Structural Studies Of The WD40 Domain Of PRPF19 And The Chromo Barrel Domain Of TIP60

Epigenetics is the study of heritable phenotype changes(DNA methylation profiles,chromatin structural states,and gene expression profiles)in intergenerational transmissions that do not involve alterations of DNA sequence.Studies have shown that histone modifications and chromatin structure affect Pre-mRNA splicing,and Pre-mRNA splicing itself also affects chromatin structure.In the mammalian genome,95%of the multiple exons produce proteins with different function through different splicing methods,and these splicing methods are closely related to human diseases.Post-translational modifications(PTMs)of histones provide a fine-tuned mechanism for regulating chromatin structure and dynamics.PTMs can alter direct interactions between histones and DNA and serve as docking sites for protein effectors,or readers,of these PTMs.Binding of the readers recruits or stabilizes various components of the nuclear signaling machinery at specific genomic sites,mediating fundamental DNA-template processes,including gene transcription and DNA recombination,replication and repair.In this study,we focused on the structural and functional studies of two readers in epigenetics,namely the WD40 repeat domain of Pre-mRNA Processing factor 19(PRPF19)and chromo barrel(CB)domain of acetyltransferase TIP60.Pre-mRNA Processing factor 19(PRPF19)is an important component in human spliceosome machinery.It has been found that PRPF19 has multiple function in DNA recombination and has important function in the connection of DNA repair in the unhomologous terminal.PRPF19 contains a WD40 repeat domain at its C-terminus,which is also conserved in yeast.Here we determined the crystal structure of the C-terminal WD40 repeat domain of hPRPF19 by X-ray crystallography.Our structural analysis revealed some significantly different structure features between the human and yeast Prp19 WD40 repeat domain.However,there are also conserved clusters of residues at the bottom surface of the fourth and the fifth WD40 repeats.Tat-interactive protein 60(TIP60),also known as KAT5 or HTATIP,is a MYST(MOZ,YBF2,SAS2,and TIP60)family of acetyltransferase and is an important member of the evolutionarily very conserved NuA4 protein complex.It comprises an N-terminal chromo barrel domain(TIP60-CB)and a C-terminal histone acetyltransferase domain.Over the past decade,studies have confirmed that TIP60 can act as a transcriptional regulator in combination with nuclear receptors or transcription factors such as c-MYC,AICD/Fe65,NCoR,E2F to activate or inhibit the expression of downstream genes.On the other hand,a series of histones or non-histones can be acetylated to regulate its activity and stability,thereby regulating some important cellular function,such as DNA damage repair response,cell cycle progression,activation of cell cycle checkpoints,apoptosis,metabolism and autophagy,etc.Therefore,histone acetylation dysfunction can cause some related diseases.It was reported that TIP60-CB recognizes trimethylated lysine at site 9 of histone H3(H3K9me3),which triggers TIP60 to acetylate and activate ATM(ataxia telangiectasia mutated)kinase,thereby promoting the DSB repair pathway.According to another study,TIP60-CB binds to monomethylated lysine at site 4 of histone H3(H3K4me1),which stabilizes TIP60 recruitment to a subset of estrogen receptor alpha(ER?)target genes,and then facilitates regulation of the associated gene transcription.However,the molecular details of these interactions remain unclear.Based on the published reports of histone binding by the chromo barrel domain of TIP60,we aimed at characterizing the interaction between human TIP60-CB and histone peptides by quantitative binding assays and crystallographic analysis.In this study,we tried to analyze the interactions between TIP60-CB and histone peptides by quantitative fluorescence polarization(FP)and isothermal titration calorimetry(ITC),however we could not detect any obvious binding.We attempted to co-crystallize TIP60-CB with its reported partners and only solved the peptide unbound structures.Analysis of the TIP60-CB apo-structure and comparison with published structures of other chromo barrel domains reveal a putative peptide binding site that might be occluded by the basic side chain of a residue in a unique ? hairpin between the two N-terminal strands of the ?barrel,leading to the inability of TIP60-CB in histone binding.This study provides a structural basis for understanding the functional differences by comparing the similarities and differences between the WD40 of human PRPF19 and the WD40 domain of Prp19 conserved in yeast and lays a structural foundation for further exploration of its function in cellular processes.In addition,this subject also made a preliminary structural analysis of TIP60-CB itself and its interacting with histone peptides.Whereas the crystal structure of TIP60-CB indicates that the formation of an aromatic cage is possible,it also suggests that access to the cage may be occluded by a basic amino acid side chain within a unique ? hairpin.This observation alone does not rule out peptide binding,as such obstruction may be transient.All in all,these studies provide the structural basis for the future functional studies and drug design of PRPF19 WD40 repeat domain and TIP60-CB.