During flower development, MADS-proteins are known to determine the identity of the floral organs (the ABC-model of flower development). TCPs on the other hand, determine organ growth by regulating cell differentiation and proliferation. Our hypothesis is that these two types of transcription factors work together to shape the organs of a flower.
Using proteomics approaches (Mass Spectrometry) and Yeast-2-Hybrid assays, we know that MADS and TCP proteins can interact with each other. This is the first evidence to support our hypothesis that they work together. As transcription factors they should control the expression of other genes. How these MADS-TCP complexes do that is still a mystery. To answer the research question whether the complex formed between a MADS and a TCP could regulate the exact size of a floral organ, we will perform the following experiments:
- Are they able to bind DNA? To study the binding of proteins to DNA we will design EMSA experiments. An EMSA (Electrophoretic Mobility Shift Assay) can determine if a protein or mixture of proteins is capable of binding to a certain DNA sequence, and can indicate if more than one protein molecule is involved in the binding complex.
- To which DNA sequence do they bind? Using EMSA assays and different DNA sequences for binding we hope to get a clue about the affinity of binding.
- The sequence to which they bind gives an opportunity to see in the Arabidopsis genome where these sequences occur, i.e. which genes they might regulate as a complex. Using bioinformatics tools we will perform a genome-wide analysis. The genes that are regulated by the MADS-TCP complex can on their turn tell us more about the exact function of this complex.
- Confirm the DNA interaction in vivo. For this we will isolate chromatin that still contains the transcription factors bound to the DNA. This is called Chromatin Immunoprecipitation (ChIP).
- Molecular cloning, sequencing
- Performing transcription/translation techniques for in vitro protein production
- Electrophoretic Mobility Shift Assay (EMSA)
- Bioinformatics tools
- Chromatin Immunoprecipitation (ChIP)