The pleomorphic adenoma gene (PLAG) family of transcription factors regulates a wide range of physiological processes, including cell proliferation, tissue-specific gene regulation, and embryonic development, although little is known regarding the mechanisms that regulate PLAG protein activity. In this study, a yeast two-hybrid screen identified PC2, a component of the Mediator complex, as a PLAGL2-binding protein. We show that PC2 cooperates with PLAGL2 and PU.1 to enhance the activity of a known PLAGL2 target promoter (NCF2). The PLAGL2-binding element in the NCF2 promoter consisted of the core sequence of the bipartite PLAG1 consensus site, but lacked the G-cluster motif, and was recognized by PLAGL2 zinc fingers 5 and 6. Promoter and PLAGL2 mutants showed that PLAGL2 and PU.1 were required to bind to their respective sites in the promoter, and PC2 knockdown demonstrated that PC2 was essential for enhanced promoter activity. Co-immunoprecipitation and promoter-reporter studies reveal that the effect of PC2 on PLAGL2 target promoter activity was conferred via the C-terminus of PLAGL2, the region that is required for PC2 binding and contains the PLAGL2 activation domain. Importantly, chromatin immunoprecipitation analysis and PC2 knockdown studies confirmed that endogenous PC2 protein associated with the NCF2 promoter in MM1 cells in the region occupied by PLAGL2, and was required for PLAGL2 target promoter activity in TNF-alpha-treated MM1 cells, respectively. Lastly, the expression of another known PLAGL2 target gene, insulin-like growth factor II (IGF-II), was greatly diminished in the presence of PC2 siRNA. Together, the data identify PC2 as a novel PLAGL2-binding protein and important mediator of PLAGL2 transactivation.

Mutations in the ABCC6 gene are known as causative factors of pseudoxanthoma elasticum (PXE), a connective tissue calcification disorder, but the molecular mechanism of pathogenesis or the physiological function of ABCC6 protein is the subject of intense debate. The ABCC6 gene expression is tightly regulated at the transcriptional level and its tissue-specific distribution is consistent with PXE being a metabolic disease caused by failure of ABCC6 function in organs distant from the diseased sites. In an effort to provide clues to its role by elucidating the mechanisms of its regulation, we identified ABCC6 as a target gene for transcriptional induction by PLAG1 and PLAGL1, transcription factors from the PLAG family of cell cycle progression-related DNA-binding proteins. Both these factors are shown to bind to the same single consensus-binding element in the ABCC6 proximal promoter in cell lines of hepatic and renal origin by reporter gene assay, electrophoretic mobility shift assay and chromatin immunoprecipitation. PLAG-mediated ABCC6 transactivation may play an important role in determining the level of tissue-specific expression of this gene. The described mechanism can also find potential application in therapeutic interventions in forms of PXE related to impaired ABCC6 expression.

Pleomorphic adenoma gene like-2 (PLAGL2), a developmentally regulated and stress inducible zinc finger protein can be post-translationally modified by small ubiquitin-like modifier peptide (SUMO-1); and SUMOylation attenuates PLAGL2 activity on the interactive promoter. Since PLAGL2 was a transactivator of the surfactant protein-C (SP-C) promoter, we hypothesized that SUMOylation down-regulated PLAGL2-activated SP-C promoter activity. Unexpectedly, the SUMO-conjugating enzyme Ubc9 enhanced, rather than reduced, PLAGL2 activated promoter activity but did not affect TTF-1 activation of the promoter. Ubc9 mutant (Ubc9-C93S) defective in SUMO-conjugating activity also enhanced PLAGL2-driven promoter activity suggesting that the stimulatory effect of Ubc9 on SP-C promoter activation was independent of its enzymatic function. PLAGL2 mutants without the K250 and/or K269 SUMOylation sites did not further improve PLAGL2 programmed transcription nor did they abolish Ubc9 enhanced promoter activity supporting the SUMOylation-independent mechanism. Chromatin immunoprecipitation (ChIP) assay demonstrated the association of PLAGL2 and Ubc9 with the SP-C promoter in vivo. Taken together, our data suggests that Ubc9 can function as a co-factor of PLAGL2, uncoupling from its enzymatic activity, to mediate PLAGL2 interactive SP-C promoter activity.

PLAG1 proto-oncogene overexpression has been causally linked to multiple tumors, highlighting its broad tumorigenic relevance. Here, the oncogenic potential of PLAG1 in mammary gland tumorigenesis was investigated in PLAG1 transgenic mice. To target mammary glands, mice of 2 independent PLAG1 transgenic strains, PTMS1 and PTMS2, in which PLAG1 expression can be modulated by Cre-mediation, were crossed with MMTV-Cre transgenic mice, resulting in P1-MCre and P2-MCre offspring, respectively. Hundred percentage of P1-MCre female mice showed mammary gland hyperplasia, caused by adenomyoepithelial adenosis, at 8 weeks. The tumorigenic process could not be studied further in P1-MCre mice, because concomitant fast-growing salivary gland tumors required euthanasia. Sixteen percentage of P2-MCre females developed mammary gland adenomyoepitheliomas within 30-45 weeks, and none displayed concomitant salivary gland tumors. To further study mammary gland tumorigenesis in PTMS1-derived mice, intercrossing with WAP-Cre transgenic mice, resulting in P1-WAPCre mice, was performed to target PLAG1 expression more specifically to mammary glands. Eighty percentage of such mice developed adenomyoepitheliomas within 53-88 weeks. All PLAG1-induced adenomyoepitheliomas revealed expression upregulation of Igf2/H19, Dlk1/Gtl2, Igfbps and Wnt signaling genes (Wnt6, Cyclin D1). Collectively, these results establish the oncogenic potential of PLAG1 in mammary glands of mice and point towards contributing roles of Igf and Wnt signaling.

OBJECTIVES To determine the expression level of the Wnt components-WIF1 (Wnt inhibitory factor 1), WNT1, and beta-catenin-in salivary gland tumor cells and to investigate the mechanisms that contribute to activation of the Wnt pathway in human salivary gland tumors. DESIGN The expression of WIF1, WNT1, and beta-catenin in salivary gland normal tissue and tumor cell lines was analyzed by reverse transcription-polymerase chain reaction and Western blot analysis. A relationship between the expression of distinct genes was determined by Pearson correlation. The presence of rearrangements involving WIF1 was evaluated by reverse transcription-polymerase chain reaction analysis. SUBJECTS Samples were obtained from 6 normal salivary glands and 10 cell lines established from primary benign and malignant salivary gland tumors. RESULTS The expression of WIF1 was high in normal salivary gland tissue but was significantly down-regulated in all salivary gland tumor cell lines analyzed (P <.001). The WIF1 rearrangements were recurrent but rare in salivary gland tumors. Expression of WNT1 protein was undetectable in normal tissue but readily detectable by Western blot analysis in all salivary gland tumor cell lines. beta-Catenin messenger RNA expression was significantly up-regulated in salivary gland tumor cells. A positive linear correlation between beta-catenin and PLAG1 (pleomorphic adenoma gene 1) gene expression was observed. CONCLUSIONS This is the first report (to our knowledge) showing down-regulation of an antagonist of the Wnt pathway, WIF1, and up-regulation of a Wnt agonist, WNT1, in salivary gland tumor cells. This dysregulation of WNT1 and WIF1 expression, coupled with the observed increase in beta-catenin transcription, may consequently promote salivary gland oncogenesis. Our data support the study of the Wnt pathway as a putative therapeutic target for salivary gland cancer.

The objective was to characterize the potential laying traits and underlying transcripts expressed in the hypothalamus and pituitary gland that were associated with egg production variability in five genetic stocks of chickens: two commercial lines, Red-(n=12) and Black-feather (n=14) Taiwan country chickens (TCCs); two selected lines of TCCs, B (high body weight/comb size; n=17) and L2 (high-egg production; n=14); and a commercial single comb White Leghorn (WL; n=17). Six laying traits, age at first egg, clutch length, pause length, oviposition lag within clutch, follicle rapid growth period, and rate of yolk accumulation were measured. The significance of differential values among five chicken stocks and correlation coefficients between laying traits and number of eggs to 50 weeks of age or laying rate after first egg, and the expression level of 33 transcripts were determined. Longer clutch length and shorter oviposition lag within clutch contributed to a higher number of eggs to 50 weeks of age or laying rate after first egg in L2 (P<0.05) and WL strains (P<0.05). However, their rate of yolk accumulation (P<0.05) and follicle rapid growth period (P<0.05) were different, indicating the accumulation of different alleles after long-term, independent selection. Across all five strains, numbers of eggs to 50 weeks of age were positive correlated with average clutch length (P<0.05) as well as the rate of yolk accumulation (P<0.05). Expressions of PLAG1, STMN2, PGDS, PARK7, ANP32A, PCDHA@, SCG2, BDH and SAR1A transcripts contributed to number of eggs to 50 weeks of age (P<0.05) or laying rate after first egg (P<0.05). Analysis of correlation coefficients indicated that PLAG1 additionally played roles in decreasing average pause length. Two transcripts, PRL and GARNL1, specifically contributed to number of eggs to 50 weeks of age or laying rate after first egg by reducing oviposition lag within clutch (P<0.05) and/or increasing average clutch length (P<0.05), respectively. Expression level of NCAM1, contributed to laying rate after first egg by association with a shorter oviposition lag within clutch (P<0.05). The current study attributed egg production phenotype in five strains into several laying traits; correlations between these traits and expression levels of underlying transcripts expressed in the hypothalamus and pituitary gland were also established.

Pleomorphic adenoma (PA), a benign mixed salivary gland tumor, has been associated with abnormal karyotypes in up to 70% of cases, with nonrandom involvement of 8q12, the locus of the pleomorphic adenoma (PLAG1) gene. In this study, cytogenetics and fluorescence in situ hybridization (FISH) were used to investigate PLAG1 involvement in PA from seven patients. There were two males and five females ranging in age from 25 to 65 years. Samples of parotid gland tissue from the tumor sites, set up as solid tumor cultures, showed a normal karyotype in two cases [46,XY;46,XX] and cytogenetic abnormalities in five cases (71%). The abnormalities comprised one variant translocation [t(1;4;8)(p32;q35;q12)], two classic translocations [t(5;8)(p13;q12)], one novel deletion [del(12)(p11.2p12.1)], and a novel insertion [ins(9;8)(p22;q12q21.1)]. FISH was performed in all cases by using two probes from the RP11 library, flanking PLAG1; a sequence 1.48 megabases (Mb) upstream and another 2.27 Mb downstream, covering a total area of 3.8 Mb. The PLAG1 gene was intact and normally situated in four cases - the 46,XY, 46,XX, del(12p), and one t(5;8). PLAG1 was disrupted in three cases - one t(5;8), ins(9;8), and t(1;4;8). In addition, genomic instability was seen in two cases, one with PLAG1 amplification in the form of a homogeneously staining region, and the other in der(8) ring formation. The data provide further unique cases showing the complexity of PLAG1 gene rearrangements in PA.

Pleiomorphic adenoma gene (PLAG) family proteins are oncogenes involved in various malignancies including lipoblastomas, hepatoblastomas, and acute myeloid leukemia. Overexpression of PLAGL2 induces cell transformation and proliferation, but little is known about how its activities are regulated. We previously showed that transcriptional activity of PLAGL2 is negatively regulated by sumoylation. Here we report that Tip60 modulates PLAGL2 functions through acetylation. Tip60 associates with PLAGL2 through its zinc finger domain and acetylates PLAGL2. Wild-type but not the histone acetyltransferase (HAT)-minus mutant form of Tip60 enhances PLAGL2-mediated transactivation. In addition, coexpression of Tip60 and PLAGL2 completely abolishes the sumoylation of PLAGL2. Both Tip60 and DN-Ubc9 increase transactivation activity of wild-type but not the sumoylation deficient form of PLAGL2 (K250, 269, 356R), indicating that Tip60 acetylates PLAGL2 and abolishes the sumoylation of PLAGL2 possibly through modification of the same lysine residues (K250, 269, 356) within PLAGL2. Tip60 effects vary between different PLAGL2 target gene promoters, suggesting that Tip60 is a novel promoter-specific coactivator of PLAGL2. This is the first demonstration that Tip60 can function as a sumoylation inhibitor in part through its intrinsic acetyltransferase activity to regulate specific gene expression.

This review summarizes the new findings on salivary gland pathology under the following categories: immunohistochemistry; molecular genetics; newly recognized tumour types; known tumour entities with new findings; and progression of salivary gland tumours. In the application of immunohistochemistry, CD117 can aid in highlighting the luminal cell component of various salivary gland tumours, whereas p63 or maspin can aid in highlighting the abluminal cell component. A high Ki67 index remains the most useful marker to predict adverse outcome in salivary gland carcinoma. Specific chromosomal translocations are recognized in pleomorphic adenoma (with translocation involving PLGA1 or HMGA2 gene) and mucoepidermoid carcinoma (with MECT1-MAML2 gene fusion). Newly recognized entities include: sclerosing polycystic adenosis (with recent molecular evidence supporting its neoplastic nature), sclerosing mucoepidermoid carcinoma with eosinophilia, keratocystoma, adenoma with additional stromal component (lymphadenoma, lipoadenoma and adenofibroma), cribriform adenocarcinoma of the tongue and signet ring adenocarcinoma of minor salivary gland. Known tumour entities with new findings include: salivary duct carcinoma (with newly recognized mucinous, micropapillary and sarcomatoid variants), intraductal carcinoma (with controversies in terminology), mucoepidermoid carcinoma (with newly proposed grading parameters and oncocytic variant), epithelial-myoepithelial carcinoma (with newly recognized morphological variants), small cell carcinoma (with most cases being related to Merkel cell carcinoma), extranodal marginal zone B-cell lymphoma (with specific chromosomal translocation) and chronic sclerosing sialadenitis (being a component of IgG4-related sclerosing disease). Progression of salivary gland tumours can take the form of malignant transformation of a benign tumour, progression from low-grade to high-grade carcinoma, dedifferentiation, or stromal invasion of an in situ carcinoma.

Lost-on-transformation 1 (LOT1)(PLAGL1/ZAC1) is a member of the novel subfamily of zinc-finger transcription factors, designated as PLAG family. The other members in this group include PLAG1 and PLAGL2, which share high homology with each other and with LOT1, particularly in their zinc-finger amino-terminal region. They are structurally similar but functionally different. For example, the LOT1 gene encodes a growth suppressor protein and is localized on human chromosome 6q24-25, a chromosomal region that is frequently deleted in many types of human cancers. The gene is maternally imprinted and is linked to developmental disorders such as growth retardation and transient neonatal diabetes mellitus (TNDM). LOT1 is a target of growth factor signaling pathway(s) and silenced by epigenetic mechanisms, as well as by the loss of heterozygosity in different tumor tissues. PLAG1 is a protooncogene that is localized on chromosome 8q12 and was found to be a target of several types of chromosomal rearrangement including the one identified in pleomorphic adenomas of the salivary gland. Since the discovery of the PLAG family members in 1997, much has been learned about their structure and function, as are summarized in this review. While the available data suggest that these proteins may play important roles in regulating normal physiological functions in the mammals, a great deal more about their signaling pathway(s), potential role in the complex pathologies such as cancer and developmental disorders, and functional relationship between different family members and splice variants still remains to be uncovered.

We have isolated and characterized two novel cDNAs encoding C2H2 zinc finger proteins showing high sequence homology to PLAG1, a protein ectopically activated by promoter swapping or promoter substitution in pleomorphic adenomas with chromosomal abnormalities at chromosome 8q12. PLAG1 and the two new PLAG1 family members (PLAGL1 and PLAGL2) constitute a novel subfamily of zinc finger proteins that recognize DNA and/or RNA. To examine the potential of the three human proteins to modulate transcription, we constructed several PLAG/GAL4 DNA binding domain fusion proteins and measured their ability to activate transcription of a reporter gene construct in different mammalian cell lines and in yeast. Although the carboxyl-terminal part of PLAGL1 shows strong overall transcriptional activity in mesenchymal (COS-1) and epithelial cells (293), both PLAG1 and PLAGL2 transactivate in mesenchymal cells only if depleted from a repressing region. This effect is less profound in epithelial cells. These data suggest that the activation in pleomorphic adenomas of PLAG1 most likely results in uncontrolled activation of downstream target genes.

We have previously shown that the PLAG1 gene on chromosome 8q12 is consistently rearranged in pleomorphic adenomas of the salivary glands with t(3;8)(p21;q12) translocations. The t(3;8) results in promoter swapping between the PLAG1 gene, which encodes a novel zinc finger protein, and the constitutively expressed gene for beta-catenin (CTNNB1), a protein with roles in cell-cell adhesion and the WG/WNT signalling pathway. In order to assess the importance of other translocation partner genes of PLAG1, and their possible relationship to CTNNB1, we have characterized a second recurrent translocation, i.e. the t(5;8)(p13;q12). This translocation leads to ectopic expression of a chimeric transcript consisting of sequences from the ubiquitously expressed gene for the leukemia inhibitory factor receptor (LIFR) and PLAG1. As for the t(3;8), the fusions occurred in the 5'-noncoding regions of both genes, exchanging regulatory control elements while preserving the coding sequences. The results of the current as well as previous studies indicate that ectopic expression of PLAG1 under the control of promoters of distinct translocation partner genes is a general pathogenetic mechanism for pleomorphic adenomas with 8q12 aberrations.

Pleiomorphic adenoma of the salivary glands is a benign epithelial tumour occurring primarily in the major and minor salivary glands. It is by far the most common type of salivary gland tumour. Microscopically, pleiomorphic adenomas show a marked histological diversity with epithelial, myoepithelial and mesenchymal components in a variety of patterns. In addition to a cytogenetic subgroup with normal karyotypes, pleiomorphic adenomas are characterized by recurrent chromosome rearrangements, particularly reciprocal translocations, with breakpoints at 8q12, 3p21, and 12q13-15, in that order of frequency. The most common abnormality is a reciprocal t(3;8)(p21;q12). We here demonstrate that the t(3;8)(p21;q12) results in promoter swapping between PLAG1, a novel, developmentally regulated zinc finger gene at 8q12, and the constitutively expressed gene for beta-catenin (CTNNB1), a protein interface functioning in the WG/WNT signalling pathway and specification of cell fate during embryogenesis. Fusions occur in the 5'-non-coding regions of both genes, exchanging regulatory control elements while preserving the coding sequences. Due to the t(3;8)(p21;q12), PLAG1 is activated and expression levels of CTNNB1 are reduced. Activation of PLAG1 was also observed in an adenoma with a variant translocation t(8;15)(q12;q14). Our results indicate that PLAG1 activation due to promoter swapping is a crucial event in salivary gland tumourigenesis.

Pleomorphic adenoma, or mixed tumor of the salivary glands, is a benign tumor originating from the major and minor salivary glands. Eighty-five percent of these tumors are found in the parotid gland, 10% in the minor (sublingual) salivary glands, and 5% in the submandibular gland. It is the most common type of salivary gland tumor, accounting for almost 50% of all neoplasms in these organs. In fact, after the first observation of recurrent loss of chromosome 22 in meningioma, this was the second type of benign tumor for which non-random chromosomal changes were reported. The rate of malignant change with the potential to metastasize has been reported to be only 2 to 3%, and only a few cases of metastasizing pleomorphic salivary gland adenomas have been described to date. The fact that these tumors arise in organs located in an ontogenetic transitional zone, a region where endoderm and ectoderm meet, might be one of the reasons for the often-problematic histopathological classification. This type of benign tumor has been cytogenetically very well-characterized, with several hundreds of tumors karyotyped. In addition to the cytogenetic subgroup with an apparently normal diploid stemline (making up approximately 30% of the cases), three major cytogenetic subgroups can be distinguished. In addition to a subgroup showing non-recurrent clonal abnormalities, another subgroup is various translocations involving 12q15. By far the largest cytogenetic subgroup, however, consists of tumors with chromosome 8 abnormalities, mainly showing translocations involving region 8q12. The most frequently encountered aberration in this group is a t(3;8)(p21;q12).

Pleomorphic adenoma gene 1 (PLAG1), a zinc finger transcription factor gene, is consistently rearranged and overexpressed in human pleomorphic adenomas of the salivary glands with 8q12 translocations. In this report, we describe the immunohistochemical localization of PLAG1 protein in pleomorphic adenomas of the salivary gland and corresponding normal tissue, in relation to cytokeratin, vimentin, and BCL-2 expression. Normal salivary gland tissue was not immunoreactive for PLAG1. In primary pleomorphic adenomas, cells strongly immunoreactive for PLAG1 were detected in the outer layer of tubulo-ductal structures, which are thought to be the origin of cells with bi-directional, epithelial, and mesenchymal phenotypes. In contrast, epithelial cells with abundant cytokeratin in the inner tubulo-ductal structures only sporadically expressed PLAG1. BCL-2 immunoreactivity was found mainly in the cells surrounding the tubulo-ductal structures and in the solid undifferentiated cellular masses, within the areas that had moderate PLAG1 immunoreactivity. The variability of PLAG1 expression in neoplastic cells seemed to reflect the morphologic heterogeneity that correlated with the stage of differentiation of the tumor cells. Immunohistochemical/cytogenetic evaluation of two pleomorphic adenomas with t(3;8)(p21;q12) or t(5;8)(p13;q12) translocations demonstrated the clonal nature of immunophenotypically diverse cells. This finding confirms the theory that pleomorphic adenoma cells share a common single-cell origin, most likely from the epithelial progenitor basal duct cells.

We have previously shown (K. Kas et al, Nat. Genet., 15: 170-174, 1997) that the developmentally regulated zinc finger gene pleomorphic adenoma gene 1 (PLAG1) is the target gene in 8q12 in pleomorphic adenomas of the salivary glands with t(3;8)(p21;q12) translocations. The t(3;8) results in promoter swapping between PLAG1 and the constitutively expressed gene for beta-catenin (CTNNB1), leading to activation of PLAG1 expression and reduced expression of CTNNB1. Here we have studied the expression of PLAG1 by Northern blot analysis in 47 primary benign and malignant human tumors with or without cytogenetic abnormalities of 8q12. Overexpression of PLAG1 was found in 23 tumors (49%). Thirteen of 17 pleomorphic adenomas with a normal karyotype and 5 of 10 with 12q13-15 abnormalities overexpressed PLAG1, which demonstrates that PLAG1 activation is a frequent event in adenomas irrespective of karyotype. In contrast, PLAG1 was overexpressed in only 2 of 11 malignant salivary gland tumors analyzed, which suggests that, at least in salivary gland tumors, PLAG1 activation preferentially occurs in benign tumors. PLAG1 over-expression was also found in three of nine mesenchymal tumors, i.e., in two uterine leiomyomas and one leiomyosarcoma. RNase protection, rapid amplification of 5'-cDNA ends (5'-RACE), and reverse transcription-PCR analyses of five adenomas with a normal karyotype revealed fusion transcripts in three tumors. Nucleotide sequence analysis of these showed that they contained fusions between PLAG1 and CTNNB1 (one case) or PLAG1 and a novel fusion partner gene, i.e., the gene encoding the transcription elongation factor SII (two cases). The fusions occurred in the 5' noncoding region of PLAG1, leading to exchange of regulatory control elements and, as a consequence, activation of PLAG1 gene expression. Because all of the cases had grossly normal karyotypes, the rearrangements must result from cryptic rearrangements. The results suggest that in addition to chromosomal translocations and cryptic rearrangements, PLAG1 may also be activated by mutations or indirect mechanisms. Our findings establish a conserved mechanism of PLAG1 activation in salivary gland tumors with and without 8q12 aberrations, which indicates that such activation is a frequent event in these tumors.

In the process of identification of the multiple endocrine neoplasia type 1 gene, which was recently published, we isolated a novel gene in the 11q13 region. This gene (named ZFPL1, for zinc-finger protein-like 1) is expressed strongly in the exocrine pancreas as a 1.4-kb polyadenylated RNA encoding a putative protein of 310 amino acids. A mouse EST contig predicts an equally sized murine protein with 91% amino acid sequence identity to the human protein. No significant homology with known proteins could be found through database screening. However, zinc-finger-like domains and leucine-zipper-like motifs in the predicted ZFPL1 protein were identified, suggesting the presence of DNA-binding and dimerization domains possibly involved in transcription regulation. This notion is supported by the presence of a putative bipartite nuclear localization signal. This paper presents the full-length cDNA sequence for this gene, its genomic structure and chromosomal orientation, and expression studies by Northern blot hybridization and RNA in situ hybridization.

In this report, the identification and molecular characterization of a novel gene, designated TM7SF2, is reported. This gene was found in the FAU neighboring area (FAUNA) to which other genes have been mapped previously. The FAUNA gene cluster is located at chromosome 11q13 between landmarks H4B and D11S2196E. The TM7SF2 gene contains eight coding exons, and their splice site consensus sequences are consistent with AG/GT rule. Northern blot analysis with a cDNA probe corresponding to TM7SF2 revealed varying expression levels of a 1.7-kb transcript in adult human heart, brain, pancreas, lung, liver, skeletal muscle, kidney, ovary, prostate, and testis, but no detectable expression in placenta, spleen, thymus, small intestine, colon (mucosal lining), or peripheral blood leukocytes. The open reading frame in the cDNA sequence codes for a protein of 590 amino acids that is rich in glycine (23%) and arginine (17%) residues in its amino-terminal half and contains seven transmembrane domains in its carboxy-terminal half. The transmembrane region of the putative TM7SF2 protein shows amino acid sequence similarity to those of the lamin B receptor and the C14/C24 sterol reductase.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant familial cancer syndrome characterized by parathyroid, pancreatic, and anterior pituitary tumors. The MEN1 locus has been previously localized to chromosome 11q13, and a 2-Mb gene-rich region flanked by D11S1883 and D11S449 has been defined. We have pursued studies to facilitate identification of the MEN1 gene by narrowing this critical region to a 900-kb interval between the VRF and D11S1783 loci through melotic mapping. This was achieved by investigating 17 cosmids for microsatellite polymorphisms, which defined two novel polymorphisms at the VRF and A0138 loci, and utilizing these to characterize recombinants in MEN1 families. In addition, we have established a 1200-kb sequence-ready contig consisting of 26 cosmids, eight BACs, and eight PACs that encompass this region. The precise locations for 19 genes and three ESTs within this contig have been determined, and three gene clusters consisting of a centromeric group (VRF, FKBP2, PNG, and PLCB3), a middle group (PYGM, ZFM1, SCG1, SCG2 (which proved to be the MEN1 gene), and PPP2R5B), and a telomeric group (H4B, ANG3, ANG2, ANG1, FON, FAU, NOF, NON, and D11S2196E) were observed. These results represent a valuable transcriptional map of chromosome 11q13 that will help in the search for disease genes in this region.

BACKGROUND: Epstein Barr virus (EBV) infects the majority of the human population, causing fatal diseases in a small proportion in conjunction with environmental factors. Following primary infection, EBV remains latent in the memory B cell population for life. Recurrent reactivation of the virus occurs, probably due to activation of the memory B-lymphocytes, resulting in viral replication and re-infection of B-lymphocytes. Methylation of the viral DNA at CpG motifs leads to silencing of viral gene expression during latency. Zta, the key viral protein that mediates the latency/reactivation balance, interacts with methylated DNA. Zta is a transcription factor for both viral and host genes. A sub-set of its DNA binding sites (ZREs) contains a CpG motif, which is recognised in its methylated form. Detailed analysis of the promoter of the viral gene BRLF1 revealed that interaction with a methylated CpG ZRE (RpZRE3) is key to overturning the epigenetic silencing of the gene. METHODOLOGY AND PRINCIPAL FINDINGS: Here we question whether we can use this information to identify which host genes contain promoters with similar response elements. A computational search of human gene promoters identified 274 targets containing the 7-nucleotide RpZRE3 core element. DNA binding analysis of Zta with 17 of these targets revealed that the flanking context of the core element does not have a profound effect on the ability of Zta to interact with the methylated sites. A second juxtaposed ZRE was observed for one promoter. Zta was able to interact with this site, although co-occupancy with the RpZRE3 core element was not observed. CONCLUSIONS/SIGNIFICANCE: This research demonstrates 274 human promoters have the potential to be regulated by Zta to overturn epigenetic silencing of gene expression during viral reactivation from latency.

The pleomorphic adenoma gene (PLAG) family of transcription factors regulates a wide range of physiological processes, including cell proliferation, tissue-specific gene regulation, and embryonic development, although little is known regarding the mechanisms that regulate PLAG protein activity. In this study, a yeast two-hybrid screen identified PC2, a component of the Mediator complex, as a PLAGL2-binding protein. We show that PC2 cooperates with PLAGL2 and PU.1 to enhance the activity of a known PLAGL2 target promoter (NCF2). The PLAGL2-binding element in the NCF2 promoter consisted of the core sequence of the bipartite PLAG1 consensus site, but lacked the G-cluster motif, and was recognized by PLAGL2 zinc fingers 5 and 6. Promoter and PLAGL2 mutants showed that PLAGL2 and PU.1 were required to bind to their respective sites in the promoter, and PC2 knockdown demonstrated that PC2 was essential for enhanced promoter activity. Co-immunoprecipitation and promoter-reporter studies reveal that the effect of PC2 on PLAGL2 target promoter activity was conferred via the C-terminus of PLAGL2, the region that is required for PC2 binding and contains the PLAGL2 activation domain. Importantly, chromatin immunoprecipitation analysis and PC2 knockdown studies confirmed that endogenous PC2 protein associated with the NCF2 promoter in MM1 cells in the region occupied by PLAGL2, and was required for PLAGL2 target promoter activity in TNF-alpha-treated MM1 cells, respectively. Lastly, the expression of another known PLAGL2 target gene, insulin-like growth factor II (IGF-II), was greatly diminished in the presence of PC2 siRNA. Together, the data identify PC2 as a novel PLAGL2-binding protein and important mediator of PLAGL2 transactivation.

Recent reports indicate that the activating transcription factor 5 (ATF5) is required for the survival of cancer cells but not for noncancer cells. However, the mechanisms by which ATF5 regulates genes and promotes cell survival are not clear. Using a cyclic amplification and selection of targets (CASTing) approach, we identified a novel ATF5 consensus DNA binding sequence. We show in C6 glioma and MCF-7 breast cancer cells that ATF5 occupies this sequence and that ATF5 activates reporter gene expression driven by this site. Conversely, reporter activity is diminished when ATF5 activity is blocked or when ATF5 expression is down-regulated by serum withdrawal. We further show that early growth response factor 1 (Egr-1), whose promoter contains two adjacent ATF5 consensus binding sites at a conserved promoter position in rat, mouse, and human, is targeted and regulated by ATF5 in C6 and MCF-7 cells. These data provide new insight on the mechanisms by which ATF5 promotes gene regulation and cancer-specific cell survival.

Mxr1p (methanol expression regulator 1) functions as a key regulator of methanol metabolism in the methylotrophic yeast Pichia pastoris. In this study, a recombinant Mxr1p protein containing the N-terminal zinc finger DNA binding domain was overexpressed and purified from E. coli cells and its ability to bind to promoter sequences of AOXI encoding alcohol oxidase was examined. In the AOX1 promoter, Mxr1p binds at six different regions. Deletions encompassing these regions result in a significant decrease in AOXI promoter activity in vivo. Based on the analysis of AOXI promoter sequences, a consensus sequence for Mxr1p binding consisting of a core 5' CYCC 3' motif was identified. When the core CYCC sequence is mutated to CYCA, CYCT or CYCM (M = 5-methylcytosine), Mxr1p binding is abolished. Though Mxr1p is the homologue of Saccharomyces cerevisiae Adr1p transcription factor, it does not bind to Adr1p binding site of S. cerevisiae alcohol dehydrogenase promoter (ADH2UAS1). However, two point mutations convert ADH2UAS1 into an Mxr1p binding site. The identification of key DNA elements involved in promoter recognition by Mxr1p is an important step in understanding its function as a master regulator of the methanol utilization pathway in P. pastoris.

CudA, a nuclear protein required for Dictyostelium prespore-specific gene expression, binds in vivo to the promoter of the cotC prespore gene. A 14 nucleotide region of the cotC promoter binds CudA in vitro and ECudA, an Entamoeba CudA homologue, also binds to this site. The CudA and ECudA DNA-binding sites contain a dyad and, consistent with a symmetrical binding site, CudA forms a homodimer in the yeast two-hybrid system. Mutation of CudA binding sites within the cotC promoter reduces expression from cotC in prespore cells. The CudA and ECudA proteins share a 120 amino acid core of homology, and clustered point mutations introduced into two highly conserved motifs within the ECudA core region decrease its specific DNA binding in vitro. This region, the presumptive DNA-binding domain, is similar in sequence to domains in two Arabidopsis proteins and one Oryza protein. Significantly, these are the only proteins in the two plant species that contain an SH2 domain. Such a structure, with a DNA-binding domain located upstream of an SH2 domain, suggests that the plant proteins are orthologous to metazoan STATs. Consistent with this notion, the DNA sequence of the CudA half site, GAA, is identical to metazoan STAT half sites, although the relative positions of the two halves of the dyad are reversed. These results define a hitherto unrecognised class of transcription factors and suggest a model for the evolution of STATs and their DNA-binding sites.

We have identified downstream promoter sequence of the PS1 gene that may be regulated by novel transcription factors. 3' deletion from +178 to +165 had no effect on PS1 transcription. 3' deletion from +178 to +140 decreased promoter activity by 50%. Further 3' deletion from +178 to +114 decreased promoter activity by 80%. Therefore, a crucial element controlling over 80% of the promoter activity in SK-N-SH cell line is located between +114 and +165. Electrophoretic mobility shift assays suggested that zinc finger proteins Sp1 and ADR1 interacted with the PS1 promoter sequence (+114 to +140) and promoter region (+140 to +165) respectively. A three base pair substitution within the core sequence (GGCGGGGA to GGCGactA) of the ADR1 consensus in the element (+140 to +165) that abolished ADR1-DNA interaction, reduced PS1 transcription by 50%. The substitution mutation in the sequence (+114 to +140) that abolished Sp1-DNA interaction had no effect on PS1 expression. These data suggest that a novel mammalian trans-activator protein ADR1 binds to the downstream element (+140 to +165) to activate PS1 transcription.

Transcriptional repression plays a critical role in development and homeostasis. The ETO family represents a group of highly conserved and ubiquitously expressed transcriptional regulatory proteins that are components of a diverse range of multi-protein repressor complexes. ETO proteins function as transcriptional repressors by interacting with a number of transcription factors that bind to their cognate consensus DNA binding sequences within the promoters of target genes. We previously reported that the classical C2H2 zinc finger DNA binding protein ZNF652 specifically and functionally interacts with the ETO protein CBFA2T3, and has a role in the suppression of breast oncogenesis. Here we report identification and validation of the ZNF652 consensus DNA binding sequence. Our results show that E-box gene HEB is a direct target of CBFA2T3-ZNF652-mediated transcriptional repression. CBFA2T3-ZNF652 complex regulates HEB expression by binding to a single ZNF652 response element located within the promoter sequence of HEB. This study also shows that the NHR3 and NHR4 domains of CBFA2T3 interact with a conserved proline-rich region located within the C-terminus of ZNF652. Our results, together with previous reports, indicate that HEB has a complex relationship with CBFA2T3; CBFA2T3 interacts with ZNF652 to repress HEB expression and in addition CBFA2T3 interacts with the HEB protein to inhibit its activator function. These findings suggest that CBFA2T3-ZNF652-mediated HEB regulation may play an important role in haematopoiesis and myogenesis.

The Nczf gene, which is identified as a target gene of Ncx, encodes a novel Kruppel-associated box (KRAB) zinc finger protein, which functions as a sequence-specific transcriptional repressor. We generated a fusion protein of the zinc finger domain of Nczf and glutathione S-transferase to identify Nczf-binding consensus DNA sequences with random oligonucleotides of 15 and 35 bases. The consensus binding sequence of core nucleotides contains (A/T/C)CTTT(A/G)TTNT. In a gel mobility shift assay, the probe containing these sequences bound to the fusion protein. In silico analysis, these consensus sequences were found on regulatory regions of the endothelin receptor B and the microphthalmia-associated transcription factor genes, which are involved in neural crest development. These results suggest that Nczf functions as a sequence-specific transcription repressor to regulate neural crest cell development.

The anticancer drug XR5944 was originally developed as a topoisomerase inhibitor and was subsequently shown to be a transcription inhibitor. It has shown exceptional anticancer activity both in vitro and in vivo and was significantly more potent than traditional topoisomerase inhibitors. The solution structure of the XR5944/DNA complex recently obtained in our laboratory indicates that XR5944 bis-intercalates at the 5'-(TpG):(CpA) site of duplex DNA, which is found in the consensus DNA-binding site of estrogen receptor (ER). Thus, we tested the ability of XR5944 to inhibit ER activity both in vitro and in cultured cells. In electrophoretic mobility shift assays, it is seen that the DNA binding of recombinant ERalpha protein, as well as ER from nuclear extracts, is inhibited by XR5944 in a dose-dependent manner. In luciferase reporter assays, XR5944 inhibited the reporter gene expression from an estrogen response element-containing promoter but not from a basal promoter sequence that lacks any cis-acting elements. In contrast, the RNA polymerase inhibitor actinomycin D inhibits the transcription from both the above-mentioned promoters. The specificity of XR5944 activity is displayed by a separate reporter assay in which the transactivation of reporter gene expression by Sp1 proteins was not inhibited by XR5944. Collectively, these data suggest that XR5944 is capable of specifically inhibiting the binding of ER to its consensus DNA sequence and its subsequent activity. This represents a novel mechanism of ER inhibition, which may allow the development of agents capable of overcoming resistance to current antiestrogens.

A novel 114-kDa zinc finger protein, ZEC, has been found by cDNA cloning and characterized. ZEC was strongly expressed in the testis, liver and kidney, and also in embryonic stem cells. Epitope-tagged experiments indicated nuclear localization of ZEC. ZEC contained 18 C2H2 zinc fingers which were organized in two clusters. A ZEC binding DNA sequence, C/GA/TA/TGGTTGGTTGC, which we have designated the GT box, was identified by random oligonucleotide binding selection assay. The GT box did not contain binding sites for other previously characterized transcription factors and thus represented a potentially novel DNA target sequence. Electrophoretic mobility shift assay (EMSA) showed that both clusters of zinc fingers bound to the same DNA sequence. Site-directed mutagenesis revealed that the core sequence TTGGTT within the GT box was essential to ZEC binding, while DNA sequences outside of the core sequence enhanced this interaction. Furthermore, co-transfection assays demonstrated that ZEC could activate a reporter luciferase gene driven by this DNA sequence.