Evolution and DNA-binding specificity of the SIX class of transcription factors
Author
Rivera Barreto, Anthony R.
Advisor
Rodríguez Martínez, José A.Type
DissertationDegree Level
Ph.D.Date
2022-12-21Metadata
Show full item recordAbstract
Transcription factors (TF) are critical for development and cellular processes and are found in all organisms. How their DNA-binding specificity changes through time has yet to be fully understood. TF DNA-binding specificity is determined by how their DNA-binding domain (DBD) interacts with DNA. TFs are identified by the sequence homology shared with described DBDs, which allows them to be classified into families. It is accepted that similar DBDs have the same DNA-binding specificity and bind to the same sequences. However, changes in a TF can lead to changes in its DNA recognition. TFs members of the sine oculis homeobox (SIX) homeodomain family are found from sponges to humans and are considered atypical members of the homeodomain (HD) family. They regulate numerous processes and phenotypic features, from eye development in flies and humans to red color patterning in Heliconius butterflies wings to human brain development. How evolutionary related TFs diversify has yet to be fully understood, especially diversification of their DNA-binding specificity. To understand the evolutionary history of this family, we performed phylogenetic inference that placed the first SIX within Porifera and the presence of the three canonical SIX (sine oculis, optix, and six4) in Cnidaria. In addition, we observe the presence of two major groups that show that optix and six4 are more evolutionary related. To determine changes in DNA-binding specificity, we performed in vitro Systematic Evolution of Ligands by Exponential Enrichment (SELEX-seq) using full length SIX TF proteins from Drosophila melanogaster, Heliconius erato, and Homo sapiens. Our data shows the majority of SIX TFs bind to the canonical binding motif (5' -TGATAC-3' ), except for six4 members, which seem to prefer (5' -TGACAC-3' ).
Interestingly, the way they bind to these motifs differs. Both sine oculis and six4 homologs require a 5' -GA dinucleotide flanking the core motif on the 5' -end. In comparison, optix related members prefer a shorter flaking region and less dependence on 5' -GA. This is interesting since optix is more evolutionarily related to six4 than to sine oculis. We also found that Heliconius erato optix can bind DNA both as a monomer and as a homodimer with a preferred spacing of 2-bp between binding sites. Using the determined DNA-binding specificity of optix, we were able to predict optix binding to cis-regulatory elements (CRE) active during wing development. optix was capable to bind to all the predicted sites, including to its own promoter. Validation of optix binding to these CREs allows to expand the search of optix gene targets and contribute to our understanding of the mechanism of wing development and red color patterns in Heliconius butterflies.
Interestingly, the way they bind to these motifs differs. Both sine oculis and six4 homologs require a 5' -GA dinucleotide flanking the core motif on the 5' -end. In comparison, optix related members prefer a shorter flaking region and less dependence on 5' -GA. This is interesting since optix is more evolutionarily related to six4 than to sine oculis. We also found that Heliconius erato optix can bind DNA both as a monomer and as a homodimer with a preferred spacing of 2-bp between binding sites. Using the determined DNA-binding specificity of optix, we were able to predict optix binding to cis-regulatory elements (CRE) active during wing development. optix was capable to bind to all the predicted sites, including to its own promoter. Validation of optix binding to these CREs allows to expand the search of optix gene targets and contribute to our understanding of the mechanism of wing development and red color patterns in Heliconius butterflies.