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Encrypted in the DNA lays most information needed for the development of an organism The transcription of this information into precise patterns of gene activity results in the development of different cell types organs and developmental structures Moreover transcriptional regulation enables an organism to respond to changing environmental conditions Essential for the regulation of transcription are DNA-binding transcription factors TFs TFs bind the DNA in a sequence-specific fashion Upon binding of a TF to its DNA binding site TFs typically activate or repress the transcription of nearby genes To better understand transcriptional regulation it is essential to study DNA binding specificity of TFs In the last decades technological advances allowed the development of high-throughput methods to study protein-DNA interactions Traditional in vitro methods study one or a few interactions while new high-throughput methods can determine TF specificity by measuring relative DNA-binding affinities against a large collection or even all possible binding sites Several high-throughput techniques to study TF-DNA interactions are discussed in Chapter 1 of this thesis These new technologies and methods resulted in a fast growing number of studies on DNA binding specificities of TFs expanding the knowledge about TF specificity A review on the current knowledge of TF DNA binding specificity is described in Chapter 1 One aspect that influences DNA binding of TFs are differences in ability to form protein-protein interactions The aim of this thesis was to study the role of protein-protein interactions in determining DNA binding specificity of a developmental regulatory MADS domain TF in Arabidopsis thaliana While the members of the MADS-box protein family have many diverse in vivo functions all members bind in vitro to a 10-bp motif called the CArG-box Moreover studies demonstrated that closely related MADS proteins are expressed in the same cells therefore encountering the same DNA accessibility and DNA methylation patterns but bind different in vivo targets Interestingly MADS domain proteins bind DNA obligatorily as homo- and heterodimers and the interactions between MADS domain proteins are highly protein specific Hence MADS domain proteins are a perfect model system to study the influence of intra-family protein interactions on DNA binding specificity To study the influence of protein-protein interactions on DNA-binding specificity this work focusses on one specific MADS domain protein FRUITFULL FUL FUL is expressed at two stages during flower development and in both stages FUL has highly diverse functions In Chapter 2 we demonstrate using RNA-seq that FUL regulates different sets of target genes in the two stages Moreover using ChIP-seq we show that FUL genomic DNA binding is partly tissue-specific These tissue-specifically bound and regulated genes are in line with the known dual functions of FUL during development Interestingly using protein complex immunoprecipitation for the two studied tissues stages we show that the interactions of FUL with other MADS domain proteins are also tissue-specific To determine whether the tissue-specific in vivo binding pattern are due to differences in DNA binding specificity of the FUL-MADS dimers we studied the DNA binding specificities of the different protein complexes using SELEX-seq The SELEX-seq results show that although all tested dimers preferably bind the canonical binding motif of MADS domain proteins different dimers have different preferences for nucleotides within and surrounding the canonical binding site Hence different MADS domain dimers have different in vitro DNA binding specificities By mapping the SELEX-seq affinities to the genome we were able to compare these results with in vivo tissue-specific ChIP-seq data This analysis revealed a strong correlation between tissue-specific dimer affinities and tissue-specific genomic binding sites of FUL Hence we show that the choice of MADS dimerization partner influences DNA binding specificity highlighting the role of intra-family protein interactions in defining DNA binding specificity To allow other researchers to determine genome-wide DNA binding of TFs Chapter 3 provides a step-by-step guide for ChIP-seq experiments and computational analysis The protocol is designed for wet-lab biologists to perform ChIP-seq experiments and analyse their own ChIP-seq data Using the genome-wide DNA binding patterns determined by ChIP-seq Chapter 4 and Chapter 5 take a more detailed look at some of the genes directly bound by FUL In Chapter 4 we demonstrate a connection between developmentally and environmentally regulated growth programs We studied a gene directly bound by FUL in pistil tissue SMALL AUXIN UPREGULATED RNA 10 SAUR10 SAUR10 expression is regulated by FUL in multiple tissues among others cauline leaves stems and branches The results show that the expression of SAUR10 at the abaxial side of branches is influenced by a combination of environmental and developmental regulated growth programs hormones light conditions and FUL binding This spatial regulation possibly affects the angle between the side branches and the main inflorescence stem Additionally we discuss several other FUL target genes involved in hormone pathways and light conditions Chapter 5 focusses on the putative direct targets of FUL in IM tissue Among the putative direct targets two genes involved in flavonoid synthesis were identified FLAVONOID SYNTHESE 1 FLS1 and UDP-GLUCOSYL TRANSFERASE 78D3 UGT78D3 Interestingly similar to the ful-7 mutant the fls1 mutant is late flowering Moreover expression data exposed an increased gene expression for both FLS1 and UGT78D3 in developing meristems and showed FLS1 expression to be influenced by light conditions We report the first link between the MADS domain protein FUL and flavonoid synthesis in Arabidopsis Moreover our results indicate a possible link between flavonoids and flowering time In Chapter 6 I discuss the findings of this thesis and make suggestions for further research Taken together the work in this thesis shows that intra-family protein interactions can influence DNA-binding specificity of a protein Thereby these protein-protein interactions can influence genome-wide binding patterns and as a result the function of a protein Moreover by studying several putative direct targets of FUL in more detail we demonstrated a connection between development and environment in growth-regulated programs Interestingly the FUL target SAUR10 is repressed by FUL in several tissues including cauline leaves inflorescence stems and branches However no influence of FUL on SAUR10 expression could be detected in the pistil So despite the binding of FUL to the promotor of SAUR10 in the pistil this binding does not result in gene regulation This finding reflects the complex relation between TF occupancy and gene regulation further research is needed to better understand this relation Moreover besides MADS domain protein interactions we found FUL to interact with several proteins of other families The role of these cross-family protein interactions in cooperative gene regulation is not fully understood and will be an important research topic in the coming years

MADS specificity : Unravelling the dual function of the MADS domain protein FRUITFULL

The baculovirus per os infectivity complex

Publication search with eiwit-eiwit interacties as keyword

MADS specificity : Unravelling the dual function of the MADS domain protein FRUITFULL

The baculovirus per os infectivity complex

Publication search
with eiwit-eiwit interacties as keyword