Mouse models for pRB growth control via E2F/DP action

描述(由申请人提供)：在人类(RB)和小鼠(RB)中，视网膜母细胞瘤肿瘤抑制因子的失活促进了肿瘤的进展。在很大程度上，pRB通过抑制E2F/DP家族复合体来调节生长，E2F/DP家族复合体中的一部分在游离时刺激细胞周期进展。相反，pRB通过调节负责特定分化程序的转录因子(例如，MyoD、C/EBP、CBFA1的激活)来刺激分化。然而，pRb的缺失并不会损害所有组织的发育，而胚系Rb或Rb突变会导致高渗透性、组织特异性的肿瘤易感性。在体外，Gl Cyclin/CDK介导的磷酸化程度控制着PRB抑制细胞周期进程的能力；然而，最近证明Gl Cyclins和CDK对大多数组织的正常发育是可有可无的；提出了一个问题：在体内，什么是正常调节PRB/E2F/DP通路的“连接”。PRb独特的肿瘤抑制功能(在pRb家族中)可能源于其协调生长抑制和特定谱系承诺的能力。只有通过确定在体内工作的信号，我们才能理解Rb的缺失如何扰乱关键组织的发育，并促进特定部位的肿瘤发生。首先，利用我们新的一系列野生型和突变型Rb启动子转基因基因和一个新的Rb启动子敲入等位基因，我们将识别正常调控Rb神经元特异性表达的激活子和抑制子，并在体外和体内测试解除Rb表达调控的功能后果(目标11)。其次，我们将通过siRNA介导的DP家族表达下调和条件DPI和/或DP2基因敲除小鼠的建立，确定整个DP家族及其单个家族成员(DPI和DPI)在细胞周期控制和胚胎发育中的要求(目标2)。

DESCRIPTION (provided by applicant): Inactivation of the retinoblastoma tumor suppressor in humans (RB) and in mice (Rb) facilitates neoplastic progression. To a large part, pRB regulates growth by repressing E2F/DP famil) complexes, a subset of which when free, stimulates cell cycle progression. Instead, pRB stimulates differentiation by regulating transcription factors responsible for specific differentiation programs (e.g., activation of MyoD, C/EBP, CBFA1). Yet, loss of pRB does not compromise the development of all tissues, and germ-line RB or Rb mutations lead to highly penetrant, tissue specific tumor predisposition. In vitro, the extent of Gl Cyclin/Cdk-mediated phosphorylation governs the ability of pRB to restrain cell cycle progression; however, the recently demonstrated dispensability of Gl Cyclins and Cdks for the normal development of most tissues; raises the question of what is the "wiring" that normally regulates the pRB/E2F/DP pathway in vivo. It is likely that the unique tumor suppressive function of pRB (among pRB family numbers) stems from its ability to coordinate growth inhibition with specific lineage commitment. Only by identifying the signals operating in vivo can we understand how loss of Rb disrupts the development of key tissues and facilitates tumorigenesis at specific sites. First, using our novel series of wildtype and mutant Rb promoter transgenics and a new Rb promoter knock-in allele, we will identify the activators and repressors that normally regulate the neuronal-specific expression of Rb and test the functional consequences of deregulating Rb expression in vitro and vivo (Aim 11. Second, we will define a requirement for the entire DP family and its individual family members (Dpi and Dpi) in cell cycle control and embryonic development through siRNA-mediated knockdown of DP family expression and the establishment of conditional Dpi and/or Dp2 knockout mice (Aim 2).