Regulation of cyclin D3 in B lymphocyte development

描述（由申请人提供）：B细胞发育是一个高度有序和调控的过程，在此过程中，谱系承诺的前B细胞产生不同的外周克隆B淋巴细胞群，显示出广泛的抗原特异性。在B淋巴形成早期，Igm与替代轻链和Iga/Igb组装形成pre-BCR，首先扩展前B细胞群，进行框内重链重排，然后启动轻链重排。在之前的研究中，我们证明了细胞周期蛋白D3是前b细胞克隆扩增所必需的（Nat Immunol 7:48 89）。细胞周期蛋白D3的积累和细胞周期的开始是由gc依赖的信号协调调节的，这些信号增强了Ccnd3 mRNA和通过pre-BCR的信号，增加了核周期蛋白D3的可用性。在初步结果和正在进行的工作中（Nat immuno1），我们证明了bcr依赖的Ras/MEK/ERK信号通路的激活协调了Ccnd3转录的终止和Igk重组的启动。在ERK的下游，特定的信号效应物介导每个离散的发育事件。Igk转录是通过ERK介导的Id蛋白的抑制和E2A的抑制来诱导的，而Aiolos的诱导使Ccnd3沉默，导致细胞周期退出。除了确定协调B细胞发育的中心通路外，这些发现还为理解前bcr和IL-7依赖信号之间的相互作用提供了一个框架。具体来说，我们证明了Igk转录是由E2A和STAT 5在内含子Igk增强子（Eki）上的竞争控制的。相反，细胞周期蛋白D3受PI-3激酶调控，PI-3激酶通过一种新的淋巴特异性机制，控制细胞周期蛋白D3相对于抑制剂p27kip1和CDK4/Rb效应复合物的亚核位置。这些观察结果唤起了一个模型，其中最初的前b细胞增殖是由STAT 5和PI-3k的协调激活介导的。stat5增强Ccnd3转录并抑制Igk重组，而PI-3k驱动生成周期蛋白D3/CDK4/Rb复合物。随后的Igk重组既需要脱离STAT 5，也需要前bcr激活ERK。该模型的中心预测将在以下具体目标中得到检验：确定E2A和STAT 5如何整合调控Igk转录。目标2。研究PI-3激酶如何调控细胞周期蛋白D3的核定位和与CDK4、Rb和p27kip1的组装。目的3：在体内确定为什么周期蛋白D3对淋巴细胞发育有独特的贡献。

DESCRIPTION (provided by applicant): B cell development is a highly ordered and regulated process during which lineage-committed pro-B cells give rise to diverse peripheral populations of clonal B lymphocytes displaying a broad repertoire of antigen specificities. Early in B lymphopoiesis, Igm assembles with surrogate light chain and Iga/Igb to form a pre-BCR that first expands pre-B cell populations bearing one in-frame heavy chain rearrangements and then initiates light chain rearrangement. In previous studies, we demonstrated that cyclin D3 is uniquely required for clonal expansion of pre-B cells (Nat Immunol 7:489). The accumulation of cyclin D3, and the initiation of cell cycle, is coordinately regulated by gc-dependent signals that enhance Ccnd3 mRNA and signals through the pre-BCR that increase the availability of nuclear cyclin D3. In Preliminary Results, and in work now in press (Nat Immunol), we demonstrate that pre-BCR dependent activation of the Ras/MEK/ERK signaling pathway coordinates the termination of Ccnd3 transcription with the initiation Igk recombination. Downstream of ERK, specific signaling effectors mediate each discrete developmental event. Igk transcription is induced by ERK- mediated suppression of the Id proteins, and derepression of E2A, while induction of Aiolos silences Ccnd3 leading to cell cycle exit. In addition to identifying a central pathway coordinating B cell development, these findings provide a framework for understanding the interplay between pre-BCR and IL-7 dependent signals. Specifically, we demonstrate that Igk transcription is controlled by competition between E2A and STAT 5 at the intronic Igk enhancer (Eki). In contrast, cyclin D3 protein is regulated by PI-3 kinase that, through a novel, lymphoid specific mechanism, controls the subnuclear location of cyclin D3 relative to the inhibitor p27kip1 and the CDK4/Rb effector complex. These observations evoke a model in which initial pre-B cell proliferation is mediated by the coordinated activation of STAT 5 and PI-3k. STAT 5 enhances Ccnd3 transcription and suppresses Igk recombination while PI-3k drives the formation of productive cyclin D3/CDK4/Rb complexes. Subsequent Igk recombination requires both escape from STAT 5 and activation of ERK by the pre-BCR. The central predictions of this model will be tested in the following Specific Aims: Aim 1. To determine how E2A and STAT 5 integrate to regulate Igk transcription. Aim 2. To determine how PI-3 kinase regulates cyclin D3 nuclear localization and assembly with CDK4, Rb and p27kip1. Aim 3: To determine, in vivo, why cyclin D3 makes a unique contribution to lymphocyte development. PUBLIC HEALTH RELEVANCE: The development of B cells in the bone marrow is determined by both cytokines present in the bone marrow and by expression of the pre-B cell antigen receptor (pre-BCR) on lymphocyte precursors destined to become B cells. In this grant application, we will determine how intracellular signals, delivered through the pre-BCR and cytokine receptors, integrate to direct complex developmental processes. These studies will lead to a greater understanding of both the generation of the immune system of those processes that lead to leukemia.