2012 Theses Doctoral
Structure-Function Analysis of Hox-cofactor Interactions during Drosophila melanogaster Embryonic Development.
Regulation of gene expression is critical to many aspects of life. From cell survival and proliferation to animal development and species propagation, improper gene regulation can have serious, often fatal, consequences. Therefore, understanding the processes that control gene expression can provide important biological insights. At the center of many of these regulatory processes are trans-acting proteins called transcription factors. Most transcription factors contain DNA-binding domains that recognize specific DNA sequences. These site-specific transcription factors target genes by recognizing binding sites in regulatory sequences called cis-regulatory modules (CRMs). However, many transcription factors recognize degenerate DNA-sequences that can be found frequently throughout the genome. Despite this potential for promiscuity, transcription factors control very specific in vivo functions. This "specificity paradox" is best understood in the context of one particular family of transcription factors: the Homeobox (Hox) proteins. Conserved in all bilaterians, Hox genes are best known for their roles in embryonic pattering and organogenesis. Characterized by a highly conserved DNA-binding domain called the homeodomain, all Hox proteins recognize similar `AT' rich sequences. One way Hox proteins achieve functional specificity is through cooperative DNA-binding with the cofactor Extradenticle (Exd) in invertebrates or Pbx in vertebrates. Using Drosophila melanogaster as a model system we conducted a structure-function analysis of three different Hox proteins, Sex combs reduced (Scr), Ultrabithorax(Ubx) and AbdominalA (AbdA) to understand how interactions with a shared cofactor can increase specificity.
To identify amino acid sequence motifs that contribute to Exd-dependent functions, we generated and tested a series of mutant Hox proteins for cooperative DNA-binding ability in vitro, and for their ability to regulate target genes in vivo. The results of these studies demonstrate that while Scr uses a single conserved motif, more posteriorly expressed Hox proteins Ubx and AbdA use multiple, sometimes unique motifs to regulate Exd-dependent functions. This discrepancy between the quantity and quality of motifs endows AbdA with the ability to outcompete Scr for DNA-binding and regulation of an Exd-dependent target. In addition, by testing the ability for AbdA mutants to carry out a variety of in vivo functions, we observed that the different modes of interaction with Exd affect functional specificity. However, in the case of Ubx, we find that despite the contribution of Exd-interaction motifs to cooperative complex formation in vitro, none of these motifs are required individually or in combination for in vivo functions. Together, these data suggest that one technique Hox proteins use to differentiate themselves when interacting with a shared cofactor is through the utilization of different interaction motifs. Furthermore, having multiple modes of interaction can expand and alter their functional specificity. However, as illustrated by Ubx, the functional interactions between Hox proteins and cofactors can be more complex and may not require cooperative DNA-binding. In conclusion, the characterization of Hox-cofactor interactions helps us better understand how transcription factors select their targets and regulate gene expression in a highly specific manner.
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