Department of Medical Genetics, School of Human Development, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
J Mol Biol. 2010 Sep 10;402(1):178-93. doi: 10.1016/j.jmb.2010.07.016. Epub 2010 Jul 17.
The transcription factor PAX3 is essential for myogenesis and neural crest development, and is one of several genes mutated in human Waardenburg syndrome. Analysis of disease-causing missense mutations in PAX3 has established the interdependence of its two DNA-binding domains, the paired domain (PD) and the homeodomain (HD), as well as defects in localization and mobility. Paradoxically, mutants that retained DNA binding activity exhibited the greatest defects in localization and mobility, regardless of the domain in which they reside. In the present study, structure-function analyses were used to determine the mechanistic basis of this effect. In the context of the isolated DNA-binding domains, HD mutants adopted an increase in mobility proportional to their loss in DNA binding, while PD mutants continued to display the inverse relationship observed in the full-length protein. At the structural level, this reflected an unexpected dependence on base-specific contacts in the PD, whereas HD mobility was more severely affected by loss of backbone contacts, as has been observed with other DNA-binding proteins. This requires that the HD switch to a base-specific mode in the full-length protein. Moreover, both domains underwent substantial reduction in mobility and altered localization when in a contiguous polypeptide with the endogenous linker segment. Notably, although the HD conferred localization to heterochromatin, this activity was masked when linked to the PD, despite the absence of determinants for subnuclear compartmentalization in the PD or linker. Last, the propensity for PAX3 heterochromatin localization was modulated by sequences at the amino and carboxy termini, supporting a model in which alternate conformations lead to unmasking of the HD. These data indicate that the PD and the HD functionally interact in vivo and behave as a single binding module whose mobility and localization are dependent on sequence-specific contacts.
转录因子 PAX3 对于肌发生和神经嵴发育是必不可少的，它是人类 Waardenburg 综合征中几个突变的基因之一。对 PAX3 致病错义突变的分析确立了其两个 DNA 结合域，即配对域（PD）和同源域（HD）的相互依赖性，以及定位和迁移缺陷。矛盾的是，尽管它们位于不同的结构域中，但保留 DNA 结合活性的突变体表现出最大的定位和迁移缺陷。在本研究中，结构功能分析用于确定这种效应的机制基础。在分离的 DNA 结合结构域的背景下，HD 突变体的迁移率增加与 DNA 结合活性的丧失成正比，而 PD 突变体则继续表现出全长蛋白中观察到的相反关系。在结构水平上，这反映了 PD 中碱基特异性接触的意外依赖性，而 HD 迁移率受骨干接触丧失的影响更为严重，这在其他 DNA 结合蛋白中也观察到。这要求 HD 在全长蛋白中切换到碱基特异性模式。此外，当与内源性连接片段的连续多肽时，两个结构域的迁移率和定位都发生了显著降低。值得注意的是，尽管 HD 将定位赋予异染色质，但当与 PD 连接时，这种活性被掩盖，尽管 PD 或连接中不存在亚核区室化的决定因素。最后，PAX3 异染色质定位的倾向由氨基和羧基末端的序列调节，支持了这样的模型，即交替构象导致 HD 去掩蔽。这些数据表明，PD 和 HD 在体内具有功能相互作用，并表现为单个结合模块，其迁移率和定位依赖于序列特异性接触。
