Mouse models for pRB growth control via E2F/DP action

描述（由申请人提供）：人（RB）和小鼠（RB）视网膜母细胞瘤肿瘤抑制因子的失活促进肿瘤进展。在很大程度上，pRB通过抑制E2F/DP家族复合物来调节生长，其中一个子集在游离时刺激细胞周期进程。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的缺失并不会影响所有组织的发育，种系RB或RB突变会导致高度渗透的、组织特异性的肿瘤易感性。在体外，Gl Cyclin/ cdk介导的磷酸化程度决定了pRB抑制细胞周期进展的能力；然而，最近证明Gl细胞周期蛋白和Cdks对于大多数组织的正常发育是不可缺少的；提出了一个问题，即在体内正常调节pRB/E2F/DP通路的“线路”是什么？pRB（在pRB家族中）独特的肿瘤抑制功能可能源于其协调生长抑制与特定谱系承诺的能力。只有通过识别在体内运作的信号，我们才能理解Rb的缺失是如何破坏关键组织的发育并促进特定部位的肿瘤发生的。首先，利用我们的一系列新的野生型和突变Rb启动子转基因基因和一个新的Rb启动子敲入等位基因，我们将确定正常调节Rb神经元特异性表达的激活因子和抑制因子，并在体外和体内测试Rb表达失调的功能后果（Aim 11）。其次，我们将通过sirna介导的DP家族表达敲低和条件Dpi和/或Dp2敲除小鼠的建立，确定整个DP家族及其个体家族成员（Dpi和Dpi）在细胞周期控制和胚胎发育中的要求（Aim 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).