1: J Dairy Res. 2005;72 Spec No:34-43. Functional study of the equine beta-casein and kappa-casein gene promoters. Lenasi T, Kokalj-Vokac N, Narat M, Baldi A, Dovc P. University of Ljubljana, Biotechnical faculty, Department of Animal Science, Groblje 3, SI-1230 Domzale, Slovenia. Casein genes are expressed in a tissue-specific and highly coordinated manner. The main goals of casein gene promoter studies are to unravel cis- and trans-acting factors involved in the complex signalling pathway controlling milk production, and to explore the possibility of using these promoters for tissue-specific production of heterologous proteins in the mammary gland. Here we present a comparative study of the equine beta-casein and kappa-casein gene proximal promoters. In order to confirm the assumption that in the horse, as in other mammalian species, casein genes are organized in a cluster located on a single chromosome, we performed in situ hybridization of pro-metaphase chromosomes with two BAC clones containing different equine casein genes. Sequence analysis of the beta-casein and kappa-casein gene proximal promoters revealed binding sites for activators (STAT5, GRE, NF1, MAF) and repressors (YY1, PMF), characteristic for casein genes. The alignments of casein gene promoters revealed the highest sequence identity in the proximal promoter region between the equine and human beta-casein gene promoters. We directly compared the activity of equine beta-casein and kappa-casein gene promoters in vitro using bovine mammary gland cell line BME-UV1. In this system, the kappa-casein gene proximal promoter activated the reporter gene expression more efficiently than the beta-casein gene promoter of approximately the same length. The 810 bp of beta-casein promoter activated the reporter gene expression more efficiently than the long fragment (1920 bp) and the 1206 bp fragment of the same promoter, which included also 396 bp of 5' UTR. PMID: 16180719 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: 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] --------------------------------------------------------------- 3: Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 1998 Sep;12(3):217-22. [YY1 and its repressive effect on human papillomavirus 16 early promoter P97 existed widely among human epithelial cell lines] [Article in Chinese] Dong X, Liu H, Pfister H. Institute of Virology, Chinese Academy of Preventive Medicine, Beijing 100052. Cellular transcription factor YY1 represses the activity of early promoter P97 of HPV 16. Removal of the specific binding sites for YY1 in HPV 16 LCR can increase the P97 activity. To analyse the expression of YY1 and the existence of the activation effect on P97 due to removing YY1 binding sites in the LCR region, we extracted nuclear proteins from four cervical cancer cell lines and two human keratinocytes. EMSA assays were carried out in vitro. At the same time, we transiently transfected the luciferase reporter plasmids, which contain HPV 16 reference and mutated LCR sequences, respectively, into the different cells and determined the luciferase expressions. The results showed that all the analysed cell lines contained biologically functional YY1 proteins. No remarkable difference in the concentration of native YY1 proteins could be detected among the tested cells. Removal of YY1 binding sites in HPV 16 LCR could elevate P97 activity in several analysed cell lines, including normal human primary kerationcytes from foreskin. It suggests that YY1 regulatory system exists widely in human epithelial cell lines. We also found that the transcription activator NF1 was more highly expressed in C33a cell than in HT3 cell, indicating an unevenly distribution of functional proteins in various cell lines. PMID: 12526319 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: J Clin Virol. 2001 Dec;23(1-2):65-77. Intratype HPV16 sequence variation within LCR of isolates from asymptomatic carriers and cervical cancers. Schmidt M, Kedzia W, Gozdzicka-Jozefiak A. Department of Molecular Virology, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Miedzychodzka 5, 60-371 Poznan, Poland. mschmidt@amu.edu.pl BACKGROUND: HPV16 is a predominant type of virus identified in genital lesions and strongly associated with the development of genital cancers. Infection with the virus is considered to be the main risk factor in the development of cervical cancer. Based on HPV16 DNA isolated from invasive cancers, a classification of intratype genetic variants was established and the strains were designated according to geographical regions. The HPV16 variants classification was based on isolates derived from cancers. OBJECTIVES: Analysis of HPV16 LCR variants isolated from asymptomatic carriers for comparison with cervical cancer isolates to examine whether a correlation can be found between cervical epithelium state and variant of HPV16 it carries. MATERIALS AND METHODS: The HPV16 LCR fragments were amplified by PCR using DNA isolated from cervical swabs and tissue sections then screened for nucleotide changes by SSCP. Polymorphic sites were analysed for regulatory protein binding properties by EMSA. RESULTS: Comparison of the two groups revealed that isolates from cervical cancers predominantly carry changes in sequences of YY1 binding sites (especially at nucleotide 7519), while variants from asymptomatic carriers contained nucleotide changes within or close to transcription binding sites for AP-1, Oct-1, NF1, Tef-1, Tef-2, Sp1, YY1 and viral E2. EMSA study showed that sequence changes in the segment alter binding and formation of transcriptional complexes in quantitative and/or qualitative manner and so they may inflict viral activity. CONCLUSION: The results of our study show that there might be HPV16 variants of decreased oncogenic potential therefore infection with such variants can recede. PMID: 11595585 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Int J Cancer. 2000 Jun 1;86(5):695-701. Analysis of mutations in the URR and E6/E7 oncogenes of HPV 16 cervical cancer isolates from central China. Stephen AL, Thompson CH, Tattersall MH, Cossart YE, Rose BR. Department of Infectious Diseases, Faculty of Medicine, University of Sydney, Sydney, Australia. High rates of cervical cancer have been reported from parts of China and this may reflect a predominance of cervical infection with particularly aggressive human papillomavirus (HPV) variants. This PCR-based investigation of cervical tumours from Sichuan province in central China demonstrated an HPV positivity rate of 88%. HPV 16 was most common (21/34, 61%), followed by HPV 18 (3/34, 9%), while types 33, 45, 58 and 59 were each identified in one specimen. Sequencing of up to 1349 bases of the 21 HPV 16-positive isolates, encompassing the enhancer/promoter of the upstream regulatory region (URR) and the E6 and E7 genes, revealed distinct patterns of genomic stability and variability. An overall mutation rate of 5% was seen in the URR. One isolate had a large deletion of 436 bases in the enhancer; while varying combinations of 21 point mutations were identified in the remainder, impacting several YY1, NF1, TEF-1 and Oct-1 sites. More sequence variations were found in E6 compared to E7 (81% vs. 52% of isolates showing at least one mutation), some of which resulted in changes to the translated amino acids. Since the E6/E7 genes encode the oncogenic proteins essential for malignant transformation, and as their expression is controlled by the URR, it is possible that some of the identified mutations altered the oncogenicity of the virus: either directly by changing amino acid sequences of the E6 or E7 oncoproteins, or indirectly through alterations to transcription factor binding sites in the URR. Copyright 2000 Wiley-Liss, Inc. PMID: 10797293 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: 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] --------------------------------------------------------------- 7: 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] --------------------------------------------------------------- 8: 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] --------------------------------------------------------------- 9: Biosystems. 1998 Jan;45(1):29-44. Computer analysis of distribution of putative cis- and trans-regulatory elements in milk protein gene promoters. Malewski T. Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Mrokow, Poland. Multiple alignment of 28 milk protein gene promoters belonging to seven protein superfamilies is described. In these gene promoters three groups of common motifs were found: group I--specific for all milk protein gene promoters; group II--specific only for one gene superfamily; and group III--motifs shared by several gene superfamilies. Motifs of group I and III do not have any preferential location in the promoters, while group II motifs are located in the proximal part, from -36 to -224. Milk protein gene promoters were analysed for presence of putative binding sites for nine transcription factors important for the expression of this group of genes. The transcription factor binding sites for C/EBP, CTF/NF1, MAF and MGF were found in all promoters investigated. The set of putative transcription factor binding sites or response elements for GRE, IRE, PMF, STR and YY1 is unique for every gene superfamily. PMID: 9492953 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: Nucleic Acids Res. 1996 Nov 1;24(21):4341-8. In simple synthetic promoters YY1-induced DNA bending is important in transcription activation and repression. Kim J, Shapiro DJ. Department of Biochemistry, University of Illinois, Urbana 61801, USA. Depending on promoter context, YY1 can activate or repress transcription, or provide a site for transcription initiation. To investigate whether the ability of YY1 to induce DNA bending influenced its ability to activate and repress transcription, simple synthetic promoters were constructed in which the YY1 binding site was inserted between the TATA box and either the NF1 or AP1 recognition sequences. In transient transfections of COS cells, the NF1YY1TATA and NF1RYY1TATA promoters exhibited a dramatic 15-20-fold increase in correctly initiated transcription. These promoters exhibited even larger 60-80-fold increases in transcription in HeLa cells. Neither multiple copies of the YY1 binding site alone, nor placement of a YY1 site upstream of the NF1 site activated transcription. Deletion of 4 bp between the NF1 and YY1 sites, which changes the phase of the DNA bends, abolished the 16-fold activation of transcription by NF1YY1TATA. Insertion of the YY1 site between the AP1 site and the TATA box decreased transcription approximately 3-fold. Replacing the YY1 binding site with an intrinsic DNA bending sequence mimicked this transcription repression. Sequences of similar length which do not bend DNA fail to repress AP1-mediated transcription. Gel mobility shift assays were used to show that binding of YY1 to its recognition sequence did not repress binding of AP1 to its recognition sequences. Our data indicate that YY1-induced DNA bending may activate and repress transcription by changing the spatial relationships between transcription activators and components of the basal transcription apparatus. PMID: 8932392 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: 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] ---------------------------------------------------------------