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Protein-DNA interactions of oxidative-stress transcription factors OxyR1 and OxyR2 in Aliivibrio fischeri
(2022-05-11)
Transcription factors (TFs) are regulatory proteins that bind tightly to specific DNA sequences 15-20 base pairs long. Being sequence-specific DNA-binding proteins, TFs bear the key to the cellular state and control how organisms respond to different environmental stresses. OxyR, a LysR-type transcription factor, binds strongly to H<sub>2</sub>O<sub>2</sub> and activates a set of genes whose main purpose is to protect bacteria against oxidative stress. The genome of Aliivibrio fischeri bacteria codes for two different OxyR proteins, OxyR1 and OxyR2. However, the collection of target genes of OxyR1 and OxyR2 in A. fischeri remains to be determined. This study aims to clone, overexpress, and purify OxyR1 and OxyR2 TFs to determine their DNA binding functionality throughout the assessment of their intrinsic DNA-binding preferences. Purified OxyR1 and OxyR2 TFs were used to determine their DNA binding specificity using Systematic Evolution of Ligands by Exponential Enrichment (SELEX-seq)....
Protein-DNA interactomes of NKX2-5 and TBX5 mutations associated to Congenital Heart Defects
(2022-12-21)
Congenital Heart Diseases (CHD) are the most common disease found in neonates, with over 100,000 new reported cases each year. CHDs are characterized by malformations in the heart’s chambers, walls, and great vessels, ...
Evolution and DNA-binding specificity of the SIX class of transcription factors
(2022-12-21)
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' ).<br />
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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....