Crystal Structure Of PHD Of Rice Chromatin Remodeling Factor CHR729

In the eukaryotic nucleus,genomic DNA is packaged into chromatin by wrapping around histone octamers.Thus the compact structure of chromatin masks the regulatory DNA sequences and inhibits specific sequences recognized by most of the transcription factors,thereby impacting on some biological processes,such as DNA replication,genetic transcription and DNA repair.To ensure the normal life activities,chromatin remodeling factors alter interactions between DNA and histone octamers by utilizing the energy from ATP hydrolysis,resulting in changes of nucleosomal DNA accessibility.Chromatin remodeling factors recognize and bind the post-translational modification of histones that target them to specific genomic loci.Rice is the main food crop in the world and becomes a model plant of monocot.The mechenism of rice chromatin remodeling factors remains unknow,especially in structural biology.CHR729 is a CHD3-type chromatin remodeling factor in rice,which contains two kinds of histone modification readers,namely the plant homeodomain(PHD)finger and chromodomain.Binding of the PHD of CHR729 to trimethylated H3K27 is first reported in the histone modifications recognized by PHD.In this study,we expressed and purified the PHD finger of CHR729,then obtained the crystal and solved its structure.Meanwhile,we tried to obtain the crystal of PHD in complex with H3K27me3 peptide.The main results are listed as follows:1.Gene cloning of the 78-123 and 67-138 fragments of CHR729-PHD into pET28b-sumo and pGEX-6p-1,respectively.2.Obtaining the PHD(78-123)and PHD(67-138)protein with high purity.3.Growing well-diffracted PHD(78-123)protein crystal.4.Structural determination of PHD(78-123).5.Quantitative characterization of PHD(67-138)interaction with H3K27me3 peptide by MST and ITC experiments.In this study,we also predicted the binding sites of CHR729-PHD interaction with H3K27me3,according to the sequence alignment,structure superimposition and the results of MST and ITC experiments.The putative binding sites need to be poved by mutational analysis for further exploration.Meanwhile,the structural determination of PHD lay a foundation for future work to obtain PHD and H3K27me3 complex crystal.