Chromatin modifications in immunoglobulin switch recombination

免疫球蛋白开关重组中的染色质修饰

• 批准号: 7967408
• 负责人: MARTIN F. GELLERT
• 金额: $ 21.51万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7967408
• 关键词: Acetylation; Allergic; Antibodies; B-Lymphocytes; Binding; CD40 Ligand; Cell Line; Cell Proliferation; Cell surface; Cells; Cytidine Deaminase; DNA Methylation; Deoxycytidine; Event; Genetic Transcription; Histone H4; Histones; Human; IL4 gene; IgE; Immunoglobulin Class Switching; Immunoglobulin Switch Recombination; Immunoglobulins; Maps; Mediating; Modification; Nucleosomes; Pattern; RNA Polymerase II; RNA Splicing; Relative (related person); Reporting; Resolution; Site; TNFRSF5 gene; Transcript; Variant; Work; chromatin modification; constant region gene; cytokine; histone modification; promoter; receptor; response

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.

切换到IgE可以通过IL4的组合（诱导该区域的转录）的组合以及诱导细胞增殖的细胞表面CD40受体的激活来激活。激活可以由抗CD40抗体或CD40配体三聚体形式产生。我们的研究集中于人类B细胞系CL-01，据报道该细胞系重组。我们已经证明，IL4在IgE开关区域上的转录本（未贴合和剪接）增加了100倍。我们还观察到了负责启动CSR的胞苷脱氨酶的AID转录的大幅度（5倍）。 已经进行了对未诱导和诱导的CL-01细胞中组蛋白修饰的高分辨率研究，重点是I-EPSILON启动子和IgE开关区域的3端之间的区域。这是与此CSR事件相关的最重要区域。发现双重修饰的乙酰基-H3K9/14（或单独的乙酰H3K9）和三甲基-H3 K4的最大增加，而包括组蛋白H4二乙酰化（K5/8/12/16）（K5/8/12/16）和H3 K27甲基化（H3 K27甲基化）在IL4诱导后并没有发生很大的改变。这种模式与该区域的广泛激活一致。此外，大多数修饰分布不均：例如，二甲基-H3K4，四乙酰基-H4和乙酰基-H3 K9/14都集中在I-EPSILON启动子上。为了帮助解释这些结果，在同一区域绘制了核小体的丰度，并且观察到I-Epsilon的相对耗竭，而没有进一步改变诱导。现在，我们将这些观察结果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。我们已经表明，用5-aza-脱氧胞苷（一种已知的脱甲基剂）治疗细胞会导致IL4刺激的转录增加。进一步的工作正在进行中，以确定该区域中辅助结合的潜在位点以及RNA聚合酶II的分布。 还在努力寻找刺激切换到IGE的条件。