Sequence of EndoA gene encoding mouse cytokeratin and its methylation state in the CpG-rich region.

A genomic clone obtained from mouse liver DNA using a mouse cytokeratin EndoA cDNA probe revealed the complete sequence of the EndoA gene. The gene is divided into nine exons and the exon-intron pattern has been conserved compared to that of other type-II cytokeratin-encoding genes. The 5' upstream, 3' downstream and first and third introns contain potential regulatory sequences, including polyoma virus enhancer motifs (PEA1 and PEA3) and AP-1 elements. The 5' regions upstream of the EndoA, EndoB and Ck8 genes contain homologous sequences surrounding the TATA boxes. In addition, a CpG dinucleotide cluster region was located around the first exon. This CpG cluster region was found to be hypomethylated in endodermal PYS-2 cells, retinoic acid-treated F9 cells, and F9 embryonal carcinoma cells, but hypermethylated in BALB/C 3T3 fibroblast cells that do not express EndoA. These findings may provide a clue to understanding the molecular mechanisms of EndoA gene expression.     

Identification of an adenosine 3',5'-monophosphate (cAMP)-responsive region in the rat growth hormone gene: evidence for independent and synergistic effects of cAMP and thyroid hormone on gene expression

Rat GH gene expression is known to be stimulated by several factors, including thyroid hormone and GRF. This effect of GRF appears to be mediated by cAMP resulting from activation of adenylate cyclase by the peptide. The elements of the rat GH gene important for thyroid hormone stimulation and cell-specific expression have been previously mapped using gene transfection techniques. Cell-specific expression of the gene is mediated by two cell-specific elements located from -137 to -107 and from -95 to -65. Sequences mediating thyroid hormone stimulation are thought to be located between -208 and -160. In this study, using three different methods to elevate cAMP levels in cells [forskolin, a direct activator of the adenylate cyclase catalytic subunit; 8-(4-chlorophenylthio)cAMP, a nonmetabolizable cAMP analog; and isobutylmethylxanthine, a phosphodiesterase inhibitor], we show that 5'-flanking DNA of the rat GH gene can mediate stimulation by cAMP (10- to 20-fold). The cAMP-responsive region was mapped to sequences between -104 and +11, which contains the proximal cell-specific element (-95/-65) important for cell-specific expression. Either the -97/-65 or the -104/-47 region of the gene, cloned upstream of a heterologous promoter, conferred only minimal or no activation by cAMP. This suggests that these sequences are not the direct target of cAMP action or that they are insufficient alone to mediate the cAMP response. The cAMP regulatory element (TGACGTCA) is not found between - 104 and +11, and cAMP activation does not appear to act via putative AP-2 elements, since phorbol esters did not stimulate expression.(ABSTRACT TRUNCATED AT 250 WORDS)     

A potent far-upstream enhancer in the mouse pro alpha 2(I) collagen gene regulates expression of reporter genes in transgenic mice

We have identified three DNase I-hypersensitive sites in chromatin between 15 and 17 kb upstream of the mouse pro alpha 2 (I) collagen gene. These sites were detected in cells that produce type I collagen but not in cells that do not express these genes. A construction containing the sequences from -17 kb to +54 bp of the mouse pro alpha 2 (I) collagen gene, cloned upstream of either the Escherichia coli beta- galactosidase or the firefly luciferase reporter gene, showed strong enhancer activity in transgenic mice when compared with the levels seen previously in animals harboring shorter promoter fragments. Especially high levels of expression of the reporter gene were seen in dermis, fascia, and the fibrous layers of many internal organs. High levels of expression could also be detected in some osteoblastic cells. When various fragments of the 5' flanking sequences were cloned upstream of the 350-bp proximal pro alpha 2(I) collagen promoter linked to the lacZ gene, the cis-acting elements responsible for enhancement were localized in the region between -13.5 and -19.5 kb, the same region that contains the three DNase I-hypersensitive sites. Moreover, the DNA segment from -13.5 to -19.5 kb was also able to drive the cell-specific expression of a 220-bp mouse pro alpha 1(I) collagen promoter, which is silent in transgenic mice. Hence, our data suggest that a far-upstream enhancer element plays a role in regulating high levels of expression of the mouse pro alpha 2(I) collagen gene.     

Developmental regulation of human gamma-globin genes in transgenic mice.

We report results showing that several gamma gene promoter elements participate in the developmental control of gamma-globin genes. Four gamma gene constructs with 5' truncated at -141, -201, -382, and -730 of the A gamma gene promoter linked to a micro locus control region (microLCR) cassette were used for production of transgenic mice and analysis of gamma gene expression during development. Mice carrying a microLCR -141 A gamma construct displayed downregulation of gamma gene expression in the adult stage of development, indicating that the proximal promoter contains elements participating in gamma gene silencing. Mice carrying a microLCR -201 A gamma or a microLCR -382 A gamma construct displayed high gamma gene expression in the fetal stage of development and complete loss of gamma gene downregulation in the adult stage, suggesting that the -141 to -201 gamma gene sequence contains elements which upregulate gamma gene expression and are dominant over the negative element 3' to -141. Extension of the promoter to -730 resulted in reappearance of gamma gene downregulation, suggesting that the -382 to -730 sequences contain an adult-stage-specific silencer. gamma gene expression in the microLCR -201 A gamma and the microLCR -382 A gamma transgenic mice was copy number dependent. All the microLCR -730 A gamma transgenic mice expressed gamma mRNA; however, gamma gene expression was copy number independent, indicating that levels of gamma gene expression were modulated by the surrounding chromatin. Our results suggest that multiple elements participate in gamma gene silencing. The findings in the microLCR-201 A gamma and microLCR -382 A gamma transgenic mice are interpreted to indicate that the LCR interacts not only with the minimal gamma gene promoter but also with sequences of the upstream promoter. We postulate that gamma gene downregulation is achieved when the interaction between LCR and the upstream promoter is disturbed by the silencer located in the -382 to -730 region. We propose that gamma gene silencing is achieved by the combined effect of negative elements located 3' to -141, the negative element located between -382 and -730, and the competition by the beta gene promoter during the adult stage of development.     

Regulation of growth differentiation factor 9 expression in oocytes in vivo: a key role of the E-box

Growth differentiation factor 9 (GDF9) is preferentially expressed in oocytes and is essential for female fertility. To identify regulatory elements that confer high-level expression of GDF9 in the ovary but repression in other tissues, we generated transgenic mice in which regions of the Gdf9 locus were fused to reporter genes. Two transgenes (-10.7/+5.6mGdf9-GFP) and (-3.3/+5.6mGdf9-GFP) that contained sequences either 10.7 or 3.3 kb upstream and 5.6 kb downstream of the Gdf9 initiation codon demonstrated expression specifically in oocytes, thereby mimicking endogenous Gdf9 expression. In contrast, transgenes -10.7mGdf9-Luc and -3.3mGdf9-Luc, which lacked the downstream 5.6-kb region, demonstrated reporter expression not only in oocytes but also high expression in male germ cells. This suggests that the downstream 5.6-kb sequence contains a testis-specific repressor element and that 3.3 kb of 5'-flanking sequence contains all the cis-acting elements for directing high expression of Gdf9 to female (and male) germ cells. To define sequences responsible for oocyte expression of Gdf9, we analyzed sequences of Gdf9 genes from 16 mammalian species. The approximately 400 proximal base pairs upstream of these Gdf9 genes are highly conserved and contain a perfectly conserved E-box (CAGCTG) sequence. When this 400-bp region was placed upstream of a luciferase reporter (-0.4mGdf9-Luc), oocyte-specific expression was observed. However, a similar transgene construct (-0.4MUT-mGdf9-Luc) with a mutation in the E-box abolished oocyte expression. Likewise, the presence of an E-box mutation in a longer construct (-3.3MUT-mGdf9-Luc) abolished expression in the ovary but not in the testis. These observations indicate that the E-box is a key regulatory sequence for Gdf9 expression in the ovary.     

Characterization of promoter elements required for cell-specific expression of the neurotensin/neuromedin N gene in a human endocrine cell line.

Expression of the gene encoding neurotensin/neuromedin N (NT/N) is mostly limited to the brain and specialized enteroendocrine cells (N cells) of the distal small intestine. We have analyzed the NT/N DNA sequences upstream of the RNA start site that direct cell-specific expression using a novel human endocrine cell line, BON, that resembles intestinal N cells in several important aspects, including NT/N precursor protein processing, ratios of different NT/N mRNA forms, and high levels of constitutive expression of the NT/N gene. Transient transfection assays with plasmids with progressive 5' deletions of the rat NT/N promoter identified the proximal 216 bp of 5' flanking sequences as essential for high-level constitutive NT/N expression in BON cells. In addition, a detailed mutational analysis defined multiple regions within the proximal 216 bp that contribute to cell-specific NT/N expression. These elements include a proximal cyclic AMP response element (CRE)/AP-1-like motif (TGACATCA) that binds c-Jun, JunD, CRE-binding (CREB), and ATF proteins, a near-consensus glucocorticoid response element, and a distal consensus AP-1 site that binds c-Fos, Fra-1, and JunD. In addition, elements contained within two 21-bp imperfect direct repeats play an important role in NT/N expression in BON cells and may bind novel factors that act as positive regulators of NT/N expression. DNase I footprinting and gel shift analyses demonstrate that the sites identified by mutational analysis, and at least one additional site, specifically bind BON cell nuclear proteins in vitro. We speculate that a complex pattern of regulation requiring interaction between a proximal CRE/AP-1-like motif and other upstream control elements play an important role in the high-level constitutive expression of NT/N in the human endocrine cell line BON. In addition, the BON cell line provides a unique model to further characterize the factors regulating cell-specific NT/N expression and to better understand the mechanisms responsible for the terminal differentiation of the N-cell lineage in the gut.     

A cell-specific enhancer of the mouse alpha 1-antitrypsin gene has multiple functional regions and corresponding protein-binding sites.

We have previously described the isolation and characterization of genomic clones corresponding to the mouse alpha 1-antitrypsin gene (Krauter et al., DNA 5:29-36, 1986). In this report, we have analyzed the DNA sequences upstream of the RNA start site that direct hepatoma cell-specific expression of this gene when incorporated into recombinant plasmids. The 160 nucleotides 5' to the cap site direct low-level expression in hepatoma cells, and sequences between -520 and -160 bp upstream of the RNA start site functioned as a cell-specific enhancer of expression both with the alpha 1-antitrypsin promoter and when combined with a functional beta-globin promoter. Within the enhancer region, three binding sites for proteins present in hepatoma nuclear extracts were identified. The location of each site was positioned, using both methylation protection and methylation interference experiments. Each protein-binding site correlated with a functionally important region necessary for full enhancer activity. These experiments demonstrated a complex arrangement of regulatory elements comprising the alpha 1-antitrypsin enhancer. Significant qualitative differences exist between the findings presented here and the cis-acting elements operative in regulating expression of the human alpha 1-antitrypsin gene (Ciliberto et al., Cell 41:531-540, 1985; De Simone et al., EMBO J. 6:2759-2766, 1987).     

Far-upstream elements are dispensable for tissue-specific proenkephalin expression using a Cre-mediated knock-in strategy

Several cis-regulatory DNA elements are present in the 5' upstream regulatory region of the enkephalin gene (ENK) promoter. To determine their role in conferring organ-specificity of ENK expression in mice and to circumvent the position effects from random gene insertion that are known to often frustrate such analysis in transgenic mice, we used a Cre-mediated gene knock-in strategy to target reporter constructs to a "safe haven" loxP-tagged locus in the hypoxanthine phosphoribosyltransferase (HPRT) gene. Here we report reliable and reproducible reporter gene expression under the control of the 5' upstream regulatory region of the mouse ENK gene in gene-modified mice using this Cre-mediated knock-in strategy. Comparison of two 5'ENK regulatory regions (one with and the other without known cis-regulatory DNA elements) in the resulting adult mice showed that conserved far-upstream cis-regulatory DNA elements are dispensable for correct organ-specific gene expression. Thus the proximal 1.4 kb of the murine ENK promoter region is sufficient for organ-specificity of ENK gene expression when targeted to a safe-haven genomic locus. These results suggest that conservation of the far-upstream DNA elements serves more subtle roles, such as the developmental or cell-specific expression of the ENK gene.