Chromatin modifications in immunoglobulin switch recombination

转换为IgE可以通过IL4和CD40受体的组合激活，IL4诱导该区域的转录，CD40受体激活细胞表面CD40受体，诱导细胞增殖。CD40抗体或CD40配体的三聚体可以激活B细胞。我们的研究主要集中在人类CL-01 B细胞系上，据报道它发生了开关重组。我们已经证明，IL4导致未剪接和剪接的转录产物在IgE切换区增加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的研究，重点是I-epsilon启动子和IgE切换区3端之间的区域，这是与CSR事件相关的最重要的区域。其中乙酰基-H3K9/14(或单独的乙酰基-H3K9)和三甲基-H3K4的表达增加最大，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，在大多数情况下，给定的修饰的分布是不均匀的：例如，二甲基H3K4、四乙酰-H4和乙酰基-H3K9/14都在i-epsilon启动子上显示了相对的峰。为了解释这些结果，在同一区域绘制了核小体的丰度图，并观察到I-epsilon的相对耗竭，诱导后没有任何进一步的变化。我们现在正在将这些观察扩展到研究组蛋白变体H3.3和H2A.Z的分布。此外，我们正在研究同一区域的DNA甲基化。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。

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 have focused on the human CL-01 line of B cells, 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, the most important region associated with this CSR event. The largest increases were found for 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, the distribution of a given modification was in most cases non-uniform: for example, dimethyl-H3K4, tetraacetyl-H4, and acetyl-H3 K9/14 all showed relative peaks over the I-epsilon promoter. To interpret 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.