Structural Insight Into ASH1L PHD Finger Recognizing Methylated Histone H3K4 And Promoting Cell Growth In Prostate Cancer

Background:Histone chemical modifications,including methylation,acetylation,phosphorylation and ubiquitination,play an important role in the epigenetic regulation of gene expression and are closely related to virtually all biological processes including DNA repair,gene transcription,cell cycle,stress response,development,differentiation,aging and so on.Histone methylation is one of the most widely studied and the most complex form of histone modification.There are one,two,or three methyl groups that are added at the arginine(R)or lysine(K)residues in the N terminus of histone H3 or H4.The histone methylation can recruit different regulatory proteins,thus activate or repress gene transcription.The methylation modification at different histone sites also has different transcriptional regulatory functions.In general,the methylation of histone H3 lysine 4(H3K4)and lysine 36(H3K36)are used for gene transcription activation,whereas the methylation of H3K9 and H3K27 are associated with gene repression.Therefore,identification of the recognition profiles of different histone modification marks may help us to understand the underlying regulatory mechanisms of epigenetic modification on target genes.ASH1L(Absent,small,or homeotic disc1-like)is a member of the Trithorax-group(Trx G)protein.ASH1L consists of an AWS(associated with SET)domain,SET(Su(var)3-9,Enhancer-of-zeste,Trithorax)domain,cysteine-rich post-SET domain,bromodomain(BrD),plant homeodomain(PHD)zinc finger,and a bromo-adjacent homology(BAH)domain.Because the conserved SET domain harbors lysine methyltransferase activity,the ASH1L can promote gene expression by methylating histone H3K4and H3K36,and counteract gene silencing mediated by Polycomb group(Pc G)protein.In mammals,ASH1L plays important roles in the normal development and function of the nervous system,muscular dystrophy pathogenesis,and immune cell development.ASH1L is also an emerging oncogene in acute leukemia,thyroid carcinoma,prostate cancer,renal cell carcinoma,and Hepatoma.In terms of treatment,the first-in-class inhibitor of the ASH1L SET domain,AS-99,has shown remarkable anti-leukemic activity against MLL1 fusion leukemia,suggesting that ASH1L can be potentially targeted for the therapeutic cancer treatment.The PHD zinc finger is a structurally conserved module of 50-80 amino acid residues.The PHD fold generally consists of two antiparallelβ-folds and a C-terminalα-helix,which is stabilized by two zinc ions in a“Cysteine4-Histidine-Cysteine3(Cys4-His-Cys3)”mode.PHD zinc fingers can be found in many nuclear proteins including transcription factors,histone modifying enzymes,and ATP-dependent chromatin remodeling complexes,and play an important role in recognizing chromatin modifications and recruiting regulatory proteins to specific genes.A subset of PHD fingers have been shown to bind histone lysine residues of different modification states on the N terminus of histone H3,such as H3K4me0,H3K4me3,H3K9me3,H3K14ac,and have different recognition preferences.Therefore,different proteins can bind and anchor at different gene loci through the recognition function of PHD structure,and regulate histone modification,gene transcription or chromosome remodeling at these sites.However,it is still not completely understood how the ASH1L PHD finger binds to methyl modification on histone H3 and functions as a platform for ASH1L in biological processes.Prostate Cancer(PCa)is a malignant tumor occurring in the prostate epithelium.It is the second most common male malignancy and one of the leading causes of mortality in the world.Although there are many treatments for local prostate cancer,such as surgical resection and radiotherapy,up to 40%of the patients will eventually relapse,and the advanced prostate cancer with recurrence or metastasis will gradually become resistant to androgen deprivation therapy(ADT),and eventually progresses to incurable castration-resistant prostate cancer(CRPC).However,the specific molecular mechanism of CRPC progression is still unclear,further research is needed to investigate the development of prostate cancer,especially CRPC.Objective:To explore the role of PHD domain in ASH1L protein in recognizing the different methyl modifications of histone H3 at different lysine sites,solve solution structures of the PHD protein in complex with di-methylated histone H3 peptide using nuclear magnetic resonance(NMR)technology,and analyze the molecular mechanism of the peptide recognition,the function of PHD domain in ASH1L protein,and explore the role of ASH1L protein in the occurrence and development of PCa.Providing new therapeutic targets for prostate cancer treatment in the field of epigenetics.Methods:(1)In terms of protein binding affinity,the PHD domain of ASH1L protein was expressed and purified by a prokaryotic cell expression system.The peptide array experiment,Nuclear Magnetic Resonance-Heteronuclear Single Quantum Coherence(NMR-HSQC)experiment,and isothermal titration calorimetry(ITC)assay were used to detect the binding selectivity of ASH1L-PHD domain to different methylated modification on the N terminus of histone H3.The binding affinity constant(K_D)was measured by ITC and NMR HSQC titration experiments.(2)In terms of protein structure,high-resolution structure of the ASH1L-PHD finger in complex with the H3K4me2(1-15)peptide were solved by multi-dimensional NMR technology.The key amino acid sites around methylated K4 in the binding pocket of PHD domain were identified by amino acid site-directed mutagenesis technology and ITC.The similarities and differences between ASH1L-PHD and other methylated H3K4 recognizing PHD domains were analyzed and compared by superimposing.(3)In terms of biological function,the levels of ASH1L protein and overall H3K36me2 modification in different prostate cancer cell lines were compared by western blot.The effect of methyltransferase ASH1L on the overall H3K36me2 modification level in cells was analyzed.The overexpression plasmids with complete C-terminus of ASH1L protein(Flag-ASH1L-C)or the C-terminus with PHD deletion(Flag-ASH1L-C-ΔPHD)were constructed and used for the study of the role of PHD domain in the normal function of ASH1L.Small interfering RNA(si RNA)technology was used to knock down the m RNA level of ASH1L in cells,and RNA sequencing technology(RNA-seq)was used to analyze the downstream genes and signaling pathways regulated by ASH1L in prostate cancer cell line PC3.The molecular mechanism of ASH1L regulating cell cycle and apoptosis was verified by qRT-PCR and western blot.The effects of ASH1L on proliferation,apoptosis,and cell viability were verified at the cellular level by Flow cytometry,clone formation assay,and MTT assay.Results:(1)ASH1L-PHD finger recognizes three methylated states of H3K4(H3K4me1,H3K4me2,H3K4me3)with comparable affinities,but shows only marginal binding to the unmodified H3K4me0peptide.It also doesn’t bind both H3K9me2 and H3K36me2 peptides.(2)The protein structure of the ASH1L-PHD finger in complex with the H3K4me2(1-15)peptide in solution was solved.(3)ASH1L-PHD finger has a narrow binding groove,and the residues in the binding pocket such as Asp2573,Ile2575,Met2588,Trp2597 and Glu2585 are necessary for proper conformational folding of the binding pocket and the binding affinity for different methylation modifications.(4)The importance of the PHD domain in ASH1L’s function as a histone methyltransferase was demonstrated.Overexpression of the C-terminal core of ASH1L increased the overall H3K36me2 level.PHD deficiency can affect the normal function of ASH1L,leading to the down-regulation of H3K36me2level.(5)Compared with androgen-sensitive LNCa P cell lines,ASH1L has a higher expression level in CRPC cell lines PC3 and DU145.Knockdown of ASH1L modulated gene expression and cellular pathways involved in apoptosis and cell-cycle regulation,and consequently induced cell-cycle arrest,cell apoptosis and reduced colony forming abilities in PC3 and DU145 cells.Conclusions:This study identifies ASH1L PHD finger as the first native reader that interacts with mono-,di-and tri-methylated states of H3K4 peptides with comparable affinities,indicating that ASH1L-PHD non-selectively recognizes to all three methylation states of H3K4.Solved NMR structures picture the ASH1L PHD finger binding to di-methylated H3K4 peptide,and characterize the side chain of K4me2 embedded in the PHD binding pocket.The narrow binding groove and residue composition in the binding pocket of ASH1L-PHD result in the specific binding strength and selectivity with methylated K4.The recognition of methylated H3K4 by the PHD finger is the key to the function of ASH1L methyltransferase,and the PHD deficiency can reduce the level of H3K36me2 in cells.In addition,we found that ASH1L protein is overexpressed in castrate-resistant prostate cancer(CRPC)PC3 and DU145 cells in comparison to prostate cancer LNCa P cells.ASH1L can promote cell cycle by upregulating CCNA1 and CCND1expression,inhibit apoptosis by decreasing cleaved-caspase3 and cleaved-PARP,promote the growth and survival of cancer cells,and play a cancer promoting role in prostate cancer.