Organ-specific DNA-binding site selection and transcriptional regulation by floral homeotic transcription factors

• Applicant: Dr. Cezary Smaczniak
• Subject Area: Plant Cell and Developmental Biology
• Term: from 2018 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 408478570

Unraveling how transcription factors interact with specific DNA sequences is crucial to understand how they control gene expression and ultimately how they exert their biological functions. In plants, MADS-domain transcription factors are prominent master regulators of flower development, acting as so-called homeotic regulators. Their role is to specify four different types of floral organs - sepals, petals, stamens and carpels - originating from meristematic stem cells. Homeotic regulators interact with each other, thereby forming organ-specific dimeric and higher-order protein complexes. Each protein complex potentially possesses a different DNA-binding specificity, resulting from DNA-binding preferences of individual dimers, preferred spacing of binding sites, interactions with non-MADS cofactors as well as DNA structure. While we previously found using SELEX-seq that DNA-binding specificities of individual MADS dimers contribute to DNA-binding specificity of floral homeotic complexes, other major determinants of DNA-binding specificity are still poorly understood. This is however crucial to understand the cis-regulatory code underlying floral organ specification in plants.In this project, I propose to functionally characterize complex-specific DNA binding sites of homeotic regulators. We will analyze the impact of binding site spacing on DNA-binding specificity of higher-order complexes using DAP-seq, and identify protein domains involved in determining DNA-binding specificity. We will also investigate the impact of interactions with NonMADS co-factors on binding site selection and regulatory specificity (activation or repression of target genes). Here, we will study how co-repressor complexes are recruited to a subset of MADS transcription factor-bound genomic regions, and how different homeotic complexes affect this recruitment using a combination of (cofactor) ChIP-seq and genetic approaches. In our analysis, we will integrate novel results with available tissue-specific binding and chromatin accessibility data. Ultimately, we will functionally characterize DNA regulatory elements in the Arabidopsis genome responsible for floral organ specification directly downstream of the homeotic regulators. For this, we will employ the CRISPR-Cas9 methodology for targeted mutagenesis of MADS-domain transcription factor binding sites and investigate flower phenotypes of the mutant plants. In addition, we will perform detailed expression analyses of novel target genes that are predicted key targets for floral organ specification. In summary, this project will combine several state-of-the art technologies to understand the cis-regulatory code underlying specification of floral organs in plants, and to determine novel key organ-specific target genes of homeotic regulators. Therefore, this project offers a conceptual basis for determining the genomic basis of developmental programming of plant stem cells, using flower development as a model system.

• DFG Programme: Research Grants