Zinc Finger Proteins and Functional Specificity of Hox Patterning

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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.