Dissection of cis-regulatory elements in the C. elegans Hox gene egl-5 promoter

Hox genes are highly conserved segmental identity genes well known for their complex expression patterns and divergent targets. Here we present an analysis of cis-regulatory elements in the Caenorhabditis elegans Hox gene egl-5, which is expressed in multiple tissues in the posterior region of the nematode. We have utilized phylogenetic footprinting to efficiently identify cis-regulatory elements and have characterized these with gfp reporters and tissue-specific rescue experiments. We have found that the complex expression pattern of egl-5 is the cumulative result of the activities of multiple tissue or local region-specific activator sequences that are conserved both in sequence and near-perfect order in the related nematode Caenorhabditis briggsae. Two conserved regulatory blocks analyzed in detail contain multiple sites for both positively and negatively acting factors. One of these regions may promote activation of egl-5 in certain cells via the Wnt pathway. Positively acting regions are repressed in inappropriate tissues by additional negative pathways acting at other sites within the promoter. Our analysis has allowed us to implicate several new regulatory factors significant to the control of egl-5 expression.     

An abundant androgen-regulated mRNA in the mouse kidney.

We have identified an abundant 20,000 dalton protein (KAP) by in vitro translation of male mouse kidney mRNA. This protein is synthesized in reduced amounts from female kidney mRNA. A KAP cDNA fragment was purified and used for nucleic acid hybridization studies. Females and castrated males have 10 and 200 fold lower levels, respectively, of KAP mRNA relative to males. The administration of testosterone to females or castrated males results in the induction of KAP mRNA to normal male levels. Testicular feminized (Tfm) mice have 3 fold lower levels of KAP mRNA relative to normal males and are not induced by testosterone. KAP mRNA is not found in significant amounts in tissues other than the kidney, and the KAP gene renatures with kinetics similar to single-copy DNA. With the rapidly expanding knowledge of mouse genetics, KAP should prove useful in determining genetic factors which regulate the inducibility and tissue specificity of a hormonally regulated gene.     

Differential effect of 5 alpha-reductase inhibition and castration on androgen-regulated gene expression in rat prostate.

Castration reduces prostate size and causes intraprostatic testosterone (T) and dihydrotestosterone (DHT) to fall to very low levels. 5 alpha-Reductase inhibition also reduces prostate size, but results in a marked increase in intraprostatic T levels. To compare the effects of 5 alpha-reductase inhibition and castration on prostate physiology, male Sprague-Dawley rats were left intact, castrated, or given the selective 5 alpha-reductase inhibitor finasteride for up to 9 days. To be sure that finasteride itself did not directly affect gene expression, an additional group of rats was castrated and given finasteride for 4 days. The prostates were weighed, intraprostatic RNA, DNA, and androgen levels were measured, and mRNAs for two androgen-regulated genes, prostate steroid-binding protein (PSBP; an androgen-induced gene) and testosterone-repressed prostate message (TRPM-2), were quantitated by Northern and slot blot analyses. Finasteride caused a 95% reduction in intraprostatic DHT levels and a 10-fold increase in intraprostatic T levels. Finasteride, as expected, caused a pronounced decrease in prostate weight (45% on day 4). DNA content fell correspondingly (48% on day 4). Intraprostatic DNA (micrograms of DNA per gland) on day 4 was 328 +/- 53 in control rats, 171 +/- 10 in finasteride-treated rats (P less than 0.001 compared to controls), 115 +/- 2 in castrated rats (P less than 0.05 compared to finasteride), and 107 +/- 43 in finasteride-treated plus castrated rats (P = NS compared to castration alone). There were no significant differences in DNA levels among the groups when expressed per mg prostate tissue, indicating that mean prostate cell size was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of the mec-3 gene by the C.elegans homeoproteins UNC-86 and MEC-3.

The mec-3 gene encodes a homeodomain protein with LIM repeats that is required for the specification of touch cell fate in Caenorhabditis elegans. Previous experiments suggested that mec-3 expression requires the product of the unc-86 gene, a POU-type homeoprotein, and mec-3 itself. We have analyzed the control of mec-3 expression by identifying potential cis regulatory elements in the mec-3 gene (by conservation in a related nematode and by DNase I footprinting using unc-86 and mec-3 proteins) and testing their importance by transforming C.elegans with mec-3lacZ fusions in which these sites have been mutagenized in vitro. Both unc-86 and mec-3 proteins bind specifically to the promoter of the mec-3 gene, suggesting that both proteins may be directly involved in the regulation of the mec-3 gene. In addition, the footprint pattern with mec-3 protein is altered in the presence of unc-86 protein. In vivo transformation experiments reveal that some of the binding regions of the two proteins are needed for general positive control and maintenance of mec-3 expression while others have no detectable, unique function. Interestingly, the unc-86 gene appears to be required not only to initiate mec-3 expression but also to maintain it.     

Sexually dimorphic expression of a laryngeal-specific, androgen-regulated myosin heavy chain gene during Xenopus laevis development.

Masculinization of the larynx in Xenopus laevis frogs is essential for the performance of male courtship song. During postmetamorphic (PM) development, the initially female-like phenotype of laryngeal muscle (slow and fast twitch fibers) is converted to the masculine form (entirely fast twitch) under the influence of androgenic steroids. To explore the molecular basis of androgen-directed masculinization, we have isolated cDNA clones encoding portions of a new Xenopus myosin heavy chain (MHC) gene. We have detected expression of this gene only in laryngeal muscle and specifically in males. All adult male laryngeal muscle fibers express the laryngeal myosin (LM). Adult female laryngeal muscle expresses LM only in some fibers. Expression of LM during PM development was examined using Northern blots and in situ hybridization. Males express higher levels of LM than females throughout PM development and attain adult levels by PM3. In females, LM expression peaks transiently at PM2. Treatment of juvenile female frogs with the androgen dihydrotestosterone masculinizes LM expression. Thus, LM appears to be a male-specific, testosterone-regulated MHC isoform in Xenopus laevis. The LM gene will permit analysis of androgen-directed sexual differentiation in this highly sexually dimorphic tissue.     

Prosaposin is a novel androgen-regulated gene in prostate cancer cell line LNCaP

Androgen-regulated genes (ARG) are implicated in normal and neoplastic growth of the prostate. Recently, we reported genomic amplification and/or overexpression of a previously known neurotrophic factor, prosaposin, in androgen-independent (AI) or metastatic prostate cancer (PCa) cells and tissues. Prosaposin and/or its known active molecular derivatives (e.g., saposin C) function as a pluripotent growth factor with diverse biological activities that favor malignant phenotypes in PCa cells. In addition, prosaposin or saposin C upregulates androgen receptor (AR) and AR-target genes (i.e., prostate-specific antigen, Probasin) expression and activity in LNCaP cells. Here, we examined prosaposin as an ARG. We report that DHT treatment of LNCaP cells increases prosaposin expression. In addition, we demonstrate androgen-responsiveness of prosaposin promoter and AR occupancy to a hormone-responsive element located in the proximal region of the prosaposin promoter. Our data for the first time identify prosaposin as an ARG. This observation, together with the pleiotropic growth factor activity of prosaposin, might suggest a role for this molecule in AR-dependent progression of prostate cancer at its early or late AI-state.     

Genomic organization and DNA sequence of the mouse kidney androgen-regulated protein (KAP) gene

The gene for kidney androgen-regulated protein (KAP) is expressed under androgenic control in the epithelial cells of the renal cortical proximal tubules. However, there is an androgen-independent component of the expression of this gene that occurs specifically in the outermedullary S3 segments of the proximal tubules. In these cells, the KAP gene is estrogen responsive and its expression is dependent on pituitary function. As a first step in correlating its interesting cell-specific and hormonal regulation with the structure of the gene, the genomic organization of the KAP gene was described and sequence of the gene and the proximal 1 kb of 5'-flanking DNA was determined. Sequence motifs were identified in the 5'-flanking DNA that may function in the regulation KAP gene expression by androgen, estrogen, and pituitary glycoprotein hormones. The gene is present in a single copy in the mouse genome and is 3,807 nucleotides in length. It contains 4 exons of 120, 177, 63, and 251 nucleotides and three intervening sequences of 1,450, 126, and 1,620 nucleotides. The gene exhibits a high degree of a genetic polymorphism as revealed by comparison of restriction digests of DNA from two highly inbred strains, BALB/c and C57BL/6.     

cis-Regulatory elements for mesophyll-specific gene expression in the C4 plant Flaveria trinervia, the promoter of the C4 phosphoenolpyruvate carboxylase gene

C(4) photosynthesis depends on the strict compartmentalization of CO(2) assimilatory enzymes. cis-regulatory mechanisms are described that ensure mesophyll-specific expression of the gene encoding the C(4) isoform of phosphoenolpyruvate carboxylase (ppcA1) of the C(4) dicot Flaveria trinervia. To elucidate and understand the anatomy of the C(4) ppcA1 promoter, detailed promoter/reporter gene studies were performed in the closely related C(4) species F. bidentis, revealing that the C(4) promoter contains two regions, a proximal segment up to -570 and a distal part from -1566 to -2141, which are necessary but also sufficient for high mesophyll-specific expression of the beta-glucuronidase reporter gene. The distal region behaves as an enhancer-like expression module that can direct mesophyll-specific expression when inserted into the ppcA1 promoter of the C(3) plant F. pringlei. Mesophyll expression determinants were restricted to a 41-bp segment, referred to as mesophyll expression module 1 (Mem1). Evolutionary and functional studies identified the tetranucleotide sequence CACT as a key component of Mem1.     

Association between the C3F gene and atherosclerotic vascular diseases.

The C3 phenotype distribution was studied in a group of patients suffering from atherosclerotic vascular diseases. A statistically significant association was found between the presence of the C3F gene and the occurrence of atherosclerosis. A relative risk incidence of the disease of 1.87 was found for the C3F-positive individuals as compared to the C3F-negative ones.     

Multiple genes coding for the androgen-regulated major urinary proteins of the mouse.

We have purified a cDNA fragment complementary to the mRNA coding for one of the major urinary proteins (MUPs) synthesized in the mouse liver. Using this cDNA as a hybridization probe, we have shown that the level of MUP mRNA is lower in the livers of females and castrated males than in those of males. The addition of testosterone to females and castrated males results in an increase in the concentration of the mRNA to levels found in males. There are approximately 15 gene per haploid genome coding for the MUPs; this allows a possible new interpretation of some of the genetic data concerning the regulation of levels of the different MUPs in the urine (Szoka and Paigen, 1978). Finally, we have shown that mouse MUP and rat alpha 2u-globulin mRNA share common sequences, but that there are surprising differences in gene number and regulation of the genes in these two closely related animals.     

Minimal enhancer elements of the leghemoglobinlba andlbc3 gene promoters fromGlycine max L. have different properties

The characteristics of the soybean leghemoglobinlba gene promoter were analyzed and important promoter elements from thelba andlbc3 promoters were compared using transgenicLotus corniculatus plants. A 5 deletion analysis of thelba promoter delimited twocis-acting elements controlling expression: a distal positive element (–1254, –884) required for expression and a proximal element (–285, –60) essential for full-level activity. In contrast to the corresponding region of thelbc3 promoter, thelba proximal element is unable to control expression from the heterologous CaMV 35S enhancer. The upstream positive element of thelba gene contains a position- and orientation-independent enhancer between positions (–1091, –788). The sequence of this enhancer region is conserved in thelbc3 gene upstream (–1333, –1132) of the previously assigned strong positive element (SPE; –1090, –947). The present analysis revealed some of the properties of this extendedlbc3 SPE element. The extended element (–1364, –947) functions in both orientations from 5 locations whereas the SPE2 subcomponent (–1364, –1154) containing the conserved sequence is only active in the correct orientation. Removal of the SPE2 by internal deletion demonstrates that the SPE2 subcomponent is indispensable for the activity of thelbc3 upstream positive element. These results indicate that the upstream positive elements of thelba andlbc3 genes possess different properties although their conserved minimal enhancer sequence has similar function. This may reflect the differential expression of the twolb genes ofGlycine max L.