Zinc Finger Proteins and Functional Specificity of Hox Patterning

虽然果蝇Hox复合体的克隆已经过去了20多年，但科学家们仍然不确定它们如何控制动物的身体图案。HOX基因编码序列特异的DNA结合蛋白，通过控制下游基因或“靶”基因来指定身体模式。不同的HOX蛋白(或它们的组合)在不同的片段中表达，因此，每个片段表达一组特定的靶基因，这导致了片段特有的形态特征。然而，在体外，所有的Hox蛋白都与类似的、相对简单的DNA序列结合，核心共识是Taat。周围的碱基可以影响结合强度，但不同HOX蛋白的DNA结合特性似乎几乎没有特异性，或者更恰当地说，选择性。有证据表明在活体中也是如此，所以很难解决单个HOX蛋白在具有相似的、相当非特异性的DNA结合特性的同时具有如此特定的发育作用的问题。此前，已有遗传学证据表明，部分冗余的C2H2锌指蛋白断开连接(Disco)和Disco-r相关(Disco-r)分别是HOx蛋白在胚胎上颌和唇部发育过程中变形(DFD)和性梳减少(SCR)的辅助因素。作为特定的辅因子，编码的锌指蛋白建立了只有某些HOX蛋白才能发挥作用的结构域。然而，这些基因似乎还有另一个作用。它们建立了一般的颌骨(或口后)节段类型。由于还有其他锌指编码基因，如teashirt(TSH)具有类似的特性，这种锌指/HOX伙伴关系模型似乎控制着整个胚胎的发育。这里描述的工作将进一步评估该模型，并在发育机制的遗传学和基因组研究领域为本科生、研究生和博士后提供极好的培训机会。实验将：(1)测定锌指-HOX配对的生化性质。将检查DNA结合和蛋白质-蛋白质相互作用，并将确定锌指和HOX蛋白在DNA结合过程中是否存在协同作用。(2)有迹象表明，同源结构域的N-末端臂对于决定哪种HOX蛋白与哪种锌指蛋白(DISCO或TSH)的功能可能是重要的。这将通过改变这个结构域的氨基酸序列来测试，并确定这是否改变了与DISCO或TSH一起发挥作用的能力。这个问题涉及HOX的功能特异性。(3)最终目标是考察通过特定的HOX-锌指身份对发育的控制。Hox和Zincinger蛋白的异位表达将被用来产生在整个胚胎中以大约单一节段类型(上颌骨与唇部；颌骨与躯干)发育的胚胎。基因表达的差异将使用微阵列基因表达谱进行比较。Hox基因控制着所有后生动物的身体模式，因此了解这一过程对于了解动物身体模式是如何在整个动物进化过程中产生和变化至关重要的。此外，编码锌指转录因子的基因是动物基因组中含量最丰富的一类。它们与许多发育和疾病有关。这项研究为了解转录因子在基因表达过程中的相互作用提供了机会。此外，这将为不同层次的学生提供机会，体验依赖于许多不同学科的互动研究计划：遗传学、细胞生物学、生物化学和基因组学。

Though it has been more than twenty years since the cloning of the Drosophila Hox complexes, scientists are still uncertain how they control animal body patterning. hox genes encode sequence-specific DNA-binding proteins that specify body pattern by controlling downstream or 'target' genes. Different Hox proteins (or combinations of them) are expressed in different segments, and, therefore, each segment expresses a specific set of target genes, which leads to segment specific morphological features. However, in vitro all Hox proteins bind to similar, relatively simple DNA sequences with a core consensus of TAAT. Surrounding bases can influence binding strength, but there appears to be little specificity or, more appropriately, selectivity, in the DNA binding properties of different Hox proteins. There is evidence that this is true in vivo as well, so it is difficult to resolve how individual Hox proteins can have such specific developmental roles while having similar, rather nonspecific DNA binding properties.Previously, genetic evidence was obtained that the partially redundant C2H2 zinc finger proteins Disconnected (Disco) and Disco-related (Disco-r) are cofactors for the Hox proteins Deformed (Dfd) and Sex combs reduced (Scr) during development of the embryonic maxillary and labial gnathal segments, respectively. As specific cofactors, the encoded zinc finger proteins establish the domain in which only certain Hox proteins can function. However, these genes appear to have another role. They establish the general gnathal (or post-oral) segment type. Since there are other zinc finger-encoding genes, such as teashirt (tsh) that have similar properties, this model of zinc finger/Hox partnerships appears to govern development throughout the embryo.The work described here will evaluate this model further and provide excellent training opportunities for undergraduate, graduate and postdoctoral students in the fields of genetic and genomic studies of developmental mechanisms. The experiments will: (1) determine the biochemical properties of the zinc finger-Hox partnership. The DNA binding and protein-protein interactions will be examined and it will be determined if the zinc finger and Hox proteins have cooperative interactions during DNA binding. (2) There are indications that the N-terminal arm of the homeodomain may be important for determining which Hox protein functions with which zinc finger protein (Disco or Tsh). This will be tested by altering the amino acid sequence of this domain and determining whether or not this changes the ability to function with Disco or Tsh. This question addresses Hox functional specificity. (3) The final goal examines the control of development by specific Hox-zinc finger identities. Ectopic expression of Hox and zincfinger proteins will be used to generate embryos that develop with approximately a singlesegment type (maxillary vs labial; gnathal vs trunk) throughout the whole embryo. Thedifferences in gene expression will be compared using microarray gene expressionprofiling.Hox genes control body patterning in all metazoans, so understanding how this isaccomplished is paramount to understanding how animal body patterns have arisen andchanged throughout animal evolution. Further, genes encoding zinc finger transcriptionfactors are the most abundant class in animal genomes. They have been implicated inmany developmental and disease conditions. This study provides an opportunity to learnabout the interactive nature of transcription factors during gene expression. Further, thiswill provide an opportunity for students at many levels to experience an interactiveresearch program that relies on many different disciplines: genetics, cell biology,biochemistry and genomics.