Elucidation of the epigenetic regulation of the antigen receptor gene rearrangement

1)E2A诱导染色质结构改变的因素分析当E2A与RAG_1/2在高效转染性的BOSC-23细胞中异位表达时，内源性免疫球蛋白κ基因座可诱导生殖系转录和重排。E2a与重组靶区结合，促进组蛋白H3和H4的乙酰化。在所检测的几种组蛋白乙酰转移酶中，p300/CBP被E2A特异性地招募到靶点。与此一致的是，p300/CBP和E2A的过表达导致组蛋白乙酰化、生殖系转录和重排增加。相反，siRNA下调内源性p300/CBP导致组蛋白乙酰化、生殖系转录和重排减少。在这些观察的基础上，发现E2A通过增加His…来招募p300/CBP到重组靶区并通过开放染色质结构来诱导重排等位基因排除的核机制和生理作用的分析抗原受体基因的等位排斥是一种现象，在这种现象中，只在一个等位染色体上发生生产性重排，并确保单个淋巴细胞只表达一个抗原受体。对于等位基因排斥的可能机制，人们推测，一旦产生了有效的重排，由抗原受体介导的负反馈信号就会阻止持续的重排。我们发现，E2A通过直接与靶基因片段结合来促进T细胞受体β基因重排，以剂量敏感的方式增加染色质的可及性。反馈信号废除了E2A结合，导致可及性下降。相反，E2A的强制表达通过推翻反馈抑制而诱导重排。因此，E2A的丰度在基因座激活中是速率限制的，反馈信号下调E2A的活性以确保等位基因排斥。较少

1)Analysis of the factors involved in chromatin structural changes induced by E2AWhen E2A was ectopically expressed with RAG1/2 in BOSC 23 cells that are highly efficient in transfection, germline transcription as well as rearrangement were induced in the endogenous immunoglobulin κ locus. E2A was found to associate with the recombination target regions and increase acetylation of histone H3 and H4. Among several histone acetyltransferases examined, p300/CBP were specifically recruited to the target sites by E2A. Consistent with this, overexpression of p300/CBP together with E2A resulted in increased histone acetylation, germline transcription and rearrangement. Conversely, down-regulation of endogenous p300/CBP by siRNA led to reduction of histone acetylation, germline transcription and rearrangement. Based on these observations, E2A was found to recruit p300/CBP to the recombination target regions and induce rearrangement by opening the chromatin structure through the increase of his … More tone acetylation.2)Analysis of the nuclear mechanisms and physiological roles for allelic exclusionAllelic exclusion of the antigen receptor genes is a phenomenon, in which a productive rearrangement takes place only on one allelic chromosome, and ensures that an individual lymphocyte expresses only one antigen receptor. It has been postulated for the possible mechanism underlying allelic exclusion that, once a productive rearrangement has been generated, a negative feedback signal mediated by the antigen receptor prevents continued rearrangement. We found that E2A promotes T cell receptor β gene rearrangement by directly binding to target gene segments to increase chromatin accessibility in a dosage-sensitive manner. Feedback signaling abrogates E2A binding, leading to a decline in accessibility. Conversely, enforced expression of E2A induces rearrangement by overriding the feedback inhibition. Thus, the abundance of E2A is rate limiting in locus activation, and feedback signaling down-regulates E2A activity to ensure allelic exclusion. Less