Structure-Activity Relationships of Cbx7 Inhibitors, Including Selectivity Studies against Other Cbx Proteins.
ABSTRACT: The five human polycomb (Pc) paralog proteins, chromobox homolog (Cbx) 2/4/6/7/8, are a family of chromodomain containing methyllysine reader proteins that are canonical readers of trimethyllysine 27 on histone 3 (H3K27me3). The aberrant expression of the Cbx7 gene is implicated in several cancers including prostate, gastric, thyroid, pancreas, and colon cancer. Previous reports on antagonizing the molecular recognition of Cbx7-H3K27me3 with chemical inhibitors showed an impact on prostate cancer cell lines. We report here on the design, synthesis, and structure-activity relationships of a series of potent peptidomimetic antagonists that were optimized on a trimethyllysine-containing scaffold to target Cbx7. The ligands were characterized using fluorescence polarization (FP) for their binding efficiency and selectivity against the Pc paralog Cbx proteins. The most selective ligand 9, as indicated by the FP data analysis, was further characterized using the isothermal titration calorimetry (ITC). Compound 9 exhibits a 220 nM potency for Cbx7 and exhibits 3.3, 1.8, 7.3 times selective for Cbx7 over Cbx2/4/8 and 28-fold selective over the HP1 family member Cbx1. Our research provides several potent and partially selective inhibitors for Cbx2/4/7 that do not contain trimethyllysine. Our models and binding data suggest that the aromatic cages of Cbx7/Cbx4 can accommodate larger alkyl groups such as diisobutyl substitution on the lysine nitrogen.
Project description:Chromobox (CBX) family proteins are canonical components in polycomb repressive complexes 1 (PRC1), with epigenetic regulatory function and transcriptionally repressing target genes via chromatin modification. A plethora of studies have highlighted the function specifications among CBX family members in various cancer, including lung cancer, colon cancer and breast cancer. Nevertheless, the functions and prognostic roles of distinct CBX family members in breast cancer (BC) remain elusive. In this study, we reported the prognostic values of CBX family members in patients with BC through analysis of a series of databases, including CCLE, ONCOMINE, Xena Public Data Hubs, and Kaplan-Meier plotter. It was found that the mRNA expression of CBX family members were noticeably higher in BC than normal counterparts. CBX2 was highly expressed in Basal-like and HER-2 subtypes, while CBX4 and CBX7 expressions were enriched in Luminal A and Luminal B subtypes of BC. Survival analysis revealed that CBX1, CBX2 and CBX3 mRNA high expression was correlated to worsen relapse-free survival (RFS) for all BC patients, while CBX4, CBX5, CBX6 and CBX7 high expression was correlated to better RFS in this setting. Noteworthily, CBX1 and CBX2 were associated with chemoresistance whereas CBX7 was associated with tamoxifen sensitivity, as well as chemosensitivity in breast tumors. Therefore, we propose that CBX1, CBX2 and CBX7 are potential targets for BC treatment. The results might be beneficial for better understanding the complexity and heterogeneity in the molecular underpinning of BC, and to develop tools to more accurately predict the prognosis of patients with BC.
Project description:The Polycomb PRC1 plays essential roles in development and disease pathogenesis. Targeting of PRC1 to chromatin is thought to be mediated by the Cbx family proteins (Cbx2/4/6/7/8) binding to histone H3 with a K27me3 modification (H3K27me3). Despite this prevailing view, the molecular mechanisms of targeting remain poorly understood. Here, by combining live-cell single-molecule tracking (SMT) and genetic engineering, we reveal that H3K27me3 contributes significantly to the targeting of Cbx7 and Cbx8 to chromatin, but less to Cbx2, Cbx4, and Cbx6. Genetic disruption of the complex formation of PRC1 facilitates the targeting of Cbx7 to chromatin. Biochemical analyses uncover that the CD and AT-hook-like (ATL) motif of Cbx7 constitute a functional DNA-binding unit. Live-cell SMT of Cbx7 mutants demonstrates that Cbx7 is targeted to chromatin by co-recognizing of H3K27me3 and DNA. Our data suggest a novel hierarchical cooperation mechanism by which histone modifications and DNA coordinate to target chromatin regulatory complexes.
Project description:Background:The chromobox (CBX) proteins CBX2, CBX4, CBX6, CBX7, and CBX8, also known as Polycomb (Pc) proteins, are canonical components of the Polycomb repressive complex 1 (PRC1). Abundant evidence indicates that abnormal expression of Pc proteins is associated with a variety of tumors, but their role in the pathogenesis of hepatocellular carcinoma (HCC) has not been fully elucidated. In the present study, we performed a case-control study to investigate the relationship between single nucleotide polymorphisms (SNPs) of CBX genes and HCC. Methods:Nine SNPs on CBX genes (rs7217395, rs2036316 of CBX2; rs3764374, rs1285251, rs2289728 of CBX4; rs7292074 of CBX6; and rs710190, rs139394, rs5750753 of CBX7) were screened and genotyped using MassARRAY technology in 334 HCC cases and 321 controls. The association between SNPs and their corresponding gene expressions was analyzed through bioinformatics methods using the Ensembl database and Blood eQTL browser online tools. Results:The results indicated that rs2289728 (G>A) of CBX4 (P = 0.03, OR = 0.56, 95% CI: 0.33-0.94) and rs139394 (C>A) of CBX7 (P = 0.02, OR = 0.55, 95% CI: 0.33-0.90) decreased the risk of HCC. Interaction between rs2036316 and HBsAg increased the risk of HCC (P = 0.02, OR = 6.88, 95% CI: 5.20-9.11), whereas SNP-SNP interaction between rs710190 and rs139394 reduced the risk of HCC (P = 0.03, OR = 0.33, 95% CI: 0.12-0.91). Gene expression analyses showed that the rs2289728 A allele and the rs139394 A allele significantly reduced CBX4 and CBX7 expression, respectively. Conclusion:Our findings suggest that CBX4 rs2289728 and CBX7 rs139394 are protective SNPs against HCC. The two SNPs may reduce the risk of HCC while suppressing the expression of CBX4 and CBX7.
Project description:The eight mammalian Cbx proteins are chromodomain-containing proteins involved in regulation of heterochromatin, gene expression, and developmental programs. They are evolutionarily related to the Drosophila HP1 (dHP1) and Pc (dPc) proteins that are key components of chromatin-associated complexes capable of recognizing repressive marks such as trimethylated Lys-9 and Lys-27, respectively, on histone H3. However, the binding specificity and function of the human homologs, Cbx1-8, remain unclear. To this end we employed structural, biophysical, and mutagenic approaches to characterize the molecular determinants of sequence contextual methyllysine binding to human Cbx1-8 proteins. Although all three human HP1 homologs (Cbx1, -3, -5) replicate the structural and binding features of their dHP counterparts, the five Pc homologs (Cbx2, -4, -6, -7, -8) bind with lower affinity to H3K9me3 or H3K27me3 peptides and are unable to distinguish between these two marks. Additionally, peptide permutation arrays revealed a greater sequence tolerance within the Pc family and suggest alternative nonhistone sequences as potential binding targets for this class of chromodomains. Our structures explain the divergence of peptide binding selectivity in the Pc subfamily and highlight previously unrecognized features of the chromodomain that influence binding and specificity.
Project description:Chromobox (CBX) family proteins control chromatin structure and gene expression. However, the functions of CBXs in cancer progression, especially breast cancer, are inadequately studied. We assessed the significance of eight CBX proteins in breast cancer. We performed immunohistochemistry and bioinformatic analysis of data from Oncomine, GEPIA Dataset, bcGenExMiner, Kaplan-Meier Plotter, and cBioPortal. We compared mRNA and protein expression levels of eight CBX proteins between breast tumor and normal tissue. The expression difference of CBX7 was the greatest, and CBX7 was downregulated in breast cancer tissues compared with normal breast tissues. The expression of CBX2 was strongly associated with tumor stage. We further analyzed the association between the eight CBX proteins and the following clinicopathological features: menopause age, estrogen receptor (ER), progesterone receptor (PR) and HER-2 receptor status, nodal status, P53 status, triple-negative status, and the Scarff-Bloom-Richardson grade (SBR) and Nottingham prognostic index (NPI). Survival analysis in the Kaplan-Meier Plotter database showed that the eight CBX proteins were significantly associated with prognosis. Moreover, CBX genes in breast cancer patients had a high net alteration frequency of 57%. There were significant co-expression correlations between the following CBX protein pairs: CBX4 positively with CBX8, CBX6 positively with CBX7, and CBX2 negatively with CBX7. We also analyzed the Gene Ontology enrichment of the CBX proteins, including biological processes, cellular components, and molecular functions. CBX 1/2/3/5/8 may be oncogenes for breast cancer, whereas CBX 6 and 7 may be tumor suppressors for breast cancer. All eight CBX proteins may be predictive for prognosis. Clinical trials are needed to confirm the significance of the eight CBX proteins in breast cancer.
Project description:The Polycomb Repressive Complex 1 (PRC1) is a chromatin-associated protein complex involved in transcriptional repression of hundreds of genes controlling development and differentiation processes, but also involved in cancer and stem cell biology. Within the canonical PRC1, members of Pc/CBX protein family are responsible for the targeting of the complex to specific gene loci. In mammals, the Pc/CBX protein family is composed of five members generating, through mutual exclusion, different PRC1 complexes with potentially distinct cellular functions. Here, we performed a global analysis of the cbx gene family in 68 teleost species and traced the distribution of the cbx genes through teleost evolution in six fish super-orders. We showed that after the teleost-specific whole genome duplication, cbx4, cbx7 and cbx8 are retained as pairs of ohnologues. In contrast, cbx2 and cbx6 are present as pairs of ohnologues in the genome of several teleost clades but as singletons in others. Furthermore, since zebrafish is a widely used vertebrate model for studying development, we report on the expression of the cbx family members during zebrafish development and in adult tissues. We showed that all cbx genes are ubiquitously expressed with some variations during early development.
Project description:Polycomb chromobox (CBX) proteins regulate gene transcription by maintaining chromatin states, which guide a variety of biological processes. Now, epigenetic regulation of innate immune response is an emerging field. However, the role of CBX proteins in innate immunity remains unclear. We confirmed that the expression of CBX family proteins, especially Cbx2, was decreased in macrophages upon viral infection, and then we investigated the role of Cbx2 in the antiviral immune response. Silencing or knockdown of Cbx2 in macrophages inhibited virus-induced production of IFN-?. Furthermore, heterozygous Cbx2 knockout were susceptible to VSV challenge. Mechanistically, Cbx2 binds to and recruits Jmjd3 to the Ifnb promoter, leading to demethylation of H3K27me3 and increased transcription of IFN-?. Together, our study reveals a non-traditional function of a Cbx protein and adds new insight into the epigenetic regulation of antiviral innate immunity.
Project description:We report the design and characterization of UNC3866, a potent antagonist of the methyllysine (Kme) reading function of the Polycomb CBX and CDY families of chromodomains. Polycomb CBX proteins regulate gene expression by targeting Polycomb repressive complex 1 (PRC1) to sites of H3K27me3 via their chromodomains. UNC3866 binds the chromodomains of CBX4 and CBX7 most potently, with a K(d) of ?100 nM for each, and is 6- to 18-fold selective as compared to seven other CBX and CDY chromodomains while being highly selective over >250 other protein targets. X-ray crystallography revealed that UNC3866's interactions with the CBX chromodomains closely mimic those of the methylated H3 tail. UNC4195, a biotinylated derivative of UNC3866, was used to demonstrate that UNC3866 engages intact PRC1 and that EED incorporation into PRC1 is isoform dependent in PC3 prostate cancer cells. Finally, UNC3866 inhibits PC3 cell proliferation, consistent with the known ability of CBX7 overexpression to confer a growth advantage, whereas UNC4219, a methylated negative control compound, has negligible effects.
Project description:Polycomb group proteins are transcriptional repressors recruited to many developmental control genes. The specificity of polycomb group protein targeting is incompletely understood. Subunits of polycomb repressive complexes (PRC) are encoded by multigene families in vertebrates. Five chromodomain-containing CBX family proteins are thought to mediate chromatin association by PRC1 complexes. We visualized the recruitment of CBX proteins to chromatin using bimolecular fluorescence complementation (BiFC) analysis, wherein fragments of fluorescent proteins fused to CBX family members and histone H3 form a fluorescent complex when the CBX proteins bind to nucleosomes. Different CBX family proteins associated with nucleosomes in different subnuclear regions in both ES cells and fibroblasts. The total populations of most CBX proteins had distributions distinct from those of the chromatin-associated complexes, indicating that most of these CBX proteins were not bound to nucleosomes. The conserved chromodomain and chromobox regions of CBX proteins were dispensable for chromatin association. The absence of H3 K27 trimethylation in EED null ES cells had minimal effects on chromatin association by CBX proteins. The BiFC complexes did not colocalize with anti-trimethyl-K27 immunofluorescence, with the exception of inactive X. Metaphase spreads derived from stable cell lines with inducible CBX fusion expression revealed reciprocal patterns of chromosome association by CBX2 and CBX6 BiFC complexes. H3.2 purified from CBX2-H3.2 BiFC complexes was enriched in trimethyl-K27, dimethyl-K4, and acetyl-K9 modifications. We conclude that different CBX proteins are recruited to distinct chromatin regions through nonconserved interactions, expanding the regulatory diversity of polycomb group proteins.
Project description:The polycomb paralogs CBX2, CBX4, CBX6, CBX7, and CBX8 are epigenetic readers that rely on "aromatic cage" motifs to engage their partners' methyllysine side chains. Each CBX carries out distinct functions, yet each includes a highly similar methyllysine-reading chromodomain as a key element. CBX7 is the only chromodomain that has yet been targeted by chemical inhibition. We report a small set of peptidomimetic agents in which a simple chemical modification switches the ligands from one with promiscuity across all polycomb paralogs to one that provides selective inhibition of CBX6. The structural basis for this selectivity, which involves occupancy of a small hydrophobic pocket adjacent to the aromatic cage, was confirmed through molecular dynamics simulations. Our results demonstrate the increases in affinity and selectivity generated by ligands that engage extended regions of chromodomain binding surfaces.