Fluorescent Reporter Systems to Investigate Chromatin Effector Proteins in Living Cells.

Epigenetic research faces the challenge of the high complexity and tight regulation in chromatin modification networks. Although many isolated mechanisms of chromatin-mediated gene regulation have been described, solid approaches for the comprehensive analysis of specific processes as parts of the bigger epigenome network are missing. In order to expand the toolbox of methods by a system that will help to capture and describe the complexity of transcriptional regulation, we describe here a robust protocol for the generation of stable reporter systems for transcriptional activity and summarize their applications. The system allows for the induced recruitment of a chromatin regulator to a fluorescent reporter gene, followed by the detection of transcriptional changes using flow cytometry. The reporter gene is integrated into an endogenous chromatin environment, thus enabling the detection of regulatory dependencies of the investigated chromatin regulator on endogenous cofactors. The system allows for an easy and dynamic readout at the single-cell level and the ability to compensate for cell-to-cell variances of transcription. The modular design of the system enables the simple adjustment of the method for the investigation of different chromatin regulators in a broad panel of cell lines. We also summarize applications of this technology to characterize the silencing velocity of different chromatin effectors, removal of activating histone modifications, analysis of stability and reversibility of epigenome modifications, the investigation of the effects of small molecule on chromatin effectors and of functional effector-coregulator relationships. The presented method allows to investigate the complexity of transcriptional regulation by epigenetic effector proteins in living cells.

CBX3 antagonizes IFNγ/STAT1/PD-L1 axis to modulate colon inflammation and CRC chemosensitivity.

As an important immune stimulator and modulator, IFNγ is crucial for gut homeostasis and its dysregulation links to diverse colon pathologies, such as colitis and colorectal cancer (CRC). Here, we demonstrated that the epigenetic regulator, CBX3 (also known as HP1γ) antagonizes IFNγ signaling in the colon epithelium by transcriptionally repressing two critical IFNγ-responsive genes: STAT1 and CD274 (encoding Programmed death-ligand 1, PD-L1). Accordingly, CBX3 deletion resulted in chronic mouse colon inflammation, accompanied by upregulated STAT1 and CD274 expressions. Chromatin immunoprecipitation indicated that CBX3 tethers to STAT1 and CD274 promoters to inhibit their expression. Reversely, IFNγ significantly reduces CBX3 binding to these promoters and primes gene expression. This antagonist effect between CBX3 and IFNγ on STAT1/PD-L1 expression was also observed in CRC. Strikingly, CBX3 deletion heightened CRC cells sensitivity to IFNγ, which ultimately enhanced their chemosensitivity under IFNγ stimulation in vitro with CRC cells and in vivo with a syngeneic mouse tumor model. Overall, this work reveals that by negatively tuning IFNγ-stimulated immune genes' transcription, CBX3 participates in modulating colon inflammatory response and CRC chemo-resistance.

MicroRNA­1224 inhibits cell proliferation by downregulating CBX3 expression in chordoma.

MicroRNAs (miRNAs/miRs) have abnormal expression in numerous tumors and are closely related to tumor development and resistance to radiotherapy and chemotherapy. However, there are few studies assessing the role and mechanism of miRNA in chordoma. The sequencing data of three pairs of chordoma and notochord tissues from the GSE56183 dataset were analyzed in the present study. Cell proliferation was assessed in vitro using Cell Counting Kit-8. Bioinformatics analysis and the dual luciferase reporter assay were used to evaluate the regulatory relationship between miR-1224 and chromobox 3 (CBX3) in chordoma. The results demonstrated that miR-1224 had a significantly lower expression level in chordoma tissues and cell lines. Overexpression of miR-1224 inhibited proliferation in the chordoma cells, while the knockdown of miR-1224 promoted proliferation of the chordoma cells. Bioinformatics analysis and the dual luciferase reporter assay confirmed that CBX3 was a direct target gene of miR-1224 and that miR-1224 induced the proliferation of chordoma cells through the inhibition of CBX3. In summary, miR-1224 reduced the proliferation of chordoma cells through inhibition of CBX3, which provides a theoretical basis for selecting a novel therapeutic target for chordoma.

m5C modification of LINC00324 promotes angiogenesis in glioma through CBX3/VEGFR2 pathway.

Angiogenesis plays a major role in tumor initiation, progression, and metastasis. This is why finding antiangiogenic targets is essential in the treatment of gliomas. In this study, NSUN2 and LINC00324 were significantly upregulated in conditionally cultured glioblastoma endothelial cells (GECs). Knockdown of NSUN2 or LINC00324 inhibits GECs angiogenesis. NSUN2 increased the stability of LINC00324 by m5C modification and upregulated LINC00324 expression. LINC00324 competes with the 3'UTR of CBX3 mRNA to bind to AUH protein, reducing the degradation of CBX3 mRNA. In addition, CBX3 directly binds to the promoter region of VEGFR2, enhances VEGFR2 transcription, and promotes GECs angiogenesis. These findings demonstrated NSUN2/LINC00324/CBX3 axis plays a crucial role in regulating glioma angiogenesis, which provides new strategies for glioma therapy.

Targeting CBX3 with a Dual BET/PLK1 Inhibitor Enhances the Antitumor Efficacy of CDK4/6 Inhibitors in Prostate Cancer.

The development of castration-resistant prostate cancer (CRPC) is a significant factor that reduces life expectancy among patients with prostate cancer. Previously, it is reported that CDK4/6 inhibitors can overcome the resistance of CRPC to BET inhibitors by destabilizing BRD4, suggesting that the combination of CDK4/6 inhibitors and BET inhibitors is a promising approach for treating CRPC. In this study, candidates that affect the combined antitumor effect of CDK4/6 inhibitors and BET inhibitors on CRPC is aimed to examine. The data demonstrates that CBX3 is abnormally upregulated in CDK4/6 inhibitors-resistant cells. CBX3 is almost positively correlated with the cell cycle in multiple malignancies and is downregulated by BET inhibitors. Mechanistically, it is showed that CBX3 is transcriptionally upregulated by BRD4 in CRPC cells. Moreover, it is demonstrated that CBX3 modulated the sensitivity of CRPC to CDK4/6 inhibitors by binding with RB1 to release E2F1. Furthermore, it is revealed that PLK1 phosphorylated CBX3 to enhance the interaction between RB1 and CBX3, and desensitize CRPC cells to CDK4/6 inhibitors. Given that BRD4 regulates CBX3 expression and PLK1 affects the binding between RB1 and CBX3, it is proposed that a dual BRD4/PLK1 inhibitor can increase the sensitivity of CRPC cells to CDK4/6 inhibitors partially through CBX3.

CBX3 promotes clear cell renal carcinoma through PI3K/AKT activation and aberrant immunity.

BACKGROUND: A chromobox homologue 3 (CBX3) is elevated in various cancers and significantly contributes to the promotion of malignant behavior; despite this, its exact involvement in clear cell renal cell carcinoma (ccRCC) is yet unknown. METHODS: The Cancer Genome Atlas database served to evaluate CBX3 production and its connection to survival in patients with ccRCC. Our team evaluated the effects of knockdown of CBX3 levels in ccRCC cell populations using in vitro together with in vivo models. CBX3, proteins related to death, and epithelial-to-mesenchymal transition (EMT)-related proteins were measured in ccRCC cells using western blotting and immunohistochemical assays. Through the analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) and GeneOntology (GO) and Gene Set Enrichment Analysis (GSEA), the biological processes and signal pathways related to CBX3 expression were identified. Immune-related activity reduced by CBX3 was assessed using various online tools. RESULTS: Both genomic and protein expression showed that CBX3 was upregulated in ccRCC. Further functional analyses revealed that CBX3 played a crucial role in enhancing cell growth, migration, and EMT in vitro along with in vivo. Moreover, the study results provided distinct mechanistic evidence that CBX3 exerts its pathological functions in ccRCC by activating the PI3K/AKT pathway. Finally, immunoassays revealed that CBX3, a possible biomarker of ccRCC, was significantly associated with immunity. CONCLUSIONS: Our results suggest that the overexpression of CBX3 promotes ccRCC advancement through PI3K/AKT activation and even immunological dysregulation, making it a potentially viable and beneficial therapeutic target.

PSMC4 promotes prostate carcinoma progression by regulating the CBX3-EGFR-PI3K-AKT-mTOR pathway.

Proteasome 26S subunit ATPase 4 (PSMC4) could regulate cancer progression. However, the function of PSMC4 in prostate carcinoma (PCa) progression requires further clarification. In the study, PSMC4 and chromobox 3 (CBX3) levels were verified by TCGA data and tissue microarrays. Cell counting kit-8, cell apoptosis, cell cycle, wound healing, transwell and xenograft tumour model assays were performed to verify biological functions of PSMC4 in PCa. RNA-seq, PCR, western blotting and co-IP assays were performed to verify the mechanism of PSMC4. Results showed that PSMC4 level was significantly increased in PCa tissues, and patients with PCa with a high PSMC4 level exhibited shorter overall survival. PSMC4 knockdown markedly inhibited cell proliferation, cell cycle and migration in vitro and in vivo, and significantly promoted cell apoptosis. Then further study revealed that CBX3 was a downstream target of PSMC4. PSMC4 knockdown markedly reduced CBX3 level, and inhibited PI3K-AKT-mTOR signalling. CBX3 overexpression markedly promoted epidermal growth factor receptor (EGFR) level. Finally, PSMC4 overexpression showed reverse effect in DU145 cells, and the effects of PSMC4 overexpression on cell proliferation, migration and clonal formation were rescued by the CBX3 knockdown, and regulated EGFR-PI3K-AKT-mTOR signalling. In conclusion, PSMC4 could regulate the PCa progression by mediating the CBX3-EGFR-PI3K-AKT-mTOR pathway. These findings provided a new target for PCa treatment.

NCAPG is transcriptionally regulated by CBX3 and activates the Wnt/ß-catenin signaling pathway to promote proliferation and the cell cycle and inhibit apoptosis in colorectal cancer.

Background: Colorectal cancer (CRC) is highly heterogeneous at the genetic and molecular level and a major contributor to cancer-death worldwide. Non-structural maintenance of chromosomes (SMC) condensin I complex subunit G (NCAPG) is a subunit of condensin I and has been shown to be associated with the prognosis of cancers. This study investigated the functional role of NCAPG in CRC and its mechanism. Methods: Messenger RNA (mRNA) and protein expressions of NCAPG and chromobox protein homolog 3 (CBX3) were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot. The proliferation, cycle, and apoptosis of HCT116 cells were analyzed by Cell Counting Kit-8 (CCK-8), flow cytometry, and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. RT-qPCR and western blot were used to determine the transfection efficacy of short hairpin (sh)-NCAPG and sh-CBX3. Western blot was used to explore cycle-, apoptosis-, and Wnt/ß-catenin signaling-related proteins, and the activity of NCAPG promoter was evaluated using a luciferase report assay. The expressions of cleaved caspase9 and cleaved caspase3 were assessed by colorimetric caspase activity assay. Results: The results showed that NCAPG expression was elevated in CRC cells. After transfection with sh-NCAPG, NCAPG expression was reduced. It was also discovered that NCAPG knockdown suppressed proliferation and the cell cycle but induced apoptosis in HCT116 cells. The Human Transcription Factor Database (HumanTFDB; http://bioinfo.life.hust.edu.cn/HumanTFDB#!/) predicted the binding sites of CBX3 and NCAPG promoters. Meanwhile, the Encyclopedia of RNA Interactomes (ENCORI) database (https://starbase.sysu.edu.cn/) revealed that CBX3 was positively correlated with NCAPG. Our results showed that NCAPG was transcriptionally regulated by CBX3. Additionally, Wnt/ß-catenin signaling was discovered to be activated by CBX3 overexpression. Further experiments showed that NCAPG transcriptionally regulated by CBX3 activated Wnt/ß-catenin signaling to regulate the proliferation, cell cycle, and apoptosis of HCT116 cells. Conclusions: Collectively, the results of our study indicated that NCAPG was transcriptionally regulated by CBX3 and activated the Wnt/ß-catenin signaling pathway to facilitate the progression of CRC.

CBX3 Regulated By YBX1 Promotes Smoking-induced Pancreatic Cancer Progression via Inhibiting SMURF2 Expression.

As a key reversible and heritable mechanism of transcriptional regulation, the epigenetic modification plays a crucial role in tumorigenesis. Of note, tobacco smoking induces epigenetic modifications to promote pancreatic cancer development. Chromobox protein homolog 3 (CBX3) acts as an epigenetic regulator, modulating gene expression of downstream targets via chromatin modifications. To date, the relationship between CBX3 and smoking in pancreatic cancer remains unknown. This study aimed to uncover the specific role and underlying mechanism of CBX3 in smoking-related pancreatic cancer. The bioinformatics analyses were conducted to identify CBX3 as a key player in tobacco-induced pancreatic cancer. The abnormal upregulation of CBX3 was associated with poor prognosis in pancreatic cancer patients. Moreover, cigarette smoke extract (CSE) exposure promoted the overexpression of Y-box-binding protein 1 (YBX1), which consequently led to upregulated CBX3 in pancreatic cancer cells. We also revealed that CBX3 enhanced pancreatic cancer progression, likely by inhibiting the expression of SMAD specific E3 ubiquitin protein ligase 2 (SMURF2) and promoting the activation of TGF-ß signaling. In summary, the YBX1/CBX3/SMURF2 signaling axis may be a promising therapeutic target in patients with smoking-related pancreatic cancer.

Analysis of Pan-Cancer Revealed the Immunological and Prognostic Potential of CBX3 in Human Tumors.

Chromobox protein homolog 3 (CBX3) has been recognized as a member of the heterochromatin protein 1 family and participate in transcriptional activation or inhibition, cell differentiation and growth. Despite more and more evidence shows that CBX3 has a critical function in the development of some tumors, no systematic extensive analysis of CBX3 has been reported. Thus, we intended to examine the prognostic significance of CBX3 in 33 tumors and investigate its potential immune function. We employed several bioinformatics methods to explore the potential carcinogenic impact of CBX3 premised on the data sets collected from tumor genome maps, human protein maps, cBioPortal, and genotype tissue expression. The approaches include assessing the link between CBX3 and prognosis of different tumors, immune cell infiltration, micro-satellite instability (MSI), DNA methylation, and tumor mutational burden (TMB). The outcomes illustrated that CBX3 was increasingly expressed in 29 tumors. Moreover, CBX3 exhibited a negative correlation with the prognosis of many tumors. The expression of CBX3 was linked to MSI in 12 tumors and TMB in 16 tumors. In 24 tumors, the expression of CBX3 was linked to DNA methylation. Moreover, the CBX3 expression exhibited a negative relationship with the infiltration level of the majority of immune cells, but showed a positive link to T gamma delta cells, central memory T cells, and T helper cells, especially when invading breast carcinoma, thymic carcinoma, colon carcinoma, cutaneous melanoma, endometrial carcinoma, and lung squamous carcinoma. Our research indicates that CBX3 might be used as a prognostic indicator for different malignant tumors due to its function in tumor genesis as well as tumor immunity.

CBX3 regulated by miR-139 promotes the development of HCC by regulating cell cycle progression.

Hepatocellular carcinoma (HCC), a major primary liver cancer, is one of the most lethal malignancies worldwide. Increasing evidence has demonstrated that chromobox protein homolog 3 (CBX3) functions as an oncogene in different cancers. However, its expression profiles and biological functions in HCC remain unknown. Data on CBX3 expression in HCC acquired from the GEO and TCGA databases were analyzed. The biological functions of CBX3 in HCC were examined by in vitro experiments. Bioinformatics analysis, qRT-PCR and western blotting were performed to explore the mechanism of CBX3 in HCC. CBX3 mRNA was upregulated in HCC tissues, and overexpression of CBX3 mRNA was negatively correlated with malignancies and poor prognosis in HCC patients. CBX3 knockdown decreased growth, migration and invasion of HCC cells in vitro. Moreover, bioinformatics analysis and experimental observation indicated that CBX3 expression was correlated with cell cycle regulatory proteins in HCC cells. Finally, starBase predicted that miR-139 could directly target CBX3 in HCC. Confirmatory experiments verified that miR-139 overexpression attenuated HCC cell proliferation and migration, and these effects could be reversed by overexpressing CBX3. Our results showed that the miR-139/CBX3 axis may be involved in HCC development by regulating cell cycle progression and may be a promising target in the treatment of HCC.

CBX3 is associated with metastasis and glutathione/glycosphingolipid metabolism in colon adenocarcinoma.

Background: Metastasis is the major cause of colon adenocarcinoma (COAD) mortality. Increasing studies demonstrated that the epigenetics and downstream expression change of pivotal genes may act as a major role in promoting COAD progression and metastasis. Therefore, identifying the dysregulation of key genes associating with COAD metastasis may provide a new strategy for the discovery of potential treatment targets. Methods: This study included a single-cell RNA sequencing profile consisting of 17,469 tumor cells derived from 23 samples, and 326 COADs available from The Cancer Genome Atlas (TCGA), etc. The study was performed using comparative analysis to characterize the role of CBX3 in COAD metastasis and progression. Results: This study revealed that the mRNA level of Chromebox homolog 3 (CBX3) in the metastatic COAD was significantly higher than that of the primary COAD and normal colon tissues (Wilcoxon's rank-sum test, P<0.05). Activation of CBX3 was involved in regulating an interaction network consisting of CCT6A, LSM5, and GGCT, etc., which may subsequently participate in glutathione metabolism. Besides, CBX3 also exhibited a negative correlation with glycosphingolipid metabolism, which may associate with the regulation of CBX3 on DNA methylation. Clinical data analysis demonstrated that patients with high CBX3 mRNA levels showed a nearly 2-fold shorter overall survival time than the control group (hazard ratio =1.59; likelihood ratio test, P=0.04). Conclusions: Our study demonstrated that CBX3 overexpression is associated with COAD metastasis. CBX3 downstream regulation network involves in TCP1 complex, LSM family, and glutathione metabolism, which may provide a potential target for suppressing tumor metastasis.

PΨFinder: a practical tool for the identification and visualization of novel pseudogenes in DNA sequencing data.

BACKGROUND: Processed pseudogenes (PΨgs) are disabled gene copies that are transcribed and may affect expression of paralogous genes. Moreover, their insertion in the genome can disrupt the structure or the regulatory region of a gene, affecting its expression level. These events have been identified as occurring mutations during cancer development, thus being able to identify PΨgs and their location will improve their impact on diagnostic testing, not only in cancer but also in inherited disorders. RESULTS: We have implemented PΨFinder (P-psy-finder), a tool that identifies PΨgs, annotates known ones and predicts their insertion site(s) in the genome. The tool screens alignment files and provides user-friendly summary reports and visualizations. To demonstrate its applicability, we scanned 218 DNA samples from patients screened for hereditary colorectal cancer. We detected 423 PΨgs distributed in 96% of the samples, comprising 7 different parent genes. Among these, we confirmed the well-known insertion site of the SMAD4-PΨg within the last intron of the SCAI gene in one sample. While for the ubiquitous CBX3-PΨg, present in 82.6% of the samples, we found it reversed inserted in the second intron of the C15ORF57 gene. CONCLUSIONS: PΨFinder is a tool that can automatically identify novel PΨgs from DNA sequencing data and determine their location in the genome with high sensitivity (95.92%). It generates high quality figures and tables that facilitate the interpretation of the results and can guide the experimental validation. PΨFinder is a complementary analysis to any mutational screening in the identification of disease-causing mutations within cancer and other diseases.

Comprehensive pan-cancer analysis on CBX3 as a prognostic and immunological biomarker.

BACKGROUND: Increased evidence supports the relationship between chromobox protein homolog 3 (CBX3) and tumorigenesis of some cancers. However, the role of CBX3 in pan-cancers remains poorly defined. In the research, we aimed to investigate the prognostic value and the immunological functions of CBX3. RESULTS: We explored the potential oncogenic roles of CBX3 in mRNA and protein levels based on the diverse databases, including the expression, the correlation with prognosis, tumor microenvironment (TME), DNA methylation, protein phosphorylation and enrichment analysis across all TCGA tumors. The results show that CBX3 is overexpressed in multiple cancers, and significant correlations exist between high expression and adverse prognosis in most tumor patients. We observed an enhanced phosphorylation level in uterine corpus endometrial carcinoma, colon cancer and lung adenocarcinoma. A distinct relationship was also found between CBX3 expression and TME, including immune infiltration of tumor-infiltrating lymphocytes and cancer-associated fibroblasts, immune score or matrix score, immune checkpoints. The correlative transcription factors and miRNAs of CBX3-binding hub genes were analyzed to investigate the molecular mechanism. Moreover, alcoholism and alteration of DNA cellular biology may be involved in the functional mechanisms of CBX3. CONCLUSION: The first pan-cancer study offers a relatively comprehensive cognition on the oncogenic roles of CBX3 as a prognostic and immunological marker in various malignant tumors.

Clinicopathological significance of CBX3 in colorectal cancer: An intensive expression study based on formalin-fixed and paraffin-embedded tissues.

CBX3 is an isoform of the heterochromatin protein 1 family, which is involved in carcinogenesis and promotes the progression of certain types of cancer. The expression level and clinicopathological significances of CBX3 in colorectal cancer (CRC) are still not well reported. In this study, we examined CBX3 protein expression in formalin-fixed and paraffin-embedded normal mucosae, hyperplastic polyps, low-and high-grade adenomas, and CRC tissue samples using immunohistochemistry. The associations of CBX3 expression levels with clinicopathological parameters, mismatch repair (MMR) protein expression, and kirsten rat sarcoma viral oncogene homolog (KRAS) and B-raf proto-oncogene (BRAF) mutations were analyzed. Our results showed that CBX3 protein was negatively expressed in normal mucosae and hyperplastic polyps, as well as in most low-grade adenomas. Interestingly, CBX3 protein was positively expressed in most high-grade adenomas and CRC tissues. CBX3 expression level was associated with tumor differentiation (p = 0.012), lymph node metastasis (p = 0.024), TNM stage (p = 0.008) and survival (p = 0.029). CBX3 expression was associated with MMR protein expression (p = 0.011) and KRAS mutation (p = 0.013), but not with BRAF mutation (p = 0.097). Our data suggest that CBX3 may be used as a molecular marker in CRC to evaluate tumor differentiation, lymph node metastasis, and pathological stage.

Targeting Cbx3/HP1γ Induces LEF-1 and IL-21R to Promote Tumor-Infiltrating CD8 T-Cell Persistence.

Immune checkpoint blockade (ICB) relieves CD8+ T-cell exhaustion in most mutated tumors, and TCF-1 is implicated in converting progenitor exhausted cells to functional effector cells. However, identifying mechanisms that can prevent functional senescence and potentiate CD8+ T-cell persistence for ICB non-responsive and resistant tumors remains elusive. We demonstrate that targeting Cbx3/HP1γ in CD8+ T cells augments transcription initiation and chromatin remodeling leading to increased transcriptional activity at Lef1 and Il21r. LEF-1 and IL-21R are necessary for Cbx3/HP1γ-deficient CD8+ effector T cells to persist and control ovarian cancer, melanoma, and neuroblastoma in preclinical models. The enhanced persistence of Cbx3/HP1γ-deficient CD8+ T cells facilitates remodeling of the tumor chemokine/receptor landscape ensuring their optimal invasion at the expense of CD4+ Tregs. Thus, CD8+ T cells heightened effector function consequent to Cbx3/HP1γ deficiency may be distinct from functional reactivation by ICB, implicating Cbx3/HP1γ as a viable cancer T-cell-based therapy target for ICB resistant, non-responsive solid tumors.

HP1γ binding pre-mRNA intronic repeats modulates RNA splicing decisions.

HP1 proteins are best known as markers of heterochromatin and gene silencing. Yet, they are also RNA-binding proteins and the HP1γ/CBX3 family member is present on transcribed genes together with RNA polymerase II, where it regulates co-transcriptional processes such as alternative splicing. To gain insight in the role of the RNA-binding activity of HP1γ in transcriptionally active chromatin, we have captured and analysed RNAs associated with this protein. We find that HP1γ is specifically targeted to hexameric RNA motifs and coincidentally transposable elements of the SINE family. As these elements are abundant in introns, while essentially absent from exons, the HP1γ RNA association tethers unspliced pre-mRNA to chromatin via the intronic regions and limits the usage of intronic cryptic splice sites. Thus, our data unveil novel determinants in the relationship between chromatin and co-transcriptional splicing.

The LncRNA RP11-279C4.1 Enhances the Malignant Behaviour of Glioma Cells and Glioma Stem-Like Cells by Regulating the miR-1273g-3p/CBX3 Axis.

Glioma is the most common type of solid tumour affecting the central nervous system, and the survival rate of patients with glioma is low. However, the mechanism associated with glioma progression remains unclear. Growing evidence suggests that lncRNAs play essential roles in the initiation and progression of tumours, including gliomas. In the present study, we identified and verified the expression of the novel lncRNA RP11-279C4.1 by analyzing the TANRIC database and performing qRT-PCR assays, the results of which revealed its upregulation in glioma tissues and cell lines. The results of multiple functional experiments demonstrated that RP11-279C4.1 knockdown inhibited glioma malignant phenotypes, including cell proliferation, migration, invasion and cell self-renew ability in vitro. In addition, RP11-279C4.1 downregulation suppressed tumour growth in vivo. Mechanistically, RP11-279C4.1 induced CBX3 activation via competitively sponging miR-1273g-3p, and rescue assay results confirmed the importance of the RP11-279C4.1/miR-1273g-3p/CBX3 axis. Overall, the results of our present study demonstrated that RP11-279C4.1 functions as an oncogene that promotes tumour progression by modulating the miR-1273g-3p/CBX3 axis in glioma, suggesting that RP11-279C4.1 may be a novel therapeutic target for glioma.

Combined genomic and proteomic approaches reveal DNA binding sites and interaction partners of TBX2 in the developing lung.

BACKGROUND: Tbx2 encodes a transcriptional repressor implicated in the development of numerous organs in mouse. During lung development TBX2 maintains the proliferation of mesenchymal progenitors, and hence, epithelial proliferation and branching morphogenesis. The pro-proliferative function was traced to direct repression of the cell-cycle inhibitor genes Cdkn1a and Cdkn1b, as well as of genes encoding WNT antagonists, Frzb and Shisa3, to increase pro-proliferative WNT signaling. Despite these important molecular insights, we still lack knowledge of the DNA occupancy of TBX2 in the genome, and of the protein interaction partners involved in transcriptional repression of target genes. METHODS: We used chromatin immunoprecipitation (ChIP)-sequencing and expression analyses to identify genomic DNA-binding sites and transcription units directly regulated by TBX2 in the developing lung. Moreover, we purified TBX2 containing protein complexes from embryonic lung tissue and identified potential interaction partners by subsequent liquid chromatography/mass spectrometry. The interaction with candidate proteins was validated by immunofluorescence, proximity ligation and individual co-immunoprecipitation analyses. RESULTS: We identified Il33 and Ccn4 as additional direct target genes of TBX2 in the pulmonary mesenchyme. Analyzing TBX2 occupancy data unveiled the enrichment of five consensus sequences, three of which match T-box binding elements. The remaining two correspond to a high mobility group (HMG)-box and a homeobox consensus sequence motif. We found and validated binding of TBX2 to the HMG-box transcription factor HMGB2 and the homeobox transcription factor PBX1, to the heterochromatin protein CBX3, and to various members of the nucleosome remodeling and deacetylase (NuRD) chromatin remodeling complex including HDAC1, HDAC2 and CHD4. CONCLUSION: Our data suggest that TBX2 interacts with homeobox and HMG-box transcription factors as well as with the NuRD chromatin remodeling complex to repress transcription of anti-proliferative genes in the pulmonary mesenchyme.

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.