Characterization of mouse epithelial protein lost in neoplasm (EPLIN) and comparison of mammalian and zebrafish EPLIN.

EPLIN is a cytoskeleton-associated protein that was initially identified as the product of a gene that is transcriptionally down-regulated in cancer cells. In human, there are two known isoforms, EPLIN-alpha and -beta, generated by alternative promoter usage from a single gene. With the exception of a single LIM (lin-11, isl-1, and mec-3) domain, the sequence of EPLIN is unique and does not provide any clues to its function. To identify conserved regions of EPLIN that may be important for its function, we have characterized mouse (m) and zebrafish (zf) EPLIN. As in human, two isoforms, the 593 aa mEPLIN-alpha (77% identity; 83% similarity) and 753 aa mEPLIN-beta (75% identity; 83% similarity), were present in mouse. mEPLIN-alpha is highly expressed in embryonic tissue and adult lung and spleen, whereas mEPLIN-beta is preferentially expressed in kidney, testis, lung and liver. The analysis of mEPLIN gene revealed that the overall organization of the exons in mouse and human are conserved. In zebrafish, there was only one form, the 629 aa zfEPLIN, corresponding to the mammalian EPLIN-beta. Like its mammalian counterparts, ectopically expressed zfEPLIN is co-localized to the actin cytoskeleton. While the overall homology between mammalian and zebrafish EPLIN was not striking (37% identity; 50% similarity), there were seven highly conserved regions, which should be useful in structure-function studies of this novel protein.

Characterization of the human EPLIN (Epithelial Protein Lost in Neoplasm) gene reveals distinct promoters for the two EPLIN isoforms.

EPLIN is a novel LIM domain protein that co-localizes to the actin stress fibers and focal adhesion plaques. We previously have demonstrated that two isoforms, the 600aa EPLIN-alpha and the 759aa EPLIN-beta, are generated from a single gene. In the majority of human breast and prostate cancer cell lines, the expression of EPLIN-alpha is significantly reduced, while the expression of EPLIN-beta is either up-regulated or unchanged. To understand the basis of this differential regulation, we have determined the organization of the human EPLIN gene. The human EPLIN100kb and consists of 11 exons. The EPLIN-beta mRNA requires all 11 exons, while the EPLIN-alpha mRNA requires Exons 4-11. The transcriptional start sites of EPLIN-alpha were mapped within the third intron by 5' RACE and S1 nuclease protection. Similarly, the 5' ends of EPLIN-beta were mapped upstream of Exon 1. The DNA sequences flanking the EPLIN-alpha or EPLIN-beta transcriptional start sites were capable of stimulating the expression of promoter reporter constructs. Interestingly, the endogenous transcription of EPLIN-alpha, but not EPLIN-beta, could be stimulated by serum, indicating that the expression of two EPLIN isoforms can be independently regulated. A consensus serum response element was present within 100bp upstream of the transcriptional start sites of EPLIN-alpha. The activity of 0.7kb EPLIN-alpha promoter reporter construct could be enhanced by activated RhoA, indicating that this serum response element is functional.

EPLIN, epithelial protein lost in neoplasm.

We have identified a novel cytoskeletal protein, EPLIN (Epithelial Protein Lost In Neoplasm), that is preferentially expressed in human epithelial cells. Two EPLIN isoforms, a 600 amino acid EPLIN-alpha and a 759 amino acid EPLIN-beta, are detected in primary epithelial cells of oral mucosa, prostate and mammary glands. The expression of EPLIN-alpha is either down-regulated or lost in the majority of oral cancer cell lines (8/8), prostate cancer cell lines (4/4) and xenograft tumors (3/3), and breast cancer cell lines (5/6). The amino acid sequence of EPLIN is characterized by the presence of a single centrally located LIM domain. Both EPLIN isoforms localize to filamentous actin and suppress cell proliferation when overexpressed. These findings indicate that the loss of EPLIN seen in cancer cells may play a role in cancer progression.

Identification of a cell type-specific enhancer in the distal 5'-region of the platelet-derived growth factor A-chain gene.

Transient transfection analysis of DNA subfragments from the distal 5'-flanking region of the human platelet-derived growth factor A-chain gene (-18.3 to -1.8 kilobase pairs (kb)) revealed enhancer and silencer elements that contribute significantly to transcriptional regulation. Two adjacent regions (-8.2 to -7.5 kb and -7.5 to -7.0 kb) enhanced transcription of both A-chain and heterologous thymidine kinase promoters, whereas repression was observed in two other nearby regions (-9.9 to -8.2 kb and -7.0 to -5. 9 kb). The -7.5 to -7.0-kb fragment, or J, was the strongest enhancer, and its activity was localized to a 66-base pair element (A-chain cell type-specific enhancer (ACE 66)). ACE66 activity was highly cell type-specific, with greatest activity seen in choriocarcinoma cell lines (4-10-fold enhancement). Progressive 5'- and 3'-deletions of the ACE66 revealed distribution of activity across the element, with nucleotides 1-33 being critical for function. Electrophoretic mobility shift assays revealed cell type-specific patterns of high affinity protein binding to the element. Ethylation interference footprinting of JEG-3 extract localized guanine contacts on nucleotides 1-18 of both strands of the ACE element, whereas more extensive contacts were made with the phosphate backbone (nucleotides 1-32). The ACE66 element is a potent transcriptional regulator in placental cells and represents a valuable model of long distance regulation in a growth factor gene.

Repression of platelet-derived growth factor A-chain gene transcription by an upstream silencer element. Participation by sequence-specific single-stranded DNA-binding proteins.

Platelet-derived growth factor A-chain is a potent mitogen expressed in a restricted number of normal and transformed cells. Transient transfection and deletion analysis in BSC-1 (African green monkey, renal epithelial) cells revealed that the -1680 to -1374 region of the A-chain gene repressed homologous and heterologous promoter activities by 60-80%. An S1 nuclease-hypersensitive region (5'SHS) was identified within this region (-1418 to -1388) that exhibited transcriptional silencer activity in BSC-1 and a variety of human tumor cell lines (U87, HepG2, and HeLa). Electrophoretic mobility shift assays conducted with 5'SHS oligodeoxynucleotide probes revealed several binding protein complexes that displayed unique preferences for binding to sense, antisense, and double-stranded forms of the element. Southwestern blot analysis revealed that the antisense strand of 5'SHS binds to nuclear proteins of molecular mass 97, 87, 44, and 17 kDa, whereas the double-stranded form of 5'SHS is recognized by a 70-kDa factor. Mutations within 5'SHS element indicated the necessity of a central 5'-GGGGAGGGGG-3' motif for protein binding and silencer function, while nucleotides flanking both sides of the motif were also critical for repression. These results support a model in which silencer function of 5'SHS is mediated by antisense strand binding proteins, possibly by stabilizing single-stranded DNA conformations required for interaction with enhancer sequences in the proximal promoter region of the A-chain gene.

Platelet-derived growth factor A-chain gene transcription is mediated by positive and negative regulatory regions in the promoter.

Platelet-derived growth factor (PDGF) is a disulphide-linked heterodimer of two polypeptide chains, the A and B chains, which are encoded by genes on separate chromosomes. The A-chain gene is transcribed in a number of transformed and non-transformed cell lines and is inducible by a wide variety of growth factors, cytokines and other mitogenic agonists. To localize DNA elements that mediate basal transcription in the promoter regulatory region of the A-chain gene, we have employed 5'-endpoint deletion mutagenesis and transient expression analysis in the renal epithelial cell line BSC-1 (African green monkey). Studies conducted in this cell line, which expresses high concentrations of PDGF A-chain mRNA, reveal a positive regulatory element (PRE) in a GC-rich stretch of the A-chain promoter between -82 and -40, relative to the transcription start site. Two discrete regions of the promoter were identified as negative regulatory elements (NREs), located between -1029 and -880 (NRE1) and between -1800 and -1029 (NRE2). The -1800 to -812 region, which contains both NREs, functions as a potent NRE when relocated in either orientation adjacent to the herpes simplex virus thymidine kinase promoter, reducing transcription activity by 60% in the positive orientation and 85% in the negative orientation. Comparison of BSC-1 cells and Saos-2 cells (human osteogenic sarcoma), which do not express significant quantities of PDGF A-chain mRNA or protein, indicates that basal transcription of the gene is determined by enhancer activity mediated by the GC-rich region rather than through de-repression of the upstream NREs. Electrophoretic gel mobility shift assays reveal a complex pattern of nuclear protein binding to the GC-rich PRE (-73 to -46). Competition studies conducted with mutant oligonucleotides that alternately disrupt consensus binding sites for Sp-1 or Egr-1 demonstrate a requirement for the presence of an Sp1-like core sequence (GGCGGG) but not Egr-1/Krox-24 [GCG(G/T)-GGGCG] for the formation of specific DNA-protein complexes. Our observations suggest that basal transcription of the A-chain gene in renal epithelial cells is achieved through active enhancement, mediated by a GC-rich PRE and nuclear proteins that bind to Sp-1-like consensus DNA sequences.