Expression Of ZBP-89 Research Articles

Wide field-of-view fluorescence imaging for organ-level lineage tracing of rare intestinal stem cell populations.

Lineage tracing using fluorescent reporters is a common tool for monitoring the expression of genes and transcription factors in stem cell populations and their progeny. The zinc-binding protein 89 (ZBP-89/Zfp148 mouse gene) is a transcription factor that plays a role in gastrointestinal (GI) stem cell maintenance and cellular differentiation and has been linked to the progression of colon cancer. While lineage tracing is a useful tool, it is commonly performed with high-magnification microscopy on a small field of view within tissue sections, thereby limiting the ability to resolve reporter expression at the organ level. Furthermore, this technique requires extensive tissue processing, which is time consuming and requires euthanizing the animal. Further knowledge could be elucidated by measuring the expression of fluorescent reporters across entire organs with minimal tissue processing. We present the application of wide-field fluorescence imaging for whole-organ lineage tracing of an inducible Zfp148-tdTomato-expressing transgenic mouse line to assess the expression of ZBP-89/Zfp148 in the GI tract. We measured tdTomato fluorescence in ex vivo organs at time points between 24h and 6 months post-induction. Fluctuations in tdTomato expression were validated by fluorescence microscopy of tissue sections. Quantification of the wide field-of-view images showed a statistically significant increase in fluorescent signal across the GI tract between transgenic mice and littermate controls. The results also showed a gradient of decreasing reporter expression from proximal to distal intestine, suggesting a higher abundance of ZBP-89 expressing stem cells, or higher expression of ZBP-89 within the stem cells, in the proximal intestine. We demonstrate that wide-field fluorescence imaging is a valuable tool for monitoring whole-organ expression of fluorescent reporters. This technique could potentially be applied in vivo for longitudinal assessment of a single animal, further enhancing our ability to resolve rare stem cell lineages spatially and temporally.

ZBP-89 negatively regulates self-renewal of liver cancer stem cells via suppression of Notch1 signaling pathway.

Liver cancer stem cells (LCSCs) initiate hepatocellular carcinoma (HCC) and contribute to its recurrence and treatment resistance. Studies have suggested ZBP-89 as a candidate tumor suppressor in HCC. We explored the role of ZBP-89 in the regulation of LCSCs. This study was performed in liver tissue samples from 104 HCC patients, 2cell lines and mouse tumor models. We demonstrated that ZBP-89 was weakly expressed in LCSCs. Patients with high expression of LCSC markers displayed reduced survivals and higher recurrence rates after curative surgical operation. The expression of ZBP-89 was predictive for decreased recurrence. LCSC markers were negatively correlated with ZBP-89 in HCC tissues and in enriched liver tumor spheres. The exogenous expression of ZBP-89 attenuated the tumor-sphere formation and secondary colony formation capabilities of LCSCs in vitro and tumorigenicity in vivo. Furthermore, the negative effect of ZBP-89 on cancer stemness was Notch1-dependent. Localized with Notch1 intracellular domain (NICD1) in the nucleus, ZBP-89 repressed the Notch1 signaling pathway by competitive binding to NICD1 with MAML1. Collectively, ZBP-89 negatively regulates HCC stemness via inhibiting the Notch1 signaling.

598 Transforming Growth Factor-Beta Signaling on Dendritic Cells Regulates Bacterial Communities and Gut Homeostasis

Background: Transcription factor Zinc-finger Binding Protein-89 (ZBP-89, ZNF148) expression is regulated by butyrate and forms protein-protein complexes with tumor suppressor factors, e.g. p53, p300, ataxia-telangiectasia mutated (ATM). To study the role of ZBP-89 in vivo, we generated a conditional knockout in the intestine and colon (ZBP-89ΔInt). Mice exhibited increased morbidity and mortality when challenged with Salmonella typhimurium, in part due to reduced tryptophan hydroxylase 1(Tph1) expression and reduced colonic antimicrobial peptide secretion (defensins). We found that β-catenin cooperates with ZBP89 to induce tryptophan hydroxylase 1(Tph1) expression in enterochromaffin cells. Aim: To determinewhether ZBP-89 interacts directly with β-catenin to prevent colonic transformation. Methods: ZBP-89ΔInt and WT littermates were treated with 7.4mg/kg azoxymethane (AOM) and water containing 2% dextran sulfate sodium (DSS). After 3 cycles, mice were sacrificed 5 weeks later for tumor evaluation, mRNA and histological analysis. Expression of β-cateninTCF gene targets was determined by rt-qPCR on colonic lysates. The ZBP-89 expression vector was co-transfected with or without β-catenin into HEK293 or SW480, a human colorectal cell line with the TCF reporter plasmid TOPFLASH to assess direct regulation of Wnt-β-catenin-TCF transcriptional activity. To demonstrate the association of ZBP-89 with β-catenin, cell lysates of SW480 were used to perform co-immunoprecipitation. Results: We observed 100% increase in tumor incidence and size in the distal colon and rectum of ZBP-89ΔInt compared to WT mice after the administration of AOM/DSS, N=16 mice/group (P<0.05). mRNA for cyclinD1, c-myc and Axin2 was significantly increased 3-fold in the ZBP89ΔInt mice after AOM/DSS treatment. Ectopic expression of ZBP-89 mitigated induction of the TOPFLASH reporter by β-catenin by 60% in both HEK293 and SW480 cell lines. Coimmunoprecipitation/western blotting showed that ZBP-89 and β-catenin formed a complex in cytoplasmic and nuclear extracts. Confocal microscopy demonstrated that ZBP-89 colocalized with β-catenin in the nucleus. In addition to protein-protein interactions, we found that knockdown of ZBP-89 by siRNA reduced β-catenin protein levels, suggesting that ZBP89 regulates β-catenin expression. Indeed, in silico analysis revealed a putative ZBP-89 binding site in the human and mouse β-catenin promoter at -83 to -78. Conclusion: Collectively, the data suggests that ZBP-98 modulates β-catenin activity and subsequently the induction of Wnt-β-catenin-TCF pathway in vivo and in vitro. Pull-down assays demonstrated that ZBP-89 modulates β-catenin activity through both protein-protein interactions and possibly transcriptional regulation of β-catenin expression, providing a possible mechanism by which this transcription factor inhibits colonic tumor formation.

High-expression of ZBP-89 correlates with distal metastasis and poor prognosis of patients in clear cell renal cell carcinoma

ZBP-89, a Krüppel-type zinc-finger transcription factor, is found to participate in tumor development, invasion and metastasis. However, the expression status of ZBP-89 in clear cell renal cell carcinoma (CCRCC) remains elusive. Using quantitative real-time-PCR and Western Blot, we found that, in fresh cancer tissues, ZBP-89 was remarkably decreased in 79.2% (19/24) and 83.3% (5/6) of CCRCC at mRNA and protein level, respectively. Immunohistochemistry also revealed a significant decline of ZBP-89 expression in CCRCC, showing that low expression of ZBP-89 was present in 73.9% (105/142) of tumorous tissues but in 48.1% (52/108) of the corresponding adjacent kidney tissues. Furthermore, ZBP-89 expression in CCRCC was significantly correlated with several clinicopathological features, including TNM stage (P=0.005) and distal metastasis (P=0.001). Further study confirmed that ZBP-89 expression was markedly higher in metastatic CCRCC than that in non-metastatic tissue (P=0.002). In addition, CCRCC patients with low ZBP-89 expression survived longer than those with high ZBP-89 expression, as indicated by the result of univariate analysis (P<0.0001). More importantly, multivariate analysis revealed that ZBP-89 was an independent predictor of overall survival (HR, 2.871; 95% CI, 1.409–5.853; P=0.004). Collectively, our study provides vigorous evidence that ZBP-89 was significantly downregulated in CCRCC and could be served as a promising biomarker for prediction of distal metastasis and prognosis of patient with CCRCC.

Increased expression of ZBP‐89 and its prognostic significance in hepatocellular carcinoma

ZBP-89 plays a role in cell growth and death. Its expression in hepatocellular carcinoma (HCC) is not well documented. This study aimed to analyse ZBP-89 expression in HCC. We examined ZBP-89 expression in five HCC cell lines and 182 HCC tissue samples by reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis and immunofluorescence staining. Our results showed that the expression of ZBP-89 was higher in HCC than adjacent non-tumour liver, at both mRNA and protein levels. ZBP-89 was localized in the nucleus in most HCC tissue samples, but was found in the cytoplasm in 11.5% of cases. Patient survival in those tumours showing high ZBP-89 expression was better than in those with low expression. High ZBP-89 expression tended to be more common in World Health Organization (WHO) grade I than grades II-IV HCC. There was a significant association between HBV positivity and high ZBP-89 expression. Colony formation was reduced dramatically in those HCC cell lines in which ZBP-89 overexpression was demonstrated; this appeared to correlate with increased apoptosis, inferred by finding elevated levels of cleaved poly(ADP-ribose)polymerases (PARP), the probable mechanisms for which may involve increased p53 or p21 expression. ZBP-89 has anti-tumour properties and is a potential biomarker for prognosis of HCC.

Characterization of a Functional ZBP-89 Binding Site That Mediates Gata1 Gene Expression during Hematopoietic Development

GATA-1 is a lineage-restricted transcription factor that plays essential roles in hematopoietic development. The Gata1 gene hematopoietic enhancer allowed Gata1 reporter expression in erythroid cells and megakaryocytes of transgenic mice. The Gata1 hematopoietic enhancer activity is strictly dependent on a GATA site located in the 5' region of the enhancer. However, the importance of the GC-rich region adjacent to the 3'-end of this GATA site has been also suggested. In this study, we show that this GC-rich region contains five contiguous deoxyguanosine residues (G(5) string) that are bound by multiple nuclear proteins. Interestingly, deletion of one deoxyguanosine residue from the G(5) string (G(4) mutant) specifically eliminates binding to ZBP-89, a Krüppel-like transcription factor, but not to Sp3 and other binding factors. We demonstrate that GATA-1 and ZBP-89 occupy chromatin regions of the Gata1 enhancer and physically associate in vitro through zinc finger domains. Gel mobility shift assays and DNA affinity precipitation assays suggest that binding of ZBP-89 to this region is reduced in the absence of GATA-1 binding to the G1HE. Luciferase reporter assays demonstrate that ZBP-89 activates the Gata1 enhancer depending on the G(5) string sequence. Finally, transgenic mouse studies reveal that the G(4) mutation significantly reduced the reporter activity of the Gata1 hematopoietic regulatory domain encompassing an 8.5-kbp region of the Gata1 gene. These data provide compelling evidence that the G(5) string is necessary for Gata1 gene expression in vivo and ZBP-89 is the functional trans-acting factor for this cis-acting region.

The Transcription Factor ZBP-89 Controls Generation of the Hematopoietic Lineage in Zebrafish and Mouse Embryonic Stem Cells.

Hematopoietic development is closely linked to that of blood vessels and the two processes are regulated in large part by transcription factors that control cell fate decisions and cellular differentiation. Both blood and blood vessels derive from a common progenitor, termed the hemangioblast, but the factors specifying development and differentiation of this stem cell population into the hematopoietic and vascular lineages remain ill defined. Here we report that knockdown of the Krüppel-like zinc finger ZBP-89 (ZNF148) in zebrafish embryos results in a bloodless phenotype, caused by disruption of both primitive and definitive hematopoiesis, leaving primary blood vessel formation intact. Expression of the primitive (Scl, Lmo2, Gata2, Gata1) and definitive (Runx1, c-Myb) hematopoietic stem cell lineage markers in ZBP-89 morphants were significantly decreased while expression of the lymphopoiesis lineage markers Ikaros and Gata3 was unaffected. These developmental HSC defects are rescued by injection of human ZBP-89 RNA, SCL RNA or ZBP-89 cDNA under control of the zebrafish Flk1 promoter. Injection of ZBP-89 RNA into cloche zebrafish embryos, which lack the hematopoietic and endothelial lineages, rescued hematopoiesis but not vasculogenesis. Overexpression of ZBP-89 in wild type zebrafish expanded hematopoietic progenitors (Scl+, Lmo2+, Gata1+) but reduced the endothelial cell markers FLK1 and TIE1. Forced expression of ZBP-89 in mouse embryonic stem cell cultures also induced the expansion of hematopoietic progenitors (c-KIT+, SCA1+) and definitive hematopoietic cell population (CD45+), but inhibited sprouting angiogenesis and the CD31+, VE-Cadherin+ endothelial cell population. These findings establish a unique regulatory role for ZBP-89, positioned at the interface between early blood and blood vessel development.

The transcription factor ZBP-89 controls generation of the hematopoietic lineage in zebrafish and mouse embryonic stem cells

Hematopoietic development is closely linked to that of blood vessels and the two processes are regulated in large part by transcription factors that control cell fate decisions and cellular differentiation. Both blood and blood vessels derive from a common progenitor, termed the hemangioblast, but the factor(s) specifying the development and differentiation of this stem cell population into the hematopoietic and vascular lineages remain ill defined. Here, we report that knockdown of the Krüppel-like transcription factor ZBP-89 in zebrafish embryos results in a bloodless phenotype, caused by disruption of both primitive and definitive hematopoiesis, while leaving primary blood vessel formation intact. Injection of ZBP-89 mRNA into cloche zebrafish embryos, which lack both the hematopoietic and endothelial lineages, rescues hematopoiesis but not vasculogenesis. Injection of mRNA for Stem Cell Leukemia (SCL), a transcription factor that directs hemangioblast development into blood cell precursors, rescues the bloodless phenotype in ZBP-89 zebrafish morphants. Forced expression of ZBP-89 induces the expansion of hematopoietic progenitors in wild-type zebrafish and in mouse embryonic stem cell cultures but inhibits angiogenesis in vivo and in vitro. These findings establish a unique regulatory role for ZBP-89, positioned at the interface between early blood and blood vessel development.

Inhibition of Growth Hormone Receptor Gene Expression by Saturated Fatty Acids: Role of Krüppel-Like Zinc Finger Factor, ZBP-89

The expression and function of the GH receptor is critical for the actions of pituitary GH in the intact animal. The role of systemic factors in the reduced expression of the GH receptor and consequent GH insensitivity in pathological states such as sepsis, malnutrition, and poorly controlled diabetes mellitus is unclear. In the current study, we demonstrate that saturated (palmitic and myristic; 50 microM) fatty acids (FA) inhibit activity of the promoter of the major (L2) transcript of the GH receptor gene; unsaturated (oleic and linoleic) FA (200 microM) do not alter activity of the promoter. Comparable effects with palmitic acid and the nonmetabolizable analog bromo-palmitic acid, and failure of triacsin C to abrogate palmitic acids effects on GH receptor expression indicate that this effect is due to direct action(s) of FA. Palmitic acid, but not the unsaturated FA linoleic acid, decreased steady-state levels of endogenous L2 mRNA and GHR protein in 3T3-L1 preadipocytes. The effect of FA was localized to two cis elements located approximately 600 bp apart on the L2 promoter. EMSA and chromatin immunoprecipitation assays established that both these cis elements bind the Krüppel-type zinc finger transcription factor, ZBP-89. Ectopic expression of ZBP-89 amplified the inhibitory effect of FA on L2 promoter activity and on steady-state levels of endogenous L2 mRNA in 3T3-L1 preadipocytes. Mutational analyses of the two ZBP-89 binding sites revealed that both the sites are essential for palmitic acid's inhibitory effect on the L2 promoter and for the enhancing effect of ZBP-89 on palmitic acid-induced inhibition of the L2 promoter. Our results establish a molecular basis for FA-induced inhibition of GH receptor gene expression in the pathogenesis of acquired GH insensitivity in pathological states such as poorly controlled diabetes mellitus and small for gestational age.

ZBP-89-induced apoptosis is p53-independent and requires JNK.

ZBP-89 induces apoptosis in human gastrointestinal cancer cells through a p53-independent mechanism. To understand the apoptotic pathway regulated by ZBP-89, we identified downstream signal transduction targets. Ectopic expression of ZBP-89 induced apoptosis through the mitochondrial pathway and was accompanied by activation of all three MAP kinase subfamilies: JNK1/2, ERK1/2 and p38 MAP kinase. ZBP-89-induced apoptosis was markedly enhanced by ERK inhibition with U0126. In contrast, inhibiting JNK with a JNK1-specific peptide inhibitor or dominant-negative JNK2 expression abrogated ZBP-89-mediated apoptosis. The p38 inhibitor SB202190 had no effect on ZBP-89-induced cell death. Protein dephosphorylation assays revealed that ZBP-89 activates JNK via repression of JNK dephosphorylation. Oligonucleotide microarray analyses revealed that ectopic expression of ZBP-89 downregulated expression of the dual-specificity phosphatase MKP6. Overexpression of MKP6 blocked ZBP-89-induced JNK phosphorylation and PARP cleavage. In addition, ectopic expression of ZBP-89 repressed Bcl-xL and Mcl-1 expression, but had no effect on Bcl-2. Silencing ZBP-89 with small interfering RNA enhanced both Bcl-xL and Mcl-1 expression. Taken together, ZBP-89-mediated apoptosis occurs via a p53-independent mechanism that requires JNK activation.

ZBP-89 Mediates Butyrate Regulation of Gene Expression

Inducible p53-independent regulation of the cyclin-dependent kinase inhibitor p21(Waf1) transcription is mediated through its proximal GC-rich sites. Prior studies have shown that Sp1, Sp3 and the histone acetyltransferase coactivator p300 are components of the complexes that bind to these sites. Although Sp1 and Sp3 collaborate with p300, a direct interaction between Sp1 and p300 does not occur. Zinc-finger binding protein-89 (ZBP-89, also known as BFCOL1, BERF-1 and ZNF-148) is a Krüppel-type zinc-finger transcription factor that binds to the same GC-rich sequences as Sp1. We sought to determine whether ZBP-89 is a target of p300 during butyrate induction of p21(Waf1). This review summarizes the evidence that supports a crucial role for ZBP-89 in butyrate regulation of p21(Waf1). Adenovirus-mediated expression of ZBP-89 in HT-29 cells reveals that ZBP-89 potentiates butyrate induction of endogenous p21(Waf1) gene expression. DNA-protein interaction assays demonstrate that Sp1, Sp3 and ZBP-89 bind the p21(Waf1) promoter at -245 to -215. Coprecipitation assays reveal that p300 preferentially binds to the N-terminus of ZBP-89. ZBP-89 also induces p21(Waf1) through stabilization of p53. Although ZBP-89 binds mutant and wild-type p53, only wild-type p53 is stabilized. Moreover, mutant p53 shifts the subnuclear location of ZBP-89 to the nuclear periphery, which is a domain rich in heterochromatin. This finding led to the conclusion that mutant p53 exerts a dominant negative effect on ZBP-89. We propose that gene silencing by mutant p53 might be mediated by sequestering ZBP-89 within heterochromatin regions at the nuclear periphery. Overall, ZBP-89 is a butyrate-regulated coactivator of p53 and is able to induce p21(Waf1) gene expression through both p53-dependent and -independent mechanisms to inhibit cell growth.

Mutation of p53 in Recurrent Hepatocellular Carcinoma and Its Association with the Expression of ZBP-89

p53 has recently been identified as a downstream target of ZBP-89, a zinc finger transcription factor. ZBP-89 promotes growth arrest through stabilization of the p53 protein. The aim of this study is to determine the status of the p53 gene in recurrent human hepatocellular carcinoma (HCC) and test the link between the expression of ZBP-89 and the p53 gene. The results showed that mutations in the p53 gene were frequently detected in recurrent HCC. The interval between surgical resection and the recurrence of HCC was significantly longer in patients with the wild-type p53 gene than those with mutations, strongly suggesting a pathological role for the mutant p53 gene in HCC recurrence. Among those positive for the p53 protein, nearly 85% (18 of 21) showed nuclear localization of the p53 protein while only about 14% (3 of 21) were positive for the p53 protein in the cytoplasm. ZBP-89 co-localized with p53 in the nucleus in about 67% (12 of 18) of all cases positive for the nuclear p53 protein, suggesting that ZBP-89 may play a role in the nuclear accumulation of the p53 protein in a subset of recurrent HCC. With accumulation of p53 protein in the nucleus, tumor cells undergo apoptosis and thus are more susceptible to radiotherapy and chemotherapy. Therefore, co-localization of p53 protein with ZBP-89 may define a subgroup of recurrent HCC that is more sensitive to treatment.

Transcription Factor ZBP-89 Cooperates with Histone Acetyltransferase p300 during Butyrate Activation of p21 waf1 Transcription in Human Cells

Inducible p53-independent regulation of the cyclin-dependent kinase inhibitor p21(waf1) transcription is mediated through proximal GC-rich sites. Prior studies have shown that Sp1, Sp3, and the histone acetylase co-activator p300 are components of the complexes binding to these sites. Although Sp1 and Sp3 collaborate with p300, a direct interaction between Sp1 and p300 does not occur. This study sought to determine whether ZBP-89 rather than Sp1 is the direct target of p300 during butyrate induction of p21(waf1). ZBP-89 (BFCOL1, BERF-1, ZNF 148) is a Krüppel-type zinc finger transcription factor that binds to GC-rich elements and represses or activates known target genes. Adenoviral-mediated expression of ZBP-89 in HT-29 cells revealed that ZBP-89 potentiates butyrate induction of endogenous p21(waf1) gene expression. Further, cotransfection of a ZBP-89 expression vector with a 2.3-kilobase p21(waf1) reporter recapitulated the potentiation by butyrate. DNase I footprinting analysis of the human p21(waf1) promoter with recombinant ZBP-89 identified a binding site at -245 to -215. Electrophoretic mobility shift assays confirmed that both recombinant and endogenous ZBP-89 and Sp1 bind to this element. The potentiation was abolished in the presence of adenoviral protein E1A. Deletion of the N-terminal domain of ZBP-89 abolished the potentiation mediated by butyrate treatment. This same deletion mutant abolished the ZBP-89 interaction with p300. Cotransfection of p300 with ZBP-89 stimulated the p21(waf1) promoter in the absence of butyrate. p300 co-precipitated with ZBP-89 but not with Sp1, whereas ZBP-89 co-precipitated with Sp1. Together, these findings demonstrate that ZBP-89 also plays a critical role in butyrate activation of the p21(waf1) promoter and reveals preferential cooperation of this four-zinc finger transcription factor with p300.