Hox/Pbx and Brn binding sites mediate Pax3 expression in vitro and in vivo
Hox/Pbx and Brn binding sites mediate Pax3 expression in vitro and in vivo
复制标题
DOI：
10.1016/j.modgep.2004.04.006
复制
发表时间：
2004-10-01
影响因子：
1.2
通讯作者：
Heinaman, R
中科院分区：
文献类型：
--
作者：
Pruitt, SC;Bussman, A;Heinaman, R
Pax 3是一种同源异型结构域类转录因子，在脊椎动物胚胎发育过程中的背腹和内侧-外侧模式中起作用。其表达定位于发育中的神经管内的背侧域和发育中的体节内的侧域。此外，其表达的调节发生沿着喙尾轴。先前的研究[Development 124（1997）617]已经将Pax 3在神经管和神经嵴中表达所需的序列元件定位于1.6kbp启动子片段。在本研究中，四个离散的DNA元件内的1.6 kbp的启动子片段显示电泳迁移率变动分析（EMSA）显示序列特异性的相互作用与蛋白质存在于细胞核提取物从P19 EC细胞诱导表达Pax 3的处理与视黄酸（RA）。基于使用DNA亲和层析或候选方法的生化纯化，鉴定在这些元件中的每一个处相互作用的蛋白质。这些鉴定通过特异性抗体在EMSA中超移位DNA-蛋白质复合物的能力得到证实。四个DNA序列元件中的两个显示与神经特异性Pou结构域III类转录因子Brn 1和Brn 2相互作用。其余的网站包含任何共识的异源二聚体的Pbx和前一组的Hox家族成员，从旁系同源组1-5，或单体的Meis的结合元件，并显示与Pbx和Meis家族的成员相互作用。Brn 2加上HoxA 1的异位表达，而不是单独的任一因子，足以在P19 EC干细胞中诱导内源性Pax 3启动子的有效表达，在所述条件下，它们不会以其他方式表达Pax 3。最后，在转基因小鼠中，Pou结构域蛋白结合位点的任一个的突变导致整个神经管中的表达降低，而Pbx/Hox结合位点的突变导致前结构域中的表达丧失，其中来自旁系同源组1-5的Hox家族成员在前结构域中表达。这些观察结果表明，神经和前-后位置特异性转录因子的结合元件介导Pax 3表达的结构域。(C)2004 Elsevier B. V.保留所有权利。
Pax3 is a paired-homeodomain class transcription factor that serves a role in dorsal-ventral and medial-lateral patterning during vertebrate embryogenesis. Its expression is localized to dorsal domains within the developing neural tube and lateral domains within the developing somite. Additionally, modulation of its expression occurs along the rostral-caudal axis. Previous studies [Development 124 (1997) 617] have localized sequence elements required for expression of Pax3 in the neural tube and neural crest to a 1.6 kbp promoter fragment. In the present study, four discrete DNA elements within the 1.6 kbp promoter fragment are shown by electrophoretic mobility shift assays (EMSA) to exhibit sequence specific interactions with proteins present in nuclear extracts from P19 EC cells induced to express Pax3 by treatment with retinoic acid (RA). Proteins interacting at each of these elements are identified based on biochemical purification using DNA affinity chromatography or a candidate approach. These identifications were confirmed by the ability of specific antibodies to supershift DNA-protein complexes in EMSA. Two of the four DNA sequence elements are shown to interact with the neural specific Pou-domain class III transcription factors Brn1 and Brn2. The remaining sites contain either consensus binding elements for heterodimers of Pbx and an anterior set of Hox family members, from paralogous groups 1-5, or monomeric Meis and are shown to interact with members of the Pbx and Meis families. Ectopic expression of Brn2 plus HoxA1 but not either factor alone, is sufficient to induce efficient expression from the endogenous Pax3 promoter in P19 EC stem cells under conditions where they would not otherwise express Pax3. Finally, in transgenic mice, mutation of either of the Pou-domain protein binding sites results in reduced expression throughout the neural tube while mutation of the Pbx/Hox binding site results in loss of expression in the anterior domain in which Hox family members from paralogous groups 1-5 are expressed. These observations demonstrate that binding elements for both neural and anterior-posterior position specific transcription factors mediate domains of Pax3 expression. (C) 2004 Elsevier B.V. All rights reserved.