| Posttranslational modification(PTM)on histones is an essential epigenetic mechanism,which plays a great role in regulating gene replication,transcription,and DNA-damage repair.Histone PTMs are dynamically and strictly regulated by their modifying and erasing enzymes(called writer and eraser),which are responsible for the adding and removing of the modifying groups and introduces chemical changes on histone,so as to directly alter the DNA-histone interaction.At the same time,histone PTMs can be recognized by specific readers to translate the histone code into functional outputs.Novel modifications have constantly been identified due to the development of high-resolution mass spectrometry technology.Histone serotonylaiton is catalyzed by the tissue glutamine transferase TGM2,and appears on the Gln5 site of histone H3(H3Q5ser),which is the first reported endogenous monoaminylation on histones.H3Q5ser may crosstalk with the neighboring H3K4me3.H3K4me3Q5ser enhances the recruitment of the transcriptional initiation TAF complex and facilitates gene expression.But the reader and eraser of H3Q5ser mark have not been identified yet.The first part of the paper focuses on identifying the reader protein of H3Q5ser mark.By applying Label free mass spectrometry assay and combining with the findings by Farrelly et al.,the well-known WD-40 repeat protein WDR5 may possibly serve as an H3Q5ser reader.The full-length and truncated(22-334)WDR5 protein were purified in vitro and carried out pull-down and Isothermal Titration Calorimetry(ITC)assays with four types of H3 peptides(unmodified H3、H3K4me3、H3Q5ser、H3K4me3Q5ser),which showed that serotonylaiton on H3Q5 site enhanced the binding of WDR5 to H3 peptide whether in the presence or absence of K4me3.To precisely decipher the H3Q5ser recognition mechanism by WDR5,the crystal structures of WDR5 in complex with H3Q5ser or H3K4me3Q5ser peptide were solved both at 1.6 A resolution.The overall structure of WDR5 in both complex structures are similar,with an RMSD of 0.1 ?.The N-terminus of the H3 peptide extends deep into the narrow channel in the center of WDR5 and is stabilized by hydrogen bonds and van der Waals contacts.Based on the crystal structure,amino acid residues which were adjacent to the serotonyl group were screened out and several WDR5 mutants were generated.The interaction between WDR5 mutants and H3 peptides were detected,which suggested that N130 and Y131 involved in the serotonyl group recognition.Meanwhile,the complex structure of WDR5N130A with H3Q5ser peptide was also determined.The co-localization of H3Q5ser with WDR5 in SK-N-SH cells were detected and they were co-enriched in the promoter regions of some cancer-promoting genes and promoted gene transcription,thus promoted SK-N-SH cell proliferation.In addition,the ability of TGM2 to catalyze the monoaminylation of other histone glutamine sites were examined using histone peptides as substrates.In the second part,I successfully constructed the recombinant expression plasmids of human and Saccharomyces cerevisiae PAF1 complex(PAF1C).The five subunits of Saccharomyces cerevisiae PAF1C were expressed in insect Sf9 cell expression system and successfully purified the well-folded PAC1C protein and carried out negative staining electron microscopy. |
