Fig 1: PRMT7 monomethylation of HSP70 depends on the open (ATP-bound) form of HSP70.a–c HSP70 structures in closed and open confirmations reveal differential accessibility of the conserved R469-containing sequence (HSPA8) monomethylated by PRMT7. Structures are color coded for domains (orange—ATP binding, blue—substrate binding, and green—lid domains). The HSP70 substrate-binding domain loop containing PRMT7 methylated arginine is colored red. a, b Closely related homolog HSPA5 structures (65% overall sequence identity to HSPA8) were analyzed to investigate the position of the arginine methylation site in the different conformations. In the ADP-bound state, the lid of the substrate-binding domain is closed (PDB 5E85), limiting accessibility of the R492 (analogous to R469 in HSPA8) residue for methylation by PRMT7. In the ATP-bound form (PDB 5E84), the arginine residue is accessible therefore permitting access by the PRMT7 enzyme. c The structure of the more closely HSPA8 related HSPA1A (86% overall sequence identity and 82% sequence identity for aa. 386–646 in the substrate-binding domain, PDB 4PO2) in the closed conformation in which R469 is occluded by the lid subdomain. d–f Kinetic analysis of HSPA8 methylation by PRMT7 in vitro. Kinetic parameters were determined for HSPA8 methylation in the presence and absence of ATP. PRMT7 had no activity in the absence of ATP. d Kinetic analysis at fixed 10 µM HSPA8 (SAM Km = 1.6 ± 0.1 µM). e Kinetic analysis at fixed 20 µM of SAM (HSPA8 Km = 10.6 ± 0.1 µM and kcat of 2.2 ± 0.1 h-1). f HSPA8–R469K mutant is not methylated by PRMT7 in vitro. The results are mean ± SEM of three technical replicates. Source data are provided as a Source Data file.
Fig 2: SGC8158 is a potent and selective PRMT7 inhibitor in vitro.a Structures of HTS hit compound SGC0911, potent compound SGC8172, active component of the chemical probe SGC8158 and its negative control SGC8158N. b SGC8158 inhibits PRMT7 in vitro with IC50 of <2.5 nM, whereas negative control compound SGC8158N has IC50 of 14 ± 2 µM, (n = 3 biological replicates, mean ± SEM). c SGC8158 is selective against a panel of 35 proteins, DNA, and RNA methyltransferases. IC50 values are represented by colored circles indicated top left of the panel. d Crystal structure of MmPRMT7 in complex with SGC8158. MmPRMT7 is shown in cartoon representation in cyan, hydrophobic pocket residues are shown in cyan sticks, and SGC8158 is in orange. The THW motif loop region is highlighted in red with dashed lines representing the unmodeled H313 and W314 residues. e For comparison, the crystal structure of MmPRMT7_SAH (PDB ID: 4C4A) is shown in cartoon representation in yellow, hydrophobic pocket residues are shown in yellow sticks, and SAH is in pink. The THW motif loop region is highlighted in green. f Comparison of the THW motif loop region of MmPRMT7_SGC8158 (in cyan) with that of PRMT5 (PDB ID: 5GQB) (in magenta), and PRMT1, 2, 3, 4, 6, and 8 (in gray) (PDB IDs: 1OR8, 5FUL, 2FYT, 2V74, 4Y30, and 5DST, respectively). SGC8158 is shown in orange sticks and SAH in gray sticks. Source data are provided as a Source Data file.
Fig 3: A candidate enhancer variant rs73613962 is screened and validated to be significantly associated with HCC risk.a Flowchart shows the candidate single-nucleotide polymorphisms (SNPs) in enhancers selected from the discovery stage and validated in two stages of replication. HRC Haplotype Reference Consortium, MAF minor allele frequency, HWE Hardy-Weinberg equilibrium, eQTL expression quantitative trait loci, TF transcription factor. b Regional association plot shows the association results (-log10 P) of all the SNPs in the region 100 kb upstream and 100 kb downstream of PRMT7 rs73613962 in Discovery stage (n = 1161 cases, n = 1353 controls). The association of each SNP with HCC risk was evaluated through logistic regression under an additive model adjusting for gender and age. As the index SNP, rs73613962 is shown in purple, and the r2 values of the remaining SNPs are indicated by color. The genes within the region are annotated and shown as arrows. c The meta forest plot shows the association results of rs73613962 in the five independent populations of the discovery stage, replication stage 1, and replication stages 2a, 2b, and 2c. The association of rs73613962 with HCC risk in each population was calculated through logistic regression under an additive model adjusting for gender and age. The MAFs in cases and controls are shown for each population. The ORs and 95% CIs were calculated by considering the major allele as a reference. The center of each square and the horizontal line show the OR and the corresponding 95% CI, respectively. The pooled OR was obtained using the fixed-effects model and is represented by a hollow diamond, where its center indicates the OR and its ends correspond to the 95% CI.
Fig 4: The enhancer variant rs73613962 modulates PRMT7 expression by regulating enhancer activity.a, b The association between the genotypes of rs73613962 and PRMT7 expression through linear regression analysis in GTEx liver tissues (a, the center white line and the black box in the plot indicate the median and the first to third quartile, respectively) and TCGA LIHC (b, the center white dot, the black limit, and the whisker represent the median, the first to third quartile, and the 95% confidence interval, respectively). c Gene editing of the region surrounding rs73613962 by CRISPR-Cas9. The above panel shows the basic principle of the Cas9 assay. The Cas9 protein binds to the neighboring sequence of the rs73613962 site guided by small guide RNAs (sgRNAs) targeting the upstream and downstream of this locus to induce the double-stranded breaks of these regions in the PRMT7 gene. qPCR detection of relative PRMT7 expression upon the controls without any mutation in these regions (Control_1, 2), and the mutations being edited in these regions (Mutation_1, 2, 3) are shown in the bottom panel (P = 0.0001, P = 0.0003, and P = 0.0231 in Mutation_1, 2, or 3 compared to Control_1, respectively). d, g Illustration of the dCas9 assay. The above diagram shows the CRISPR-interference (CRISPRi) assay. DCas9-KRAB binds to the upstream and downstream of rs73613962 guided by sgRNAs to inhibit PRMT7 expression (d). The bottom diagram shows the CRISPR-activation (CRISPRa) assay to activate the expression of PRMT7 guided by sgRNAs (g). e, f RNA level of PRMT7 detected by qPCR in the CRISPRi assay in the QGY-7703 (e, P = 0.0038 in dCas9-KRAB_gRNA (K_gRNA) compared to dCas9-KRAB_Control (K_Ctrl)) and HepG2 (f, P = 0.0011 in K_gRNA compared to K_Ctrl) cell lines. h, i RNA level of PRMT7 detected by qPCR in the CRISPRa assay in the QGY-7703 (h, P = 0.0007 in dCAS9-VP48_gRNA (V_gRNA) compared to dCAS9-VP48_Control (V_Ctrl)) and HepG2 (i, P = 0.0300 in V_gRNA compared to V_Ctrl) cell lines. Values are expressed as the mean ± SD, n = 3 in c, e, f, h, and i. *, **, and *** mean P-values less than 0.05, 0.01, and 0.001, respectively (two-sided student’ t-test).
Fig 5: The risk allele G of rs73613962 enhances the binding ability of transcription factor HNF4A to promote PRMT7 expression.a The result of the competitive EMSA. The binding affinity of protein to DNA oligos is demonstrated by adding unlabeled-G or unlabeled-T to the reaction. The light blue arrow indicates the protein-biotin-G-oligo complexes. This experiment is replicated two times at least and similar results are observed. b The enrichment of HNF4A on the rs73613962-containing region examined by ChIP. The left panel shows the diagram for HNF4A-ChIP. The antibody of HNF4A recognizes and binds to HNF4A; thus, the DNA sequence bound by HNF4A is pulled down from the fragmented chromatin. Then, the enriched DNA sequence is detected by the following qPCR as shown in the right panel. P = 0.0001 in HNF4A compared to IgG. c ChIP-PCR results. The agarose gel electrophoresis of PCR products for Input and ChIP, respectively, in the above panel. Light blue arrows indicate the marker bands. Source data are provided as a Source Data file. The relative gray value of each PCR band is analyzed using ImageJ in the bottom panel. P = 0.7769 and P < 0.0001 in HNF4A compared to IgG in Input and ChIP, respectively. d The dual-luciferase assay of enhancer activity for the enhancer-T and enhancer-G before and after the downregulation of HNF4A. P = 0.0046 in Enhancer-G compared to Enhancer-T without HNF4A knockdown, and P = 0.1493 and P = 0.0002 in Enhancer-T or Enhancer-G with HNF4A knockdown to Enhancer-T or Enhancer-G without HNF4A knockdown, respectively. e The result of the blocking EMSA of HNF4A. The amount of anti-HNF4A antibody in lane 3 and lane 4 is 1 µg and 2 µg, respectively. The IgG amount in lane 5 is 2 µg. The light blue arrow indicates the HNF4A-biotin-G-oligo complexes. We repeat this experiment two times at least and always observe the similar results. f qPCR detection of HNF4A and PRMT7 expressions after the downregulation of HNF4A in cells through two siRNAs. For detection of HNF4A expression level, P = 0.0017 and 0.0037 in HNF4A_small interfering RNA (HNF4A_si)1 or HNF4A_si2 compared to small interfering RNA negative control (siNC), respectively. For detection of PRMT7 expression level, P = 0.0001 and P = 0.0002 in HNF4A_si1 or HNF4A_si2 compared to siNC, respectively. All the above experiments were conducted in the QGY-7703 cell line (a–f). g–i The correlation of PRMT7 and HNF4A expressions showed in all HCC subjects (g), and subjects with the rs73613962 TT homozygote (h), or rs73613962 G allele carriers (i) in TCGA LIHC. TPM, transcripts per million. Values are expressed as the mean ± SD, n = 3 in b–d, and f. ‘ns’ means not significant; ** and *** mean P values less than 0.01, and 0.001, respectively (two-sided student’ t-test).
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