Desenvolvimento e aplicação de métodos bioquímicos e bioinformáticos para buscar interações regulatórias: Tead4 como modelo

Abstract

Yeast two-hybrid method consists of a genetic trap that selects for prey cDNA products within a library that interact with a bait protein of interest. Here we report two protocols of liquid screening that further improve this valuable method and eliminate the laborious classic screening in solid medium. To evaluate the accuracy of the new protocol we used two Saccharomyces cerevisiae yeast strains, KGY37 and Y190, three different baits: m-RTel1, m-Trim2 or h-TRIM32 and their respective cDNA libraries: mouse testis, mouse brain and human skeletal muscle. These combinations had been previously screened with the classic method allowing for a direct comparison to the novel method. The new screen resulted in the selection of interactions for all the bait proteins used, the number of positive clones obtained in the assays is comparable to that of the solid medium and the liquid screening is a faster, more economic and more efficient approach. Currently, the generation of protein interaction data and gene regulation occurs in a large scale. The integration of these data and generation of pathways and networks contained in databases such as KEGG Pathway is essential for the comprehension of complex biological processes. We noticed the absence of a chart or pathway describing the largely studied preimplantation development stages; furthermore, not all genes involved in the process have entries in KEGG Orthology. In this work we sought to develop this regulatory pathway using text-mining tools such as Medline Ranker and PESCADOR to reveal biointeractions among the genes involved in this process. The genes present in the resulting pathway were also used as seed in software developed by our group called SeedServer to create clusters of homologous genes. These homologs allowed the determination of the last common ancestor for each gene and revealed that the preimplantation development pathway consists of an ancient fraction with the addition of modern elements. One of the transcription factors essential for this developmental stage is Tead4, participating in several regulations during preimplantation. The only known interaction partner for Tead4 is YAP, co-activator of the differentiation of trophectoderm lineage. In pursuit of new partners that interact with Tead4 we used this protein as bait in a two-hybrid screen with our previously reported protocol. The cDNA library used was mouse seven-day embryo and one of the recovered clones was characterized as Thioredoxin1, a protein with other reported associations to transcription factors.