NuA4

Our objective is to decipher the structure and the function of the transcriptional coactivator NuA4 and its nucleosome remodeling partner SWR1 by combining molecular biology and biochemical approaches, structural biology and particularly electron cryo-microscopy (cryo-EM), and chemical biology. The regulation of gene expression at the transcriptional level is a vital mechanism that determines cell fate and influences all physiological and pathophysiological aspects of life. Transcription of protein coding genes is tightly controlled and requires the coordinated action of a large number of proteins that trigger the assembly of the Pre-Initiation Complex (PIC) at the promoter of the transcribed genes, leading to correct transcription initiation. Sequence-specific transcriptional activators and post-translational modifications of nucleosomal histones contribute to the recruitment of multi-subunit coactivator complexes acting as bridging factors between the transcriptional activators and the PIC. Co-activators modify the chromatin structure around the promoter region and coordinate PIC assembly, epigenetic chromatin modifications and activator-mediated cellular signaling events. Our goal is to understand the functional role, the structural organization and the mode of action of the yeast transcriptional co-activator NuA4, a multi protein complex containing 13 different subunits with for a total molecular weight of 1.0 MDa. NuA4 integrates epigenetic signaling by reading and writing post-translational histone modifications, interacts with transcriptional activators and incorporates specific histone variant. The Esa1 subunit of NuA4 is an acetyl transferase capable of modifying histone H4 and is essential for yeast growth. The large 430 kDa Tra1 subunit is involved in transcriptional activator binding thus making the link with the cellular signaling pathways. NuA4 also participates in the recruitment of the histone variant H2A.Z, a chromatin marker that bookmarks active gene promoters. To perform this function NuA4 interacts with the chromatin remodeler SWR1. In humans, the homologous NuA4 and SWR1 are physically associated to form the TIP60/p400 complex, which encompasses both histone acetyltransferase (TIP60) and histone exchange (p400) activities. Despite their key biological importance, the structural organization of the full NuA4 and SWR1 complexes is poorly understood and we aim at solving their high-resolution structure by cryo-EM. The mechanism by which these factors are recruited to gene promoters is currently unknown and we will form supramolecular complexes between NuA4 and, both transcriptional activators and nucleosomes to understand the network of interactions that target NuA4 to active genes. With the help of chemists we will measure binding constants of NuA4 with small molecule inhibitors, histone tails and activation domains in order to quantify the interaction network of the NuA4 complex. To stabilize the conformation of these molecular machineries, a prerequisite to achieve near atomic resolution, we will synthesize bi-functional chemical reagents in order to establish connections between flexible protein domains. Finally, even though the histone H2A.Z exchange activity is central for regulated gene expression and for DNA repair, the association of NuA4 with the chromatin remodeler SWR1 has not been studied so far at the structural level. We will analyze the structure of the NuA4/SWR1 complex by cryo-EM as well as structural intermediates of the exchange reaction. We will also analyze the structure and the function of the homologous human TIP60/p400 complexes in order to better understand the H2A.Z exchange mechanism.