Structural Analysis Of Budding Yeast Eaf3 Protein-related Complexes

Eaf3 is a chromatin-binding protein involved in recruiting various chromatinassociated complexes that regulate gene transcription.Eaf3 contains an N-terminal chromo-barrel domain(CBD)and a C-terminal conserved MRG domain.In Saccharomyces cerevisiae,Eaf3 is shared between the two functionally opposite Rpd3S and NuA4 complexes.The Rpd3S complex couples with Eaf3 and the Rco1 subunit to recognize and bind nucleosomes in the wake of the RNA polymerase II,which is recruited to the coding region to suppress cryptic transcription initiation and maintain chromatin structure integrity.However,the molecular mechanism of how Rcol recognizes the N-terminus of histone H3 is still unknown.In the context of NuA4,Eaf3 also binds Eaf5 and Eaf7 to form Eaf3/5/7 submodule in the NuA4 complex.The submodule is conserved in eukaryotes and is named TINTIN.Because TINTIN is prone to dissociation from NuA4 complexes,the reported cryo-electron microscopy structures of NuA4 complexes lack this functional module.It is still unclear how Eaf3 regulates chromatin interactions in the context of TINTIN and how this regulatory mode different from the observed Eaf3 in Rpd3S.We focused on the structure and function of chromatin binding protein Eaf3 in histone deacetylase complex Rpd3S and histone acetyl transferase complex NuA4.Two structures of the Eaf3 MRG domain were obtained,and its structure and function were verified in vivo and in vitro.The PHD1 domain of Rcol was first identified to recognize the unmodified N-terminal of histone H3,as well as the direct interaction between the MRG domain of Eaf3 and the SID domain of Rco1.Subsequently,the crystal structure of the ternary complex of Eaf3MRG-RcolPHD1-SID complex and histone H3 peptide was obtained through structural biology techniques.In the Eaf3MRG-RcolPHD1-SID-H3 ternary complex structure,only the first six amino acids of the H3 peptide are visible and bound to the surface of RcolPHD1.RcolPHD1 specifically recognizes H3 unmethylated Lys4(H3K4)through the negative charge cavity formed by its conserved aspartic acid and adjacent hydrophilic amino acids.Rco1PHD1 also forms multiple hydrogen bond interactions with the second arginine(H3 R2)of H3,which plays a maj or role in substrate recognition.In addition,in vitro biochemistry experiments and in vivo cryptic reporter assays with mutants impairing Eaf3-Rco1 interaction further validated the interface and demonstrated its critical role in Rpd3S-mediated gene regulation.In the context of Rpd3S or TINTIN,Eaf3 is assembled to the complex through its conserved MRG domain.According to sequences alignment,both Eaf7 and Rcol contain the Phe-x-Leu-Pro(FxLP)motif,which is contained in all MRG-binding proteins identified in the higher eukaryotes.The structural analysis suggests that Eaf7 and Rco1 interact with Eaf3 in a mutually exclusive manner by binding to the same surface on Eaf3MRG,and the FxLP motif is crucial for binding to Eaf3MRG.Together,we obtained the crystal structure of the ternary complex of Eaf3MRGRct1PHD1-SID complex and histone H3 peptide,revealing the molecular mechanism by which the PHD1 domain of Rcol recognizes histone H3,and elucidating the biological ftunction and mechanism of action of Rco1 at the molecular level.In addition,we also preliminarily explored the structure and function of TINTIN composite,which complements our understanding of how Eaf3 assembles with other subunits in NuA4.