Function of synthetic Hox transcription factors "in vivo" and the quantitative study of their molecular interactions with nuclear DNA at the single-molecule level in live cells

Over a century of research, invested in providing an answer to the fundamental question of how a single diploid cell constructs a whole organism, has placed Drosophila in the fore scene of developmental biology and has allowed us to merge the knowledge acquired from embryology with that of molecular biology and genetics. The assignment of differential morphological and functional characteristics to the different body parts during development is largely mastered by Homeobox (Hox) genes which are involved in the specification of segmental identity along the anterior-posterior (AP) axis of bilateral animals. Hox genes encode transcription factors that contain the Homeodomain (HD), a helix-turn-helix DNA-binding domain, the conservation of which spans long evolutionary distances as the one between yeast and humans. The biological function of Hox transcription factors has been studied extensively to date and much is known about their role in development at the genetic, molecular and structural level. The molecular interactions of various HDs with their DNA binding sites have been precisely dissected in vitro and numerous developmentally important Hox downstream genes (effector genes) have been identified, providing the link between segmental specification and pattern formation, originally in Drosophila and subsequently in other animals. However, the conservation of the HD in metazoans, albeit providing meaningful evidence for the interpretation of molecular evolutionary relationships between animal phyla, has been proven an obstacle in explaining the specificity of Hox target selection in vivo. Since all Hox orthologs and paralogs bind very similar regulatory sequences in vivo, it is challenging to explain how they simultaneously regulate differentially the formation of diverse body parts during development. The finding of a limited number of Hox cofactors has allowed us to take first steps towards solving this riddle, but a satisfactory explanation has not been provided yet. In the present study we have constructed synthetic Drosophila Hox genes which encode a small carboxy-terminal (C-terminal) portion [tyrosine-proline-tryptophan-methionine (YPWM) motif, HD and C-terminus] of the full-length protein and have examined their function as transcriptional regulators genetically and their interactions with nuclear DNA in live cells, using methods with single-molecule sensitivity, quantitative imaging and Fluorescence Correlation Spectroscopy (FCS). We have found that both Antennapedia (Antp) and Sex combs reduced (Scr) synthetic genes are functional in vivo and that the synthetic transcription factors find their binding sites primarily by multiple association/dissociation events, the rapidity of which is largely owed to electrostatic interactions. Synthetic Scr genes exhibited specific function in vivo by inducing homeotic transformations in the embryo (allowing the formation of an additional pair of salivary glands) and in the adult fly (transforming the adult antenna into a prothoracic tarsus). They repressed antennal-specific genes, ectopically activated leg-specific genes in the antennal imaginal disc and bound DNA specifically both in vitro and in vivo. Their transformation capacity was found to be enhanced as compared to the full-length protein, suggesting that the amino-terminal (N-terminal) portion of the protein contributes quantitative, rather than qualitative, effects in Scr-mediated transcription. Having proven their functionality in vivo, we used the synthetic Scr genes as tools for the study of HD-DNA interactions in live salivary gland cells, which express Scr normally during development, and thus represent a native Scr environment. By means of quantitative imaging, using Avalanche Photo-Diodes (APDs) with single-molecule sensitivity, we studied the molecular distribution and dynamics of synthetic transcription factors in polytene nuclei. We could distinguish wild type from mutant peptides directly by APD imaging, on the basis of their differential association with nuclear DNA, and study by FCS their movement and interactions with chromatin at the molecular level. This has been possible using the expression “leakage” of the heat shock protein 70 (hsp70)-minimal promoter of the Upstream Activating Sequences (UAS)-constructs and its responsiveness to heat-shock, features, which facilitated measurements at low, physiologically-relevant levels. Thus, we were able to titrate the concentration of the transcription factor in live nuclei and to construct transcription factor-DNA binding curves in order to analyze the underlying chemical interactions kinetically. Using a simple two-step model in which non-specific interactions are followed by specific interactions, we derived experimentally the in vivo macroscopic equilibrium dissociation constant of the HD-DNA complex for specific and non-specific interactions, as well as the corresponding DNA-binding constants by numerical simulations. Functional analysis was also performed using synthetic Antp genes. These were also able to confer gene-specific homeotic transformations during embryonic and larval development, such as head-to-trunk transformations reflected in the embryonic cuticle, repression and activation of markers in the embryo, as well as antenna-to-mesothoracic-tarsus transformations in the adult, mediated by repression of antennal-specific genes and ectopic activation of leg-specific genes in the antennal imaginal disc. At the same time we examined the role of the Antp YPWM motif in transcriptional regulation and found it important for both activation and repression. We also investigated the importance of linker size between the YPWM motif and the HD (naturally varying in Antp splicing variants) and showed that linkers of different size preferentially favor or limit the function of the protein either as a transcriptional repressor or as an activator. Taken together, our results show that synthetic Hox genes are functional in vivo. They exhibit gene-specific phenotypes, comparable to or enhanced over their full-length counterparts. This suggests that the long N-terminal portion of Hox transcription factors is not required for specificity, but for enhancing or limiting the strength of the transcriptional response. Our study also proposes synthetic genes as important tools for synthetic biology, since smaller but functional peptides might bear advantages for biomedically relevant applications, as compared to larger proteins. In addition, we establish an experimental platform for the quantitative study of transcription factor-DNA binding in vivo, devoid of overexpression and/or destructive observation of molecules and molecular interactions. We derive by FCS and quantitative imaging for the first time the in vivo HD-DNA binding constant and unravel the molecular interactions of the HD with chromatin in unprecedented detail. Our work might be used as a starting point for the dissection of transcription factor-DNA interactions in vivo which are relevant for development and disease.