Zinc Finger And BTB Domain Containing 16 Research Articles

Identification of E3 ubiquitin ligase-associated prognostic genes and construction of a prediction model for uterine cervical cancer based on bioinformatics analysis.

E3 ligases are engaged in a variety of physiological processes within cells and use ubiquitin-labeled substrates to control their activity and stability. Although some research has indicated that E3 ligases or particular substrates have an impact on the treatment that cervical cancer patients get after their diagnosis, The exact purpose of these enzymes in the occurrence and evolution of cancer of the cervical region (CC) is not clear. In order to extract and analyze relevant mRNA gene expression data as well as clinical patient data, we used open databases. A reliable risk prediction model was developed by applying the least absolute shrinkage and selection operator (LASSO) technique in conjunction with Cox regression analysis. Column-line plots were combined to analyze the predictive model, and the GSE44001 dataset served as an external validation.Four gene models:proteasome (prosome, macropain) 26S subunit, non-ATPase, 14(PSMD14),proteasome (prosome, macropain) subunit, alpha type, 4(PSMA4,),zinc finger and BTB domain containing 16(ZBTB16),and ankyrin repeat domain 9(ANKRD9). Gene expression levels in both healthy and cancerous tissues have been confirmed by the HPA database. Next, the investigation focused on immunological state and tumor mutation load. The high-risk group and Cluster B had distinct levels of immune cell infiltration and a worse prognosis. Additionally, KEGG and GO analyses of differentially expressed genes (DEGs) between the high- and low-risk groups were performed, as well as tumor microenvironment (TME) investigations. Targeting E3 ligases may be an efficient strategy to treat cervical cancer (CC), according to a novel and comprehensive E3 ubiquitination ligase-associated gene model that has been presented.

Long non-coding RNA LINC00930 targeting miR-6792-3p/ZBTB16 regulates the proliferation and EMT of pancreatic cancer

Emerging evidence suggests the dysregulation of long non-coding RNAs (lncRNAs) involved in pancreatic cancer (PC). However, the function of LINC00930 in PC has not been elaborated. In this study, we found that LINC00930 was significantly down-regulated in PC cell lines and tissues, and associated with tumor size, lymphatic metastasis, TNM stage and poor prognosis. According to the bioinformatics database, the downregulation of LINC00930 was a common event in PC associated with prognosis and EMT. Overexpression of LINC00930 inhibited the aggressive cancer phenotypes including proliferation, metastasis and epithelial-mesenchymal transition (EMT) of PC in vitro and in vivo. Bioinformatics and dual-luciferase reporter assay indicated that miR-6792-3p could directly bind to LINC00930. Additionally, the Zinc finger and BTB domain containing 16 (ZBTB16) was significantly declined in PC, which was predicted to be the downstream gene of miR-6792-3p. MiR-6792-3p mimic rescued the decreased proliferation, metastasis and EMT caused by ZBTB16 in PC cells. The LINC00930/miR-6792-3p/ZBTB16 axis was associated with the malignant progression and process of PC. The relative expression of LINC00930 was negatively correlated with the expression of miR-6792-3p and was closely linked with ZBTB16 levels in PC. LINC00930 might serve as a potential prognostic biomarker and therapeutic target for PC.

Enhanced ZBTB16 Levels by Progestin-Only Contraceptives Induces Decidualization and Inflammation.

Progestin-only long-acting reversible-contraceptive (pLARC)-exposed endometria displays decidualized human endometrial stromal cells (HESCs) and hyperdilated thin-walled fragile microvessels. The combination of fragile microvessels and enhanced tissue factor levels in decidualized HESCs generates excess thrombin, which contributes to abnormal uterine bleeding (AUB) by inducing inflammation, aberrant angiogenesis, and proteolysis. The- zinc finger and BTB domain containing 16 (ZBTB16) has been reported as an essential regulator of decidualization. Microarray studies have demonstrated that ZBTB16 levels are induced by medroxyprogesterone acetate (MPA) and etonogestrel (ETO) in cultured HESCs. We hypothesized that pLARC-induced ZBTB16 expression contributes to HESC decidualization, whereas prolonged enhancement of ZBTB16 levels triggers an inflammatory milieu by inducing pro-inflammatory gene expression and tissue-factor-mediated thrombin generation in decidualized HESCs. Thus, ZBTB16 immunostaining was performed in paired endometria from pre- and post-depo-MPA (DMPA)-administrated women and oophorectomized guinea pigs exposed to the vehicle, estradiol (E2), MPA, or E2 + MPA. The effect of progestins including MPA, ETO, and levonorgestrel (LNG) and estradiol + MPA + cyclic-AMP (E2 + MPA + cAMP) on ZBTB16 levels were measured in HESC cultures by qPCR and immunoblotting. The regulation of ZBTB16 levels by MPA was evaluated in glucocorticoid-receptor-silenced HESC cultures. ZBTB16 was overexpressed in cultured HESCs for 72 h followed by a ± 1 IU/mL thrombin treatment for 6 h. DMPA administration in women and MPA treatment in guinea pigs enhanced ZBTB16 immunostaining in endometrial stromal and glandular epithelial cells. The in vitro findings indicated that: (1) ZBTB16 levels were significantly elevated by all progestin treatments; (2) MPA exerted the greatest effect on ZBTB16 levels; (3) MPA-induced ZBTB16 expression was inhibited in glucocorticoid-receptor-silenced HESCs. Moreover, ZBTB16 overexpression in HESCs significantly enhanced prolactin (PRL), insulin-like growth factor binding protein 1 (IGFBP1), and tissue factor (F3) levels. Thrombin-induced interleukin 8 (IL-8) and prostaglandin-endoperoxide synthase 2 (PTGS2) mRNA levels in control-vector-transfected HESCs were further increased by ZBTB16 overexpression. In conclusion, these results supported that ZBTB16 is enhanced during decidualization, and long-term induction of ZBTB16 expression by pLARCs contributes to thrombin generation through enhancing tissue factor expression and inflammation by enhancing IL-8 and PTGS2 levels in decidualized HESCs.

ZBTB16 eases lipopolysaccharide‑elicited inflammation, apoptosis and degradation of extracellular matrix in chondrocytes during osteoarthritis by suppressing GRK2 transcription.

Osteoarthritis (OA) is a chronic degenerative disease of the bone that is a major contributor of disability in the elderly population. Zinc finger and BTB domain-containing 16 (ZBTB16) is a transcription factor that has been previously revealed to be impaired in human OA tissues. The present study was designed to elaborate the potential impact of ZBTB16 on OA and to possibly assess any latent regulatory mechanism. ZBTB16 expression in human OA tissues was examined using the Gene Expression Series (GSE) database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE169077) whereas ZBTB16 expression in chondrocytes was examined using reverse transcription-quantitative PCR (RT-qPCR) and western blotting. Cell viability was examined using a Cell Counting Kit-8 assay. A TUNEL assay and western blotting were used to assess cell apoptosis and apoptosis-related markers, including Bcl-2, Bax and cleaved caspase-3. The levels and expression of inflammatory factors, including TNF-α, IL-1β and IL-6, were determined by ELISA and western blotting. RT-qPCR and western blotting were also used to analyze the expression levels of extracellular matrix (ECM)-degrading enzymes, including MMP-13, a disintegrin-like and metalloproteinase with thrombospondin type-1 motifs-5, aggrecan and collagen type II α1. After the potential binding of ZBTB16 with the G protein coupled receptor kinase type 2 (GRK2) promoter was predicted using the Cistrome DB database, GRK2 expression was confirmed by RT-qPCR and western blotting. Chromatin immunoprecipitation and luciferase reporter assays were then used to determine the potential interaction between ZBTB16 and the GRK2 promoter. Following GRK2 overexpression in ZBTB16-overexpressing chondrocytes by co-transfection of GRK2 and ZBTB16 overexpression plasmids, the aforementioned functional experiments were performed again. ZBTB16 expression was found to be reduced in human OA tissues compared with in normal cartilage tissues and lipopolysaccharide (LPS)-stimulated chondrocytes. ZBTB16 overexpression increased cell viability whilst decreasing apoptosis, inflammation and ECM degradation by LPS-treated chondrocytes. In addition, GRK2 expression was found to be increased in LPS-stimulated chondrocytes. ZBTB16 successfully bound to the GRK2 promoter, which negatively modulated GRK2 expression. GRK2 upregulation reversed the effects of ZBTB16 overexpression on the viability, apoptosis, inflammation and ECM degradation by LPS-challenged chondrocytes. In conclusion, these data suggest that ZBTB16 may inhibit the development of OA through the transcriptional inactivation of GRK2.

GCN5 regulates ZBTB16 through acetylation, mediates osteogenic differentiation, and affects orthodontic tooth movement.

In the process of orthodontic tooth movement (OTM), periodontal ligament fibroblasts (PDLFs) must undergo osteogenic differentiation. OTM increased the expression of Zinc finger and BTB domain-containing 16 (ZBTB16), which is implicated in osteogenic differentiation. Our goal was to investigate the mechanism of PDLF osteogenic differentiation mediated by ZBTB16. The OTM rat model was established, and PDLFs were isolated and exposed to mechanical force. Hematoxylin-eosin staining, Alizarin Red staining, immunofluorescence, and immunohistochemistry were carried out. The alkaline phosphatase (ALP) activity was measured. Dual-luciferase reporter gene assay and chromatin immunoprecipitation assay were conducted. In OTM models, ZBTB16 was significantly expressed. Additionally, there was an uneven distribution of PDLFs in the OTM group, as well as an increase in fibroblasts and inflammatory infiltration. ZBTB16 interference hindered PDLF osteogenic differentiation and decreased Wnt and β-catenin levels. Meanwhile, ZBTB16 activated the Wnt/β-catenin pathway. ZBTB16 also enhanced the expression of the osteogenic molecules osterix, osteocalcin (OCN), osteopontin (OPN), and bone sialo protein (BSP) at mRNA and protein levels. The interactions between Wnt1 and ZBTB16, as well as GCN5 and ZBTB16, were also verified. The adeno-associated virus-shZBTB16 injection also proved to inhibit osteogenic differentiation and reduce tooth movement distance in in vivo tests. ZBTB16 was up-regulated in OTM. Through acetylation modification of ZBTB16, GCN5 regulated the Wnt/β-catenin signaling pathway and further mediated PDLF osteogenic differentiation.

The role of ZBTB16 in odontogenic differentiation of dental pulp stem cells

ObjectiveOdontogenic differentiation of dental pulp stem cells (DPSCs) is highly controlled by the activation of several transcription factors. The zinc finger and BTB domain-containing 16 (ZBTB16) gene encodes a BTB/POZ domain and zinc finger containing transcription factors and is involved in several biological processes, but little is known about its role in odontogenic differentiation. The main goal of the current study was to determine the role of ZBTB16 in odontogenic differentiation of DPSCs. DesignZBTB16, runt-related transcription factor 2 (RUNX2), and osterix (OSX) were silenced via small-hairpin RNA (shRNA) lentivirus. The odontoblastic differentiation of DPSCs was detected by alkaline phosphatase (ALP) staining, activity measurement, and alizarin red S staining in vitro. The gene and protein expression levels were assessed by RT-qPCR and western blotting. Further, an ectopic implantation experiment was performed to explore the role of ZBTB16 in mineralization regulation in vivo followed by histological examination. ResultsThe silencing of ZBTB16 attenuated ALP activity and mineralized nodules formation by DPSCs. In addition, knockdown of ZBTB16 impaired the expression of markers involved in odontogenic differentiation, including dentin sialophosphoprotein, dentin matrix acidic phosphoprotein 1, and collagen 1 in vitro and vivo. Silencing the OSX gene suppressed ZBTB16 expression and, in turn, OSX expression decreased after ZBTB16 knockdown. However, shRUNX2 did not suppress ZBTB16 expression and shZBTB16 did not affect RUNX2 expression. ConclusionsZBTB16 may play an important role in modulating the odontoblastic differentiation of DPSCs and act as a regulator of OSX in a possible feed-back cycle independent of RUNX2.

Suppressive role of E3 ubiquitin ligase FBW7 in type I diabetes in non-obese diabetic mice through mediation of ubiquitination of EZH2

The current study tried to uncover the molecular mechanism of E3 ubiquitin ligase F-box and WD repeat domain-containing 7 (FBW7) in a heritable autoimmune disease, type I diabetes (T1D). After streptozotocin-induced T1D model establishment in non-obese diabetic (NOD) mouse, the protein expression of FBW7, enhancer of zeste homolog 2 (EZH2), and Zinc finger and BTB domain containing 16 (ZBTB16) was quantified. Next, splenocytes and pancreatic beta cells were isolated to measure the production of pro-inflammatory cytokines in splenocytes, as well as islet beta-cell apoptosis. Additionally, the stability of EZH2 induced by FBW7 was analyzed by cycloheximide chase assay. The binding affinity of FBW7 and EZH2 and the consequence of ubiquitination were monitored by co-immunoprecipitation assay. Last, a chromatin immunoprecipitation assay was employed to analyze the accumulation of EZH2 and H3K27me3 at the ZBTB16 promoter region. Our study demonstrated downregulated FBW7 and ZBTB16 and upregulated EZH2 in diabetic NOD mice. Overexpression of FBW7 in the NOD mice inhibited pro-inflammatory cytokine release in the splenocytes and the apoptosis of islets beta cells. FBW7 destabilized EZH2 and accelerated ubiquitin-dependent degradation. EZH2 and H3K27me3 downregulated the ZBTB16 expression by accumulating in the ZBTB16 promoter and methylation. FBW7 upregulates the expression of ZBTB16 by targeting histone methyltransferase EZH2 thus reducing the occurrence of T1D.

Leveraging Northern European population history: novel low-frequency variants for polycystic ovary syndrome.

STUDY QUESTIONCan we identify novel variants associated with polycystic ovary syndrome (PCOS) by leveraging the unique population history of Northern Europe?SUMMARY ANSWERWe identified three novel genome-wide significant associations with PCOS, with two putative independent causal variants in the checkpoint kinase 2 (CHEK2) gene and a third in myosin X (MYO10).WHAT IS KNOWN ALREADYPCOS is a common, complex disorder with unknown aetiology. While previous genome-wide association studies (GWAS) have mapped several loci associated with PCOS, the analysis of populations with unique population history and genetic makeup has the potential to uncover new low-frequency variants with larger effects.STUDY DESIGN, SIZE, DURATIONA population-based case–control GWAS was carried out.PARTICIPANTS/MATERIALS, SETTING, METHODSWe identified PCOS cases from national registers by ICD codes (ICD-10 E28.2, ICD-9 256.4, or ICD-8 256.90), and all remaining women were considered controls. We then conducted a three-stage case–control GWAS: in the discovery phase, we had a total of 797 cases and 140 558 controls from the FinnGen study. For validation, we used an independent dataset from the Estonian Biobank, including 2812 cases and 89 230 controls. Finally, we performed a joint meta-analysis of 3609 cases and 229 788 controls from both cohorts. Additionally, we reran the association analyses including BMI as a covariate, with 2169 cases and 160 321 controls from both cohorts.MAIN RESULTS AND THE ROLE OF CHANCETwo out of the three novel genome-wide significant variants associating with PCOS, rs145598156 (P = 3.6×10−8, odds ratio (OR) = 3.01 [2.02–4.50] minor allele frequency (MAF) = 0.005) and rs182075939 (P = 1.9×10−16, OR = 1.69 [1.49–1.91], MAF = 0.04), were found to be enriched in the Finnish and Estonian populations and are tightly linked to a deletion c.1100delC (r2 = 0.95) and a missense I157T (r2 = 0.83) in CHEK2. The third novel association is a common variant near MYO10 (rs9312937, P = 1.7 × 10−8, OR = 1.16 [1.10–1.23], MAF = 0.44). We also replicated four previous reported associations near the genes Erb-B2 Receptor Tyrosine Kinase 4 (ERBB4), DENN Domain Containing 1A (DENND1A), FSH Subunit Beta (FSHB) and Zinc Finger And BTB Domain Containing 16 (ZBTB16). When adding BMI as a covariate only one of the novel variants remained genome-wide significant in the meta-analysis (the EstBB lead signal in CHEK2 rs182075939, P = 1.9×10−16, OR = 1.74 [1.5–2.01]) possibly owing to reduced sample size.LARGE SCALE DATAThe age- and BMI-adjusted GWAS meta-analysis summary statistics are available for download from the GWAS Catalog with accession numbers GCST90044902 and GCST90044903.LIMITATIONS, REASONS FOR CAUTIONThe main limitation was the low prevalence of PCOS in registers; however, the ones with the diagnosis most likely represent the most severe cases. Also, BMI data were not available for all (63% for FinnGen, 76% for EstBB), and the biobank setting limited the accessibility of PCOS phenotypes and laboratory values.WIDER IMPLICATIONS OF THE FINDINGSThis study encourages the use of isolated populations to perform genetic association studies for the identification of rare variants contributing to the genetic landscape of complex diseases such as PCOS.STUDY FUNDING/COMPETING INTEREST(S)This work has received funding from the European Union’s Horizon 2020 research and innovation programme under the MATER Marie Skłodowska-Curie grant agreement No. 813707 (N.P.-G., T.L., T.P.), the Estonian Research Council grant (PRG687, T.L.), the Academy of Finland grants 315921 (T.P.), 321763 (T.P.), 297338 (J.K.), 307247 (J.K.), 344695 (H.L.), Novo Nordisk Foundation grant NNF17OC0026062 (J.K.), the Sigrid Juselius Foundation project grants (T.L., J.K., T.P.), Finska Läkaresällskapet (H.L.) and Jane and Aatos Erkko Foundation (H.L.). The funders had no role in study design, data collection and analysis, publishing or preparation of the manuscript. The authors declare no conflicts of interest.

Activation of transcriptional factor ZBTB16 expression during osteogenic differentiation of mesenchymal stem cells

Aim.Calcified aortic valve stenosis is the third leading cause of cardiovascular disease. The mechanisms underlying this process remain unclear, however, it is known that they are largely similar to the formation of bone tissue during embryonic development, as well as in the postnatal period during regeneration. There is evidence for the involvement of Zinc Finger and BTB Domain Containing 16 (ZBTB16) in skeletal development. At the same time, a number of studies carried out on different types of cell cultures indicate a contradictory and ambiguous effect of ZBTB16 on RUNX2 expression. Thus, the aim of this study was to investigate the dynamic variability of ZBTB16 expression, as well as its role in aortic valve calcification.Methods.The study used different types of mesenchymal cells cultures - aortic valve interstitial cells, umbilical cord mesenchymal stem cells, ligament stem cells and dental pulp stem cells. Changes in ZBTB16 and RUNX2 expression levels under the influence of osteogenic stimuli, as well as during exogenous activation of ZBTB16, were analyzed using real-time PCR. Expression levels of some osteogenic markers - BMP2,4, COL1A1, IBSP, DLX2, PDK4 - were analyzed in the interstitial cells of the aortic valve.Results.The results of the study indicate that a significant increase in the expression of ZBTB16 is observed during the induction of osteogenic differentiation of various cell cultures - interstitial cells of the aortic valve, mesenchymal stem cells of the umbilical cord, stem cells of the ligaments and dental pulp. Apparently, the processes of osteogenic differentiation of aortic valve interstitial cells, in the presence of dexamethasone in cultivation medium, are provided through RUNX2-dependent signaling for the further activation of osteogenic markers.Conclusion.The study of modulation of cellular signals by ZBTB16, when activating or suppressing the work of a transcriptional factor, in the future may bring us closer to the ability to enhance the regenerative abilities of bone tissue cells or, conversely, prevent calcification of the aortic valve tissues.

Fibronectin within Sodium Alginate Microcapsules Improved Osteogenic Differentiation of BMMSCs in Dose Dependent Manner by Targeting SP7, OCN, CDK1, ZBTB16, and Twist1 Expression.

Purpose: Insoluble fibronectin as an extracellular matrix (ECM) protein has the potential to promote proliferation, differentiation, and migration of mesenchymal stem cells (MSCs). However, there is limited information about the effects of fibronectin various concentrations on bone marrow-derived MSCs (BMMSCs) function and differentiation. Methods: In this experimental study, using a gel injection device, BMMSCs were encapsulated in sodium alginate microcapsules containing 1.25% alginate, 1% gelatin, and fibronectin (0.01, 0.05, 0.1, and 0.2 μg/ml). MTT assay was used to examine the proliferation of BMMSCs. Also, BMMSCs apoptosis were analyzed using Annexin-V/PI staining and fluorescence activated cell sorting (FACS). Alkaline phosphatase (ALP) test was conducted to assess BMMSCs osteogenic differentiation potential. Finally, mRNA expression levels of the SP7, osteocalcin (OCN), Twist Family BHLH Transcription Factor 1 (Twist1), Peroxisome proliferator‐activated receptor γ2 (PPARγ2), Cyclin-dependent kinase 1 (CDK1), and Zinc Finger and BTB Domain Containing 16 (ZBTB16), following exposure with fibronectin 0.1 μg/ml. Results: According to results, fibronectin had the potential to promote proliferation rates of the BMMSCs, in particular at 0.1 and 0.2 μg/ml concentrations. we showed that the fibronectin was not able to modify apoptosis rates of the BMMSCs. ALP test results approved the notable potential of the fibronectin, to trigger osteogenic differentiation of the BMMSCs. Also, RT-PCR results indicated that fibronectin 0.1 μg/ml could augment osteogenic differentiation of cultured BMMSCs through targeting of OCN, SP7, Twist1, CDK1, and ZBTB16, strongly or slightly. Conclusion: Results showed that fibronectin can improve proliferation and osteogenic differentiation of BMMSCs without any effect on these cells' survival.

Zbtb16 regulates social cognitive behaviors and neocortical development

Zinc finger and BTB domain containing 16 (ZBTB16) play the roles in the neural progenitor cell proliferation and neuronal differentiation during development, however, how the function of ZBTB16 is involved in brain function and behaviors unknown. Here we show the deletion of Zbtb16 in mice leads to social impairment, repetitive behaviors, risk-taking behaviors, and cognitive impairment. To elucidate the mechanism underlying the behavioral phenotypes, we conducted histological analyses and observed impairments in thinning of neocortical layer 6 (L6) and a reduction of TBR1+ neurons in Zbtb16 KO mice. Furthermore, we found increased dendritic spines and microglia, as well as developmental defects in oligodendrocytes and neocortical myelination in the prefrontal cortex (PFC) of Zbtb16 KO mice. Using genomics approaches, we identified the Zbtb16 transcriptome that includes genes involved in neocortical maturation such as neurogenesis and myelination, and both autism spectrum disorder (ASD) and schizophrenia (SCZ) pathobiology. Co-expression networks further identified Zbtb16-correlated modules that are unique to ASD or SCZ, respectively. Our study provides insight into the novel roles of ZBTB16 in behaviors and neocortical development related to the disorders.

Molecular characteristics of primary pulmonary lymphoepithelioma-like carcinoma based on integrated genomic analyses

Primary pulmonary lymphoepithelioma-like carcinoma (pLELC) is a rare non-small cell lung cancer (NSCLC) subtype. Clinical features have been described in our previous report, but molecular characteristics remain unclear. Herein, pLELC genomic features were explored. Among 41,574 lung cancers, 128 pLELCs and 162 non-pLELC NSCLCs were enrolled. Programmed cell death ligand 1 (PD-L1) and protein 53 (p53) expression was detected in 47 surgically resected pLELC samples by immunohistochemical assays. Multiomics genomic analyses, including whole-genome sequencing (WGS), RNA whole-transcriptome sequencing (RNA-seq), and Epstein-Barr virus (EBV) integration analyses, were performed on eight frozen pLELC tissues and compared with 50 lung adenocarcinomas (LUADs) and 50 lung squamous cell carcinomas (LUSCs) from The Cancer Genome Atlas (TCGA) and another 26 EBV-positive nasopharynx cancers (EBV+-NPCs). Progression-free survival (PFS) and overall survival (OS) of pLELC patients were better than those of non-pLELC patients. High PD-L1 or p53 expression was associated with extended disease-free survival (DFS). pLELC had 14 frequently mutated genes (FMGs). Somatically mutated genes and enrichment of genetic lesions were found, which differed from observations in LUAD, LUSC, and EBV+-nasopharyngeal carcinoma (NPC). Three tumor-associated genes, zinc finger and BTB domain-containing 16 (ZBTB16), peroxisome proliferator activated receptor gamma (PPARG), and transforming growth factor beta receptor 2 (TGFBR2), were downregulated with copy number variation (CNV) loss. EBV was prone to integrating into intergenic and intronic regions with two upregulated miR-BamH1-A rightward transcripts (BARTs), BART5-3P and BART20-3P. Our findings reveal that pLELC has a distinct genomic signature. Three tumor-associated genes with CNV loss and two miR-BARTs might be involved in pLELC tumorigenesis.

Epigenetic mechanisms are behind the regulation of the key genes associated with the osteoblastic differentiation of the mesenchymal stem cells: The role of zoledronic acid on tuning the epigenetic changes.

Mesenchymal stem cells (MSCs) are multipotent stem cells and show distinct features such as capability for self-renewal and differentiation into several lineages of cells including osteoblasts, chondrocytes, and adipocytes. In this study, the methylation status of the promoter region of zinc finger and BTB domain containing 16 (ZBTB16), twist-related protein 1(Twist1), de novo DNA methyltransferases 3A (DNMT3A), SRY-box 9 (Sox9), osteocalcin (OCN), and peroxisome proliferator-activated receptor γ2 (PPARγ2) genes and their messenger RNA (mRNA) expression levels were evaluated during the osteoblastic differentiation of MSCs (ODMSCs). We planned two experimental groups including zoledronic acid (ZA)-treated and nontreated cells (negative control) which both were differentiated into the osteoblasts. Methylation level of DNA in the promoter regions was assayed by methylation-specific-quantitative polymerase chain reaction (MS-qPCR), and mRNA levels of the target inhibitory/stimulatory genes during osteoblastic differentiation of MSCs were measured using real-time PCR. During the experimental induction of ODMSCs, the mRNA expression of the OCN gene was upregulated and methylation level of its promoter region was decreased. Moreover, Sox9 and PPARγ2 mRNA levels were attenuated and their promoter regions methylation levels were significantly augmented. However, the mRNA expression of the DNMT3A was not affected during the ODMSCs though its methylation rate was increased. In addition, ZA could enhance the expression of the ZBTB16 and decrease its promoter regions methylation and on the opposite side, it diminished mRNA expression of Sox9, Twist1, and PPARγ2 genes and increased their methylation rates. Intriguingly, ZA did not show a significant impact on gene expression and methylation levels the OCN and DNMT3A. We found that methylation of the promoter regions of Sox9, OCN, and PPARγ2 genes might be one of the main mechanisms adjusting the genes expression during the ODMSCs. Furthermore, we noticed that ZA can accelerate the MSCs differentiation to the osteoblast cells via two regulatory processes; suppression of osteoblastic differentiation inhibitor genes including Sox9, Twist1, and PPARγ2, and through promotion of the ZBTB16 expression.

Functions of promyelocytic leukaemia zinc finger (Plzf) in male germline stem cell development and differentiation.

Promyelocytic leukaemia zinc finger (Plzf), also known as zinc finger and BTB domain containing 16 (ZBTB16) or zinc-finger protein 145 (ZFP145), is a critical zinc finger protein of male germline stem cells (mGSCs). Multiple lines of evidence indicate that Plzf has a central role in the development, differentiation and maintenance of many stem cells, including mGSCs, and Plzf has been validated as an essential transcription factor for mammalian testis development and spermatogenesis. This review summarises current literature focusing on the significance of Plzf in maintaining and regulating self-renewal and differentiation of mGSCs, especially goat mGSCs. The review summarises evidence of the specificity of Plzf expression in germ cell development stage, the known functions of Plzf and the microRNA-mediated mechanisms that control Plzf expression in mGSCs.

ZBTB16 and Metabolic Syndrome: a Network Perspective

Metabolic syndrome is a prevalent, complex condition. The search for genetic determinants of the syndrome is currently undergoing a paradigm enhancement by adding systems genetics approaches to association studies. We summarize the current evidence on relations between an emergent new candidate, zinc finger and BTB domain containing 16 (ZBTB16) transcription factor and the major components constituting the metabolic syndrome. Information stemming from studies on experimental models with altered Zbtb16 expression clearly shows its effect on adipogenesis, cardiac hypertrophy and fibrosis, lipid levels and insulin sensitivity. Based on current evidence, we provide a network view of relations between ZBTB16 and hallmarks of metabolic syndrome in order to elucidate the potential functional links involving the ZBTB16 node. Many of the identified genes interconnecting ZBTB16 with all or most metabolic syndrome components are linked to immune function, inflammation or oxidative stress. In summary, ZBTB16 represents a promising pleiotropic candidate node for metabolic syndrome.

Role of PLZF as a tumor suppressor in prostate cancer

The promyelocytic leukemia zinc finger (PLZF), also known as ZBTB16 (Zinc Finger And BTB Domain Containing 16), is a transcription factor involved in the regulation of diverse biological processes, including cell proliferation, differentiation, organ development, stem cell maintenance and innate immune cell development. A number of recent studies have now implicated PLZF in cancer progression as a tumor suppressor. However, in certain cancer types, PLZF may function as an oncoprotein. Here, we summarize our current knowledge on the role of PLZF in various cancer types, in particular prostate cancer, including its deregulation, genomic alterations and potential functions in prostate cancer progression.

DDX4-EGFP transgenic rat model for the study of germline development and spermatogenesis.

Spermatogonial stem cells (SSC) are essential for spermatogenesis and male fertility. In addition, these adult tissue stem cells can be used as vehicles for germline modification in animal models and may have application for treating male infertility. To facilitate the investigation of SSCs and germ lineage development in rats, we generated a DEAD-box helicase 4 (DDX4) (VASA) promoter-enhanced green fluorescent protein (EGFP) reporter transgenic rat. Quantitative real-time polymerase chain reaction and immunofluorescence confirmed that EGFP was expressed in the germ cells of the ovaries and testes and was absent in somatic cells and tissues. Germ cell transplantation demonstrated that the EGFP-positive germ cell population from DDX4-EGFP rat testes contained SSCs capable of establishing spermatogenesis in experimentally infertile mouse recipient testes. EGFP-positive germ cells could be easily isolated by fluorescence-activated cells sorting, while simultaneously removing testicular somatic cells from DDX4-EGFP rat pup testes. The EGFP-positive fraction provided an optimal cell suspension to establish rat SSC cultures that maintained long-term expression of zinc finger and BTB domain containing 16 (ZBTB16) and spalt-like transcription factor 4 (SALL4), two markers of mouse SSCs that are conserved in rats. The novel DDX4-EGFP germ cell reporter rat described here combined with previously described GCS-EGFP rats, rat SSC culture and gene editing tools will improve the utility of the rat model for studying stem cells and germ lineage development.

ZBTB16 as a Downstream Target Gene of Osterix Regulates Osteoblastogenesis of Human Multipotent Mesenchymal Stromal Cells.

ABSTRACTHuman multipotent mesenchymal stromal cells (hMSCs) possess the ability to differentiate into osteoblasts, and they can be utilized as a source for bone regenerative therapy. Osteoinductive pretreatment, which induces the osteoblastic differentiation of hMSCs in vitro, has been widely used for bone tissue engineering prior to cell transplantation. However, the molecular basis of osteoblastic differentiation induced by osteoinductive medium (OIM) is still unknown. Therefore, we used a next‐generation sequencer to investigate the changes in gene expression during the osteoblastic differentiation of hMSCs. The hMSCs used in this study possessed both multipotency and self‐renewal ability. Whole‐transcriptome analysis revealed that the expression of zinc finger and BTB domain containing 16 (ZBTB16) was significantly increased during the osteoblastogenesis of hMSCs. ZBTB16 mRNA and protein expression was enhanced by culturing the hMSCs with OIM. Small interfering RNA (siRNA)‐mediated gene silencing of ZBTB16 decreased the activity of alkaline phosphatase (ALP); the expression of osteogenic genes, such as osteocalcin (OCN) and bone sialoprotein (BSP), and the mineralized nodule formation induced by OIM. siRNA‐mediated gene silencing of Osterix (Osx), which is known as an essential regulator of osteoblastic differentiation, markedly downregulated the expression of ZBTB16. In addition, chromatin immunoprecipitation (ChIP) assays showed that Osx associated with the ZBTB16 promoter region containing the GC‐rich canonical Sp1 sequence, which is the specific Osx binding site. These findings suggest that ZBTB16 acts as a downstream transcriptional regulator of Osx and can be useful as a late marker of osteoblastic differentiation. J. Cell. Biochem. 117: 2423–2434, 2016. © 2016 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals, Inc.

ZBTB16: a novel sensitive and specific biomarker for yolk sac tumor

Although the function of zinc finger and BTB domain containing 16 (ZBTB16) in spermatogenesis is well documented, expression of ZBTB16 in germ cell tumors has not yet been studied. The aim of this study was to investigate the immunohistochemical expression and diagnostic utility of ZBTB16 in germ cell tumors. A total of 67 adult germ cell tumors were studied (62 testicular germ cell tumors, 2 ovarian yolk sac tumors, 1 mediastinal yolk sac tumor, and 2 retroperitoneal metastatic yolk sac tumors). The 62 testicular primary germ cell tumors are as follows: 34 pure germ cell tumors (20 seminomas, 8 embryonal carcinomas, 2 teratomas, 1 choriocarcinoma, 1 carcinoid, and 2 spermatocytic tumors) and 28 mixed germ cell tumors (composed of 13 embryonal carcinomas, 15 yolk sac tumors, 15 teratomas, 7 seminomas, and 3 choriocarcinomas in various combinations). Thirty-five cases contained germ cell neoplasia in situ. Yolk sac tumor was consistently reactive for ZBTB16. Among the 15 testicular yolk sac tumors in mixed germ cell tumors, all displayed moderate to diffuse ZBTB16 staining. ZBTB16 reactivity was present regardless of the histologic patterns of yolk sac tumor and ZBTB16 was able to pick up small foci of yolk sac tumor intermixed/embedded in other germ cell tumor subtype elements. Diffuse ZBTB16 immunoreactivity was also observed in 2/2 metastatic yolk sac tumors, 1/1 mediastinal yolk sac tumor, 2/2 ovarian yolk sac tumors, 2/2 spermatocytic tumors, 1/1 carcinoid, and the spermatogonial cells. All the other non-yolk sac germ cell tumors were nonreactive, including seminoma (n=27), embryonal carcinoma (n=21), teratoma (n=17), choriocarcinoma (n=4), and germ cell neoplasia in situ (n=35). The sensitivity and specificity of ZBTB16 in detecting yolk sac tumor among the germ cell tumors was 100% (20/20) and 96% (66/69), respectively. In conclusion, ZBTB16 is a highly sensitive and specific marker for yolk sac tumor.

AZFc deletions do not affect the function of human spermatogonia in vitro.

Azoospermic factor c (AZFc) deletions are the underlying cause in 10% of azoo- or severe oligozoospermia. Through extensive molecular analysis the precise genetic content of the AZFc region and the origin of its deletion have been determined. However, little is known about the effect of AZFc deletions on the functionality of germ cells at various developmental steps. The presence of normal, fertilization-competent sperm in the ejaculate and/or testis of the majority of men with AZFc deletions suggests that the process of differentiation from spermatogonial stem cells (SSCs) to mature spermatozoa can take place in the absence of the AZFc region. To determine the functionality of AZFc-deleted spermatogonia, we compared in vitro propagated spermatogonia from six men with complete AZFc deletions with spermatogonia from three normozoospermic controls. We found that spermatogonia of AZFc-deleted men behave similar to controls during culture. Short-term (18 days) and long-term (48 days) culture of AZFc-deleted spermatogonia showed the same characteristics as non-deleted spermatogonia. This similarity was revealed by the same number of passages, the same germ cell clusters formation and similar level of genes expression of spermatogonial markers including ubiquitin carboxyl-terminal esterase L1 (UCHL1), zinc finger and BTB domain containing 16 (ZBTB16) and glial cell line-derived neurotrophic factor family receptor alpha 1 (GFRA1), as well as germ cell differentiation markers including signal transducer and activator of transcription 3 (STAT3), spermatogenesis and oogenesis specific basic helix-loophelix 2 (SOHLH2), v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) and synaptonemal complex protein 3 (SYCP3). The only exception was melanoma antigen family A4 (MAGEA4) which showed significantly lower expression in AZFc-deleted samples than controls in short-term culture while in long-term culture it was hardly detected in both AZFc-deleted and control spermatogonia. These data suggest that, at least in vitro, spermatogonia of AZFc-deleted men are functionally similar to spermatogonia from non-deleted men. Potentially, this enables treatment of men with AZFc deletions by propagating their SSCs in vitro and autotransplanting these SSCs back to the testes to increase sperm counts and restore fertility.