Zbtb16 mediates a switch between Fgf signalling regimes in the developing hindbrain.

Developing tissues are sequentially patterned by extracellular signals that are turned on and off at specific times. In the zebrafish hindbrain, fibroblast growth factor (Fgf) signalling has different roles at different developmental stages: in the early hindbrain, transient Fgf3 and Fgf8 signalling from rhombomere 4 is required for correct segmentation, whereas later, neuronal Fgf20 expression confines neurogenesis to specific spatial domains within each rhombomere. How the switch between these two signalling regimes is coordinated is not known. We present evidence that the Zbtb16 transcription factor is required for this transition to happen in an orderly fashion. Zbtb16 expression is high in the early anterior hindbrain, then gradually upregulated posteriorly and confined to neural progenitors. In mutants lacking functional Zbtb16, fgf3 expression fails to be downregulated and persists until a late stage, resulting in excess and more widespread Fgf signalling during neurogenesis. Accordingly, the spatial pattern of neurogenesis is disrupted in Zbtb16 mutants. Our results reveal how the distinct stage-specific roles of Fgf signalling are coordinated in the zebrafish hindbrain.

Zbtb16 increases susceptibility of atrial fibrillation in type 2 diabetic mice via Txnip-Trx2 signaling.

Atrial fibrillation (AF) is the most prevalent sustained cardiac arrhythmia, and recent epidemiological studies suggested type 2 diabetes mellitus (T2DM) is an independent risk factor for the development of AF. Zinc finger and BTB (broad-complex, tram-track and bric-a-brac) domain containing 16 (Zbtb16) serve as transcriptional factors to regulate many biological processes. However, the potential effects of Zbtb16 in AF under T2DM condition remain unclear. Here, we reported that db/db mice displayed higher AF vulnerability and Zbtb16 was identified as the most significantly enriched gene by RNA sequencing (RNA-seq) analysis in atrium. In addition, thioredoxin interacting protein (Txnip) was distinguished as the key downstream gene of Zbtb16 by Cleavage Under Targets and Tagmentation (CUT&Tag) assay. Mechanistically, increased Txnip combined with thioredoxin 2 (Trx2) in mitochondrion induced excess reactive oxygen species (ROS) release, calcium/calmodulin-dependent protein kinase II (CaMKII) overactivation, and spontaneous Ca2+ waves (SCWs) occurrence, which could be inhibited through atrial-specific knockdown (KD) of Zbtb16 or Txnip by adeno-associated virus 9 (AAV9) or Mito-TEMPO treatment. High glucose (HG)-treated HL-1 cells were used to mimic the setting of diabetic in vitro. Zbtb16-Txnip-Trx2 signaling-induced excess ROS release and CaMKII activation were also verified in HL-1 cells under HG condition. Furthermore, atrial-specific Zbtb16 or Txnip-KD reduced incidence and duration of AF in db/db mice. Altogether, we demonstrated that interrupting Zbtb16-Txnip-Trx2 signaling in atrium could decrease AF susceptibility via reducing ROS release and CaMKII activation in the setting of T2DM.

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.

Tanshinone IIA alleviates bleomycin-induced pulmonary fibrosis by inhibiting Zbtb16.

Pulmonary fibrosis is a complex disease that can occur in a variety of clinical settings. The Zinc Finger and BTB Domain Containing 16 (Zbtb16) is a transcription factor and has not been studied in pulmonary fibrosis. Lung tissues from rats which were treated with bleomycin and Tanshinone IIA (Tan IIA) were collected for mRNA sequencing. Zbtb16, a differentially expressed gene, was screened. Using adeno-associated virus to knock down Zbtb16 in rats, it was found that the lung index and the content of hydroxyproline in lung tissue were decreased. HE and Masson staining revealed that pathological symptoms of lung histopathology were relieved after Zbtb16 knockdown. Protein expressions of α-SMA, Collagen I and Fibronectin were significantly decreased after Zbtb16 knockdown in vivo and in vitro. Meanwhile, the protein content of TGF-ß1 and the phosphorylation of Smad2/3 were inhibited by Zbtb16 knockdown. Conversely, under the treatment of Tan IIA and TGF-ß1, overexpression of Zbtb16 improved cell viability, increased the expression of fibrosis-related proteins, and promoted the phosphorylation of Smad 2/3. All above demonstrates that Zbtb16 inhibition ameliorates pulmonary fibrosis and suppresses the TGF-ß/Smad pathway. Furthermore, Zbtb16 mediates the inhibitory process of Tan IIA on pulmonary fibrosis. This study provides a novel candidate therapeutic target for pulmonary fibrosis.

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 ß-catenin levels. Meanwhile, ZBTB16 activated the Wnt/ß-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/ß-catenin signaling pathway and further mediated PDLF osteogenic differentiation.

Hyperthermia promotes degradation of the acute promyelocytic leukemia driver oncoprotein ZBTB16/RARα.

The acute promyelocytic leukemia (APL) driver ZBTB16/RARα is generated by the t(11;17) (q23;q21) chromosomal translocation, which is resistant to combined treatment of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) or conventional chemotherapy, resulting in extremely low survival rates. In the current study, we investigated the effects of hyperthermia on the oncogenic fusion ZBTB16/RARα protein to explore a potential therapeutic approach for this variant APL. We showed that Z/R fusion protein expressed in HeLa cells was resistant to ATO, ATRA, and conventional chemotherapeutic agents. However, mild hyperthermia (42 °C) rapidly destabilized the ZBTB16/RARα fusion protein expressed in HeLa, 293T, and OCI-AML3 cells, followed by robust ubiquitination and proteasomal degradation. In contrast, hyperthermia did not affect the normal (i.e., unfused) ZBTB16 and RARα proteins, suggesting a specific thermal sensitivity of the ZBTB16/RARα fusion protein. Importantly, we found that the destabilization of ZBTB16/RARα was the initial step for oncogenic fusion protein degradation by hyperthermia, which could be blocked by deletion of nuclear receptor corepressor (NCoR) binding sites or knockdown of NCoRs. Furthermore, SIAH2 was identified as the E3 ligase participating in hyperthermia-induced ubiquitination of ZBTB16/RARα. In short, these results demonstrate that hyperthermia could effectively destabilize and subsequently degrade the ZBTB16/RARα fusion protein in an NCoR-dependent manner, suggesting a thermal-based therapeutic strategy that may improve the outcome in refractory ZBTB16/RARα-driven APL patients in the clinic.

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.

Identification of fusion genes and characterization of transcriptome features in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a clonal malignancy of immature T cells. Recently, the next-generation sequencing approach has allowed systematic identification of molecular features in pediatric T-ALL. Here, by performing RNA-sequencing and other genomewide analysis, we investigated the genomic landscape in 61 adult and 69 pediatric T-ALL cases. Thirty-six distinct gene fusion transcripts were identified, with SET-NUP214 being highly related to adult cases. Among 18 previously unknown fusions, ZBTB16-ABL1, TRA-SALL2, and involvement of NKX2-1 were recurrent events. ZBTB16-ABL1 functioned as a leukemogenic driver and responded to the effect of tyrosine kinase inhibitors. Among 48 genes with mutation rates >3%, 6 were newly found in T-ALL. An aberrantly overexpressed short mRNA transcript of the SLC17A9 gene was revealed in most cases with overexpressed TAL1, which predicted a poor prognosis in the adult group. Up-regulation of HOXA, MEF2C, and LYL1 was often present in adult cases, while TAL1 overexpression was detected mainly in the pediatric group. Although most gene fusions were mutually exclusive, they coexisted with gene mutations. These genetic abnormalities were correlated with deregulated gene expression markers in three subgroups. This study may further enrich the current knowledge of T-ALL molecular pathogenesis.

The rapamycin analog Everolimus reversibly impairs male germ cell differentiation and fertility in the mouse†.

Sirolimus, also known as rapamycin, and its closely related rapamycin analog (rapalog) Everolimus inhibit "mammalian target of rapamycin complex 1" (mTORC1), whose activity is required for spermatogenesis. Everolimus is Food and Drug Administration approved for treating human patients to slow growth of aggressive cancers and preventing organ transplant rejection. Here, we test the hypothesis that rapalog inhibition of mTORC1 activity has a negative, but reversible, impact upon spermatogenesis. Juvenile (P20) or adult (P>60) mice received daily injections of sirolimus or Everolimus for 30 days, and tissues were examined at completion of treatment or following a recovery period. Rapalog treatments reduced body and testis weights, testis weight/body weight ratios, cauda epididymal sperm counts, and seminal vesicle weights in animals of both ages. Following rapalog treatment, numbers of differentiating spermatogonia were reduced, with concomitant increases in the ratio of undifferentiated spermatogonia to total number of remaining germ cells. To determine if even low doses of Everolimus can inhibit spermatogenesis, an additional group of adult mice received a dose of Everolimus ∼6-fold lower than a human clinical dose used to treat cancer. In these animals, only testis weights, testis weight/body weight ratios, and tubule diameters were reduced. Return to control values following a recovery period was variable for each of the measured parameters and was duration and dose dependent. Together, these data indicate rapalogs exerted a dose-dependent restriction on overall growth of juvenile and adult mice and negative impact upon spermatogenesis that were largely reversed; following treatment cessation, males from all treatment groups were able to sire offspring.

Verification of a ZBTB16 variant in polycystic ovary syndrome patients.

RESEARCH QUESTION: This study investigated whether rs1784692 is a risk factor for polycystic ovary syndrome (PCOS) in Han Chinese women. DESIGN: A case-control study was conducted in Han Chinese women, involving 526 PCOS patients and 522 control participants. A TaqMan MGB probe assay was used to genotype the variant rs1784692. Dominant and additive models were employed for genotype-phenotype association analysis in the PCOS and control samples. RESULTS: The minor allele C of rs1784692 is protective against PCOS (odds ratio [OR] 0.556, 95% confidence interval [CI] 0.408-0.759, P = 1.83 × 10-4), even after adjustment for body mass index (BMI) and age (ORadj 0.539, 95% CI 0.391-0.743, Padj= 1.62 × 10-4). Genotype-phenotype analysis of the dominant model showed that mean BMI in the CC+CT group was higher than in the TT group in the PCOS group (27.12 ± 5.82 versus 24.57 ± 4.52, P = 1.0 × 10-3), but not in the control groups, indicating that the minor allele C of rs1784692 associates with BMI in women with PCOS. The mean LH (luteinizing hormone) concentration in the CC+CT group was lower than in the TT group in PCOS and control participants (9.33 ± 5.08 versus 10.93 ± 5.91, P = 0.036; 4.39 ± 1.66 versus 4.89 ± 2.07, P = 0.021). Genotype-phenotype analysis of additive model showed that mean BMI in TC group was higher than in the TT group in PCOS patients compared with control participants (27.14 ± 5.81 versus 24.57 ± 4.52, P = 3.06 × 10-3). CONCLUSIONS: The SNP rs1784692 in gene ZBTB16 is protective against PCOS but is associated with increased BMI in Han Chinese women with PCOS.

Differential localization of histone variant TH2B during the first round compared with subsequent rounds of spermatogenesis.

BACKGROUND: Male germ cells are unique because they express a substantial number of variants of the general DNA binding proteins, known as histones, yet the biological significance of these variants is still unknown. In the present study, we aimed to address the expression pattern of the testis-specific histone H2B variant (TH2B) and the testis-specific histone H2A variant (TH2A) within the neonatal mouse testis. RESULTS: We demonstrate that TH2B and TH2A are present in a testis-enriched for undifferentiated spermatogonia. Co-localization studies with an undifferentiated marker, ZBTB16, revealed that TH2B and ZBTB16 co-localize in the neonatal testis. Upon the appearance of the primary spermatocytes, TH2B no longer co-localized with the ZBTB16 positive spermatogonia but were instead detected within the differentiating spermatogonia. This pattern of expression where TH2B and ZBTB16 no longer co-localize was maintained in the adult testis. CONCLUSION: These findings are in contrast to previous studies, which demonstrated that TH2B and TH2A were found only in adult spermatocytes. Our data are in support of a switch in the expression of these variants following the first round of spermatogonial differentiation. These studies reinforce current understandings that spermatogonia within the neonatal mouse testis are inherently different from those residing within the adult testis.

A protein interaction network centered on leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) regulates growth factor receptors.

Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is a tumor suppressor and a negative regulator of several receptor tyrosine kinases. The molecular mechanisms by which LRIG1 mediates its tumor suppressor effects and regulates receptor tyrosine kinases remain incompletely understood. Here, we performed a yeast two-hybrid screen to identify novel LRIG1-interacting proteins and mined data from the BioPlex (biophysical interactions of ORFeome-based complexes) protein interaction data repository. The putative LRIG1 interactors identified in the screen were functionally evaluated using a triple co-transfection system in which HEK293 cells were co-transfected with platelet-derived growth factor receptor α, LRIG1, and shRNAs against the identified LRIG1 interactors. The effects of the shRNAs on the ability of LRIG1 to down-regulate platelet-derived growth factor receptor α expression were evaluated. On the basis of these results, we present an LRIG1 protein interaction network with many newly identified components. The network contains the apparently functionally important LRIG1-interacting proteins RAB4A, PON2, GAL3ST1, ZBTB16, LRIG2, CNPY3, HLA-DRA, GML, CNPY4, LRRC40, and LRIG3, together with GLRX3, PTPRK, and other proteins. In silico analyses of The Cancer Genome Atlas data sets revealed consistent correlations between the expression of the transcripts encoding LRIG1 and its interactors ZBTB16 and PTPRK and inverse correlations between the transcripts encoding LRIG1 and GLRX3. We further studied the LRIG1 function-promoting paraoxonase PON2 and found that it co-localized with LRIG1 in LRIG1-transfected cells. The proposed LRIG1 protein interaction network will provide leads for future studies aiming to understand the molecular functions of LRIG1 and the regulation of growth factor signaling.

Gene expression of TWIST1 and ZBTB16 is regulated by methylation modifications during the osteoblastic differentiation of mesenchymal stem cells.

BACKGROUND: Osteoblastic differentiation of mesenchymal stem cells (MSCs) is the principal stage during the restoration and regeneration of bone tissue. Epigenetic modifications such as DNA methylation play a key role in the differentiation process of stem cells. In this study, the methylation status of the promoter region of ZBTB16 and Twist1 genes and their role in controlling osteoblastic differentiation in MSCs was investigated during the osteoblastic differentiation of MSCs. METHODS: The MSCs were cultured under standard conditions and differentiated into the osteoblasts. We had three treatment groups including 5-azacytidine (methylation inhibitor), metformin (Twist-inhibitor), and procaine (Wnt/ß-catenin inhibitor) and a non-treated group (control). Methylation level of DNA in the promoter regions was monitored by methylation specific-quantitative polymerase chain reaction (PCR). Also, the mRNA levels of key genes in osteoblastic differentiation were measured using real-time PCR. RESULTS: ZBTB16 gene expression was upregulated, and promoter methylation was decreased. For Twist1 messenger RNA (mRNA) level decreased and promoter methylation increased during osteoblastic differentiation of MSCs. 5-Azacytidine caused a significant reduction in methylation and increased the mRNA expression of ZBTB16 and Twist1. Metformin repressed the Twist1 expression, and therefore osteoblastic differentiation was increased. On the opposite side, procaine could block the WNT/ß-catenin signaling pathway, as a consequence the gene expression of key genes involved in osteoblastic differentiation was declined. CONCLUSION: We found that methylation of DNA in the promoter region of ZBTB16 and Twist1 genes might be one of the main mechanisms that controlling the gene expression during osteoblastic differentiation of MSCs. Also, we could find an association between regulation of Twist1 and ZBTB16 genes and osteoblastic differentiation in MSCs by showing the relation between their expression and some key genes involved in osteoblastic differentiation. In addition, we found a connection between the Twist1 expression level and osteoblastic differentiation by using a Twist-inhibitor (metformin).

ZBTB16 Overexpression Enhances White Adipogenesis and Induces Brown-Like Adipocyte Formation of Bovine White Intramuscular Preadipocytes.

BACKGROUND/AIMS: Our study aims to characterize functions of ZBTB16 gene in the process of intramuscular fat (IMF) deposition and metabolism of bovine, thereby providing insights into mechanisms for the use of ZBTB16 in fat management. METHODS: Primary preadipocytes derived from bovine IMF tissue were isolated and used as the in vitro cell model. An adenovirus Ad-ZBTB16 was transfected into bovine preadipocytes to overexpress the ZBTB16 gene. By using real-time quantitative PCR (RT-qPCR), western blotting, Oil Red-O staining, glycerol-3-phosphate dehydrogenase (GPDH) activity assay, and cell counting kit-8 (CCK-8) test, adipogenic and proliferative signals in adipocytes were monitored to investigate effects of ZBTB16 on adipogenesis of bovine preadipocytes. RESULTS: After transfection, mRNA and protein levels of ZBTB16 gene were significantly increased. Enhanced ZBTB16 significantly promoted preadipocyte differentiation, as evidenced by accelerated lipid accumulation, enhanced GPDH activity, consistently increased mRNA expressions of adipogenic key transcription factors PPARγ, C/EBPα, FABP4, and ADIPOQ, and markedly increased protein expressions of PPARγ and FABP4. No difference was observed concerning proliferation of preadipocytes after treatment with Ad-ZBTB16. Furthermore, relative mRNA levels of brown adipocyte selective genes (PRDM16, UCP1, Cidea, Cox8b, and PGC-1α) and beige adipocyte selective genes (CD137, TMEM26, and Tbx1) as well as UCP1 protein expression were significantly increased by Ad-ZBTB16. Meanwhile, Ad-ZBTB16 treatment remarkably induced mitochondrial biogenesis and increased relative mitochondrial DNA (mtDNA) copy number in bovine adipocytes. CONCLUSION: These results suggest that ZBTB16 overexpression can promote white adipogenesis and induce brown-like adipocyte formation for bovine white intramuscular preadipocytes.

ZBTB16 is a sensitive and specific marker in detection of metastatic and extragonadal yolk sac tumour.

AIMS: Accurate histological diagnosis and classification of germ cell tumours (GCTs) is key to informing successful therapeutic and surveillance strategy. The modern therapeutic approach for yolk sac tumour (YST) is highly curative. Because YST takes on a large morphological spectrum, it can be confused for other GCT subtypes as well as somatic carcinomas, particularly when YST presents in an extragonadal or a metastatic setting. Currently available immunohistochemical markers are limited by suboptimal sensitivity and specificity. We reported recently that ZBTB16 is a sensitive and specific marker for testicular YST. ZBTB16 is absent in other GCTs and in most common somatic carcinomas, including those of gastrointestinal, pancreatobillary, respiratory, genitourinary and gynaecological tracts. The purpose of this study is to investigate the diagnostic utility of ZBTB16 in the settings of metastatic and extragonadal YST. METHODS AND RESULTS: We studied 32 archived metastatic and four extragonadal primary YSTs as well as 51 somatic malignancies for their immunohistochemical expression of ZBTB16. For comparison, α-fetoprotein (AFP) and glypican-3 were also studied in parallel. Our results demonstrated an overall sensitivity of 91.6% for ZBTB16 in detecting metastatic and extragonadal YSTs. The non-YST elements (teratoma and embryonal carcinoma) in 15 YST-containing metastatic mixed GCTs were non-reactive. With the exception of occasional myoepithelial cells of salivary gland carcinoma, all the 51 somatic malignancies were negative for ZBTB16. CONCLUSIONS: ZBTB16 is a sensitive and specific marker for YST and is diagnostically superior to AFP and glypican-3 in metastatic and extragonadal settings.

Investigating the association of previously identified genome-wide significant loci (rs10739076 and rs1784692) with PCOS susceptibility and its related traits in Indian women.

OBJECTIVE(S): Polycystic ovary syndrome (PCOS) is a multifactorial endocrinopathy with an enigmatic etiology. Hallmark features include irregular menstrual cycles, insulin resistance and hyperandrogenemia and affected women are prone to development of adverse reproductive and cardiometabolic outcomes like anovulatory infertility, impaired glucose tolerance, type 2 diabetes, dyslipidemia, metabolic syndrome and cardiovascular disease. Genetic underpinnings of PCOS have been investigated extensively using genome-wide association studies, which have led to the identification of several novel susceptibility loci. However, as ethnic diversity contributes to phenotypic and genetic heterogeneity, we undertook the first genetic association study to determine the association of rs10739076 of PLGRKT and rs1784692 of ZBTB16 with PCOS susceptibility and its related traits in Indian women. STUDY DESIGN: The present case-control study comprised 497 women with PCOS diagnosed according to the Rotterdam criteria and 233 age matched healthy women as controls. All participants were characterized in terms of anthropometric, hormonal and metabolic parameters and the variants were investigated by direct sequencing. Genotypic and genotype-phenotype association of these variants with PCOS susceptibility and its related biochemical and hormonal traits was analyzed with appropriate statistical tests. RESULTS: The genotypic and allelic frequencies of rs1784692 of ZBTB16 were significantly decreased in lean women with PCOS only, and this variant was associated with lowered insulin levels, HOMA-IR, LH:FSH ratio along with increased ApoA1 levels and QUICKI in them. Although, the PLGRKT variant, rs10739076, showed similar frequency distribution in both lean and obese groups, it was found to be associated with reduced fasting glucose in all women with PCOS. CONCLUSION(S): To the best of our knowledge, this is the first study to demonstrate that ZBTB16 variant showed significant association with reduced PCOS susceptibility in lean rather than obese Indian women, highlighting the impact of obesity on determining genetic predisposition to PCOS in Indian women. In contrast, PLGRKT variant did not influence PCOS risk in lean or obese women. Importantly, both variants exerted a protective effect on glucose metabolism, insulin resistance, gonadotropin and lipid levels in women with PCOS. Determination of susceptibility variants for PCOS demand population specific replication studies to ascertain best candidate loci for PCOS.

Gibberellic acid targeting ZBTB16 reduces NF-κB dependent inflammatory stress in sepsis-induced neuroinflammation.

OBJECTIVE: Sepsis is frequently complicated by neuroinflammation. Gibberellic acid (GA3) is recognized for its anti-inflammatory properties. In this study, our objective was to investigate whether GA3 could alleviate Nuclear factor-kappa B (NF-κB) -dependent inflammatory stress in sepsis-induced neuroinflammation. METHODS: C57BL/6 J mice were administered 10 mg/kg lipopolysaccharide (LPS) to induce sepsis. BV2 cells were pre-incubated with GA3 and subjected lipopolysaccharide stimulation to replicate the inflammatory microglia during sepsis. Subsequently, we assessed the release of IL-6, TNF-α, and IL-1ß, along with the expression of Zbtb16, NF-κB, and IκB. To investigate whether any observed anti-inflammatory effects of GA3 were mediated through a Zbtb16-dependent mechanism, Zbtb16 was silenced using siRNA. RESULTS: GA3 improved the survival of sepsis mice and alleviated post-sepsis cognitive impairment. Additionally, GA3 attenuated microglial M1 activation (pro-inflammatory phenotype), inflammation, and neuronal damage in the brain. Moreover, GA3 inhibited the release of TNF-α, IL-6, and IL-1ß in microglia stimulated with LPS. The NF-κB signaling pathway emerged as one of the key molecular pathways associated with the impact of GA3 on LPS-stimulated microglia. Lastly, GA3 upregulated Zbtb16 expression in microglia that had been downregulated by LPS. The inhibitory effects of GA3 on microglial M1 activation were partially reversed through siRNA knockdown of Zbtb16. CONCLUSIONS: Pre-incubation of microglia with GA3 led to the upregulation of the NF-κB regulator, Zbtb16. This process counteracted LPS-induced microglial M1 activation, resulting in an anti-inflammatory effect upon subsequent LPS stimulation.

Human cortical neurogenesis is altered via glucocorticoid-mediated regulation of ZBTB16 expression.

Glucocorticoids are important for proper organ maturation, and their levels are tightly regulated during development. Here, we use human cerebral organoids and mice to study the cell-type-specific effects of glucocorticoids on neurogenesis. We show that glucocorticoids increase a specific type of basal progenitors (co-expressing PAX6 and EOMES) that has been shown to contribute to cortical expansion in gyrified species. This effect is mediated via the transcription factor ZBTB16 and leads to increased production of neurons. A phenome-wide Mendelian randomization analysis of an enhancer variant that moderates glucocorticoid-induced ZBTB16 levels reveals causal relationships with higher educational attainment and altered brain structure. The relationship with postnatal cognition is also supported by data from a prospective pregnancy cohort study. This work provides a cellular and molecular pathway for the effects of glucocorticoids on human neurogenesis that relates to lasting postnatal phenotypes.

Direct reprogramming of non-limb fibroblasts to cells with properties of limb progenitors.

The early limb bud consists of mesenchymal limb progenitors derived from the lateral plate mesoderm (LPM). The LPM also gives rise to the mesodermal components of the flank and neck. However, the cells at these other levels cannot produce the variety of cell types found in the limb. Taking advantage of a direct reprogramming approach, we find a set of factors (Prdm16, Zbtb16, and Lin28a) normally expressed in the early limb bud and capable of imparting limb progenitor-like properties to mouse non-limb fibroblasts. The reprogrammed cells show similar gene expression profiles and can differentiate into similar cell types as endogenous limb progenitors. The further addition of Lin41 potentiates the proliferation of the reprogrammed cells. These results suggest that these same four factors may play pivotal roles in the specification of endogenous limb progenitors.