Candidate cis-elements for human renin gene expression in the promoter region

The regulation of renin gene expression, the rate‐limiting enzyme of the system, is thought to be fundamental to the total system. Previously, we mapped six putative cis‐elements in the promoter region of the human renin gene with nuclear proteins from human chorionic cells and human renal cortex by DNase I protection assay (footprint A–F). Each footprint contains Ets motif like site (A), HOXñPBX recognition sequence (B), unknown sequence as DNA binding consensus (C), CRE (D), COUP‐TFII (ARP‐1) motif like site (E), and AGE3 like site (F). Footprint D has been characterized by means of functional studies as the genuine human renin gene CRE interacting with CREB in cooperation with the site of footprint B. To obtain further clues to the specific expression in the promoter region, these putative cis‐elements were conducted to a consensus‐specific binding assay to compare renin‐producing and non‐renin‐producing cells by EMSA and electromobility super‐shift assay. Different sequence‐specific DNA/protein binding was obtained among the different cell lines with footprint B site, with COUP‐TFII (ARP‐1) motif like site and possibly with footprint F site. The results implicate these putative cis‐elements and each corresponding trans‐factor in the specific expression of the human renin gene in the promoter region. Further functional characterization of these elements would provide important data for a better understanding of human renin gene expression. © 2004 Wiley‐Liss, Inc.     

Functional cis-element sequence requirements for suppression of gene expression by the TNPA protein of the Zea mays transposon En/Spm

TNPA, one of the two transposition proteins encoded by the En/Spm transposable elements of Zea mays, suppresses the expression of genes that contain an appropriate cis element. Suppression can be monitored in tobacco protoplasts in a transient expression assay as follows. The plant promoter-driven expression of the Escherichia coli-glucuronidase (GUS)-encoding gene, uidA, is repressed in the presence of TNPA if the GUS gene contains a functional cis element in the untranslated RNA leader sequence. Earlier, we found that the minimal cis element is composed of two 12 by sequences in a tail-to-tail inverted orientation. Each 12 by sequence is sufficient to bind TNPA in vitro and can be thought of as a half-site in the cis element. Here, we investigated the sequence requirements of the minimal cis element. Our observations support our expectations that a functional cis element must provide a template to which two TNPA molecules can bind in the correct orientation. Sequences within the half-sites can be altered as long as the eight bases that make up the consensus binding sites are not changed. However, we found the following unexpected sequence specificities. Firstly, some changes to the consensus binding sequence can be tolerated in one half-site, as long as the other site matches the consensus. Secondly, although the region between the half-sites can vary in sequence and in length between two and four bases, a thymidine residue is not tolerated directly 5 preceding the second half-site. Since many variants of the cis element sequence remain functional, the suppressor response element provides a flexible tool for artificially manipulating the expression of genes.     

Complex patterns of cis‐regulatory polymorphisms in ebony underlie standing pigmentation variation in Drosophila melanogaster

Pigmentation traits in adult Drosophila melanogaster were used in this study to investigate how phenotypic variations in continuous ecological traits can be maintained in a natural population. First, pigmentation variation in the adult female was measured at seven different body positions in 20 strains from the Drosophila melanogaster Genetic Reference Panel (DGRP) originating from a natural population in North Carolina. Next, to assess the contributions of cis‐regulatory polymorphisms of the genes involved in the melanin biosynthesis pathway, allele‐specific expression levels of four genes were quantified by amplicon sequencing using a 454 GS Junior. Among those genes, ebony was significantly associated with pigmentation intensity of the thoracic segment. Detailed sequence analysis of the gene regulatory regions of this gene indicated that many different functional cis‐regulatory alleles are segregating in the population and that variations outside the core enhancer element could potentially play important roles in the regulation of gene expression. In addition, a slight enrichment of distantly associated SNP pairs was observed in the ~10 kb cis‐regulatory region of ebony, which suggested the presence of interacting elements scattered across the region. In contrast, sequence analysis in the core cis‐regulatory region of tan indicated that SNPs within the region are significantly associated with allele‐specific expression level of this gene. Collectively, the data suggest that the underlying genetic differences in the cis‐regulatory regions that control intraspecific pigmentation variation can be more complex than those of interspecific pigmentation trait differences, where causal genetic changes are typically confined to modular enhancer elements.     

Species‐specific coordinated gene expression and trans‐regulation of larval color pattern in three swallowtail butterflies

SUMMARY The diversity of butterfly larval color pattern has been attracted to people since Darwin's time; however, its molecular mechanisms still remain largely unknown. Larval body markings often differ completely between closely related species under natural selection. The final instar larvae of the swallowtail butterflies Papilio xuthus and Papilio polytes show a green camouflage pattern, whereas those of Papilio machaon show a warning color pattern, although P. xuthus and P. machaon are closely related species. To identify the genes that contribute to species divergence, we compared the expression pattern of eight pigment‐associated genes between three Papilio species. The spatial expression pattern of melanin‐related genes coincided with the species‐specific cuticular markings. We newly found that the combination of bilin‐binding protein and yellow‐related gene (YRG) correlated perfectly with larval blue, yellow, and green coloration. To distinguish whether the interspecific differences in pigment‐associated genes are caused by cis‐regulatory changes or distribution differences in trans‐regulatory proteins, we compared species‐specific mRNA expression in an F1 hybrid specimen. Px‐YRG and Pp‐YRG showed a similar expression pattern, suggesting that the change in expression of YRG is caused mainly by changes in the distribution of trans‐regulatory proteins. Our findings shed light on the gene regulatory networks for butterfly larval color pattern.