DE1, a 12-base pair cis-regulatory element sufficient to confer dark-inducible and light down-regulated expression to a minimal promoter in pea

We found a cis-regulatory element of 12 base pairs (bp) (GGATTTTACAGT) capable of conferring light responsiveness to a minimal promoter, CaMV 35S46, in pea. The 12-bp sequence is located in the 5' upstream region of the light down-regulated gene pra2, which encodes a small GTPase belonging to the YPT/rab family. Here we examined gain-of-function analyses using synthetic promoter-luciferase constructs in a transient assay and found that the 12-bp element alone was sufficient to confer dark induction, as well as light down-regulation on the minimal promoter. We named this dark inducible element DE1. Effects of various light conditions on the reporter gene activity showed that DE1 received signals from phytochrome A, phytochrome B, and blue light photoreceptors. Using phytochrome-deficient mutants, we showed that the pra2 protein level in seedlings was also regulated by these photoreceptors. The changes in the immunoblotting pattern of the pra2 protein in these mutants were correlated with the changes in epicotyl elongation. Results from transient assays using these mutants showed that the DE1 received signals from phytochromes A and B, demonstrating that this element is indeed a light-responsive element. To our knowledge, this is the first cis-element that by itself confers light responsiveness to a minimal promoter.     

The 5''-proximal region of the wheat Cab-1 gene contains a 268-bp enhancer-like sequence for phytochrome response.

We have previously reported that the expression of the wheat Cab-1 gene is subject to phytochrome regulation and a 1.8-kb 5' upstream sequence of this gene is sufficient for the regulated expression. To delineate sequences for the phytochrome response we analyzed a series of 5' deletion mutants as well as chimeric gene constructs comprising different sequences of the Cab-1 upstream region in transgenic tobacco seedlings. We found that a deletion mutant containing a 357-bp 5' upstream sequence still exhibits maximal levels of phytochrome-regulated expression. A 268-bp enhancer-like element, located between -89 and -357, is responsible for the phytochrome response of the Cab-1 gene; sequences upstream from -357 to -843 and downstream from -124 to +1100 are probably not involved. Finally, we show that the Cab-1 mRNA stability is not regulated by phytochrome.     

BLG-e1 - a novel regulatory element in the distal region of the beta-lactoglobulin gene promoter

beta-Lactoglobulin (BLG) is a major ruminant milk protein. A regulatory element, termed BLG-e1, was defined in the distal region of the ovine BLG gene promoter. This 299-bp element lacks the established cis-regulatory sequences that affect milk-protein gene expression. Nevertheless, it alters the binding of downstream BLG sequences to histone H4 and the sensitivity of the histone-DNA complexes to trichostatin A treatment. In mammary cells cultured under favorable lactogenic conditions, BLG-e1 acts as a potent, position-independent silencer of BLG/luciferase expression, and similarly affects the promoter activity of the mouse whey acidic protein gene. Intragenic sequences upstream of BLG exon 2 reverse the silencing effect of BLG-e1 in vitro and in transgenic mice.     

A 27 bp cis-acting sequence is essential for L-type calcium channel alpha(1C) subunit expression in vascular smooth muscle cells

Expression of L-type calcium channels in cardiac myocytes and vascular smooth muscle cells (VSMC) critically regulates the contractile state of these cells. In order to discover the elements in the promoter region of the Ca(v)1.2 gene encoding the vascular/cardiac calcium channel alpha(1C) subunit that are important for the basal gene expression, approximately 2 kb of the 5'-flanking sequence of the Ca(v)1.2 gene has been cloned in our lab. In this study, using various lengths of the 5'-flanking DNA fused with a luciferase gene as a reporter, we have defined a 493-bp fragment of the cis-regulatory DNA which carries the majority of promoter activity in pulmonary artery smooth muscle (PAC1) cells. DNase I footprinting analysis of this 493-bp DNA using nuclear extracts from PAC1 cells revealed a 27-bp DNA sequence that contains a c-Ets like motif (CAGGATGC). Mutation of the Ets-like site and the respective flanking sequence within the DNase I footprinting protection region induced a marked change in the promoter activity in PAC1 cells. Electrophoretic mobility shift assays (EMSA) confirmed the presence of specific binding factor(s) in PAC1 cells' nuclear extracts for this 27-bp DNA. Competition studies with the wild-type and mutated DNA fragments established the importance of the 27 bp DNA sequence for high-affinity binding of the nuclear proteins to the promoter. We conclude that there is a 27 bp region in the promoter of the Ca(v)1.2 gene to which nuclear proteins from VSMC bind and strongly regulate the basal promoter activity.     

Characterization of the S-RNase promoters from sweet cherry (Prunus avium L.)

Genomic DNA fragments containing the S(3)-, S(4)-, and S(6)-RNase genes were isolated from the sweet cherry (Prunus avium L.) and sequenced. Comparison of the 5'-flanking sequences of these three S-RNases indicated that a highly conserved region (designated CR) existed just upstream from the putative TATA boxes. We postulate that CR contains cis-regulatory element(s) involved in pistil expression. To examine the activity of the isolated S-RNase promoters of sweet cherry in the pistil, we transiently introduced approximately 650-bp fragments of the S(4)- and S(6)-RNase promoters fused to beta-glucuronidase (GUS) gene into the pistil of the petunia using a particle bombardment technique. Histochemical analysis showed that the 5'-flanking region of each S-RNase was active in the pistil. This suggests that cis-regulatory element(s) for pistil-specific expression may exist(s) within the 650-bp region upstream from the TATA box in the sweet cherry S-RNase promoter.     

Regulation of phytochrome A mRNA abundance in parsley seedlings and cell-suspension cultures

A cDNA clone encoding the apoprotein of a parsley phytochrome was isolated and classified as parsley PHYA phytochrome, on the basis of a sequence homology comparison with all available phytochrome sequences. Red light pulses led to a phytochrome-dependent down-regulation of PHYA mRNA abundance in etiolated parsley seedlings to a level of 10–20% compared with the dark control. The PHYA mRNA abundance in a parsley cell suspension culture was also down-regulated by light pulses. Transient expression assays in parsley protoplasts showed light regulation of a chimeric pea PHYA promoter uidA-gene construct.     

Quantitative statistical analysis of cis-regulatory sequences in ABA/VP1- and CBF/DREB1-regulated genes of Arabidopsis

We have developed a simple quantitative computational approach for objective analysis of cis-regulatory sequences in promoters of coregulated genes. The program, designated MotifFinder, identifies oligo sequences that are overrepresented in promoters of coregulated genes. We used this approach to analyze promoter sequences of Viviparous1 (VP1)/abscisic acid (ABA)-regulated genes and cold-regulated genes, respectively, of Arabidopsis (Arabidopsis thaliana). We detected significantly enriched sequences in up-regulated genes but not in down-regulated genes. This result suggests that gene activation but not repression is mediated by specific and common sequence elements in promoters. The enriched motifs include several known cis-regulatory sequences as well as previously unidentified motifs. With respect to known cis-elements, we dissected the flanking nucleotides of the core sequences of Sph element, ABA response elements (ABREs), and the C repeat/dehydration-responsive element. This analysis identified the motif variants that may correlate with qualitative and quantitative differences in gene expression. While both VP1 and cold responses are mediated in part by ABA signaling via ABREs, these responses correlate with unique ABRE variants distinguished by nucleotides flanking the ACGT core. ABRE and Sph motifs are tightly associated uniquely in the coregulated set of genes showing a strict dependence on VP1 and ABA signaling. Finally, analysis of distribution of the enriched sequences revealed a striking concentration of enriched motifs in a proximal 200-base region of VP1/ABA and cold-regulated promoters. Overall, each class of coregulated genes possesses a discrete set of the enriched motifs with unique distributions in their promoters that may account for the specificity of gene regulation.     

Loss-of-function mutation in a repressor module of human-specifically activated enhancer HACNS1

The cis-regulatory element contributed to gaining humanness is of great interest in human evolutionary studies. A human-accelerated region exceeding neutral evolutionary rates, termed HACNS1, was recently reported as a positively selected sequence acquiring novel TF-binding sites responsible for human-specific gain of limb enhancer function. However, another possibility is loss of function in repressor element in HACNS1. Signature of the human substitutions in the 81-bp region infers that a GC-biased gene conversion (BGC) might create these seemingly excessive substitutions. To evaluate the 81-bp function, we performed transgenic mouse assay of the HACNS1 construct lacking the 81-bp region. The deleted construct showed similar enhancer activity to the intact human HACNS1, suggesting that the function of the human 81-bp region is not an activating enhancer but rather a disrupted repressor. This result infers that loss of function in the HACNS1 81-bp region, possibly via a BGC, played an important role in human-specific evolution.     

Short 5'-flanking regions of the Amy gene of Drosophila kikkawai affect amylase gene expression and respond to food environments

Evolution of the duplicated genes and regulation in gene expression is of great interest, especially in terms of adaptation. Molecular population genetic and evolutionary studies on the duplicated amylase genes of Drosophila species have suggested that their 5'-flanking (cis-regulatory) regions play an important role in evolution of these genes. For better understanding of evolution of the duplicated amylase genes and gene expression, we studied functional significance of the Amy1 gene of Drosophila kikkawai using in vitro deletion mutagenesis followed by P-element-mediated germline transformation. We found that a 1.6-kb of the 5'-flanking region can produce strikingly higher level of larval amylase activity on starch food compared with that on glucose food. We found two cis-regulatory elements, which increase larval amylase activity on starch food. We also found a larval cis-regulatory element, which responds to the food difference. This food-response element is necessary for the function of the element increasing larval activity on starch food. A 5-bp deletion in a putative GRE caused high amylase activity, indicating a cis-regulatory element decreasing amylase activity. These cis-regulatory elements identified in the 5'-flanking region could be the targets of natural selection.