1: Eur J Cell Biol. 2005 Jun;84(5):543-53. Targeted inhibition of the transcription factor YY1 in an embryonal carcinoma cell line results in retarded cell growth, elevated levels of p53 but no increase in apoptotic cell death. Bain M, Sinclair J. Department of Medicine, University of Cambridge, PO Box 157, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ, UK. mb231@mole.bio.cam.ac.uk The ubiquitous cellular transcription factor Yin Yang-1 (YY1) is involved in the transcriptional regulation of many cellular and viral genes. It is known to bind to, and repress the activity of, the major immediate-early promoter of human cytomegalovirus (HCMV) in non-permissive T2 cells. Thus, YY1 is at least partly responsible for the lack of productive lytic infection of these cells. In this study, we have used short interfering RNA (siRNA) to specifically knock down YY1 expression in T2 cells. We wished to assess whether the removal of this negatively acting factor would render these ordinarily non-permissive cells permissive for infection. We show that we can potently inhibit YY1 expression but that this knock down has dramatic effects on the normal biology of the cells. In particular, we noted growth retardation, altered morphology and increased levels of p53. However, the cells do not undergo apoptosis, are not induced to differentiate, do not exhibit excessive levels of DNA damage, and synthesise DNA normally. PMID: 16003908 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: J Virol. 2005 Apr;79(7):4246-56. Silencing of integrated human papillomavirus type 18 oncogene transcription in cells expressing SerpinB2. Darnell GA, Antalis TM, Rose BR, Suhrbier A. Queensland Institute of Medical Research, University of Queensland, Brisbane, Queensland, Australia. The serine protease inhibitor SerpinB2 (PAI-2), a major product of differentiating squamous epithelial cells, has recently been shown to bind and protect the retinoblastoma protein (Rb) from degradation. In human papillomavirus type 18 (HPV-18)-transformed epithelial cells the expression of the E6 and E7 oncoproteins is controlled by the HPV-18 upstream regulatory region (URR). Here we illustrate that PAI-2 expression in the HPV-18-transformed cervical carcinoma line HeLa resulted in the restoration of Rb expression, which led to the functional silencing of transcription from the HPV-18 URR. This caused loss of E7 protein expression and restoration of multiple E6- and E7-targeted host proteins, including p53, c-Myc, and c-Jun. Rb expression emerged as sufficient for the transcriptional repression of the URR, with repression mediated via the C/EBPbeta-YY1 binding site (URR 7709 to 7719). In contrast to HeLa cells, where the C/EBPbeta-YY1 dimer binds this site, in PAI-2- and/or Rb-expressing cells the site was occupied by the dominant-negative C/EBPbeta isoform liver-enriched transcriptional inhibitory protein (LIP). PAI-2 expression thus has a potent suppressive effect on HPV-18 oncogene transcription mediated by Rb and LIP, a finding with potential implications for prognosis and treatment of HPV-transformed lesions. PMID: 15767426 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Eur J Cancer. 2005 Mar;41(5):807-15. Human papillomavirus-16 associated squamous cell carcinoma of the head and neck (SCCHN): a natural disease model provides insights into viral carcinogenesis. Ferris RL, Martinez I, Sirianni N, Wang J, Lopez-Albaitero A, Gollin SM, Johnson JT, Khan S. UPCI Research Pavilion, The Hillman Cancer Center, 5117 Centre Avenue, Room 1.19d, Pittsburgh, PA 15213-1863, USA. ferrisrl@upmc.edu Uncertainty regarding the causality of human papillomaviruses (HPVs) in squamous cell carcinoma of the head and neck (SCCHN) necessitates better in vitro models. We carried out molecular analyses of a novel, naturally HPV-16-transformed SCCHN cell line (UPCI:SCC090) and show high copy number of HPV-16 DNA, present in a head to tail, tandemly repeated integrated state. Sequence analysis of the HPV-16 long control region (LCR) in UPCI:SCC090 revealed a deletion of 163 bp, removing a portion of the enhancer sequence, including the binding sites for the transcription factors YY1 and NF1. The E6 and E7 oncogenes of HPV-16 are expressed at high levels in this cell lines, as determined by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). UPCI:SCC090 contains wild-type tumour suppressor TP53 gene, and undetectable p53 protein, except after treatment with cisplatin, specific proteasome inhibitors or by E6 RNA interference, suggesting E6-dependent degradation of p53 in this cell line. The results of our studies are consistent with a causative role of HPV-16 in the pathogenesis of SCCHN. PMID: 15763658 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: Proc Natl Acad Sci U S A. 2004 Aug 17;101(33):12165-70. Epub 2004 Aug 4. YY1 inhibits the activation of the p53 tumor suppressor in response to genotoxic stress. Gronroos E, Terentiev AA, Punga T, Ericsson J. Biomedical Center, Ludwig Institute for Cancer Research, Box 595, Husargatan 3, S-751 24 Uppsala, Sweden. The tumor suppressor p53 regulates cell-cycle progression and apoptosis in response to genotoxic stress, and inactivation of p53 is a common feature of cancer cells. The levels and activity of p53 are tightly regulated by posttranslational modifications, including phosphorylation, ubiquitination, and acetylation. Here, we demonstrate that the transcription factor Yin Yang 1 (YY1) interacts with p53 and inhibits its transcriptional activity. We show that YY1 disrupts the interaction between p53 and the coactivator p300 and that expression of YY1 blocks p300-dependent acetylation and stabilization of p53. Furthermore, expression of YY1 inhibits the accumulation of p53 and the induction of p53 target genes in response to genotoxic stress. YY1 also interacts with Mdm2 and the expression of YY1 promotes the assembly of the p53-Mdm2 complex. Consequently, YY1 enhances Mdm2-mediated ubiquitination of p53. Inactivation of endogenous YY1 enhances the accumulation of p53 as well as the expression of p53 target genes in response to DNA damage, and it sensitizes cells to DNA damage-induced apoptosis. Hence, our results demonstrate that YY1 regulates the transcriptional activity, acetylation, ubiquitination, and stability of p53 by inhibiting its interaction with the coactivator p300 and by enhancing its interaction with the negative regulator Mdm2. YY1 may, therefore, be an important negative regulator of the p53 tumor suppressor in response to genotoxic stress. PMID: 15295102 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Cell. 2004 Jun 25;117(7):859-72. Yin Yang 1 is a negative regulator of p53. Sui G, Affar el B, Shi Y, Brignone C, Wall NR, Yin P, Donohoe M, Luke MP, Calvo D, Grossman SR, Shi Y. Department of Pathology, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA. Yin Yang 1 (YY1) is a transcription factor that plays an essential role in development. However, the full spectrum of YY1's functions and mechanism of action remains unclear. We find that YY1 ablation results in p53 accumulation due to a reduction of p53 ubiquitination in vivo. Conversely, YY1 overexpression stimulates p53 ubiquitination and degradation. Significantly, recombinant YY1 is sufficient to induce Hdm2-mediated p53 polyubiquitination in vitro, suggesting that this function of YY1 is independent of its transcriptional activity. We identify direct physical interactions of YY1 with Hdm2 and p53 and show that the basis for YY1-regulating p53 ubiquitination is its ability to facilitate Hdm2-p53 interaction. Importantly, the tumor suppressor p14ARF compromises the Hdm2-YY1 interaction, which is important for YY1 regulation of p53. Taken together, these findings identify YY1 as a potential cofactor for Hdm2 in the regulation of p53 homeostasis and suggest a possible role for YY1 in tumorigenesis. PMID: 15210108 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: Biochem Biophys Res Commun. 2004 May 28;318(2):615-24. YY1 binding to a subset of p53 DNA-target sites regulates p53-dependent transcription. Yakovleva T, Kolesnikova L, Vukojevic V, Gileva I, Tan-No K, Austen M, Luscher B, Ekstrom TJ, Terenius L, Bakalkin G. Department of Clinical Neuroscience, Karolinska Institute, Stockholm, Sweden. The tumor suppressor protein p53 regulates gene transcription through binding to specific DNA-target sites. We here demonstrate that a subset of these sites is targeted by another DNA-binding factor. Binding specificity, reactivity with specific antibodies, and experiments with purified protein identified the factor as the multifunctional transcription regulator YY1. The YY1 core binding sequence ACAT is present in the center of p53-half-binding sites in the p21 and GADD45 genes regulating growth arrest and DNA repair, respectively, but is absent in those of the Bax gene critical for apoptosis. In transfection experiments YY1 inhibits p53-activated transcription from the p53-binding site that contains the ACAT sequence. YY1 and p53 are colocalized around the nucleoli and in discrete nuclear domains in PC12 cells undergoing apoptosis. YY1 might attenuate p53-dependent transcription from a subset of p53-target genes and this may be relevant for directing cells either to growth arrest or apoptosis upon p53 activation. PMID: 15120643 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 2000 Sep;14(3):205-8. [The immortalized cell lines induced by human papillomavirus type 16 (HPVS 16) YY1 binding-site mutants have some characteristics of transformed cells] Liu H, Dong X, Zhou W. Beijing Stomatological Hospital, The Capital University of Medical Sciences, Beijing 100050, China. OBJECTIVE: To study the biological characteristics of the immortalized cell lines of human keratinocytes from foreskin induced by human papillomavirus type 16 (HPV 16) YY1 binding-site mutants. METHODS: The cellular extracts of immortalized cell lines were prepared and the cellular endogenous p53 proteins were determined with Western blot. The cellular telomerase activities were analyzed with TRAP. The immortalized cells were incubated in a soft agarose medium containing 10% FCS and the anchorage-independent growth abilities of the tested cell lines were evaluated. RESULTS: Western blot showed that the endogenous p53 protein in all 4 tested clones were undetectable. The telomerases were clearly identified within the 4 tested cell lines, and showed an increasing activity along with the passages of the cells. All the clones were unable to grow in the soft-agarose medium during the earlier passages, but three of them showed anchorage-independent-growth at the passages of 30-35. CONCLUSIONS: The immortalized cell lines induced by the HPV 16 YY1 binding-site mutants have active telomerase activity and ability of anchorage-independent growth. PMID: 11498679 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: J Biol Chem. 2001 May 11;276(19):15650-8. Epub 2001 Feb 23. p53 Latency. C-terminal domain prevents binding of p53 core to target but not to nonspecific DNA sequences. Yakovleva T, Pramanik A, Kawasaki T, Tan-No K, Gileva I, Lindegren H, Langel U, Ekstrom TJ, Rigler R, Terenius L, Bakalkin G. Experimental Alcohol and Drug Addiction Research Section, Department of Clinical Neuroscience, Stockholm University, Stockholm, Sweden. The p53 transcription factor is either latent or activated through multi-site phosphorylation and acetylation of the negative regulatory region in its C-terminal domain (CTD). How CTD modifications activate p53 binding to target DNA sequences via its core domain is still unknown. It has been proposed that nonmodified CTD interacts either with the core domain or with DNA preventing binding of the core domain to DNA and that the fragments of the CTD regulatory region activate p53 by interfering with these interactions. We here characterized the sequence and target specificity of p53 activation by CTD fragments, interaction of activating peptides with p53 and target DNA, and interactions of "latent" p53 with DNA by a band shift assay and by fluorescence correlation spectroscopy. In addition to CTD fragments, several long basic peptides activated p53 and also transcription factor YY1. These peptides and CTD aggregated target DNA but apparently did not interact with p53. The potency to aggregate DNA correlated with the ability to activate p53, suggesting that p53 binds to target sequences upon interactions with tightly packed DNA in aggregates. Latent full-length p53 dissociated DNA aggregates via its core and CTD, and this effect was potentiated by GTP. Latent p53 also formed complexes via both its core and CTD with long nontarget DNA molecules. Such p53-DNA interactions may occur if latent p53 binding to DNA via CTD prevents the interaction of the core domain with target DNA sites but not with nonspecific DNA sequences. PMID: 11279079 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: J Biol Chem. 2000 Jul 7;275(27):20488-95. The phosphotyrosyl phosphatase activator gene is a novel p53 target gene. Janssens V, Van Hoof C, De Baere I, Merlevede W, Goris J. Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit Leuven, Herestraat 49, B-3000 Leuven, Belgium. The minimal promoter of the phosphotyrosyl phosphatase activator (PTPA) gene, encoding a regulator of protein phosphatase 2A contains two yin-yang 1 (YY1)-binding sites, positively regulating promoter activity. We now describe a role for p53 in the regulation of PTPA expression. Luciferase reporter assays in Saos-2 cells revealed that p53 could down-regulate PTPA promoter activity in a dose-dependent manner, whereas four different p53 mutants could not. The p53-responsive region mapped to the minimal promoter. Overexpression of YY1 reverses the repressive effect of p53, suggesting a functional antagonism between p53 and YY1. The latter does not involve competition for YY1 binding, but rather direct control of YY1 function. Inhibition of PTPA expression by endogenous p53 was demonstrated in UVB-irradiated HepG2 cells, both on the mRNA and protein level. Also basal PTPA levels are higher in p53-negative (Saos-2) versus p53-positive (HepG2, U2OS) cells, suggesting "latent" p53 can control PTPA expression as well. The higher PTPA levels in U2OS cells, programmed to overexpress constitutively a dominant-negative p53 mutant, corroborate this finding. Thus, PTPA expression is negatively regulated by p53 in normal conditions and in conditions where p53 is up-regulated, via an as yet unknown mechanism involving the negative control of YY1. PMID: 10787423 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: Mol Biol Rep. 1999 Dec;26(4):223-30. Differential binding of NF1 transcription factor to P53 gene promoter and its depletion in human breast tumours. Nayak BK, Das BR. Molecular Biology Division, Institute of Life Sciences, Bhubaneswar, India. Different transcription factors activate and repress the p53 gene expression. Recently, a tissue specific binding of NF1/YY1 to p53 promoter has been reported and further, it has been demonstrated that NF1/YY1 activates p53 promoter activity. The deregulated expression of p53 appears to be a central feature of malignant transformation and the basis of this deregulation is not well defined. Hence, an attempt has been made to know the binding of NF1/YY1 to p53 promoter taking breast tumour as a model system. Results have indicated a differential binding of NF1 to p53 promoter and a depletion or low level of NF1 in majority of breast tumour samples. Further, a correlation between NF1 and p53 has indicated the presence of p53 RNA even without NF1. Hence it is assumed that p53 expression is not NF1-dependent in breast tumours. However, the results clearly demonstrate a deregulation of NF1 transcription factor in breast tumours. PMID: 10634504 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: Biochem Cell Biol. 1999;77(3):209-14. A 40-kDa NF1-like protein, not YY1, binds to the rat p53 promoter for transactivation in various rat organs. Lee M, Song H, Yu S, Lee K, Park JS. Department of Chemistry, Seoul National University, Korea. Two recognition motifs of a 40-kDa NF1-like protein were previously identified in the rat p53 promoter. One is located between -296 and -312 (NF1-like element 1) and the other between -195 and -219 (NF1-like element 2). The latter one was also identified as a NF1/YY1 recognition motif in the human p53 promoter. NF1 or YY1 binds to the motif and regulates the expression of the human p53 gene in a tissue-specific manner. In this study, we investigated the binding protein for NF1-like element 2 in various rat tissues. Unlike the human p53 transcription, an NF1-like protein, not YY1, bound to the motif in every tested tissue: thymus, kidney, and spleen. In vitro transcription assay also confirmed that the NF1-like protein regulated the p53 transcription in rat spleen, although the human p53 transcription was regulated by YY1 in that organ. The molecular mass of the binding protein was determined to be 40 kDa, which was the same as that of the NF1-like protein identified in liver. Therefore, the 40-kDa NF1-like protein may be a universal transcription regulator for the rat p53 gene. PMID: 10505791 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 12: Biol Chem. 1998 Nov;379(11):1333-40. Transcription of the rat p53 gene is mediated by factor binding to two recognition motifs of NF1-like protein. Lee M, Song H, Park S, Park J. Department of Chemistry, Seoul National University, Korea. In this study we analyzed the ratp53 promoter by electrophoretic mobility shift assay (EMSA) and DNase I footprinting analysis. As a result we identified two protein binding elements (element 1: -296 to -312, element 2: -195 to -219) with sequence homology to each other. The two identified elements bind to the same kind of protein. To identify the protein binding to these elements, competition assays were carried out with double stranded oligonucleotides containing NF1, YY1, and CRE consensus motifs. Only the NF1 consensus motif competed with element 1 and 2. Element 2 is conserved between the rat, human, and mouse p53 promoters, and has an NF1 consensus motif. However, the sequences of element 1 are comparatively variable between the species. Only the element 1 region of the rat p53 promoter has partial homology to the NF1 consensus motif. This suggests that the element 1 is specific for the rat p53 gene. The molecular mass of the binding protein, determined by Southwestern blotting analysis, was 40 kDa, which is different from that of NF1. In EMSA with an anti-NF1 antibody, DNA-protein complexes were neither supershifted nor decreased. The 40 kDa protein was also detected in rat spleen and lung, but not in kidney. The binding protein was purified by sequence-specific DNA affinity chromatography and it was confirmed that the purified protein binds to the two regions. It was also proved that the identified two elements are required for basal level transcription of the rat p53 gene by in vitro transcription assay. PMID: 9865606 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 13: Mol Cell Biol. 1996 Oct;16(10):5933-45. YY1 and NF1 both activate the human p53 promoter by alternatively binding to a composite element, and YY1 and E1A cooperate to amplify p53 promoter activity. Furlong EE, Rein T, Martin F. Pharmacology Department, University College Dublin, Ireland. A novel transcription factor binding element in the human p53 gene promoter has been characterized. It lies about 100 bp upstream of the major reported start site for human p53 gene transcription. On the basis of DNase I footprinting studies, electromobility shift assay patterns, sequence specificity of binding, the binding pattern of purified transcription factors, effects of specific antibodies, and methylation interference analysis we have identified the site as a composite element which can bind both YY1 and NF1 in an independent and mutually exclusive manner. The site is conserved in the human, rat, and mouse p53 promoters. The occupancy of the site varies in a tissue-specific manner. It binds principally YY1 in nuclear extracts of rat testis and spleen and NF1 in extracts of liver and prostate. This may facilitate tissue-specific control of p53 gene expression. When HeLa cells were transiently transfected with human p53 promoter-chloramphenicol acetyltransferase reporter constructs, a mutation in this composite element which disabled YY1 and NF1 binding caused a mean 64% reduction in basal p53 promoter activity. From mutations which selectively impaired YY1 or NF1 binding and the overexpression of YY1 or NF1 in HeLa cells we concluded that both YY1 and NF1 function as activators when bound to this site. In transient cotransfections E1A could induce the activity of the p53 promoter to a high level; 12S E1A was threefold as efficient as 13S E1A in this activity, and YY1 bound to the composite element was shown to mediate 55% of this induction. Overexpressed YY1 was shown to be able to synergistically activate the p53 promoter with E1A when not specifically bound to DNA. Deletion of an N-terminal domain of E1A, known to be required for direct E1A-YY1 interaction and E1A effects mediated through transcriptional activator p300, blocked the E1A induction of p53 promoter activity. PMID: 8816507 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------