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Immune checkpoint inhibitors have substantial advanced tumor treatment, but their limited benefits and strong responses in only a subset of patients remain challenging. In this study, we explored the immunomodulatory function of contactin-4 (CNTN4). CNTN4 was highly expressed in tumor tissues, and expression impaired the antitumor function of T cells. CNTN4 bound to amyloid precursor protein (APP) on T cells, which attenuated conjugation between cancer cells and T cells, and diminished T cell receptor signaling cascades. We developed an anti-CNTN4 antibody (GENA-104A16) and an anti-APP antibody (5A7) that blocked the binding between CNTN4 and APP. Administration of either GENA-104A16 or 5A7 promoted antitumor T cell responses in a syngeneic mouse model and increased tumor-infiltrating lymphocytes in vivo. Furthermore, elevated CNTN4 levels were associated with poor prognosis and negatively correlated with various cytotoxic immune-related markers. These results suggest that CNTN4-APP is an inhibitory checkpoint in T cells and represents a promising therapeutic strategy for cancer immunotherapy. Editor's summary: Contactin-4 (CNTN4) is an adhesion molecule known to engage amyloid precursor protein (APP) in the brain, but the function of this axis in T cells and antitumor immunity is not known. Jeon et al. explored the immunomodulatory role of CNTN4-APP, finding that CNTN4 expressed on tumor cells prevents T cell activation by engaging APP on T cells. They developed an anti-CNTN4 antibody (GENA-104A16) and an anti-APP antibody (5A7) and found that blocking the CNTN4-APP interaction promoted tumor killing. Expression of APP on T cells negatively correlated with response to immune checkpoint inhibitors in patients with cancer. These findings indicate that CNTN4-APP functions as an inhibitory checkpoint in T cells. —Hannah Isles [ABSTRACT FROM AUTHOR]

Immune checkpoint inhibitor (ICI) clinically benefits cancer treatment. However, the ICI responses are only achieved in a subset of patients, and the underlying mechanisms of the limited response remain unclear. 160 patients with non‐small cell lung cancer treated with anti‐programmed cell death protein‐1 (anti‐PD‐1) or anti‐programmed death ligand‐1 (anti‐PD‐L1) are analyzed to understand the early determinants of response to ICI. It is observed that high levels of intracellular adhesion molecule‐1 (ICAM‐1) in tumors and plasma of patients are associated with prolonged survival. Further reverse translational studies using murine syngeneic tumor models reveal that soluble ICAM‐1 (sICAM‐1) is a key molecule that increases the efficacy of anti‐PD‐1 via activation of cytotoxic T cells. Moreover, chemokine (CXC motif) ligand 13 (CXCL13) in tumors and plasma is correlated with the level of ICAM‐1 and ICI efficacy, suggesting that CXCL13 might be involved in the ICAM‐1‐mediated anti‐tumor pathway. Using sICAM‐1 alone and in combination with anti‐PD‐1 enhances anti‐tumor efficacy in anti‐PD‐1‐responsive tumors in murine models. Notably, combinatorial therapy with sICAM‐1 and anti‐PD‐1 converts anti‐PD‐1‐resistant tumors to responsive ones in a preclinical study. These findings provide a new immunotherapeutic strategy for treating cancers using ICAM‐1. [ABSTRACT FROM AUTHOR]

Disruption of the skin microbial balance can exacerbate certain skin diseases and affect prognosis and treatment. Changes in the distribution and prevalence of certain microbial species on the skin, such as Staphylococcus aureus (SA), can impact the development of severe atopic dermatitis (AD) or psoriasis (Pso). A dysfunctional skin barrier develops in AD and Pso due to SA colonization, resulting in keratinization and chronic or progressive chronic inflammation. Disruption of the skin barrier following SA colonization can elevate the production of T helper 2 (Th2)-derived cytokines, which can cause an imbalance in Th1, Th2, and Th17 cells. This study examined the ability of potential therapeutic skin microbiomes, such as Cutibacterium avidum R-CH3 and Staphylococcus hominis R9, to inhibit SA biofilm formation and restore skin barrier function-related genes through the activation of the aryl hydrocarbon receptor (AhR) and the nuclear factor erythroid-2-related factor 2 (Nrf2) downstream target. We observed that IL-4/IL-13-induced downregulation of FLG, LOR, and IVL induced by SA colonization could be reversed by dual AhR/Nrf2 activation. Further, OVOL1 expression may be modulated by functional microbiomes via dual AhR/Nrf2 activation. Our results suggest that our potential therapeutic skin microbiomes can prevent SA-derived Th2-biased skin barrier disruption via IL-13 and IL-4-dependent FLG deregulation, STAT3 activation, and AhR-mediated STAT6 expression. [ABSTRACT FROM AUTHOR]

Dysregulated matrix metalloproteinase (MMP) gene expression is a major cause of the degradation of lung tissue that is integral to emphysema pathogenesis. Cigarette smoking (CS) increases MMP gene expression, a major contributor to emphysema development. We previously reported that Zbtb7c is a transcriptional repressor of several Mmp genes (Mmps‐8, ‐10, ‐13, and ‐16). Here, we show that Zbtb7c knockout mice have mild emphysema‐like phenotypes, including alveolar wall destruction, enlarged alveoli, and upregulated Mmp genes. Alveolar size and Mmp gene expression in Zbtb7c−/− mouse lungs are increased more severely upon exposure to CS, compared to those of Zbtb7c+/+ mouse lungs. These observations suggest that Zbtb7c degradation or absence may contribute to the pathogenesis of emphysema. [ABSTRACT FROM AUTHOR]

The protein deacetylase SIRT1 is crucial to numerous physiological processes, such as aging, metabolism, and autoimmunity, and is repressed by various transcription factors, including HIC1. Conversely, we found that HIC2, which is highly homologous to HIC1, is a transcriptional activator of SIRT1 due to opposite activity of the intermediate domains of the two homologs. Importantly, this relationship between HIC2 and SIRT1 could be important for cardiac development, where both proteins are implicated. Here, we assessed whether ectopic expression of HIC2, and subsequent upregulation of SIRT1, might decrease apoptosis in H9c2 cardiomyocytes under simulated ischemia/reperfusion (I/R) injury conditions. Our results demonstrate that unlike its structural homolog HIC1, HIC2 is a pivotal transcriptional activator of SIRT1 and, consequently, may protect the heart from I/R injury. [ABSTRACT FROM AUTHOR]

Gluconeogenesis is essential for blood glucose homeostasis during fasting and is regulated by various enzymes, which are encoded by gluconeogenic genes. Those genes are controlled by various transcription factors. Zinc finger and BTB domain–containing 7c (Zbtb7c, also called Kr-pok) is a BTB-POZ family transcription factor with proto-oncogenic activity. Previous findings have indicated that Zbtb7c is involved in the regulation of fatty acid biosynthesis, suggesting an involvement also in primary metabolism. We found here that fasting induced Zbtb7c expression in the mouse liver and in primary liver hepatocytes. We also observed that Zbtb7c-knockout mice have decreased blood glucose levels, so we investigated whether Zbtb7c plays a role in gluconeogenesis. Indeed, differential gene expression analysis of Zbtb7c-knockout versus wild type mouse livers showed downregulated transcription of gluconeogenic genes encoding the glucose 6-phosphatase catalytic subunit (G6pc) and phosphoenolpyruvate carboxykinase 1 (Pck1), while Zbtb7c expression upregulated these two genes, under fasting conditions. Mechanistically, we found that when complexed with histone deacetylase 3 (Hdac3), Zbtb7c binds insulin response elements (IREs) within the G6pc and Pck1 promoters. Moreover, complexed Zbtb7c deacetylated forkhead box O1 (Foxo1), thereby increasing Foxo1 binding to the G6pc and Pck1 IREs, resulting in their transcriptional activation. These results demonstrate Zbtb7c to be a crucial metabolic regulator of blood glucose homeostasis, during mammalian fasting. • Zbtb7c is induced and plays a critical role in gluconeogenesis by activating G6pc and Pck1 gene transcription during mice fasting. • Zbtb7c complexed with Hdac3, and deacetylated Foxo1, which increases DNA binding of Foxo1. • Zbtb7c is critical in increasing Foxo1 binding to the IREs. [ABSTRACT FROM AUTHOR]

Expression of the POK family protein ZNF509L, and -its S1 isoform, is induced by p53 upon exposure to genotoxic stress. Due to alternative splicing of the ZNF509 primary transcript, ZNF509S1 lacks the 6 zinc-fingers and C-terminus of ZNF509L, resulting in only one zinc-finger. ZNF509L and -S1 inhibit cell proliferation by activating p21/CDKN1A and RB transcription, respectively. When cells are exposed to severe DNA damage, p53 activates PUMA ( p 53- u pregulated m odulator of a poptosis) transcription. Interestingly, apoptosis due to transcriptional activation of PUMA by p53 is attenuated by ZNF509S1. Thus we investigated the molecular mechanism(s) underlying the transcriptional attenuation and anti-apoptotic effects of ZNF509S1. We show that ZNF509S1 modulation of p53 activity is important in PUMA gene transcription by modulating post-translational modification of p53 by p300. ZNF509S1 directly interacts with p53 and inhibits p300-mediated acetylation of p53 lysine K382, with deacetylation of p53 K382 leading to decreased DNA binding at the p53 response element 1 of the PUMA promoter. ZNF509S1 may play a role not only in cell cycle arrest, by activating RB expression, but also in rescuing cells from apoptotic death by repressing PUMA expression in cells exposed to severe DNA damage. [ABSTRACT FROM AUTHOR]

Kr-POK (ZBTB7c) is a kidney cancer-related POK transcription factor that not only represses transcription of CDKN1A but also increases expression of FASN . However, precisely how Kr-POK affects cell metabolism by controlling gene expression in response to an energy source in rapidly proliferating cells remains unknown. In this study, we characterized the molecular and functional features of Kr-POK in the context of tumor growth and glutamine metabolism. We found that cells expressing Kr-POK shRNA exhibited more severe cell death than control cells in glucose-deprived medium, and that knockdown of Kr-POK decreased glutamine uptake. Glutamine is critical for tumor cell proliferation. Glutaminase (GLS1), which is activated by p-STAT1, catalyzes the initial reaction in the pathway of glutaminolysis. Kr-POK interacts with PIAS1 to disrupt the interaction between PIAS1 and p-STAT1, and free p-STAT1 can activate GLS1 transcription through an interaction with p300. Kr-POK can be also sumoylated by PIAS1, facilitating Kr-POK degradation by the ubiquitin-mediated proteasomal pathway. Finally, we showed that repression of Kr-POK inhibited tumor growth in vivo in a xenograft model by repressing GLS1 expression. Taken together, our data reveal that Kr-POK activates GLS1 transcription and increases glutamine uptake to support rapid cancer cell proliferation. [ABSTRACT FROM AUTHOR]

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases that play roles in cell proliferation, migration, differentiation, angiogenesis, and apoptosis. The expression of MMP gene is tightly regulated and shows cell- and tissue-specific expression patterns. Despite their differential expression, MMP genes have AP-1 (activator protein-1) binding elements within their promoters. Interestingly, c-JUN phosphorylation by cytokine signaling decreased its interaction with NCoR, but increased its interaction with p300, resulting in activation of MMP gene transcription. Here, we found that Zbtb7c (Kr-pok) is a critical component of a transcriptional repressor complex containing c-Jun and NCoR. c-Jun, bound at AP-1, interacts with Zbtb7c, which in turn recruits an NCoR/Hdac3 complex to repress several Mmp (-8, -10, -13, and -16) genes. The molecular interaction between c-Jun and Zbtb7c also prevents phosphorylation of c-Jun by p-Jnk, However, Zbtb7c phosphorylation by p-Jnk (induced by TNFα), and its (Zbtb7c) subsequent degradation by the ubiquitin-mediated proteasomal pathway, leads to c-Jun phosphorylation by p-Jnk. Promoter-bound p-c-Jun then recruits the coactivator p300 to upregulate Mmp gene. Overall, these findings show that Zbtb7c is a key molecule that recruits an NCoR/Hdac3 complex to inhibit phosphorylation of c-Jun, and thereby repress Mmp gene expression. [ABSTRACT FROM AUTHOR]

KAISO, a member of the POK protein family, is induced by DNA-damaging agents to enhance apoptosis in a p53-dependent manner. Previously, we found that p53 interacts with KAISO, and acetylation of p53 lysine residues by p300 is modulated by KAISO. APAF1, the core molecule of the apoptosome, is transcriptionally activated by KAISO only in cells expressing p53, which binds to APAF1 promoter p53-response elements (p53REs). APAF1 transcriptional upregulation is further enhanced by KAISO augmentation of p53 binding to the APAF1 promoter distal p53RE#1 (bp, − 765 to − 739). Interestingly, a NF-κB response element, located close to the p53RE#1, mediates APAF1 transcriptional repression by affecting interaction between KAISO and p53. Ectopic RelA/p65 expression led to depletion of nuclear KAISO, with KAISO being mainly detected in the cytoplasm. RelA/p65 cytoplasmic sequestration of KAISO prevents its nuclear interaction with p53, decreasing APAF1 transcriptional activation by a p53–KAISO–p300 complex in cells exposed to genotoxic stresses. While KAISO enhances p53-dependent apoptosis by increasing APAF1 gene expression, RelA/p65 decreases apoptosis by blocking interaction between KAISO and p53. These findings have relevance to the phenomenon of cancer cells' diminished apoptotic capacity and the onset of chemotherapy resistance. [ABSTRACT FROM AUTHOR]

Simple Summary: The efficacy of cancer therapeutics depends on several factors, including the tumor genome, epigenome, and transcriptome. In addition, the tumor microenvironment, which consists mainly of immune cells, can influence cancer treatment outcomes. Hence, effectively leveraging host immunity is an important aspect of cancer treatment strategies. The human gut microbiome is involved in the regulation of the immune responses and affects the efficacy of chemotherapeutic and immunotherapeutic agents, including oxaliplatin, cyclophosphamide, and immune checkpoint inhibitors. This study reveals an additional dimension to the Bifidobacterium strain-specific determination of anti-cancer therapeutic efficacy using flow cytometry and transcriptome analysis of bacterial strain-fed mice and bacterial whole transcriptome analysis. We hope that our work will contribute to leveraging the gut microbiome to improve anti-cancer therapies. Colorectal cancer (CRC) is among the leading causes of cancer-related death in the world. The development of CRC is associated with smoking, diet, and microbial exposure. Previous studies have shown that dysbiosis of the gut microbiome affects cancer development, because it leads to inflammation and genotoxicity. Supplementation with specific microbiota induces anti-tumor effects by enhancing of anti-tumor immunity. Here, we observed that supplementation with either of two B. breve strains reduces tumor growth in MC38 colon carcinoma-bearing mice. Interestingly, only one B. breve strain boosted the efficacy of cancer therapeutics, including oxaliplatin and PD-1 blockade. Extensive immune profiling and transcriptomic analysis revealed that the boosting B. breve strain augments lymphocyte-mediated anti-cancer immunity. Our results suggest that supplementation with B. breve strains could potentially be used as a strategy to enhance the efficacy of CRC therapeutics. [ABSTRACT FROM AUTHOR]

Correct temporal and spatial control of actin dynamics is essential for the cytotoxic T cell effector function against tumor cells. However, little is known whether actin engineering in tumor-targeted T cells can enhance their antitumor responses, thereby potentiating the adoptive T cell therapy. Here, we report that TAGLN2, a 22-KDa actin-stabilizing protein which is physically associated with lymphocyte function-associated antigen-1 (LFA-1), potentiates the OTI TCR CD8+ T cells to kill the intercellular adhesion molecule-1 (ICAM-1)-positive/OVA-presenting E0771 cells, but not ICAM-1-negative OVA-B16F10 cells, suggesting an ‘inside-out’ activation of LFA-1, which causes more efficient immunological synapse formation between T cells and tumor cells. Notably, recombinant TAGLN2 fused with the protein transduction domain (TG2P) overcame the disadvantages of viral gene delivery, leading to a significant reduction in tumor growth in mice. TG2P also potentiated the CD19-targeted, chimeric antigen receptor (CAR)-modified T cells to kill Raji B-lymphoma cells. Our findings indicate that activating the TAGLN2-actin-LFA-1 axis is an effective strategy to potentiate the adoptive T-cell immunotherapy. [ABSTRACT FROM AUTHOR]

Immune checkpoint inhibitor (ICI) clinically benefits cancer treatment. However, the ICI responses are only achieved in a subset of patients, and the underlying mechanisms of the limited response remain unclear. 160 patients with non-small cell lung cancer treated with anti-programmed cell death protein-1 (anti-PD-1) or anti-programmed death ligand-1 (anti-PD-L1) are analyzed to understand the early determinants of response to ICI. It is observed that high levels of intracellular adhesion molecule-1 (ICAM-1) in tumors and plasma of patients are associated with prolonged survival. Further reverse translational studies using murine syngeneic tumor models reveal that soluble ICAM-1 (sICAM-1) is a key molecule that increases the efficacy of anti-PD-1 via activation of cytotoxic T cells. Moreover, chemokine (CXC motif) ligand 13 (CXCL13) in tumors and plasma is correlated with the level of ICAM-1 and ICI efficacy, suggesting that CXCL13 might be involved in the ICAM-1-mediated anti-tumor pathway. Using sICAM-1 alone and in combination with anti-PD-1 enhances anti-tumor efficacy in anti-PD-1-responsive tumors in murine models. Notably, combinatorial therapy with sICAM-1 and anti-PD-1 converts anti-PD-1-resistant tumors to responsive ones in a preclinical study. These findings provide a new immunotherapeutic strategy for treating cancers using ICAM-1., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Gluconeogenesis is essential for blood glucose homeostasis during fasting and is regulated by various enzymes, which are encoded by gluconeogenic genes. Those genes are controlled by various transcription factors. Zinc finger and BTB domain-containing 7c (Zbtb7c, also called Kr-pok) is a BTB-POZ family transcription factor with proto-oncogenic activity. Previous findings have indicated that Zbtb7c is involved in the regulation of fatty acid biosynthesis, suggesting an involvement also in primary metabolism. We found here that fasting induced Zbtb7c expression in the mouse liver and in primary liver hepatocytes. We also observed that Zbtb7c-knockout mice have decreased blood glucose levels, so we investigated whether Zbtb7c plays a role in gluconeogenesis. Indeed, differential gene expression analysis of Zbtb7c-knockout versus wild type mouse livers showed downregulated transcription of gluconeogenic genes encoding the glucose 6-phosphatase catalytic subunit (G6pc) and phosphoenolpyruvate carboxykinase 1 (Pck1), while Zbtb7c expression upregulated these two genes, under fasting conditions. Mechanistically, we found that when complexed with histone deacetylase 3 (Hdac3), Zbtb7c binds insulin response elements (IREs) within the G6pc and Pck1 promoters. Moreover, complexed Zbtb7c deacetylated forkhead box O1 (Foxo1), thereby increasing Foxo1 binding to the G6pc and Pck1 IREs, resulting in their transcriptional activation. These results demonstrate Zbtb7c to be a crucial metabolic regulator of blood glucose homeostasis, during mammalian fasting., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Even in the face of physiological DNA damage or expression of the tumor suppressor protein p53, B cell CLL/lymphoma 6 (BCL6) increases proliferation and antagonizes apoptotic responses in B cells. BCL6 represses TP53 transcription and also appears to inactivate p53 at the protein level, and additional findings have suggested negative mutual regulation between BCL6 and p53. Here, using Bcl6 -/- knockout mice, HEK293A and HCT116 p53 -/- cells, and site-directed mutagenesis, we found that BCL6 interacts with p53 and thereby inhibits acetylation of Lys-132 in p53 by E1A-binding protein p300 (p300), a modification that normally occurs upon DNA damage-induced cellular stress and whose abrogation by BCL6 diminished transcriptional activation of p53 target genes, including that encoding caspase-1. Conversely, we also found that BCL6 protein is degraded via p53-induced, caspase-mediated proteolytic cleavage, and the formation of a BCL6-p53-caspase-1 complex. Our results suggest that p53 may block oncogenic transformation by decreasing BCL6 stability via caspase-1 up-regulation, whereas aberrant BCL6 expression inactivates transactivation of p53 target genes, either by inhibiting p53 acetylation by p300 or repressing TP53 gene transcription. These findings have implications for B cell development and lymphomagenesis., (© 2019 Kim et al.)

Expression of the POK family protein ZNF509L, and -its S1 isoform, is induced by p53 upon exposure to genotoxic stress. Due to alternative splicing of the ZNF509 primary transcript, ZNF509S1 lacks the 6 zinc-fingers and C-terminus of ZNF509L, resulting in only one zinc-finger. ZNF509L and -S1 inhibit cell proliferation by activating p21/CDKN1A and RB transcription, respectively. When cells are exposed to severe DNA damage, p53 activates PUMA (p53-upregulated modulator of apoptosis) transcription. Interestingly, apoptosis due to transcriptional activation of PUMA by p53 is attenuated by ZNF509S1. Thus we investigated the molecular mechanism(s) underlying the transcriptional attenuation and anti-apoptotic effects of ZNF509S1. We show that ZNF509S1 modulation of p53 activity is important in PUMA gene transcription by modulating post-translational modification of p53 by p300. ZNF509S1 directly interacts with p53 and inhibits p300-mediated acetylation of p53 lysine K382, with deacetylation of p53 K382 leading to decreased DNA binding at the p53 response element 1 of the PUMA promoter. ZNF509S1 may play a role not only in cell cycle arrest, by activating RB expression, but also in rescuing cells from apoptotic death by repressing PUMA expression in cells exposed to severe DNA damage., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Kr-POK (ZBTB7c) is a kidney cancer-related POK transcription factor that not only represses transcription of CDKN1A but also increases expression of FASN. However, precisely how Kr-POK affects cell metabolism by controlling gene expression in response to an energy source in rapidly proliferating cells remains unknown. In this study, we characterized the molecular and functional features of Kr-POK in the context of tumor growth and glutamine metabolism. We found that cells expressing Kr-POK shRNA exhibited more severe cell death than control cells in glucose-deprived medium, and that knockdown of Kr-POK decreased glutamine uptake. Glutamine is critical for tumor cell proliferation. Glutaminase (GLS1), which is activated by p-STAT1, catalyzes the initial reaction in the pathway of glutaminolysis. Kr-POK interacts with PIAS1 to disrupt the interaction between PIAS1 and p-STAT1, and free p-STAT1 can activate GLS1 transcription through an interaction with p300. Kr-POK can be also sumoylated by PIAS1, facilitating Kr-POK degradation by the ubiquitin-mediated proteasomal pathway. Finally, we showed that repression of Kr-POK inhibited tumor growth in vivo in a xenograft model by repressing GLS1 expression. Taken together, our data reveal that Kr-POK activates GLS1 transcription and increases glutamine uptake to support rapid cancer cell proliferation., (Copyright © 2017. Published by Elsevier B.V.)

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases that play roles in cell proliferation, migration, differentiation, angiogenesis, and apoptosis. The expression of MMP gene is tightly regulated and shows cell- and tissue-specific expression patterns. Despite their differential expression, MMP genes have AP-1 (activator protein-1) binding elements within their promoters. Interestingly, c-JUN phosphorylation by cytokine signaling decreased its interaction with NCoR, but increased its interaction with p300, resulting in activation of MMP gene transcription. Here, we found that Zbtb7c (Kr-pok) is a critical component of a transcriptional repressor complex containing c-Jun and NCoR. c-Jun, bound at AP-1, interacts with Zbtb7c, which in turn recruits an NCoR/Hdac3 complex to repress several Mmp (-8, -10, -13, and -16) genes. The molecular interaction between c-Jun and Zbtb7c also prevents phosphorylation of c-Jun by p-Jnk, However, Zbtb7c phosphorylation by p-Jnk (induced by TNFα), and its (Zbtb7c) subsequent degradation by the ubiquitin-mediated proteasomal pathway, leads to c-Jun phosphorylation by p-Jnk. Promoter-bound p-c-Jun then recruits the coactivator p300 to upregulate Mmp gene. Overall, these findings show that Zbtb7c is a key molecule that recruits an NCoR/Hdac3 complex to inhibit phosphorylation of c-Jun, and thereby repress Mmp gene expression., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Amelogenin (AMELX) is the main component of the developing tooth enamel matrix and is essential for enamel thickness and structure. However, little is known about its transcriptional regulation. Using gene expression analysis, we found that MIZ-1, a potent transcriptional activator of CDKN1A , is expressed during odontoblastic differentiation of hDPSCs (human dental pulp stem cells), and is essential for odontoblast differentiation and mineralization. We also investigated how MIZ-1 regulates gene expression of AMELX . Oligonucleotide-pull down assays showed that MIZ-1 binds to an MRE (MIZ-1 binding element) of the AMELX proximal promoter region (bp, -170 to -25). Combined, our ChIP, transient transcription assays, and promoter mutagenesis revealed that MIZ-1 directly binds to the MRE of the Amelx promoter recruits p300 and induces Amelx gene transcription. Finally, we show that the zinc finger protein MIZ-1 is an essential transcriptional activator of Amelx , which is critical in odontogenesis and matrix mineralization in the developing tooth.

KAISO, a member of the POK protein family, is induced by DNA-damaging agents to enhance apoptosis in a p53-dependent manner. Previously, we found that p53 interacts with KAISO, and acetylation of p53 lysine residues by p300 is modulated by KAISO. APAF1, the core molecule of the apoptosome, is transcriptionally activated by KAISO only in cells expressing p53, which binds to APAF1 promoter p53-response elements (p53REs). APAF1 transcriptional upregulation is further enhanced by KAISO augmentation of p53 binding to the APAF1 promoter distal p53RE#1 (bp, -765 to -739). Interestingly, a NF-κB response element, located close to the p53RE#1, mediates APAF1 transcriptional repression by affecting interaction between KAISO and p53. Ectopic RelA/p65 expression led to depletion of nuclear KAISO, with KAISO being mainly detected in the cytoplasm. RelA/p65 cytoplasmic sequestration of KAISO prevents its nuclear interaction with p53, decreasing APAF1 transcriptional activation by a p53-KAISO-p300 complex in cells exposed to genotoxic stresses. While KAISO enhances p53-dependent apoptosis by increasing APAF1 gene expression, RelA/p65 decreases apoptosis by blocking interaction between KAISO and p53. These findings have relevance to the phenomenon of cancer cells' diminished apoptotic capacity and the onset of chemotherapy resistance., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Promyelocytic leukemia zinc finger (PLZF) is a transcription repressor that was initially isolated as a fusion protein with retinoic acid receptor α. PLZF is aberrantly overexpressed in various human solid tumors, such as clear cell renal carcinoma, glioblastoma, and seminoma. PLZF causes cellular transformation of NIH3T3 cells and increases cell proliferation in several cell types. PLZF also increases tumor growth in the mouse xenograft tumor model. PLZF may stimulate cell proliferation by controlling expression of the genes of the p53 pathway (ARF, TP53, and CDKN1A). We found that PLZF can directly repress transcription of CDKN1A encoding p21, a negative regulator of cell cycle progression. PLZF binds to the proximal Sp1-binding GC-box 5/6 and the distal p53-responsive elements of the CDKN1A promoter to repress transcription. Interestingly, PLZF interacts with Sp1 or p53 and competes with Sp1 or p53. PLZF interacts with corepressors, such as mSin3A, NCoR, and SMRT, thereby deacetylates Ac-H3 and Ac-H4 histones at the CDKN1A promoter, which indicated the involvement of the corepressor·HDACs complex in transcription repression by PLZF. Also, PLZF represses transcription of TP53 and also decreases p53 protein stability by ubiquitination. PLZF may act as a potential proto-oncoprotein in various cell types., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

HKR3 (Human Krüppel-related 3) is a novel POK (POZ-domain Krüppel-like zinc-finger) family transcription factor. Recently, some of the POK (POZ-domain Krüppel-like zinc finger) family proteins have been shown to play roles in cell cycle arrest, apoptosis, cell proliferation, and oncogenesis. We investigated whether HKR3, an inhibitor of cell proliferation and an uncharacterized POK family protein, could regulate the cell cycle by controlling expression of genes within the p53 pathway (ARF-MDM2-TP53-p21WAF/CDKN1A). HKR3 potently activated the transcription of the tumor suppressor gene ARF by acting on the proximal promoter region (bp, -149∼+53), which contains Sp1 and FBI-1 binding elements (FREs). HKR3 interacted with the co-activator p300 to activate ARF transcription, which increased the acetylation of histones H3 and H4 within the proximal promoter. Oligonucleotide pull-down assays and ChIP assays revealed that HKR3 interferes with the binding of the proto-oncogenic transcription repressor FBI-1 to proximal FREs, thus derepressing ARF transcription.

The human POZ domain and Krüppel-like zinc finger (POK) family proteins play important roles in the regulation of apoptosis, cell proliferation, differentiation, development, oncogenesis, and tumor suppression. A novel POK family transcription factor, BTB/POZ and zinc finger domains factor on chromosome 1 (BOZF-1; also called ZBTB8A), contains a POZ domain and two C2H2-type Krüppel-like zinc fingers and is localized at nuclear speckles. Compared with paired normal tissues, BOZF1 expression is increased in cancer tissues of the prostate, breast, and cervix. BOZF1 repressed the transcription of p21WAF/CDKN1A by acting on the proximal promoter concentrated with Sp1-binding GC boxes. BOZF1 competed with Sp1 in binding to GC boxes 1-5/6 of the CDKN1A proximal promoter. In addition, BOZF1 interacted with p53 and decreased the acetylation of p53 by p300, which reduced the DNA binding activity of p53 at the far distal p53-binding element. BOZF1 blocked the two major molecular events that are important in both constitutive and inducible transcription activation of CDKN1A. BOZF1 is unique in that it bound to all the proximal GC boxes to repress transcription, and it inhibited p53 acetylation without affecting p53 stability. BOZF1 might be a novel proto-oncoprotein that stimulates cell proliferation.

Kr-pok (kidney cancer-related POZ domain and Krüppel-like protein) is a new proto-oncogenic POZ-domain transcription factor. Fatty acid synthase gene (FASN) encodes one of the key enzymes in fatty acids synthesis and is the only enzyme that synthesizes fatty acids in cancer cells. Sp1 and SREBP-1c are the two major transcription activators of FASN. We investigated whether Kr-pok modulates transcription of the FASN. FASN expression is significantly decreased in Kr-pok knockout murine embryonic fibroblasts. Coimmunoprecipitation, GST fusion protein pull-down, and immunocytochemistry assays show that the zinc-finger domain of Kr-pok interacts directly with the bZIP DNA binding domain of SREBP-1. Electrophoretic mobility shift assay, oligonucleotide pull-down, and chromatin immunoprecipitation assays showed that Kr-pok changes the transcription factor binding dynamics of Sp1 and SREBP-1c to the SRE/E-box elements of the proximal promoter. We found that Kr-pok expression increased during 3T3-L1 preadipocyte differentiation and that FASN expression is decreased by the knockdown of Kr-pok. Kr-pok facilitates the SREBP-1c-mediated preadipocyte differentiation and/or fatty acid synthesis. Kr-pok may act as an important regulator of fatty acid synthesis and may induce rapid cancer cell proliferation by increasing palmitate synthesis.

Transcriptional regulation by p53 is thought to play a role in its ability to suppress tumorigenesis. However, there remain gaps in understanding about how p53 regulates transcription and how disrupting this function may promote cancer. Here we report a role in these processes for the kidney cancer-related gene KR-POK (ZBTB7C), a POZ domain and Krüppel-like zinc finger transcription factor that we found to physically interact with p53. Murine embryonic fibroblasts isolated from genetically deficient mice (Kr-pok(-/-) MEFs) exhibited a proliferative defect relative to wild-type mouse embryonic fibroblasts (MEF). The zinc finger domain of Kr-pok interacted directly with the DNA binding and oligomerization domains of p53. This interaction was essential for Kr-pok to bind the distal promoter region of the CDKN1A gene, an important p53 target gene encoding the cell-cycle regulator p21WAF1, and to inhibit p53-mediated transcriptional activation of CDKN1A. Kr-pok also interacted with the transcriptional corepressors NCoR and BCoR, acting to repress histone H3 and H4 deacetylation at the proximal promoter region of the CDKN1A gene. Importantly, Kr-pok(-/-) MEFs displayed an enhancement in CDKN1A transactivation by p53 during the DNA damage response, without any parallel changes in transcription of either the p53 or Kr-pok genes themselves. Furthermore, Kr-pok promoted cell proliferation in vitro and in vivo, and its expression was increased in more than 50% of the malignant human kidney cancer cases analyzed. Together, our findings define KR-POK as a transcriptional repressor with a pro-oncogenic role that relies upon binding to p53 and inhibition of its transactivation function.

Transcriptional repression through chromatin remodeling and histone deacetylation has been postulated as a driving force for tumorigenesis. We isolated and characterized a novel POZ domain Krüppel-like zinc finger transcription repressor, ZBTB5 (zinc finger and BTB domain-containing 5). Serial analysis of gene expression (SAGE) analysis showed that ZBTB5 expression is higher in retinoblastoma and muscle cancer tissues. Immunocytochemistry showed that ZBTB5 was localized to the nucleus, particularly nuclear speckles. ZBTB5 directly repressed transcription of cell cycle arrest gene p21 by binding to the proximal GC-box 5/6 elements and the two distal p53-responsive elements (bp -2323 approximately -2299; bp -1416 approximately -1392). Chromatin immunoprecipitation assays showed that ZBTB5 and p53 competed with each other in occupying the p53 binding elements. ZBTB5 interacted with co-repressor-histone deacetylase complexes such as BCoR (BCL-6-interacting corepressor), NCoR (nuclear receptor corepressor), and SMRT (silencing mediator for retinoid and thyroid receptors) via its POZ domain. These interactions resulted in deacetylation of histones Ac-H3 and Ac-H4 at the proximal promoter, which is important in the transcriptional repression of p21. MTT (3-(4,5-di meth yl thi azol-2-yl)-2,5-diphenyltetrazolium bromide) assays and fluorescent-activated cell sorter analysis revealed that ZBTB5 stimulated both cell proliferation and cell cycle progression, significantly increasing the number of cells in S-phase. Overall, our data suggest that ZBTB5 is a potent transcription repressor of cell cycle arrest gene p21 and a potential proto-oncogene stimulating cell proliferation.

We found that ZBTB2, a POK family transcription factor, is a potent repressor of the ARF-HDM2-p53-p21 pathway important in cell cycle regulation. ZBTB2 repressed transcription of the ARF, p53, and p21 genes, but activated the HDM2 gene. In particular, ZBTB2 repressed transcription of the p21 gene by acting on the two distal p53 binding elements and the proximal Sp1 binding GC-box 5/6 elements. ZBTB2 directly interacted with Sp1 via its POZ domain and zinc fingers, which was important in the repression of transcription activation by Sp1. ZBTB2 and Sp1 competed with each other in binding to the GC-box 5/6 elements and the two p53 binding elements. ZBTB2 directly interacted with p53 via its zinc fingers, inhibiting p53 binding and repressing transcription activation by p53. The POZ domain, required for transcription repression, interacted with corepressors such as BCoR, NCoR, and SMRT. The interactions deacetylated histones Ac-H3 and -H4 at the proximal promoter. Although ectopic ZBTB2 stimulated cell proliferation, knock-down of ZBTB2 expression decreased cell proliferation and DNA synthesis. Overall, our data suggest that ZBTB2 is a potential proto-oncogenic master control gene of the p53 pathway and, in particular, is a potent transcription repressor of the cell cycle arrest gene p21 by inhibiting p53 and Sp1.

Aberrant transcriptional repression through chromatin remodeling and histone deacetylation has been postulated as the driving force for tumorigenesis. FBI-1 (formerly called Pokemon) is a member of the POK family of transcriptional repressors. Recently, FBI-1 was characterized as a critical oncogenic factor that specifically represses transcription of the tumor suppressor gene ARF, potentially leading indirectly to p53 inactivation. Our investigations on transcriptional repression of the p53 pathway revealed that FBI-1 represses transcription of ARF, Hdm2 (human analogue of mouse double minute oncogene), and p21CIP1 (hereafter indicated as p21) but not of p53. FBI-1 showed a more potent repressive effect on p21 than on p53. Our data suggested that FBI-1 is a master controller of the ARF-Hdm2-p53-p21 pathway, ultimately impinging on cell cycle arrest factor p21, by inhibiting upstream regulators at the transcriptional and protein levels. FBI-1 acted as a competitive transcriptional repressor of p53 and Sp1 and was shown to bind the proximal Sp1-3 GC-box and the distal p53-responsive elements of p21. Repression involved direct binding competition of FBI-1 with Sp1 and p53. FBI-1 also interacted with corepressors, such as mSin3A, NCoR, and SMRT, thereby deacetylating Ac-H3 and Ac-H4 histones at the promoter. FBI-1 caused cellular transformation, promoted cell cycle proliferation, and significantly increased the number of cells in S phase. FBI-1 is aberrantly overexpressed in many human solid tumors, particularly in adenocarcinomas and squamous carcinomas. The role of FBI-1 as a master controller of the p53 pathway therefore makes it an attractive therapeutic target.

FBI-1, a member of the POK (POZ and Kruppel) family of transcription factors, plays a role in differentiation, oncogenesis, and adipogenesis. eEF1A is a eukaryotic translation elongation factor involved in several cellular processes including embryogenesis, oncogenic transformation, cell proliferation, and cytoskeletal organization. CCS-3, a potential cervical cancer suppressor, is an isoform of eEF1A. We found that eEF1A forms a complex with FBI-1 by co-immunoprecipitation, SDS-PAGE, and MALDI-TOF Mass analysis of the immunoprecipitate. GST fusion protein pull-downs showed that FBI-1 directly interacts with eEF1A and CCS-3 via the zinc finger and POZ-domain of FBI-1. FBI-1 co-localizes with either eEF1A or CCS-3 at the nuclear periplasm. CCS-3 enhances transcriptional repression of the p21CIP1 gene (hereafter referred to as p21) by FBI-1. The POZ-domain of FBI-1 interacts with the co-repressors, SMRT and BCoR. We found that CCS-3 also interacts with the co-repressors independently. The molecular interaction between the co-repressors and CCS-3 at the POZ-domain of FBI-1 appears to enhance FBI-1 mediated transcriptional repression. Our data suggest that CCS-3 may be important in cell differentiation, tumorigenesis, and oncogenesis by interacting with the proto-oncogene FBI-1 and transcriptional co-repressors., (Copyright 2009 S. Karger AG, Basel.)

FBI-1 (also called Pokemon/ZBTB7A) is a BTB/POZ-domain Krüppel-like zinc-finger transcription factor. Recently, FBI-1 was characterized as a proto-oncogenic protein, which represses tumor suppressor ARF gene transcription. The expression of FBI-1 is increased in many cancer tissues. We found that FBI-1 potently represses transcription of the Rb gene, a tumor suppressor gene important in cell cycle arrest. FBI-1 binds to four GC-rich promoter elements (FREs) located at bp -308 to -188 of the Rb promoter region. The Rb promoter also contains two Sp1 binding sites: GC-box 1 (bp -65 to -56) and GC-box 2 (bp -18 to -9), the latter of which is also bound by FBI-1. We found that FRE3 (bp -244 to -236) is also a Sp1 binding element. FBI-1 represses transcription of the Rb gene not only by binding to the FREs, but also by competing with Sp1 at the GC-box 2 and the FRE3. By binding to the FREs and/or the GC-box, FBI-1 represses transcription of the Rb gene through its POZ-domain, which recruits a co-repressor-histone deacetylase complex and deacetylates histones H3 and H4 at the Rb gene promoter. FBI-1 inhibits C2C12 myoblast cell differentiation by repressing Rb gene expression.

FBI-1 (Pokemon/ZBTB7A) is a proto-oncogenic transcription factor of the BTB/POZ (bric-à-brac, tramtrack, and broad complex and pox virus zinc finger) domain family. Recent evidence suggested that FBI-1 might be involved in adipogenic gene expression. Coincidentally, expression of FBI-1 and fatty-acid synthase (FASN) genes are often increased in cancer and immortalized cells. Both FBI-1 and FASN are important in cancer cell proliferation. SREBP-1 is a major regulator of many adipogenic genes, and FBI-1 and SREBP-1 (sterol-responsive element (SRE)-binding protein 1) interact with each other directly via their DNA binding domains. FBI-1 enhanced the transcriptional activation of SREBP-1 on responsive promoters, pGL2-6x(SRE)-Luc and FASN gene. FBI-1 and SREBP-1 synergistically activate transcription of the FASN gene by acting on the proximal GC-box and SRE/E-box. FBI-1, Sp1, and SREBP-1 can bind to all three SRE, GC-box, and SRE/E-box. Binding competition among the three transcription factors on the GC-box and SRE/E-box appears important in the transcription regulation. FBI-1 is apparently changing the binding pattern of Sp1 and SREBP-1 on the two elements in the presence of induced SREBP-1 and drives more Sp1 binding to the proximal promoter with less of an effect on SREBP-1 binding. The changes induced by FBI-1 appear critical in the synergistic transcription activation. The molecular mechanism revealed provides insight into how proto-oncogene FBI-1 may attack the cellular regulatory mechanism of FASN gene expression to provide more phospholipid membrane components needed for rapid cancer cell proliferation.

Pokemon 1 is a proto-oncogenic transcriptional regulator that contains a POZ domain at the N-terminus and four Kruppel-like zinc fingers at the C-terminus. Pokemon 1 plays an important role in adipogenesis, osteogenesis, oncogenesis, and transcription of NF-kB responsive genes. Recent reports have shown that biological activities of transcription factors are regulated by sumolylation. We investigated whether Pokemon 1 is post-translationally modified by sumoylation and whether the modification affects Pokemon 1's transcriptional properties. We found that Pokemon 1 is sumoylated in vitro and in vivo. Upon careful analysis of the amino acid sequence of Pokemon 1, we found ten potential sumoylation sites located at lysines 61, 354, 371, 379, 383, 396, 486, 487, 536 and 539. We mutated each of these amino acids into arginine and tested whether the mutation could affect the transcriptional properties of Pokemon 1 on the Pokemon 1 responsive genes, such as ADH5/FDH and pG5-FRE-Luc. Wild-type Pokemon 1 potently represses transcription of ADH5/FDH. Most of the mutants, however, were weaker transcription repressors and repressed transcription 1.3-3.3 fold less effective. Although potential sumoylation sites were located close to the DNA binding domain or the nuclear localization sequence, the mutations did not alter nuclear localization or DNA binding activity. In addition, on the pG5-FRE-Luc test promoter construct, ectopic SUMO-1 repressed transcription in the presence of Pokemon 1. The sumoylation target lysine residue at amino acid 61, which is located in the middle of the POZ-domain, is important because K61R mutation resulted in a much weaker molecular interaction with corepressors. Our data suggest that Pokemon 1's activity as a transcription factor may involve sumoylation, and that sumoylation might be important in the regulation of transcription by Pokemon 1.

Sp1 activates the transcription of many cellular and viral genes with the GC-box in either the proximal promoter or the enhancer. Sp1 is composed of several functional domains, such as the inhibitory domain (ID), two serine/threonine-rich domains, two glutamine-rich domains, three C2H2-type zinc finger DNA binding domains (ZFDBD), and a C-terminal D domain. The ZDDBD is the most highly conserved domain among the Sp-family transcription factors and plays a critical role in GC-box recognition. In this study, we investigated the protein-protein interactions occurring at the Sp1ZFDBD and the Sp1ID, and the molecular mechanisms controlling the interaction. Our results found that Sp1ZFDBD and Sp1ID repressed transcription once they were targeted to the proximal promoter of the pGal4 UAS reporter fusion gene system, suggesting molecular interaction with the repressor molecules. Indeed, mammalian two-hybrid assays, GST fusion protein pull-down assays, and co-immunoprecipitation assays showed that Sp1ZFDBD and Sp1ID are able to interact with corepressor proteins such as SMRT, NcoR, and BCoR. The molecular interactions appear to be regulated by MAP kinase/Erk kinase kinase (MEK). The molecular interactions between Sp1ID and the corepressor might explain the role of Sp1 as a repressor under certain circumstances. The siRNA-induced degradation of the corepressors resulted in an up-regulation of Sp1-dependent transcription. The cellular context of the corepressors and the regulation of molecular interaction between corepressors and Sp1ZFDBD or Sp1ID might be important in controlling Sp1 activity.