Proto-oncoprotein Zbtb7c and SIRT1 repression: implications in high-fat diet-induced and age-dependent obesity

Zbtb7c is a proto-oncoprotein that controls the cell cycle and glucose, glutamate, and lipid metabolism. Zbtb7c expression is increased in the liver and white adipose tissues of aging or high-fat diet-fed mice. Knockout or knockdown of Zbtb7c gene expression inhibits the adipocyte differentiation of 3T3-L1 cells and decreases adipose tissue mass in aging mice. We found that Zbtb7c was a potent transcriptional repressor of SIRT1 and that SIRT1 was derepressed in various tissues of Zbtb7c-KO mice. Mechanistically, Zbtb7c interacted with p53 and bound to the proximal promoter p53RE1 and p53RE2 to repress the SIRT1 gene, in which p53RE2 was particularly critical. Zbtb7c induced p53 to interact with the corepressor mSin3A-HADC1 complex at p53RE. By repressing the SIRT1 gene, Zbtb7c increased the acetylation of Pgc-1α and Pparγ, which resulted in repression or activation of Pgc-1α or Pparγ target genes involved in lipid metabolism. Our study provides a molecular target that can overexpress SIRT1 protein in the liver, pancreas, and adipose tissues, which can be beneficial in the treatment of diabetes, obesity, longevity, etc.ope

Transcriptional Activity of Sp1 Is Regulated by Molecular Interactions between the Zinc Finger DNA Binding Domain and the Inhibitory Domain with Corepressors, and This Interaction Is Modulated by MEK

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.ope

Kr-pok increases FASN expression by modulating the DNA binding of SREBP-1c and Sp1 at the proximal promoter

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.ope

Two ZNF509 (ZBTB49) isoforms induce cell-cycle arrest by activating transcription of p21/CDKN1A and RB upon exposure to genotoxic stress

ZNF509 is unique among POK family proteins in that four isoforms are generated by alternative splicing. Short ZNF509 (ZNF509S1, -S2 and -S3) isoforms contain one or two out of the seven zinc-fingers contained in long ZNF509 (ZNF509L). Here, we investigated the functions of ZNF509 isoforms in response to DNA damage, showing isoforms to be induced by p53. Intriguingly, to inhibit proliferation of HCT116 and HEK293 cells, we found that ZNF509L activates p21/CDKN1A transcription, while ZNF509S1 induces RB. ZNF509L binds to the p21/CDKN1A promoter either alone or by interacting with MIZ-1 to recruit the co-activator p300 to activate p21/CDKN1A transcription. In contrast, ZNF509S1 binds to the distal RB promoter to interact and interfere with the MIZF repressor, resulting in derepression and transcription of RB. Immunohistochemical analysis revealed that ZNF509 is highly expressed in normal epithelial cells, but was completely repressed in tumor tissues of the colon, lung and skin, indicating a possible role as a tumor suppressorope

KR-POK interacts with p53 and represses its ability to activate transcription of p21WAF1/CDKN1A.

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.ope

The proto-oncoprotein FBI-1 interacts with MBD3 to recruit the Mi-2/NuRD-HDAC complex and BCoR and to silence p21WAF/CDKN1A by DNA methylation

The tumour-suppressor gene CDKN1A (encoding p21Waf/Cip1) is thought to be epigenetically repressed in cancer cells. FBI-1 (ZBTB7A) is a proto-oncogenic transcription factor repressing the alternative reading frame and p21WAF/CDKN1A genes of the p53 pathway. FBI-1 interacts directly with MBD3 (methyl-CpG–binding domain protein 3) in the nucleus. We demonstrated that FBI-1 binds both non-methylated and methylated DNA and that MBD3 is recruited to the CDKN1A promoter through its interaction with FBI-1, where it enhances transcriptional repression by FBI-1. FBI-1 also interacts with the co-repressors nuclear receptor corepressor (NCoR), silencing mediator for retinoid and thyroid receptors (SMRT) and BCL-6 corepressor (BCoR) to repress transcription. MBD3 regulates a molecular interaction between the co-repressor and FBI-1. MBD3 decreases the interaction between FBI-1 and NCoR/SMRT but increases the interaction between FBI-1 and BCoR. Because MBD3 is a subunit of the Mi-2 autoantigen (Mi-2)/nucleosome remodelling and histone deacetylase (NuRD)-HDAC complex, FBI-1 recruits the Mi-2/NuRD-HDAC complex via MBD3. BCoR interacts with the Mi-2/NuRD-HDAC complex, DNMTs and HP1. MBD3 and BCoR play a significant role in the recruitment of the Mi-2/NuRD-HDAC complex– and the NuRD complex–associated proteins, DNMTs and HP. By recruiting DNMTs and HP1, Mi-2/NuRD-HDAC complex appears to play key roles in epigenetic repression of CDKN1A by DNA methylation.ope

ZBTB2 increases PDK4 expression by transcriptional repression of RelA/p65

The NF-κB is found in almost all animal cell types and is involved in a myriad of cellular responses. Aberrant expression of NF-κB has been linked to cancer, inflammatory diseases and improper development. Little is known about transcriptional regulation of the NF-κB family member gene RelA/p65. Sp1 plays a key role in the expression of the RelA/p65 gene. ZBTB2 represses transcription of the gene by inhibiting Sp1 binding to a Sp1-binding GC-box in the RelA/p65 proximal promoter (bp, -31 to -21). Moreover, recent studies revealed that RelA/p65 directly binds to the peroxisome proliferator-activated receptor-γ coactivator1α (PGC1α) to decrease transcriptional activation of the PGC1α target gene PDK4, whose gene product inhibits pyruvate dehydrogenase (PDH), a key regulator of TCA cycle flux. Accordingly, we observed that RelA/p65 repression by ZBTB2 indirectly results in increased PDK4 expression, which inhibits PDH. Consequently, in cells with ectopic ZBTB2, the concentrations of pyruvate and lactate were higher than those in normal cells, indicating changes in glucose metabolism flux favoring glycolysis over the TCA cycle. Knockdown of ZBTB2 in mouse xenografts decreased tumor growth. ZBTB2 may increase cell proliferation by reprogramming glucose metabolic pathways to favor glycolysis by upregulating PDK4 expression via repression of RelA/p65 expression.ope

새로운 ZNF509 단백질의 동정과 세포 증식 조절

Dept. of Medical Science/박사Cell proliferation is a process which is precisely controlled by interaction between a number of regulatory proteins. In cancer cells, the aberrant expression of oncogenes and/or tumor suppressor genes causes dramatic changes in regulatory programs controlling cell proliferation. Recently, the POK family of transcription factors was characterized as important oncogenes or tumor suppressors that regulate the expression of cell cycle control genes.In this study, ZNF509, a novel POK family of transcription factors that is induced by p53, was characterized to be a transcriptional activator of the CDKN1A cell cycle arrest gene. It was shown that ZNF509 directly bound to the CDKN1A proximal promoter by EMSA, Oligonucleotide pull-down, and ChIP assays. Interaction of ZNF509 and MIZ-1 activated the transcription of CDKN1A synergistically. This interaction between ZNF509 and MIZ-1 recruited the co-activator p300, and the ZNF509-MIZ-1-p300 complex increased histone Ac-H3 and -H4 levels at the CDKN1A proximal promoter. Knockdown of endogenous ZNF509 expression caused decrease in CDKN1A transcription, and resulted in increasing cell proliferation.ZNF509 not only arrested cell cycle progression but also inhibited apoptosis when cells were exposed to DNA damaging agents. Expression of p53 was increased by etoposide treatment regardless of ZNF509 expression and, in turn, p53 increases PUMA gene expression. Expression of PUMA gene decreased moderately only in the presence of ZNF509. IP, GST pull-down, Oligonucleotide pull-down, and ChIP assays showed that ZNF509 directly interacted with p53 via its zinc-fingers, resulting in inhibition p53 binding to regulatory elements of the PUMA promoter and suppression of PUMA transcription.These results suggest that ZNF509 serves important functions in cell fate decision directly toward cell survival. First, ZNF509 potently activates transcription of CDKN1A through interaction with MIZ-1. Second, ZNF509 inhibits apoptosis by repressing the PUMA gene expression by inhibiting DNA binding of p53 to the PUMA promoter.ope

종양 억제 유전자 p53의 분자적인 기능을 조절하는 새로운 proto-oncogene Zbtb7c

Dept. of Medical Science/석사[한글]우리 연구팀은 POZ domain 계열 FBI-1의 세포 기능 조절에 대한 연구를 진행하고 있다. FBI-1과 밀접한 관계의 새로운 BTB/POZ domain protein에 관한 연구를 진행하기 위해 GeneBank database를 기초로 사람의 모든 BTB/POZ domain protein을 검색하여 BTB/POZ domain protein간의 amino acid sequence의 similarity를 분석하여 BTB/POZ polypeptides를 31개의 주요 그룹으로 분류해 phylogenic tree를 완성했다. 우리는 FBI-1과 가장 높은 homology를 가지고 있는 Zbtb7c를 발견하게 되었고 POZ domain 및 zinc finger motif만을 비교했을 때 각각 90%이상의 homology를 가지고 있는 것을 확인했다. Zbtb7c는 총 619개의 amino acid로 이루어져 있으며 구조적으로 FBI-1과 거의 유사하게 N-terminus에 POZ domain, C-terminus에 4개의 Kruppel-like zinc finger motif, 그리고 nuclear localization signal을 가지고 있었다.Zbtb7c는 human papillomavirus (HPV)68의 E6와 E7 gene이 숙주세포의 chromosome 18q21에 해당되는 곳에 integration된 자궁 암세포에서 처음 분리되었고, 그 발현의 조직분포가 제한적인 것으로 생각되었지만, 우리는 RT-PCR을 통해 mouse에서 spleen을 제외한 거의 모든 tissue에서 Zbtb7c가 발현되고 있음을 확인하게 되었다. 최근 FBI-1이 세포주기를 촉진시킨다는 보고로 proto-oncogene으로서 주목 받고 있는데 우리는 아마도 Zbtb7c 역시 세포주기에 관계되는 Arf-Mdm2-p53-p21Waf/Cip1과 RB의 promoter에 영향을 끼칠 것으로 예측하고, luciferase assay를 실시하였다. 흥미롭게도, FBI-1이 거의 모든 promoter의 전사를 억제하는 것에 반해 Zbtb7c는 오직 Arf와 p21의 전사만을 억제하는 것을 관찰하였다.특히 우리는 p21이 세포주기조절에 있어 최종적으로 기여하는 바가 크다고 판단하여 p21의 전사를 억제하는 Zbtb7c 의 정확한 작용기전을 연구하고자 하였다. 그래서 promoter reporter assay, EMSA, ChIP, GST-pull down, 그리고 Immunoprecipitation assay등을 실시한 결과 Zbtb7c는 그것의 zinc finger를 통해 p21 promoter상의 distal부분에 결합하는 p53과 상호작용하는 것으로 나타났다. 마찬가지 위와 같은 실험방법을 통해 Zbtb7c는 BTB/POZ domain을 통해 NCoR나 BCoR와 같은 corepressor와의 상호작용을 하는 것으로 나타났고 이를 통해 최종적으로 p21 proximal 부분의 Histone을 deacetylation 시킴으로써 전사를 억제시킨다는 것을 알게 되었다. 뿐만 아니라, 이러한 전사적 억제효과가 현상적으로 잘 드러나는지를 관찰하기 위해 foci formation, FACS, BrdUrd, 그리고 MTT assay를 실시한 결과 Zbtb7c가 과발현 하게되면 세포주기중 S phase의 양이 급격히 증가함으로써 세포주기가 급속히 촉진되는 것을 확인할 수 있었다.최종적으로 이런 현상이 mouse의 종양성장에 어떤 영향을 미치는지 알아보고자 인위적으로 mouse에 종양을 형성시키고 Zbtb7c를 과발현 시킬 수 있는 virus를 infection시켰을 때 그 종양의 크기가 대조군에 비해 현저하게 커진 것을 관찰할 수 있었으며 반대로 Zbtb7c의 발현을 억제시키는 virus를 infection시켰을 때는 종양의 크기가 감소하는 것을 관찰할 수 있었다.이렇듯 위와 같은 Zbtb7c에 관한 본 연구는 세포주기조절에 관한 작용점의 파악과 발암 과정의 이해증진, 그리고 새로운 치료 방법의 도출에 필요한 중요한 연구 자료가 될 것으로 생각된다. [영문]Zbtb7c is a ubiquitously expressed BTB/POZ-domain class transcription factor. We investigated whether Zbtb7c has an effect on cell cycle progression and oncogenesis through regulated gene expression of key cell cycle regulatory genes in the Arf-Mdm2-p53-p21Waf/Cip1 pathway. Zbtb7c repressed transcription of p21Waf/Cip1 and p19Arf, which requires p53. Zbtb7c interacts with p53 bound on the p21Waf/Cip1 distal promoter via its zinc-finger domain and also interacts with transcriptional corepressors, such as NCoR and BCoR, causing the deacetylation of histones at the p21Waf/Cip1 proximal promoter. Adenoviral delivery of the Zbtb7c gene potently promotes tumor growth in mice transplanted with U343 cancer cells. In contrast, RNA that interfered with Zbtb7c mRNA abolished tumor growth. The molecular interactions among p53, Zbtb7c, and corepressors at the distal p53 binding element are important in the transcriptional repression of p21Waf/Cip1, oncogenic cellular transformation, cell proliferation, and tumor growth. Zbtb7c is one of the key control genes of the Arf-Mdm2-p53-p21Waf/Cip1 pathway as it acts at both transcription and protein levels. Our study also suggests that p53 can act as an oncogene in the presence of Zbtb7c.ope

Reciprocal negative regulation between the tumor suppressor protein p53 and B cell CLL/lymphoma 6 (BCL6) via control of caspase-1 expression

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.restrictio