Chromobox Proteins Research Articles

Single-cell RNA analysis of chromodomain-encoding genes in mesenchymal stromal cells of the mouse dental pulp.

The chromodomain helicase DNA-binding (CHD) and chromobox (CBX) families of proteins play crucial roles in cell fate decisions, differentiation, and cell proliferation in a broad variety of tissues and cell types. CHD proteins are ATP-dependent epigenetic enzymes actively engaged in transcriptional regulation, DNA replication, and DNA damage repair, whereas CBX proteins are transcriptional repressors mainly involved in the formation of heterochromatin. The pleiotropic effects of CHD and CBX proteins are largely dependent on their versatility to interact with other key components of the epigenetic and transcriptional machinery. Although the function and regulatory modes of CHD and CBX factors are well established in many cell types, little is known about their roles during osteogenic differentiation. A single-cell RNA-sequencing analysis of the mouse incisor dental pulp revealed distinct spatiotemporal expression patterns of CHD- and CBX-encoding genes within different clusters of mesenchymal stromal cells (MSCs) representing various stages of osteogenic differentiation. Additionally, genes encoding interaction partners of CHD and CBX proteins, such as subunits of the trithorax-COMPASS and polycomb chromatin remodeling complexes, exhibited differential co-expression behaviors within MSC subpopulations. Thus, CHD- and CBX-encoding genes show partially overlapping but distinct expression patterns in MSCs, suggesting their differential roles in osteogenic cell fate decisions.

How CBX proteins regulate normal and leukemic blood cells.

Hematopoietic stem cell (HSC) fate decisions are dictated by epigenetic landscapes. The Polycomb Repressive Complex 1 (PRC1) represses genes that induce differentiation, thereby maintaining HSC self-renewal. Depending on which chromobox (CBX) protein (CBX2, CBX4, CBX6, CBX7, or CBX8) is part of the PRC1 complex, HSC fate decisions differ. Here, we review how this occurs. We describe how CBX proteins dictate age-related changes in HSCs and stimulate oncogenic HSC fate decisions, either as canonical PRC1 members or by alternative interactions, including non-epigenetic regulation. CBX2, CBX7, and CBX8 enhance leukemia progression. To target, reprogram, and kill leukemic cells, we suggest and describe multiple therapeutic strategies to interfere with the epigenetic functions of oncogenic CBX proteins. Future studies should clarify to what extent the non-epigenetic function of cytoplasmic CBX proteins is important for normal, aged, and leukemic blood cells.

CBX8 promotes lung adenocarcinoma growth and metastasis through transcriptional repression of CDKN2C and SCEL.

Dysregulation of polycomb group (PcG) proteins that mediate epigenetic gene silencing contributes to tumorigenesis. As core components of the polycomb repressive complex 1 (PRC1), chromobox (CBX) proteins recognize H3K27me3 to recruit PRC1 to maintain a repressive transcriptional state. However, the individual biological functions of these CBX proteins in tumorigenesis warrant in-depth investigation. In this study, we analyzed the mRNA expression of CBX family genes across multiple cancers using The Cancer Genome Atlas data and found different expression patterns of the five CBX genes in different types of cancer. This analyses together with the result of immunohistochemistry indicated that CBX8 expression was significantly higher in lung adenocarcinoma (LUAD) tissues compared to adjacent nontumor tissues. Overexpression approaches demonstrated that CBX8 facilitated LUAD cell proliferation and migration in vitro. Consistently, CBX8 knockdown reduced LUAD cell proliferation and migration in both cell culture and mouse models. RNA sequencing combined with real-time RT-PCR assays revealed CDKN2C and SCEL as target genes of CBX8. Furthermore, chromatin immunoprecipitation assays indicated that CBX8 directly bound to the promoters of CDKN2C and SCEL to establish H2AK119ub. CBX8 depletion reduced the enrichment of H2AK119ub on CDKN2C and SCEL promoters. Moreover, depletion of CDKN2C and SCEL restored the repressed growth and invasion ability of LUAD cells caused by CBX8 knockdown. These findings demonstrate that CBX8 promotes LUAD growth and metastasis through the transcriptional repression of CDKN2C and SCEL. Our study uncovers the oncogenic role of CBX8 in LUAD progression and provides a new target for the diagnosis and therapy of LUAD.

Subcellular expression pattern and clinical significance of CBX2 and CBX7 in breast cancer subtypes.

Chromobox (CBX)2 and CBX7, members of CBX family protein, show diverse expression patterns and contrasting roles in certain cancers. We aimed to investigate the subcellular expression patterns and clinical significances of CBXs in breast cancer (BC) subtypes, which have heterogeneous clinical course and therapeutic responses. Among the subtypes, the triple-negative BC (TNBC) is a heterogeneous group that lacks specific markers. We categorized TNBC into quadruple-negative BC (QNBC) and TNBC, based on androgen receptor (AR) status, to make the groups more homogeneous. Immunohistochemistry for CBX proteins was performed on 323 primary invasive BC tissues and their clinical significances were analyzed. Cytoplasmic CBX2 (CBX2-c) was linked to adverse clinicopathological factors and TNBC and QNBC subtypes. In contrast, nuclear CBX7 (CBX7-n) was associated with favorable parameters and luminal A subtype. CBX2-c expression increased progressively from that in benign lesions to that in in situ carcinomas and invasive cancers, whereas CBX7-n and AR expressions showed sequential downregulation. AR was lower in metastatic tissues compared to matched primary cancer tissues. We speculate that the upregulation of CBX2-c and downregulation of CBX7-n could play a role in breast oncogenesis and an adverse clinical course, suggesting them as potential prognostic markers and therapeutic targets in invasive BCs.

Identification of the Prognostic Biomarkers CBX6 and CBX7 in Bladder Cancer

Chromobox (CBX) proteins are essential components of polycomb group proteins and perform essential functions in bladder cancer (BLCA). However, research on CBX proteins is still limited, and the function of CBXs in BLCA has not been well illustrated. We analyzed the expression of CBX family members in BLCA patients from The Cancer Genome Atlas database. By Cox regression analysis and survival analysis, CBX6 and CBX7 were identified as potential prognostic factors. Subsequently, we identified genes associated with CBX6/7 and performed enrichment analysis, and they were enriched in urothelial carcinoma and transitional carcinoma. Mutation rates of TP53 and TTN correlate with expression of CBX6/7. In addition, differential analysis indicated that the roles played by CBX6 and CBX7 may be related to immune checkpoints. The CIBERSORT algorithm was used to screen out immune cells that play a role in the prognosis of bladder cancer patients. Multiplex immunohistochemistry staining confirmed a negative correlation between CBX6 and M1 macrophages, as well as a consistent alteration in CBX6 and regulatory T cells (Tregs), a positive correlation between CBX7 and resting mast cells, and a negative correlation between CBX7 and M0 macrophages. CBX6 and CBX7 expression levels may assist in predicting the prognosis of BLCA patients. CBX6 may contribute to a poor prognosis in patients by inhibiting M1 polarization and promoting Treg recruitment in the tumor microenvironment, while CBX7 may contribute to a better prognosis in patients by increasing resting mast cell numbers and decreasing macrophage M0 content.

Comprehensive analysis reveals the value of the expression of chromobox family members for bladder urothelial carcinoma prognosis.

Bladder urothelial carcinoma (BLCA) accounts for 95% of all cases of bladder cancer worldwide, with a high incidence and poor prognosis. Chromobox (CBX) proteins play a key role in numerous malignant tumors; however, the role of CBX in BLCA remains unknown. Herein, the present study found that, compared with in normal bladder tissues, the expression levels of CBX1, CBX2, CBX3, CBX4 and CBX8 were markedly increased in BLCA tissues, as determined by Tumor Immune Estimation Resource, UALCAN and ONCOMINE analyses, whereas CBX6 and CBX7 were decreased in BLCA tissues. Furthermore, evident hypomethylation in the promoters of CBX1, and CBX2, as well as significant hypermethylation in the promoters of CBX5, CBX6 and CBX7, was detected in BLCA tissues compared with in normal bladder tissues. The expression of CBX1, CBX2 and CBX7 was involved in the prognosis of patients with BLCA. Low CBX7 expression was strongly associated with poorer overall survival in patients with BLCA, whereas high CBX1 and CBX2 expression was associated with poorer progression-free survival. Besides, significant associations were determined between the expression of CBXs and immune cell infiltration, including dendritic cells, neutrophils, macrophages, CD4+ T cells, CD8+ T cells and B cells. Overall, the current results may provide a rationale for developing new targets and prognostic markers for BLCA therapy.

Comprehensive Analysis of the Expression and Prognosis of chromobox Family Members in Breast Cancer

Background: Chromobox proteins are canonical components of the Polycomb group family and play pivotal roles in several cancers. However, little is known about the function, prognostic value and drug sensitivity of CBX family members in breast cancer.Methods: In this study we investigated the expression, prognosis value and drug sensitivity of CBX family in breast cancer using the ONCOMINE, GEPIA, Human Protein Atlas and Kaplan-Meier Plotter databases, etc. and preliminary verified the expression of CBX family in breast cancer cell lines by RT-qPCR.Results: We found that the expression levels of CBX1/2/3/4/8 members were elevated in breast cancer tissues compared to adjacent normal breast tissues, while the expression levels of CBX6/7 genes were reduced in breast cancer tissue. In vitro qRT-PCR validated the expression differences of CBX1/2/3/4/8 in breast cancer cell lines. Further analysis showed expression of CBX family members was remarkably correlated with cancer subgroups. As nodal metastasis status increased, the mRNA expression of CBX1/2/3/4/8 members tended to be higher, while CBX6/7 tended to be lower. The expression of CBX1/2/3 was higher in patients with TP53 mutation and CBX6/7 expression tended to be lower in patients with TP53 mutation groups. High transcription levels of CBX2/3 were significantly associated with shorter overall survival in breast cancer patients, while lower expression of CBX4/5/6/7 members was associated with unfavorable overall survival. Moreover, a high mutation rate of CBX gene members (43%) was observed in breast cancer patients, and genetic alterations in CBX genes was associated with poor prognosis.Conclusion: Taken together, our results indicated that CBX2/3/6/7/8 could be considered prognostic and therapeutic biomarkers of breast cancer and are worthy of further study.

CBXs-related prognostic gene signature correlates with immune microenvironment in gastric cancer.

Background: Chromobox (CBX) proteins are important Polycomb family proteins in the development of gastric cancer. Nonetheless, the relationship between CBXs and gastric cancer microenvironment remains unclear.Methods: Multiple databases were used for the analysis of CBXs expression and clinical value in gastric cancer patients. A Cox regression analysis was used to evaluate the prognostic importance of CBXs. Thereafter, regression analysis of LASSO Cox was used to construct the prognostic model. Spearman's correlation between risk score and immune infiltration was analyzed using the McP-counter algorithm. A predicted nomogram was developed to predict the overall survival of gastric cancer patients after 1, 2, and 3 years.Results: In contrast with normal tissues, mRNA and protein expression levels of CBX2/3 were significantly high in gastric cancer tissues, whereas those of CBX6/7 were low. CBXs significantly correlated with immune subtypes and molecular subtypes. A prognostic gene model based on five CBX genes (CBX1, CBX2, CBX3, CBX7, and CBX8) predicted the overall survival of gastric cancer patients. A significant correlation was noted between the risk score of the CBXs-related prognostic gene model and immune-cell infiltration. Low risk patients could achieve a better response to immune checkpoint inhibitors. A predictive nomogram constructed using the above five CBX genes revealed that overall survival rates over 1, 2, and 3 years could be reasonably predicted. Therefore, the roles of CBXs were associated with chromatin modifications and histone methylation, etc.Conclusion: In summary, we identified a prognostic CBXs model comprising five genes (CBX1, CBX2, CBX3, CBX7, and CBX8) for gastric cancer patients through bioinformatics analysis.

Reprogramming CBX8-PRC1 function with a positive allosteric modulator

SUMMARYCanonical targeting of Polycomb repressive complex 1 (PRC1) to repress developmental genes is mediated by cell-type-specific, paralogous chromobox (CBX) proteins (CBX2, 4, 6, 7, and 8). Based on their central role in silencing and their dysregulation associated with human disease including cancer, CBX proteins are attractive targets for small-molecule chemical probe development. Here, we have used a quantitative and target-specific cellular assay to discover a potent positive allosteric modulator (PAM) of CBX8. The PAM activity of UNC7040 antagonizes H3K27me3 binding by CBX8 while increasing interactions with nucleic acids. We show that treatment with UNC7040 leads to efficient and selective eviction of CBX8-containing PRC1 from chromatin, loss of silencing, and reduced proliferation across different cancer cell lines. Our discovery and characterization of UNC7040 not only reveals the most cellularly potent CBX8-specific chemical probe to date, but also corroborates a mechanism of Polycomb regulation by non-specific CBX nucleotide binding activity.

Prognostic and immune infiltrates for the Chromobox (CBX) protein family in human pancreatic adenocarcinoma.

Globally, pancreatic adenocarcinoma (PAAD) is among the most prevalent malignant tumors. The Chromobox (CBX) protein family is a vital component of epigenetic regulatory complexes that have vital biological roles. The biological functions, immune infiltration, expression levels, and the prognostic significance of CBX proteins in PAAD have not yet been established. Using bioinformatics tools, such as the Gene Expression Profiling Interactive Analysis (GEPIA), Oncomine, Kaplan-Meier plotter, GeneMANIA, cBioPortal, TIMER and R, we evaluated the prognostic importance, expression levels, gene alterations, risk factors, and immune cell infiltration levels of CBXs in PAAD patients. The expression levels of CBX3 in clinical-pathological samples were also confirmed by immunohistochemistry. In PAAD tissues, CBX1, CBX3, CBX5, and CBX8 expressions were high. High CBX1, CBX5, CBX6, and CBX7 levels were correlated with tumor stages. Elevated CBX2, CBX6, CBX7, and CBX8 messenger ribonucleic acid (mRNA) levels were markedly correlated with better overall survival (OS) outcomes for pancreatic cancer patients, while elevated CBX3 was markedly correlated with short OS outcomes. In particular, we have validated the differential expression of CBX3 on clinical specimens using immunohistochemical methods. A multivariate logistic analysis revealed that elevated mRNA expression levels of CBX3 and suppressed CBX8 levels were independently associated with short OS outcomes for pancreatic cancer patients. The roles of CBXs and their neighboring proteins are associated with a negatively regulated cell cycle, histone binding, polycomb group protein complexes, and the regulation of pluripotent stem cell signaling pathways. Additionally, CBX levels were found to be markedly associated with immune infiltrates, and found that the immune infiltration score of CBX3 was differentially expressed in cell lines such as CD8 T cells, NK cells, Mast cells and T helper cells. CBX3/8 is a potential marker for prognostic outcomes in PAAD patients.

Expression and prognostic significance of CBX2 in colorectal cancer: database mining for CBX family members in malignancies and vitro analyses

BackgroundThe Chromobox (CBX) domain protein family, a core component of polycomb repressive complexes 1, is involved in transcriptional repression, cell differentiation, and program development by binding to methylated histone tails. Each CBX family member plays a distinct role in various biological processes through their own specific chromatin domains, due to differences in conserved sequences of the CBX proteins. It has been demonstrated that colorectal cancer (CRC) is a multiple-step biological evolutionary process, whereas the roles of the CBX family in CRC remain largely unclear.MethodsIn the present study, the expression and prognostic significance of the CBX family in CRC were systematically analyzed through a series of online databases, including Cancer Cell Line Encyclopedia (CCLE), Oncomine, Human Protein Atlas (HPA), and Gene Expression Profiling Interactive Analysis (GEPIA). For in vitro verification, we performed cell cloning, flow cytometry and transwell experiments to verify the proliferation and invasion ability of CRC cells after knocking down CBX2.ResultsMost CBX proteins were found to be highly expressed in CRC, but only the elevated expression of CBX2 could be associated with poor prognosis in patients with CRC. Further examination of the role of CBX2 in CRC was performed through several in vitro experiments. CBX2 was overexpressed in CRC cell lines via the CCLE database and the results were verified by RT-qPCR. Moreover, the knockdown of CBX2 significantly suppressed CRC cell proliferation and invasion. Furthermore, the downregulation of CBX2 was found to promote CRC cell apoptosis.ConclusionsBased on these findings, CBX2 may function as an oncogene and potential prognostic biomarker. Thus, the association between the abnormal expression of CBX2 and the initiation of CRC deserves further exploration.

Evaluation of the prognostic value of CBXs in gastric cancer patients

Chromobox (CBX) proteins were suggested to exert epigenetic regulatory and transcriptionally repressing effects on target genes and might play key roles in the carcinogenesis of a variety of carcinomas. Nevertheless, the functions and prognostic significance of CBXs in gastric cancer (GC) remain unclear. The current study investigated the roles of CBXs in the prognosis of GC using the Oncomine, The Gene Expression Profiling Interactive Analysis (GEPIA), UALCAN, The Cancer Genome Atlas (TCGA), and cBioPortal databases. CBX1/2/3/4/5 were significantly upregulated in GC tissues compared with normal tissues, and CBX7 was downregulated. Multivariate analysis showed that high mRNA expression levels of CBX3/8 were independent prognostic factors for prolonged OS in GC patients. In addition, the genetic mutation rate of CBXs was 37% in GC patients, and genetic alterations in CBXs showed no association with OS or disease-free survival (DFS) in GC patients. These results indicated that CBX3/8 can be prognostic biomarkers for the survival of GC patients.

CBX7 suppresses urinary bladder cancer progression via modulating AKR1B10\u2013ERK signaling

The chromobox (CBX) proteins mediate epigenetic gene silencing and have been implicated in the cancer development. By analyzing eight CBX family members in TCGA dataset, we found that chromobox 7 (CBX7) was the most strikingly downregulated CBX family member in urinary bladder cancer (UBC), as compared to normal tissues. Though dysregulation of CBX7 has been reported in multiple cancers, its specific role and clinical relevance in UBC remain unclear. Herein, we found that frequent downregulation of CBX7 in UBC specimens, which was due to its promoter hypermethylation, was correlated with poor prognosis. The ectopic expression of CBX7 suppressed UBC cell proliferation, migration, invasion, and cancer stemness, whereas CBX7 depletion promoted cancer cell aggressiveness. Importantly, CBX7 overexpression in UBC cells inhibited tumorigenicity, whereas CBX7 depletion promoted the tumor development, indicating its tumor-suppressive role in UBC. Using RNA-seq and chromosome immunoprecipitation (ChIP) assays, we identified aldo-keto reductase family 1 member 10 (AKR1B10) as a novel downstream target of CBX7, which was negatively modulated by CBX7 in a PRC1-dependent manner and involved in stimulating ERK signaling. Consistently, AKR1B10 overexpression induced cancer cell aggressiveness, whereas suppression of AKR1B10 by siRNA or its small molecular inhibitor, oleanolic acid, reversed the CBX7 deficiency-induced cellular effects. AKR1B10 overexpression was negatively associated with CBX7 downregulation and predicted poor clinical outcomes in UBC patients. Taken together, our results indicate that CBX7 functions as a tumor suppressor to downregulate AKR1B10 and further inactivates ERK signaling. This CBX7/AKR1B10/ERK signaling axis may provide a new therapeutic strategy against UBC.

Bioinformatic analysis of the expression and prognostic value of chromobox family proteins in human breast cancer

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.

Biological functions of chromobox (CBX) proteins in stem cell self-renewal, lineage-commitment, cancer and development.

Epigenetic regulatory proteins support mammalian development, cancer, aging and tissue repair by controlling many cellular processes including stem cell self-renewal, lineage-commitment and senescence in both skeletal and non-skeletal tissues. We review here our knowledge of epigenetic regulatory protein complexes that support the formation of inaccessible heterochromatin and suppress expression of cell and tissue-type specific biomarkers during development. Maintenance and formation of heterochromatin critically depends on epigenetic regulators that recognize histone 3 lysine trimethylation at residues K9 and K27 (respectively, H3K9me3 and H3K27me3), which represent transcriptionally suppressive epigenetic marks. Three chromobox proteins (i.e., CBX1, CBX3 or CBX5) associated with the heterochromatin protein 1 (HP1) complex are methyl readers that interpret H3K9me3 marks which are mediated by H3K9 methyltransferases (i.e., SUV39H1 or SUV39H2). Other chromobox proteins (i.e., CBX2, CBX4, CBX6, CBX7 and CBX8) recognize H3K27me3, which is deposited by Polycomb Repressive Complex 2 (PRC2; a complex containing SUZ12, EED, RBAP46/48 and the methyl transferases EZH1 or EZH2). This second set of CBX proteins resides in PRC1, which has many subunits including other polycomb group factors (PCGF1, PCGF2, PCGF3, PCGF4, PCGF5, PCGF6), human polyhomeotic homologs (HPH1, HPH2, HPH3) and E3-ubiquitin ligases (RING1 or RING2). The latter enzymes catalyze the subsequent mono-ubiquitination of lysine 119 in H2A (H2AK119ub). We discuss biological, cellular and molecular functions of CBX proteins and their physiological and pathological activities in non-skeletal cells and tissues in anticipation of new discoveries on novel roles for CBX proteins in bone formation and skeletal development.

Comprehensive Analysis of Prognostic Value and Immune Infiltration of Chromobox Family Members in Colorectal Cancer.

Objective: Colorectal cancer (CRC) is one of the common malignant tumors worldwide. Chromobox (CBX) family proteins are important components of epigenetic regulation complexes and are implicated in the development of multiple cancers by blocking differentiation and promoting proliferation. However, little is known about the function of distinct CBX proteins in colorectal cancer.Methods: Oncomine, Gene Expression Profiling Interactive Analysis (GEPIA), Kaplan-Meier plotter, cBioPortal, GeneMANIA, and TIMER were utilized to analyze differential expression, prognostic value, genetic alteration and immune cell infiltration of CBXs in colorectal cancer patients.Results: The expression levels of CBX1/2/3/4/5 and CBX8 were significantly elevated in CRC tissues, whereas the expression levels of CBX6/7 were reduced. CBX3 was significantly associated with the clinical cancer stage and short disease-free survival (DFS) in CRC patients. High mRNA expression of CBX5/6 was associated with short overall survival (OS) in rectal cancer patients. CBX3/5/6 could be potential prognostic biomarkers for the survival of CRC patients. Moreover, the functions of the differentially expressed CBXs were primarily related to the SUMOylation of DNA methylation proteins and chromatin organization and may regulate the pluripotency of stem cells. The expression of CBXs were significantly correlated with the infiltration of diverse immune cells, including six types of CD4+ T cells, macrophages, neutrophils, B cells, CD8+ T cells, and dendritic cells in colon cancers and rectal cancers.Conclusions: Our study may provide novel insights for the selection of prognostic biomarkers of CBX family in colorectal cancer.

Epigenetic Control of Osteoblast Differentiation by Chromobox 3 (Cbx3) Protein 3

Skeletal development is controlled by epigenetic mechanisms that regulate chromatin compaction and suppress access to gene regulatory sequences. Post‐translational modifications in the N‐termini of histone 3 (H3), including tri‐methylated H3 lysine 9 (H3K9Me3) or lysine 27 (H3K27Me3) represent repressive chromatin marks that are recognized by a family of heterochromatin‐associated Chromobox (Cbx) proteins (i.e., Cbx1 to Cbx8). We show using RNA‐seq analysis that each of these Cbx isoforms is actively expressed in bone and osteoblasts with variable expression of distinct members (between 1 and 100 reads per million). The most prominently expressed Cbx member is Cbx3, which encodes a member of the heterochromatin protein 1 (HP1) family that interacts with H3K9me3. Cbx3 is known to be important for normal development while promoting neurogenesis, as well as renal and smooth muscle development. Therefore, we postulated that CBx3 may perform a similar role in osteoblastogenesis. We tested this hypothesis by both transient and stable knock‐down of Cbx3 by respectively siRNA or shRNA. RNA interference in MC3T3 osteoblasts effectively decreases Cbx3 mRNA levels by 2 to 5 fold, while increasing expression of bone‐related markers by RT‐qPCR (e.g., Alpl/alkaline phosphatase, Ibsp/bone sialoprotein, Bglap/osteocalcin, and Col1a1/Collagen Type I α1 chain). In addition, Cbx3 depletion increases mineral deposition as measured by Alizarin Red staining. Thus, Cbx3 normally appears to suppress osteoblastic differentiation, unlike its essential function in the neuronal differentiation. Because Cbx3 recognizes H3K9me3, we examined whether Cbx3 controls the levels of this histone mark. Loss of Cbx3 did not affect total H3K9me3 levels unless Cbx1 and Cbx5 were simultaneously depleted by siRNA. We propose that Cbx3 may suppress osteoblastogenesis by controlling select loci with H3K9me3 marks.

CBX4 transcriptionally suppresses KLF6 via interaction with HDAC1 to exert oncogenic activities in clear cell renal cell carcinoma.

BackgroundDysregulation of polycomb chromobox (CBX) proteins that mediate epigenetic gene silencing contributes to the progression of human cancers. Yet their roles in clear cell renal cell carcinoma (ccRCC) remain to be explored.MethodsThe expression of CBX4 and its clinical significance were determined by qRT-PCR, western blot, immunohistochemistry and statistical analyses. The biological function of CBX4 in ccRCC tumor growth and metastasis and the underlying mechanism were investigated using in vitro and in vivo models.FindingsCBX4 exerts oncogenic activities in ccRCC via interaction with HDAC1 to transcriptionally suppress tumor suppressor KLF6. CBX4 expression is increased in ccRCC and correlated with poor prognosis in two independent cohorts containing 840 patients. High CBX4 expression is significantly associated with Fuhrman grade and tumor lymph node invasion. CBX4 overexpression promotes tumor growth and metastasis, whereas CBX4 knockdown results in the opposite phenotypes. Mechanistically, CBX4 downregulates KLF6 via repressing the transcriptional activity of its promoter. Further studies show that CBX4 physically binds to HDAC1 to maintain its localization on the KLF6 promoter. Ectopic expression of KLF6 or disruption of CBX4-HDAC1 interaction attenuates CBX4-mediated cell growth and migration. Furthermore, CBX4 depletion markedly enhances the histone deacetylase inhibitor (HDACi)-induced cell apoptosis and suppression of tumor growth.InterpretationOur data suggest CBX4 as an oncogene with prognostic potential in ccRCC. The newly identified CBX4/HDAC1/KLF6 axis may represent a potential therapeutic target for the clinical intervention of ccRCC.

Transcriptional expressions of Chromobox 1/2/3/6/8 as independent indicators for survivals in hepatocellular carcinoma patients.

Chromobox (CBX) proteins are important components of epigenetic regulation complexes known to play key roles in hepatocellular carcinoma (HCC). Little is known about the function of distinct CBXs in HCC. To address this issue, the study investigated the roles of CBXs in the prognosis of HCC using ONCOMINE, UALCAN, Human Protein Atlas, Kaplan-Meier Plotter, c-BioPortal databases. Over expressions of 8 CBXs members were found to be significantly associated with clinical cancer stages and pathological tumor grades in HCC patients. Besides, higher mRNA expressions of CBX1/2/3/6/8 were found to be significantly associated with shorter overall survival (OS) in HCC patients, while higher mRNA expression of CBX7 was associated with favorable OS. Multivariate analysis also showed that high mRNA expressions of CBX1/2/3/6/8 were independent prognostic factors for shorter OS of HCC patients. Moreover, high mutation rate of CBXs (51%) was also observed in HCC patients, and genetic alteration in CBXs was associated with shorter OS and disease-free survival (DFS) in HCC patients. Taken together, these results indicated that CBX1/2/3/6/8 could be prognostic biomarkers for survivals of HCC patients.

Polycomb chromobox Cbx2 enhances antiviral innate immunity by promoting Jmjd3-mediated demethylation of H3K27 at the Ifnb promoter

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-β. 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-β. Together, our study reveals a non-traditional function of a Cbx protein and adds new insight into the epigenetic regulation of antiviral innate immunity.