Evolutionary dynamics of DNA-binding sites and direct target genes of a floral master regulatory transcription factor [RNA-Seq]
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ABSTRACT:
OTHER RELATED OMICS DATASETS IN: PRJNA267853PRJNA267851PRJNA267854
ORGANISM(S): Arabidopsis thaliana Arabidopsis lyrata
PROVIDER: GSE63462 | GEO | 2015/10/11
SECONDARY ACCESSION(S): PRJNA267853
REPOSITORIES: GEO
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Project description:In this study, we compare the DNA binding specifify and affinity of SEPALLATA3 and AGAMOUS complexes The MADS transcription factors, SEPALLATA3 (SEP3) and AGAMOUS (AG), are required for floral organ identity and determinacy of the floral meristem in Arabidopsis. Dimerization is obligatory for their DNA binding, however SEP3 and SEP3-AG also form tetrameric complexes. The goal of this study is to understand how homo and hetero-dimerization and tetramerization of MADS TFs affect genome-wide DNA-binding patterns. Using a modified sequential DNA affinity purification sequencing protocol (seq-DAP-seq), we selectively purified SEP3 homomeric and SEP3-AG heteromeric complexes, including the dimeric SEP3 tet-AG complex and the tetrameric SEP3-AG complex, and determined their genome-wide binding.
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Project description:These analyses allowed the identification of more than 2000 genes regulated by the MADS TF, SEPALLATA3 (SEP3). Although the involvement of SEP3 in all flower organ development is well established, the single sep3 mutant shows no flower phenotype, due to redundancy with other SEPALLATA genes. The triple mutant, sep1sep2sep3, shows no floral organ development except sepals, while the sep1sep2 mutant showed characteristic WT flower with, from the outer to the inner whorl of the flower, 4 sepals, 4 petals, 6 stamens and one gynoecium. We performed RNA-seq analysis on sep1sep2, sep1sep2sep3 and on sep1sep2sep3 lines complemented with SEP3 to highlight genes involved in floral organ development and controlled by SEP3. For this purpose we compared gene expression between the triple and the double mutant, and between triple mutant lines complemented with SEP3 and the triple mutant. Genes were considered regulated by SEP3 when the log FC was between 1 and -1 and the FDR <0.05. Using these criteria, we obtained a list of >2000 genes regulated by SEP3.
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Project description:The molecular mechanisms by which floral homeotic genes act as major developmental switches to specify the identity of floral organs, are still largely unknown. Floral homeotic genes encode transcription factors of the MADS-box family, which are supposed to assemble in a combinatorial fashion into organ-specific multimeric protein complexes. Major mediators of protein interactions are MADS-domain proteins of the SEPALLATA subfamily, which play a crucial role in the development of all types of floral organs. In order to characterize the roles of the SEPALLATA3 transcription factor complexes at the molecular level, we analyzed genome-wide the direct targets of SEPALLATA3. We used chromatin immunoprecipitation followed by ultrahigh-throughput sequencing or hybridization to whole-genome tiling arrays to obtain genome-wide DNA-binding patterns of SEPALLATA3. The results demonstrate that SEPALLATA3 binds to thousands of sites in the genome. Most potential target sites that were strongly bound in wild-type inflorescences, are also bound in the floral homeotic agamous mutant, which displays only the perianth organs, sepals and petals. Characterization of the target genes shows that SEPALLATA3 integrates and modulates different growth-related and hormonal pathways in a combinatorial fashion with other MADS-box proteins and possibly with non-MADS transcription factors. In particular, the results suggest multiple links between SEPALLATA3 and auxin signaling pathways. Our gene expression analyses link the genomic binding site data with the phenotype of plants expressing a dominant repressor version of SEPALLATA3, suggesting that it modulates auxin response to facilitate floral organ outgrowth and morphogenesis. Furthermore, the binding of the SEPALLATA3 protein to cis-regulatory elements of other MADS-box genes and expression analyses reveal that this protein is a key component in the regulatory transcriptional network underlying the formation of floral organs. ChIP experiments were performed on Arabidopsis wildtype and agamous mutant inflorescences using an antibody raised against a C-terminal peptide of SEP3. As control, ChIP experiments were performed on the sep3 mutant.
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