Functional characterization of a novel POK family transcription factor, ZBTB5, in the transcriptional regulation of cell cycle arrest p21CIP1 gene

의학과/석사[한글]인간 게놈 프로젝트를 통해 알려지게 된 많은 유전자들은 최근 기능 동정을 중심으로 많은 연구가 진행되고 있다. 이 계열의 단백질은 단백질-단백질간의 상호작용 모티프로 작용하는 BTB/POZ domain을 가지며 다양한 세포기능 조절에 관여한다. 세포 기능조절 단백질은 수십-수백 개의 아미노산으로 구성된 조각(모듈: module)로 구성되어 있으며, 모듈을 중심으로 일어나는 단백질-단백질 상호작용에 의하여 고유의 기능을 발휘한다. 주로 POZ-도메인을 갖는 단백질들은 세포 죽음, 염색체 구조조절, 암 발생, 유전자 발현조절 등에 관련되어 있다. 이에 우리 팀은 BTB/POZ 도메인중 하나인 ZBTB5를 분리, 동정하였으며, RT-PCR을 통해 생쥐에서 심장과 갈색지방을 제외한 거의 모든 조직에서 ZBTB5가 발현되고 있음을 확인하게 되었다. SAGE 분석 결과 안암과 근육암에서 ZBTB5의 발현량이 현저하게 증가되어 있음을 알 수 있다. 최근 BTB/POZ 도메인 단백질 중 하나인 FBI-1이 세포주기를 촉진시킨다는 보고로 proto-oncogene으로서 주목을 받고 있는데, 우리는 아마도 ZBTB5 역시 세포주기에 관계되는 Arf-Mdm2-p53-p21의 promoter에 영향을 끼칠 것으로 예상하고 luciferase assay를 실시하였다. 흥미롭게도 p53 경로의 하위단계 조절인자인 p21의 전사만 억제하는 것을 관찰하였다. 이는 상위단계의 조절여부와는 상관없이 하위단계 인자를 조절하게 때문에 세포주기 조절에 기여하는 바가 크다고 판단하여 p21의 전사를 억제 하는 ZBTB5의 작용기전을 연구하였다. ZBTB5 는 직접적으로 세포주기 조절인자인 p21 유전자 프로모터의 근접조절 부위의 GC Box 5/6 번 자리와 Distal 부위의 p53 결함부위에 징크핑거를 통하여 결합하여 p21 의 발현을 감소시킴을 GST pull- down, EMSA, ChIP을 통하여 밝히었다. 또한, 기존의 알려진 p53과 Sp1 과의 관계는 ChIP(Chromatin Immunoprecipitation Assay)를 통해 p53과는 결합자리를 두고 경쟁하며, Sp1의 결합은 증가시킴을 알 수 있었다. 이와 같은 방법으로 ZBTB5는 POZ와 징크핑거 도메인을 통하여 corepressor 히스톤 디아세틸 복합체 중 BCoR, NCoR 그리고 SMRT와 상호작용을 하는 것으로 나타났고 최종적으로 p21 프로모터 근접조절 부분의 히스톤을 디아세틸화 시킴으로써 전사를 억제시킨다. 이러한 전사 억제효과가 세포기능 조절에 어떠한 영향을 미치는가를 관찰하기 위하여 MTT 와 FACS 분석을 실시하였다. 그 결과 ZBTB5가 세포성장과 세포주기의 조절에 중요하며 특히, 세포주기중 S 기의 세포수가 현저히 증가함을 관찰하였다. 결과적으로, ZBTB5는 세포주기를 멈추게 하는 p21 의 발현을 감소시킴으로써 세포의 증식을 촉진할 수 있는 원암 유전자의 성격을 가지고 있음을 발견하였다 [영문]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 Kruppel-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 ~ -2299; bp -1416 ~ -1392). Chromatin immunoprecipitation assays showed that ZBTB5 and p53 competed with each other in occupying the p53 binding elements. ZBTB5 interacted with corepress or 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-dimethylthiazol-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.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

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

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

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

KAISO 유전자와 Kaiso Knockout mice 의 기능 동정 : 세포사멸과 고환 발생

Dept. of Medical Science/박사An unresolved issue regarding the response to genotoxic stress is identification of the regulatory protein(s) induced and determination of how they activate p53 to determine cell fate. KAISO has been previously described as a protein that represses transcription following binding to methylated DNA. In this study, I determined that its expression is induced by DNA damage prior to p53 expression. KAISO interacts with the p53-p300 complex to increase acetylation of the K320 and K382 lysine residues of p53, but not of the K381 residue. Acetylated p53 shows an increase in DNA binding specificity and activity of the p53 binding RRRCWWGYYY repeated motifs with no spacer and potently induces apoptosis by activating transcription of the CDKN1A and apoptotic genes. In Kaiso knockout (KO) MEF cells, transcription of apoptotic genes is significantly compromised. KAISO may therefore be a critical regulator of apoptosis in response to various genotoxic stresses in the mammalian cell. Spermatogenesis is a highly organized cyclic process with distinct phases: mitosis, meiosis and spermiogenesis. However, the molecular mechanisms of how spermatogonia, mitotic germ cells of the testis, self-renew and differentiate into sperm remain largely unknown. Here, we show that male Kaiso knockout mice are show testicular atrophy and infertile, and that Kaiso plays a major role in spermatogenesis by repressing Bcl6 transcription in spermatocytes. In adult Kaiso mouse knockout testes, Bcl6 expression is derepressed and represses transcription of the genes of the CREM pathway in spermatocytes, which results in without differentiation into spermatid and spermatozoa. Our study shows that Kaiso plays a critical role in spermatogenesis and may have significant implications to male infertility.ope

Cell fate decisions by c-Myc depend on ZBTB5 and p53

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Temporal and differential regulation of KAISO-controlled transcription by phosphorylated and acetylated p53 highlights a crucial regulatory role of apoptosis

Transcriptional regulator KAISO plays a critical role in cell cycle arrest and apoptosis through modulation of p53 acetylation by histone acetyltransferase p300. KAISO potently stimulates apoptosis in cells expressing WT p53, but not in p53-mutant or p53-null cells. Here, we investigated how KAISO transcription is regulated by p53, finding four potential p53-binding sites (p53-responsive DNA elements; p53REs) located in a distal 5'-upstream regulatory element, intron 1, exon 2 coding sequence, and a 3'-UTR region. Transient transcription assays of pG5-p53RE-Luc constructs with various p53REs revealed that p53 activates KAISO (ZBTB33) transcription by acting on p53RE1 (-4326 to -4227) of the 5'-upstream region and on p53RE3 (+2929 to +2959) of the exon 2 coding region during early DNA damage responses (DDRs). ChIP and oligonucleotide pulldown assays further disclosed that p53 binds to the p53RE1 and p53RE3 sites. Moreover, ataxia telangiectasia mutated (ATM) or ATM-Rad3-related (ATR) kinase-mediated p53 phosphorylation at Ser-15 or Ser-37 residues activated KAISO transcription by binding its p53RE1 or p53RE3 sites during early DDR. p53RE1 uniquely contained three p53-binding half-sites, a structural feature important for transcriptional activation by phosphorylated p53 Ser-15·Ser-37. During the later DDR phase, a KAISO-mediated acetylated p53 form (represented by a p53QRQ acetyl-mimic) robustly activated transcription by acting on p53RE1 in which this structural feature is not significant, but it provided sufficient KAISO levels to confer a p53 "apoptotic code." These results suggest that the critical apoptosis regulator KAISO is a p53 target gene that is differently regulated by phosphorylated p53 or acetylated p53, depending on DDR stage.restrictio

Transcriptional activation of APAF1 by KAISO (ZBTB33) and p53 is attenuated by RelA/p65

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.ope

Role of MIZ-1 in AMELX gene expression

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.restrictio