C21, a Transcriptional Regulator in Hematopoiesis

描述（由申请人提供）：C21基因编码一个在转录调控中起作用的蛋白质家族。小鼠造血细胞中C21的过度表达会改变骨髓发育，这表明该家族成员参与调节干细胞分化。在3T3成纤维细胞中过表达C21增加其对凋亡刺激的抗性。C21与一类核共阻遏物形成复合物，其中最著名的哺乳动物形式是N-CoR（核受体共阻遏物）和SMRT（类视黄醇和甲状腺受体的沉默介质）。C21与共阻遏物结合，干扰泛素介导的共阻遏物的蛋白水解，导致共阻遏物浓度升高。该家族的成员在胎儿造血组织以及许多造血细胞系中都有高水平的表达。像许多WD40蛋白一样，C21家族成员似乎作为适配器或桥梁，促进不直接相互作用的蛋白质的相互作用。这一建议涉及四个目标。目的1。鉴定与C21相互作用的蛋白质。C21家族蛋白作为接头，将必须相互作用但彼此缺乏内在亲和力的分子聚集在一起。相互作用分子的鉴定对于理解C21家族如何调节转录至关重要。目标2。确定C21如何改变转录调控。目标3。分析C21与阻遏物复合物相互作用的后果，确定这种相互作用所调节的途径。微芯片表达阵列将用从有条件过表达C21的成纤维细胞、造血细胞系和胚胎干（ES）细胞中获得的cdna进行探测，以确定响应C21表达改变的途径。目标4。确定体内相互作用的功能后果。研究C21 cDNA过表达和靶向缺失在转基因小鼠和培养中的作用。我们将利用小鼠胚胎干细胞（ESC）分析过表达C21 cDNA对早期造血发育的影响，并通过研究维甲酸诱导的HL60和U937骨髓白血病细胞分化来探讨对髓细胞发育的影响。

DESCRIPTION (provided by applicant): The C21 gene encodes a family of proteins that play a role in transcriptional regulation. Over-expression of C21 in mouse hematopoietic cells alters myeloid development and suggest that members of this family are involved in regulating stem cell differentiation. Over-expressing C21 in 3T3 fibroblasts increases their resistance to apoptotic stimuli. C21 forms a complex with the class of nuclear co repressors of which the best known mammalian forms are N-CoR (nuclear receptor co-repressor) and SMRT (silencing mediator of retinoid and thyroid receptor). C21 binds to the co-repressors and appears to interfere with the ubiquitin-mediated proteolysis of the co-repressors, causing an elevation of the co-repressor concentration. Members of the family are expressed at high levels in fetal hematopoietic tissues as well as in many hematopoietic cell lines. Like many WD40 proteins, C21 family members appear to act by serving as an adaptor or bridge, facilitating the interaction of proteins that do not interact directly. Four Aims are addressed in this proposal. Aim 1. To identify the proteins that interact with C21. C21 family proteins act as adaptors, bringing together molecules that must interact functionally but that lack intrinsic affinity for each other. The identification of the interacting molecules is critical for any understanding of how C21 family to regulate transcription. Aim 2. To determine how C21 alters transcriptional regulation. Aim 3. To analyze the consequences of the interaction of C21 with the repressor complex by identifying the pathways regulated by this interaction. Microchip expression arrays will be probed with cDNAs obtained from fibroblasts, hematopoietic cell lines and embryonic stem (ES) cells that conditionally over-express C21 to identify the pathways that respond to alterations in C21 expression. Aim 4. To identify the functional consequences of the interaction in vivo. The effects of over-expression and targeted deletion of C21 cDNA in transgenic mice and in culture will be studied. Mouse embryonic stem cells (ESC) will be used to analyze the effects of over-expression of C21 cDNA on early hematopoietic development and the effects on myeloid development will be examined by studying the retinoic acid-induced differentiation of HL60 and U937 myeloid leukemia cells.