胞系CL-01上，据报道它正在进行开关重组，最近又包括了原代人类B细胞。我们已经证明，在CL=01细胞中，IL4导致未剪接和剪接的转录物比IgE开关区增加100倍。我们还观察到AID的转录显著增加(5倍)，AID是一种胞苷脱氨酶，负责启动CSR。 对未诱导和诱导的CL-01细胞的组蛋白修饰进行了高分辨率研究，重点研究了I-epsilon启动子和IgE切换区3端之间的区域。这是与本次CSR活动相关的最重要地区。经IL4诱导后，乙酰基-H3K9/14(或单独的乙酰基-H3K9)和三甲基-H3K4的双重修饰增加最大，而组蛋白H4的四乙酰化(K5/8/12/16)和H3K27的三甲基化等修饰在IL4诱导后变化不大。这一模式与该地区的广泛激活是一致的。此外，大多数修饰是不均匀分布的：例如，二甲基H3K4、四乙酰-H4和乙酰基-H3K9/14都集中在i-epsilon启动子上。为了帮助解释这些结果，核小体的丰度被绘制在同一区域，并观察到I-epsilon的相对耗竭，诱导后没有任何进一步的变化。我们已经证明，用已知的去甲基化试剂5-氮杂脱氧胞苷处理细胞，会导致IL4刺激的转录增加。我们还绘制了RNA聚合酶II的分布图，以及该区域可能的AID结合位点。 与我们在英国的合作者合作，我们还研究了几个受试者原代人类B细胞中的组蛋白修饰。这些模式与CL-01细胞的平均模式相似，但个体之间存在显著差异，这将是未来工作的主题。 在完成这一系列实验后，手稿的制作需要几个作者组之间的大量努力和协调。这部作品现在已经出版了。

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.