Identification of cis-acting DNA elements involved in the regulation of angiotensinogen gene expression.

Angiotensinogen is the precursor molecule of one of the most potent vasoactive substances, angiotensin-II. Angiotensinogen is normally synthesized in the liver and secreted into the plasma where it is converted into angiotensin-II by the combined proteolytic action of renin and angiotensin converting enzyme. Angiotensinogen levels in the plasma are modulated by a number of pathological and physiological factors. In order to understand the regulation of angiotensinogen gene expression, we have constructed an expression vector in which 688 bp of the 5'-flanking region of the rat angiotensinogen gene were attached to the chloramphenicol acetyl transferase (CAT) coding sequence. We have also obtained 5'-sequential deletion mutants from the rat angiotensinogen promoter attached to the CAT gene, and have identified multiple cis-acting DNA sequences involved in the regulation of angiotensinogen gene expression by transient transfection of these recombinant DNA molecules in human hepatoma cell lines, Hep3B, and HepG2.     

GRK1 and GRK7: unique cellular distribution and widely different activities of opsin phosphorylation in the zebrafish rods and cones

Retinal cone cells exhibit distinctive photoresponse with a more restrained sensitivity to light and a more rapid shutoff kinetics than those of rods. To understand the molecular basis for these characteristics of cone responses, we focused on the opsin deactivation process initiated by G protein-coupled receptor kinase (GRK) 1 and GRK7 in the zebrafish, an animal model suitable for studies on retinal physiology and biochemistry. Screening of the ocular cDNAs identified two homologs for each of GRK1 (1A and 1B) and GRK7 (7-1 and 7-2), and they were classified into three GRK subfamilies, 1 A, 1B and 7 by phylogenetic analysis. In situ hybridization and immunohistochemical studies localized both GRK1B and GRK7-1 in the cone outer segments and GRK1A in the rod outer segments. The opsin/GRKs molar ratio was estimated to be 569 in the rod and 153 in the cone. The recombinant GRKs phosphorylated light-activated rhodopsin, and the Vmax value of the major cone subtype, GRK7-1, was 32-fold higher than that of the rod kinase, GRK1A. The reinforced activity of the cone kinase should provide a strengthened shutoff mechanism of the light-signaling in the cone and contribute to the characteristics of the cone responses by reducing signal amplification efficiency.     

A single enhancer regulating the differential expression of duplicated red-sensitive opsin genes in zebrafish

A fundamental step in the evolution of the visual system is the gene duplication of visual opsins and differentiation between the duplicates in absorption spectra and expression pattern in the retina. However, our understanding of the mechanism of expression differentiation is far behind that of spectral tuning of opsins. Zebrafish (Danio rerio) have two red-sensitive cone opsin genes, LWS-1 and LWS-2. These genes are arrayed in a tail-to-head manner, in this order, and are both expressed in the long member of double cones (LDCs) in the retina. Expression of the longer-wave sensitive LWS-1 occurs later in development and is thus confined to the peripheral, especially ventral-nasal region of the adult retina, whereas expression of LWS-2 occurs earlier and is confined to the central region of the adult retina, shifted slightly to the dorsal-temporal region. In this study, we employed a transgenic reporter assay using fluorescent proteins and P1-artificial chromosome (PAC) clones encompassing the two genes and identified a 0.6-kb "LWS-activating region" (LAR) upstream of LWS-1, which regulates expression of both genes. Under the 2.6-kb flanking upstream region containing the LAR, the expression pattern of LWS-1 was recapitulated by the fluorescent reporter. On the other hand, when LAR was directly conjugated to the LWS-2 upstream region, the reporter was expressed in the LDCs but also across the entire outer nuclear layer. Deletion of LAR from the PAC clones drastically lowered the reporter expression of the two genes. These results suggest that LAR regulates both LWS-1 and LWS-2 by enhancing their expression and that interaction of LAR with the promoters is competitive between the two genes in a developmentally restricted manner. Sharing a regulatory region between duplicated genes could be a general way to facilitate the expression differentiation in duplicated visual opsins.     

Gelatinase A expression in endothelial cells is regulated by at least two cis-acting promoter elements.

Increased expression of gelatinase A is associated with both angiogenesis and alterations in blood vessel structure. Heart-derived endothelial cells derived from spontaneously hypertensive rats (SHR) were found to express significantly more gelatinase A in culture, both at the protein and mRNA level, than endothelial cells from normotensive Wistar-Kyoto (WKY) rats. Other matrix metalloproteinases, as well as their tissue inhibitors, were not differentially regulated. A 1683 bp gelatinase A promoter fragment linked to a luciferase reporter demonstrated up to 40-fold more activity when transfected into SHR-derived cells versus WKY-derived cells. The promoter region between -1324 and -1272, previously termed RE1, contributed up to a five-fold increase in basal promoter activity in both cells, but contributed only 12% of the promoter activity in SHR-derived cells compared to 85% in WKY-derived cells. In SHR-derived cells, but not in WKY-derived cells, a second region between -1435 and -1375, termed RE2, contributed 60% of the total activity of the 1683 bp promoter fragment. Both electrophoretic mobility shift assays and Southwestern blots demonstrated differences in RE2-specific binding factors in nuclear extracts derived from the two cell types. SHR-derived endothelial cells thus represent a new model system to study the regulation of gelatinase A expression, which itself may contribute to the abnormal vascular structure seen in the SHR.     

Identification of regulatory elements recapitulating early expression of L-plastin in the zebrafish enveloping layer and embryonic periderm

We have cloned and characterized an intronic fragment of zebrafish lymphocyte cytosolic protein 1 (lcp1, also called L-plastin) that drives expression to the zebrafish enveloping layer (EVL). L-plastin is a calcium-dependent actin-bundling protein belonging to the plastin/fimbrin family of proteins, and is necessary for the proper migration and attachment of several adult cell types, including leukocytes and osteoclasts. However, in zebrafish lcp1 is abundantly expressed much earlier, during differentiation of the EVL. The cells of this epithelial layer migrate collectively, spreading vegetally over the yolk. L-plastin expression persists into the larval periderm, a transient epithelial tissue that forms the first larval skin. This finding establishes that L-plastin is activated in two different embryonic waves, with a distinct regulatory switch between the early EVL and the later leukocyte. To better study L-plastin expressing cells we attempted CRISPR/Cas9 homology-driven recombination (HDR) to insert a self-cleaving peptide (Cre-P2A-EGFP-CAAX) downstream of the native lcp1 promoter. This produced a stable zebrafish line expressing Cre recombinase in EVL nuclei and green fluorescence in EVL cell membranes. In vivo tracking of these labeled cells provided enhanced views of EVL migration behavior, membrane extensions, and mitotic events. Finally, we experimentally dissected key elements of the targeted lcp1 locus, discovering a ∼300 bp intronic sequence sufficient to drive EVL expression. The lcp1: Cre-P2A-EGFP-CAAX zebrafish should be useful for studying enveloping layer specification, gastrulation movements and periderm development in this widely used vertebrate model. In addition, the conserved regulatory sequences we have isolated predict that L-plastin orthologs may have a similar early expression pattern in other vertebrate embryos.     

Characterization of a cis-acting element involved in cell-specific expression of the zebrafish brain aromatase gene

The cytochrome P450 Aromatase is the key enzyme catalyzing the conversion of androgens into estrogens. In zebrafish, the brain aromatase is encoded by cyp19b. Expression of cyp19b is restricted to radial glial cells bordering forebrain ventricles and is strongly stimulated by estrogens during development. At the promoter level, we have previously shown that an estrogen responsive element (ERE) is required for induction by estrogens. Here, we investigated the role of ERE flanking regions in the control of cell-specific expression. First, we show that a 20 bp length motif, named G x RE (glial x responsive element), acts in synergy with the ERE to mediate the estrogenic induction specifically in glial cells. Second, we demonstrate that, in vitro, this sequence binds factors exclusively present in glial or neuro-glial cells and is able to confer a glial specificity to an artificial estrogen-dependent gene. Taken together, these results contribute to the understanding of the molecular mechanisms allowing cyp19b regulation by estrogens and allowed to identify a promoter sequence involved in the strong estrogen inducibility of cyp19b which is specific for glial cells. The exceptional aromatase activity measured in the brain of teleost fish could rely on such mechanisms.     

Identification and expression analysis of mical family genes in zebrafish

Mical(molecule interacting with CasL)represent a conserved family of cytosolic multidomain proteins that has been shown to be associated with a variety of cellular processes,including axon guidance,cell movement,cell-cell junction formation,vesicle trafficking and cancer cell metastasis.However,the expression and function of these genes during embryonic development have not been comprehensively characterized,especially in vertebrate species,although some limited in vivo studies have been carried out in neural and musculature systems of Drosophila and in neural systems of vertebrates.So far,no mica/family homologs have been reported in zebrafish,an ideal vertebrate model for the study of developmental processes.Here we report eight homologs of m/ca/family genes in zebrafish and their expression profiles during embryonic development.Consistent with the findings in Drosophila and mammals,most zebrafish mical family genes display expression in neural and musculature systems.In addition,five mica/homologs are detected in heart,and one,micall2a,in blood vessels.Our data established an important basis for further functional studies of mica/family genes in zebrafish,and suggest a possible role for mica/genes in cardiovascular development.     

Pollen-specific regulation of vacuolar H+-PPase expression by multiple cis-acting elements

We dissected the regulatory region of the AVP1 gene encoding the vacuolar H⁺-pyrophosphatase (V-PPase) of Arabidopsis thaliana by using a GUS-reporter assay system. The cloned 1.4 kb 5-regulatory region in the GUS-reporter transgenic plants was sufficient for the light-induced repression. Furthermore, the 1.4 kb regulatory region was active in all tissues examined and its activity was especially enhanced in pollen, whereas the shorter 0.4 kb regulatory region was active only in pollen. Further detailed analyses revealed that the GUS activity in pollen was regulated by at least three cis-acting regions in an additive or synergetic manner. These findings establish a distinct mechanism of the tissue-specific regulation of V-PPase expression in developing pollen, and imply the biological significance of the V-PPase in pollen maturation.