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.
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. Keywords: ChIP-chip
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.
Project description:Development of eukaryotic organisms is controlled by transcription factors that trigger specific and global changes in gene expression programmes. In plants, MADS-domain transcription factors act as master regulators of developmental switches and organ specification. However, the mechanisms by which these factors dynamically regulate the expression of their target genes at different developmental stages are still poorly understood. Here, we characterize the dynamic relationship of chromatin accessibility, gene expression and DNA-binding of two MADS-domain proteins during Arabidopsis flower development. The developmental dynamics of DNA-binding of APETALA1 and SEPALLATA3 is largely independent of chromatin accessibility, and our findings suggest that AP1 acts as 'pioneer factor' that modulates chromatin accessibility, thereby facilitating access of other transcriptional regulators to their target genes. Our data provide a primer to the idea that cellular differentiation in plants can be associated to dynamic changes in chromatin accessibility, as consequence of the action of master transcription factors.
Project description:Development of eukaryotic organisms is controlled by transcription factors that trigger specific and global changes in gene expression programmes. In plants, MADS-domain transcription factors act as master regulators of developmental switches and organ specification. However, the mechanisms by which these factors dynamically regulate the expression of their target genes at different developmental stages are still poorly understood. Here, we characterize the dynamic relationship of chromatin accessibility, gene expression and DNA-binding of two MADS-domain proteins during Arabidopsis flower development. The developmental dynamics of DNA-binding of APETALA1 and SEPALLATA3 is largely independent of chromatin accessibility, and our findings suggest that AP1 acts as ‘pioneer factor’ that modulates chromatin accessibility, thereby facilitating access of other transcriptional regulators to their target genes. Our data provide a primer to the idea that cellular differentiation in plants can be associated to dynamic changes in chromatin accessibility, as consequence of the action of master transcription factors.
Project description:Development of eukaryotic organisms is controlled by transcription factors that trigger specific and global changes in gene expression programmes. In plants, MADS-domain transcription factors act as master regulators of developmental switches and organ specification. However, the mechanisms by which these factors dynamically regulate the expression of their target genes at different developmental stages are still poorly understood. Here, we characterize the dynamic relationship of chromatin accessibility, gene expression and DNA-binding of two MADS-domain proteins during Arabidopsis flower development. The developmental dynamics of DNA-binding of APETALA1 and SEPALLATA3 is largely independent of chromatin accessibility, and our findings suggest that AP1 acts as 'pioneer factor' that modulates chromatin accessibility, thereby facilitating access of other transcriptional regulators to their target genes. Our data provide a primer to the idea that cellular differentiation in plants can be associated to dynamic changes in chromatin accessibility, as consequence of the action of master transcription factors. We used the AP1-GR system to conduct chromatin immunoprecipitation experiments with SEP3-specific antibodies and GR atibodies followed by deep-sequencing (ChIP-Seq) in order to determine SEP3 and AP1 binding sites on a genome-wide scale. Samples were generated from tissue in which the AP1-GR protein was induced using a treatment of 1 uM DEX to the shoot apex. The material was collect 2, 4 and 8 days after treatment. As control, we performed ChIP experiments using pre-immune serum at the different time points. Experiments were done in two biological replicates for 4 days and 8 days time-points while one biological replicate was done for control samples and 2 days time-point. The GSE47981 includes expression data that are complementary to the data in the GSE46986 and GSE46894.