Positive Regulatory Region Research Articles

Large-scale mutational analysis identifies UNC93B1 variants that drive TLR-mediated autoimmunity in mice and humans.

Nucleic acid-sensing Toll-like receptors (TLR) 3, 7/8, and 9 are key innate immune sensors whose activities must be tightly regulated to prevent systemic autoimmune or autoinflammatory disease or virus-associated immunopathology. Here, we report a systematic scanning-alanine mutagenesis screen of all cytosolic and luminal residues of the TLR chaperone protein UNC93B1, which identified both negative and positive regulatory regions affecting TLR3, TLR7, and TLR9 responses. We subsequently identified two families harboring heterozygous coding mutations in UNC93B1, UNC93B1+/T93I and UNC93B1+/R336C, both in key negative regulatory regions identified in our screen. These patients presented with cutaneous tumid lupus and juvenile idiopathic arthritis plus neuroinflammatory disease, respectively. Disruption of UNC93B1-mediated regulation by these mutations led to enhanced TLR7/8 responses, and both variants resulted in systemic autoimmune or inflammatory disease when introduced into mice via genome editing. Altogether, our results implicate the UNC93B1-TLR7/8 axis in human monogenic autoimmune diseases and provide a functional resource to assess the impact of yet-to-be-reported UNC93B1 mutations.

Phenazopyridine promotes RPS23RG1/Rps23rg1 transcription and ameliorates Alzheimer-associated phenotypes in mice.

Alzheimer's disease (AD) is the most common form of dementia with no effective treatment options. A complete elucidation of its underlying molecular mechanisms, including the transcription regulation of genes critically involved in AD, may shed light on new therapeutic development. RPS23RG1 is a newly identified AD-associated gene, whose expression is decreased in AD and restoration can attenuate AD-like phenotypes in animal models. However, the transcription regulation of RPS23RG1 remains unknown. In this study, we explored the promoter of RPS23RG1 and identified its transcription initiation site (TSS) at 1525 bp upstream of the ATG translation start codon. Progressive deletion analysis determined the presence of a negative regulatory region and a positive regulatory region within nucleotide positions +1127 to +1187 and +732 to +1127 relative to the TSS (+1), respectively. We conducted a reporter system to screen for compounds that increase RPS23RG1 expression through antagonizing its negative regulatory elements and identified phenazopyridine. Importantly, we demonstrated that phenazopyridine not only promoted RPS23RG1/Rps23rg1 expression, but also reduced AD-like pathologies and cognitive impairments in the APP/PS1 AD model mice. We also determined a critical negative regulatory domain of RPS23RG1 within nucleotide positions +1177 to +1187 and found that the transcription factor SMAD3 bound to this domain. Inhibition of SMAD3 promoted RPS23RG1 expression. Moreover, phenazopyridine reduced SMAD3 binding to the RPS23RG1 promoter without affecting SMAD3 phosphorylation and nuclear localization. Taken together, our results determine the transcription regulation mechanism of RPS23RG1 and show that phenazopyridine has potential for AD treatment through regulating RPS23RG1 transcription.

Characterization of Transcription Regulatory Domains of OsMADS29: Identification of Proximal Auxin-Responsive Domains and a Strong Distal Negative Element.

OsMADS29 (M29) is a seed-specific MADS-box transcription factor involved in programmed cell death of nucellar tissue and maintaining auxin:cytokinin homeostasis. It affects embryo and endosperm development and starch filling during seed development in rice. Its expression seems to be tightly regulated by developmental, spatial, and temporal cues; however, cis- and trans-regulatory factors that affect its expression are largely unknown. In silico analysis of the 1.7 kb upstream regulatory region (URR) consisting of 1,290 bp promoter and 425 bp 5′-UTR regions revealed several auxin-responsive and seed-specific cis-regulatory elements distributed across the URR. In this study, the analysis of four URR deletions fused to a downstream β-glucuronidase (GUS) reporter in transgenic rice has revealed the presence of several proximal positive elements and a strong distal negative element (NE). The promoter regions containing auxin-responsive elements responded positively to the exogenous application of auxins to transgenic seedlings. The proximal positive elements are capable of driving reporter expression in both vegetative and reproductive tissues. In contrast, the NE strongly suppresses reporter gene expression in both vegetative and reproductive tissues. In a transient onion peel assay system, the NE could reduce the efficacy of a 2x CaMV 35S promoter by ∼90%. Our results indicate the existence of a complex array of positive and negative regulatory regions along with auxin-responsive elements guiding the development-dependent and spatial expression of M29.

PRDM16 Regulating Adipocyte Transformation and Thermogenesis: A Promising Therapeutic Target for Obesity and Diabetes.

Given that obesity and diabetes have been major public health concerns and that disease morbidities have been rising continuously, effective treatment for these diseases is urgently needed. Because adipose tissue metabolism is involved in the progression of obesity and diabetes, it might be efficient to target adipocyte metabolic pathways. Positive regulatory domain zinc finger region protein 16 (PRDM16), a transcription factor that is highly expressed in adipocytes, plays a key role in adipose tissue metabolism, such as the browning and thermogenesis of adipocytes, the beigeing of adipocytes, the adipogenic differentiation of myoblasts, and the conversion of visceral adipocytes to subcutaneous adipocytes. Furthermore, clinical and basic studies have shown that the expression of PRDM16 is associated with obesity and diabetes and that PRDM16 signaling participates in the treatment of the two diseases. For example, metformin promotes thermogenesis and alleviates obesity by activating the AMPK/αKG/PRDM16 signaling pathway; rosiglitazone alleviates obesity under the synergistic effect of PRDM16; resveratrol plays an antiobesity role by inducing the expression of PRDM16; liraglupeptide improves insulin resistance by inducing the expression of PRDM16; and mulberry leaves play an anti-inflammatory and antidiabetes role by activating the expression of brown fat cell marker genes (including PRDM16). In this review, we summarize the evidence of PRDM16 involvement in the progression of obesity and diabetes and that PRDM16 may be a promising therapy for obesity and diabetes.

Testis-specific expression pattern of dmrt1 and its putative regulatory region in the sea urchin (Mesocentrotus nudus).

Sea urchin (Mseocentrotus nudus) is an economically important mariculture species in several Asian countries. The growth rate and immunocompetence differ by sex in this species. However, the mechanisms of sex determination in M. nudus have remain unclear. In the present study, we focus on the dmrt1 gene of M. nudus (Mndmrt1) to investigate its dynamic expression pattern during different developmental stages. Real-time quantitative PCR (RT-qPCR) revealed that Mndmrt1 exhibits testis-specific expression and undetectable during the whole embryogenesis. With the development of ontogenetic, Mndmrt1 transcripts are first detected at 9months post-fertilization (mpf). In addition, both the transcripts and protein of Mndmrt1 gene were specifically expressed in spermatogonia and spermatocytes, indicating that it might be a male germ cells marker in sea urchin. Significantly, the 1441bp promoter sequence of Mndmrt1 gene was obtained by DNA walking, and one positive regulatory region at -1197/ -968 in the promoter, as well as one negative regulatory region at -1441/ -1198 have been identified by promoter activity analysis. Moreover, two regulatory regions contain multiple putative binding sites for transcription factors, including Sp1, Egr1, Sox5, CEBP, GATA and SRY. These findings suggest that Mndmrt1 may be related to testis differentiation and spermatogenesis in sea urchin and will provide an insight into understanding the regulatory mechanism of the dmrt1 gene.

Isolation and functional analysis of OsAOS1promoter for resistance to Nilaparvata lugens Stål infestation in rice.

Insect pests have a great impact on the yield and quality of crops. Insecticide applications are an effective method of pest control, however, they also have adverse effects on the environment. Using insect-inducible promoters to drive insect-resistant genes in transgenic crops is a potential sustainable pest management strategy, but insect-inducible promoters have been rarely reported. In this study, we found rice allene oxide synthase gene (AOS, LOC_Os03g12500) can be highly upregulated following brown planthopper (Nilaparvata lugens Stål, BPH) infestation. Then, we amplified the promoter of OsAOS1 and the β- glucuronidase reporter gene was used to analyze the expression pattern of the promoter. Through a series of 5' truncated assays, three positive regulatory regions in response to BPH infestation in the promoter were identified. The transgenic plants, P1R123-min 35S and P1TR1-min 35S promoter-driven snowdrop lectin (Galanthus nivalis agglutinin, GNA) gene, demonstrated the highest expression levels of GNA and lowest BPH survival. Our work identified a BPH-inducible promoter and three positive regions within it. Transgenic rice with GNA driven by OsAOS1 promoter and positive regions exhibited an expected lethal effect on BPH. This study proved the application potential of BPH-inducible promoter and provided a novel path for the selection of insect-resistant tools in the future.

Cloning and functional identification of a Chilo suppressalis-inducible promoter of rice gene, OsHPL2.

Promoters play a key role in driving insect-resistant genes during breeding of transgenic plants. In current transgenic procedures for breeding rice resistance to striped stem borer (Chilo suppressalis Walker, SSB), the constitutive promoter is used to drive the insect-resistant gene. To reduce the burden of constitutive promoters on plant growth, isolation and identification of insect-inducible promoters are particularly important. However, few promoters are induced specifically by insect feeding. We found rice hydroperoxide lyase gene (OsHPL2) (LOC_Os02g12680) was upregulated after feeding by SSB. We subsequently cloned the promoter of OsHPL2 and analysed its expression pattern using the β-glucuronidase (GUS) reporter gene. Histochemical assays and quantitative analyses of GUS activity confirmed that P HPL2 :GUS was activated by SSB, but did not respond to brown planthopper (Nilaparvata lugens Stål, BPH) infestation, mechanical wounding or phytohormone treatments. A series of 5' truncated assays were conducted and three positive regulatory regions (-1452 to -1213, -903 to -624, and -376 to -176) induced by SSB infestation were identified. P2R123-min 35S and P2TR2-min 35S promoters linked with cry1C of transgenic plants showed the highest levels of Cry1C protein expression and SSB larval mortality. We identified an SSB-inducible promoter and three positive internal regions. Transgenic rice plants with the OsHPL2 promoter and its positive regions driving cry1C exhibited the expected larvicidal effect on SSB. Our study is the first report of an SSB-inducible promoter that could be used as a potential resource for breeding insect-resistant transgenic crops. © 2020 Society of Chemical Industry.

Two promoter regions confer heat-induced activation of SlDREBA4 in Solanum lycopersicum

Dehydration-responsive element binding (DREB) transcription factors activate the expression of downstream functional genes in combination with a dehydration-responsive element (DRE), and thereby improve the resistance of plants to abiotic stresses such as heat. However, the upstream regulatory mechanism of DREB genes under heat is unclear. A DREBA4 subfamily transcription factor (SlDREBA4), which is heat-responsive and improves heat resistance, was isolated from Solanum lycopersicum ‘Microtom’. In this study, promoter truncation experiments were performed to verify changes in β-glucuronidase (GUS) enzyme activity and GUS gene expression levels in transgenic plants with different lengths of promoter fragments under heat and to identify specific regions in the promoter that respond to heat. Our results showed that the GUS reporter gene was constitutively expressed in tissues of the full-length promoter transgenic ‘Microtom’ plants, with higher expression in conducting tissues of root, stem, and leaf, as well as sepals of flowers and fruits. Under heat treatment, GUS enzyme activity and GUS gene expression levels in tissues of the full-length promoter transgenic plants increased. Promoter deletion analysis identified two positive regulatory regions (−1095 to −730 bp and −162 to −38 bp) responsible for the promoter’s response to heat. These results indicated that the heat shock element (HSE) and MYC recognition sequences may cooperate in heat-induced activation of SlDREBA4 promoter.

Activity and transcriptional regulatory elements of the promoter in Arctic fox (Vulpes lagopus) β-defensin103 gene

The aim of this study was to screen the active regions and transcription factor binding sites in the promoter of the CBD103 gene related to Arctic fox coat color, and to provide a basis for revealing the molecular genetic mechanism of CBD103 gene regulating the coat color formation. The 5'-flanking region fragment 2 123 bp of Arctic fox CBD103 gene was cloned, and 4 truncated promoter reporter vectors of different lengths were constructed. The promoter activity was detected by the dual-luciferase reporter assay system. Point mutations were performed on the 3 predicted specificity protein 1 (Sp1) transcription factor binding sites in the highest promoter active region, and 3 mutant vectors were constructed. The activity was then detected by the dual-luciferase reporter assay system. The results showed that the region 1 656 (-1 604/+51) had the highest activity in the 4 truncated promoters of different lengths, and the promoter activity of the three mutant vectors constructed in this region were significantly lower than that of the wild type (fragment 1 656). The region of -1 604 /+51 was the core promoter region of CBD103 gene in Arctic fox and -1 552/-1 564, -1 439/-1 454 and -329/-339 regions were positive regulatory regions. This study successfully obtained the core promoter region and positive regulation regions of the Arctic fox CBD103 gene, which laid a foundation for further study on the molecular genetic mechanism of this gene regulating Arctic fox coat color.

Specific activation of the hb4 gene in the Xenopus oocyte through a Nobox-binding element located at the proximal promoter sequence.

We isolated and characterized Xenopus tropicalis hb4 flanking DNA and showed that the -3076/+29 sequence was able to drive stage-specific transcription in the developmental process. Transgenic reporter analysis indicated that green fluorescent protein was expressed in the ovaries of female frogs at 3 months of age and in both the ovaries and testis of frogs at 6 months of age. A series of experiments with deletion of the flanking sequence and a subsequent luciferase reporter assay revealed that there were two positive regulatory regions and that the most proximal sequence of the promoter region had a certain level of transcriptional activity in oocytes. Subsequently, we showed that a conserved sequence containing Nobox-binding element (NBE) was essential for transcriptional activation and that Nobox expressed in the ovary had a crucial role in hb4 transcription through the NBE sequence.

Transcriptional Regulation of Selenoprotein F by Heat Shock Factor 1 during Selenium Supplementation and Stress Response.

Changes of Selenoprotein F (SELENOF) protein levels have been reported during selenium supplementation, stressful, and pathological conditions. However, the mechanisms of how these external factors regulate SELENOF gene expression are largely unknown. In this study, HEK293T cells were chosen as an in vitro model. The 5′-flanking regions of SELENOF were analyzed for promoter features. Dual-Glo Luciferase assays were used to detect promoter activities. Putative binding sites of Heat Shock Factor 1 (HSF1) were predicted in silico and the associations were further proved by chromatin immunoprecipitation (ChIP) assay. Selenate and tunicamycin (Tm) treatment were used to induce SELENOF up-regulation. The fold changes in SELENOF expression and other relative proteins were analyzed by Q-PCR and western blot. Our results showed that selenate and Tm treatment up-regulated SELENOF at mRNA and protein levels. SELENOF 5′-flanking regions from −818 to −248 were identified as core positive regulatory element regions. Four putative HSF1 binding sites were predicted in regions from −1430 to −248, and six out of seven primers detected positive results in ChIP assay. HSF1 over-expression and heat shock activation increased the promoter activities, and mRNA and protein levels of SELENOF. Over-expression and knockdown of HSF1 showed transcriptional regulation effects on SELENOF during selenate and Tm treatment. In conclusion, HSF1 was discovered as one of the transcription factors that were associated with SELENOF 5′-flanking regions and mediated the up-regulation of SELENOF during selenate and Tm treatment. Our work has provided experimental data for the molecular mechanism of SELENOF gene regulation, as well as uncovered the involvement of HSF1 in selenotranscriptomic for the first time.

Functional analysis of the promoter of the dmrt1 gene in Chinese tongue sole, Cynoglossus semilaevis

The dmrt1 (doublesex and mab-3 related transcription factor 1) gene is considered to play an essential role in testis differentiation and development among metazoan species. As an economically important marine fish in China, Chinese tongue sole (Cynoglossus semilaevis) possess a ZW/ZZ sexdetermining system, and the females usually grow two to four times bigger than males. Previous studies have confirmed that Z-linked dmrt1 gene is the male determining gene in C. semilaevis and TALENs-mediated knocking-out individuals showed increased growth phenotype. In order to investigate the function of dmrt1 regulatory regions and its potential application, we have cloned the promoter of C. semilaevis dmrt1. The acquired sequence consisted of a 5'-upstream sequence and a partial exon1; functional analysis showed that there were two positive regulatory regions in the promoter, as well as one negative region and one neutral region. These positive regions were then combined to obtain several newly constructed promoters. Both in vitro and in vivo analysis confirmed one of them (dmrt1 - Δ3+Δ4) showed elevated promoting activity compared to the full-length promoter and could drive exogenous gene expression in tongue sole gonads. Our results will provide useful information for understanding the regulatory mechanism of dmrt1 gene during C. semilaevis sex determination, and the endogenous promoter will facilitate the transgenic research for analyzing sex- and growth-related genes.

Identification of the core promoter of the pmel gene of Bashang long-tail chickens

To explore the activity of the pmel core promoter of Bashang long-tail chickens, we constructed dual-luciferase expression vectors and transiently transfected into DF1 cells with Lipofectamine 2000. We measured the luciferase activity with the dual-luciferase detection kit. The 1 268 bp fragment in 5-flanking region of the pmel gene in Bashang long-tail chickens was cloned. The region from -1 200 bp to +68 bp included 2 CpG islands and multiple transcription factor binding sites. We constructed 9 expression vectors with different promoter regions and a mutant vector of the core promoter region of the pmel gene of Bashang long-tail chickens. The core promoter region from -840 bp to +68 bp was identified in the pmel gene. The region from -590 to -525 bp negatively regulated the pmel gene during the transcription process. The -840--590 bp and -525--266 bp regions were positive regulatory regions. The polymorphic sites (-456, -435, -410, -374 and -341) had a significant effect on the promoter activity of the pmel gene.

Transforming growth factor-β1 and lysophosphatidic acid activate integrin β6 gene promoter in Hep-3B cells.

Although it is difficult to detect αvβ6 integrin (αvβ6) in normal epithelia cells, its expression is upregulated during wound healing and carcinogenesis. Overexpression of αvβ6 has been demonstrated in epithelial cell carcinomas, such as adenocarcinoma of the colon and ovary. However, the expression of αvβ6 has not been reported in hepatocellular carcinoma (HCC). We previously indicated that LPA may induce αvβ6-mediated TGF-β1 signaling mechanisms during the pathogenesis of lung injury and fibrosis. In addition, transforming growth factor-β1 (TGF-β1) and lysophosphatidic acid (LPA) have been demonstrated to participate in the progression of HCC. In the present study, we hypothesized that TGF-β1 and LPA would serve a key role in the subunit integrin β6 (Itgβ6) transcriptional regulatory mechanism in HCC. It was identified that human HCC tissues and Hep-3B cells expressed Itgβ6. Treatment of Hep-3B with TGF-β1 or LPA increased the expression of Itgβ6. Furthermore, truncation experiments indicated a positive regulatory region at -326 to -157 bp of the Itgβ6 promoter. TGF-β1 and LPA increased transcriptional activation at this regulatory region. To the best of our knowledge, the present study was the first to demonstrate Itgβ6 expression in HCC, and the data indicate that TGF-β1 and LPA regulate Itgβ6 expression through the Itgβ6 gene promoter, which is an important factor in the development of HCC.

Mitochondrial Ferritin Is a Hypoxia-Inducible Factor 1α-Inducible Gene That Protects from Hypoxia-Induced Cell Death in Brain

Aims: Mitochondrial ferritin (protein [FtMt]) is preferentially expressed in cell types of high metabolic activity and oxygen consumption, which is consistent with its role of sequestering iron and preventing oxygen-derived redox damage. As of yet, the mechanisms of FtMt regulation and the protection FtMt affords remain largely unknown. Results: Here, we report that hypoxia-inducible factor 1α (HIF-1α) can upregulate FtMt expression. We verify one functional hypoxia-response element (HRE) in the positive regulatory region and two HREs possessing HIF-1α binding activity in the minimal promoter region of the human FTMT gene. We also demonstrate that FtMt can alleviate hypoxia-induced brain cell death by sequestering uncommitted iron, whose levels increase with hypoxia in these cells. Innovation: In the absence of FtMt, this catalytic metal excess catalyzes the production of cytotoxic reactive oxygen species. Conclusion: Thus, the cell ability to increase expression of FtMt during hypoxia may be a skill to avoid tissue damage derived from oxygen limitation.

Cloning, expression profiling and promoter functional analysis of bone morphogenetic protein 2 in the tongue sole (Cynoglossus semilaevis)

Bone morphogenetic proteins (BMPs) play crucial roles in vertebrate developmental process and are associated with the mechanisms which drive early skeletal development. As a first approach to elucidating the role of BMPs in regulating fish bone formation and growth, we describe the cloning, expression profiling and promoter functional analysis of bmp6 and bmp7 in tongue sole (Cynoglossus semilaevis). The full length of bmp6 and bmp7 cDNA sequences is 1939 and 1836 bp, which encodes a protein of 428 and 427 amino acids, respectively. Tissue expression distribution of bmp6 and bmp7 was examined in 14 tissues of mature individuals by quantitative real-time PCR (qRT-PCR). The results revealed that bmp6 was predominantly expressed in the gonad, and bmp7 exhibited the highest expression level in the dorsal fin. Further comparison of bmp6 expression levels between female and male gonads showed that the expression in the ovary was significantly higher than in the testis. Moreover, bmp6 and bmp7 expression levels were detected at 15 sampling time points of early developmental stages (egg, larva, juvenile and fingerling stages). The highest expression level of bmp6 was observed in the egg stage (multi-cell and gastrula stage); while bmp7 exhibited the highest expression in the larva stage (1–4 days old). The high expression levels of BMP6 in the ovary as well as at early embryonic stages indicated that the maternally stored transcripts of bmp6 might play a role in early embryonic development. Whole-mount in situ hybridization showed that bmp6 and bmp7 exhibited similar spatial expression patterns. Both bmp6 and bmp7 signals were first detected in the head and anterior regions in newly hatched larvae, and then, the mRNAs appeared in the crown-like larval fin, jaw, operculum and fins (pectoral, dorsal, pelvic and anal) along with early development. Subsequently, we characterized the 5′-flanking regions of bmp6 and bmp7 by testing the promoter activity by luciferase reporter assays. Positive regulatory regions were, respectively, detected at the location of −272 to +28 and −740 to −396 in bmp6 and bmp7 gene. The predicted transcription factor binding sites (CREB, AP1 and methyl-CpG-binding protein) in the regions might participate in the transcriptional regulation of these two genes.

Promoter Polymorphism rs886205 Genotype Interacts With DNA Methylation of the ALDH2 Regulatory Region in Alcohol Dependence.

Aldehyde dehydrogenase 2 (ALDH2) protects cells from ethanol toxicity by metabolizing acetaldehyde. We studied the single nucleotide polymorphism (SNP) rs886205s located between a negative and a positive regulating promoter element in the ALDH2 gene. The negative regulatory region was already associated with differential DNA methylation in the two allele variations of rs886205 SNP. Another CpG island, in the positive regulatory region of the ALDH2 promoter, extends through the SNP rs886205 and a nuclear receptor response element. We assessed rs886305 genotype and DNA methylation using bisulfite sequencing in a cohort of 83 male alcohol-dependent patients undergoing detoxification treatment (Days 1, 7 and 14) and in 33 male age-matched controls. Luciferase reporter assays were performed to address the functional significance of genotype and methylation. We observed a higher methylation in alcohol-dependent patients compared to controls. Patients with AA (n = 52) or GG/GA (n = 31) genotype differed significantly in baseline methylation levels as well as in methylation kinetics during withdrawal. AA carriers display an increase in methylation from low baseline levels while GG/GA showed the inverse pattern. The reporter gene assays corroborate these data by showing a significant effect of genotype on ALDH2 expression as well as an interaction between genotype and methylation. Our results describe a new regulatory role of rs886205 in the methylation of ALDH2 promoter region and provide additional insight into the complex regulation of ALDH2 under the condition of alcohol dependence. Genetic variations have been described to influence DNA promoter methylation of various genes. We investigated the association between the polymorphism rs886205, located on ALDH2 promoter and methylation kinetics of the neighboring CpG island in alcohol-dependent patients. Luciferase reporter assays showed functional significance of genotype, methylation and a genotype-epigenotype interaction in vitro.

Cloning, expression profiling and promoter functional analysis of Bone morphogenetic protein 6 and 7 in tongue sole (Cynoglossus semilaevis).

Bone morphogenetic proteins (BMPs) play crucial roles in vertebrate developmental process and are associated with the mechanisms which drive early skeletal development. As a first approach to elucidating the role of BMPs in regulating fish bone formation and growth, we describe the cloning, expression profiling and promoter functional analysis of bmp6 and bmp7 in tongue sole (Cynoglossus semilaevis). The full length of bmp6 and bmp7 cDNA sequences is 1939 and 1836bp, which encodes a protein of 428 and 427 amino acids, respectively. Tissue expression distribution of bmp6 and bmp7 was examined in 14 tissues of mature individuals by quantitative real-time PCR (qRT-PCR). The results revealed that bmp6 was predominantly expressed in the gonad, and bmp7 exhibited the highest expression level in the dorsal fin. Further comparison of bmp6 expression levels between female and male gonads showed that the expression in the ovary was significantly higher than in the testis. Moreover, bmp6 and bmp7 expression levels were detected at 15 sampling time points of early developmental stages (egg, larva, juvenile and fingerling stages). The highest expression level of bmp6 was observed in the egg stage (multi-cell and gastrula stage); while bmp7 exhibited the highest expression in the larva stage (1-4days old). The high expression levels of BMP6 in the ovary as well as at early embryonic stages indicated that the maternally stored transcripts of bmp6 might play a role in early embryonic development. Whole-mount in situ hybridization showed that bmp6 and bmp7 exhibited similar spatial expression patterns. Both bmp6 and bmp7 signals were first detected in the head and anterior regions in newly hatched larvae, and then, the mRNAs appeared in the crown-like larval fin, jaw, operculum and fins (pectoral, dorsal, pelvic and anal) along with early development. Subsequently, we characterized the 5'-flanking regions of bmp6 and bmp7 by testing the promoter activity by luciferase reporter assays. Positive regulatory regions were, respectively, detected at the location of -272 to +28 and -740 to -396 in bmp6 and bmp7 gene. The predicted transcription factor binding sites (CREB, AP1 and methyl-CpG-binding protein) in the regions might participate in the transcriptional regulation of these two genes.

Cross-talk between freezing response and signaling for regulatory transcriptions of MIR475b and its targets by miR475b promoter in Populus suaveolens.

MicroRNAs (miRNAs) are small, non-coding RNAs that play important roles in post-transcriptional regulation of their target genes, yet the transcriptional regulation of plant miRNAs by promoter is poorly understood. Here, we firstly clone pri-miR475b cDNA and its native promoter from P. suaveolens, and characterize Psu-MIR475b as class-II gene transcribed by RNA polymerase II. By 5′ deletion analysis of Psu-miR475b promoter in a series of promoter-GUS chimeric vectors, we functionally identify three positive regulatory regions and multiple cis-acting elements responsible for Psu-miR475b promoter activity in response to freezing stress and exogenous hormone treatment. Moreover, the Psu-miR475b promoter activity displays a tissue-specific manner, negatively regulated by freezing stress and positively by MeJA, SA or GA treatment. Importantly, we comparatively analyze the time-course transcriptional profiles of Psu-miR475b and its targets in Psu-miR475b over-expression transgenic plants controlled by Psu-miR475b-specific promoter or CaMV 35S constitutive promoter, and explore the regulatory mechanism of Psu-miR475b promoter controlling transcriptional expressions of Psu-MIR475b and its targets in response to freezing stress and exogenous hormone treatment. Our results reveal that Psu-miR475b promoter-mediated transcriptions of Psu-MIR475b and its targets in response to freezing stress may be involved in a cross-talk between freezing response and stress signaling process.

Regulation of Smad ubiquitination regulatory factor 1 expression by NF-κB

To identify a nuclear factor κB (NF-κB) responsive element within the Smad ubiquitination regulatory factor 1 (SMURF1) gene promoter, and to demonstrate its role in the regulation of SMURF1 expression. A series of truncated luciferase reporter plasmids of the SMURF1 promoter were constructed and transfected into hepatic cancer Hep G2 cells. Luciferase assays were carried out to assess the activities of such promoters. DNA binding and chromatin immunoprecipitation (ChIP) assays were used to identify an NF-κB responsive element within the SMURF1 promoter. Lucifease plasmid with mutated NF-κB site was constructed and its activity was assessed. The expression of SMURF1 in Hep G2 cells was detected after transfection of NF-κB specific small interfering RNA (siRNA). The SMURF1 promoter showed a high transcription activity, and the region of -519 to -378 was demonstrated to be a positive regulatory region. -411 to -420 of the SMURF1 promoter was an NF-κB responsive element, and NF-κB may specifically bind to this site. Mutation of this element may prominently decrease the activity of the promoter. Transfection of NF-κB siRNA evidently down-regulated SMURF1 expression. NF-κB can specifically bind to the -411 to -420 region of the SMURF1 promoter and plays an essential role in the expression of SMURF1.