Maximal Transcription Research Articles

Promoter selectivity of the RhlR quorum-sensing transcription factor receptor in Pseudomonas aeruginosa is coordinated by distinct and overlapping dependencies on C4-homoserine lactone and PqsE.

Quorum sensing is a mechanism of bacterial cell-cell communication that relies on the production and detection of small molecule autoinducers, which facilitate the synchronous expression of genes involved in group behaviors, such as virulence factor production and biofilm formation. The Pseudomonas aeruginosa quorum sensing network consists of multiple interconnected transcriptional regulators, with the transcription factor, RhlR, acting as one of the main drivers of quorum sensing behaviors. RhlR is a LuxR-type transcription factor that regulates its target genes when bound to its cognate autoinducer, C4-homoserine lactone, which is synthesized by RhlI. RhlR function is also regulated by the metallo-β-hydrolase enzyme, PqsE. We recently showed that PqsE binds RhlR to alter its affinity for promoter DNA, a new mechanism of quorum-sensing receptor activation. Here, we perform ChIP-seq analyses of RhlR to map the binding of RhlR across the P. aeruginosa genome, and to determine the impact of C4-homoserine lactone and PqsE on RhlR binding to different sites across the P. aeruginosa genome. We identify 40 RhlR binding sites, all but three of which are associated with genes known to be regulated by RhlR. C4-homoserine lactone is required for maximal binding of RhlR to many of its DNA sites. Moreover, C4-homoserine lactone is required for maximal RhlR-dependent transcription activation from all sites, regardless of whether it impacts RhlR binding to DNA. PqsE is required for maximal binding of RhlR to many DNA sites, with similar effects on RhlR-dependent transcription activation from those sites. However, the effects of PqsE on RhlR specificity are distinct from those of C4-homoserine lactone, and PqsE is sufficient for RhlR binding to some DNA sites in the absence of C4-homoserine lactone. Together, C4-homoserine lactone and PqsE are required for RhlR binding at the large majority of its DNA sites. Thus, our work reveals three distinct modes of activation by RhlR: i) when RhlR is unbound by autoinducer but bound by PqsE, ii) when RhlR is bound by autoinducer but not bound by PqsE, and iii) when RhlR is bound by both autoinducer and PqsE, establishing a stepwise mechanism for the progression of the RhlR-RhlI-PqsE quorum sensing pathway in P. aeruginosa.

Optimization of adeno-associated virus (AAV) gene delivery into human bone marrow stem cells (hBMSCs).

Efficiently delivering nucleic acid into mammalian cells is essential to overexpress genes for assessing gene functions. Human bone marrow stem cells (hBMSCs) are the most studied tissue-derived stem cells. Adeno-associated viruses (AAVs) have been used to deliver DNA into hBMSCs for various purposes. Current literature reported that transduction efficiencies of up to 65% could be achieved by AAV gene delivery into hBMSCs. Further improvement of efficiency is needed and possible. This study tested a selection of AAV serotypes for high-efficient DNA delivery into hBMSCs. hBMSCs from different donors were infected with different serotypes of AAVs containing the enhanced green fluorescence protein (eGFP) reporter gene driven by the CMV promoter. Green fluorescence was monitored in the infected cells at five-day intervals. Cells were collected at designated time points after the infection for reverse-transcription polymerase chain reaction (RT-PCR) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) to assess eGFP mRNA transcription. The results indicated that the order of transduction efficiency of the AAV serotypes was AAV2 > AAV2.7m8 > AAV6 > AAV6.2 > AAV1 > AAV-DJ. AAV2 could achieve almost 100% transduction at the multiplicity of infection (MOI) greater than 100K. Over 90% of cells could be transduced at 20K to 50K MOI. About 80% transduction was seen at MOIs of 10K and 15K. RT-PCR analysis showed that eGFP mRNA could be detected from day 5 to day 30 post-AAV infection. The differences in the observed transduction efficiencies of the hBMSCs from different patients indicate donor-to-donor variability, and increased eGFP mRNA was generally seen after day 15 post-AAV2 infection. Maximal eGFP transcription was detected on day 30 post-infection. We conclude that AAV2 and AAV2.7m8 at an MOI of 100K or greater can efficiently deliver transgene into hBMSCs with up to near 100% transduction efficiency for sustained expression over one month. However, donor-to-donor variation exists in transduction efficiency and transgene expression, especially at MOIs less than 100K.

Genome-Wide Identification and Expression Analysis of the Aquaporin Gene Family in Lycium barbarum during Fruit Ripening and Seedling Response to Heat Stress.

Plant-water relations mediated by aquaporins (AQPs) play vital roles in both key plant growth processes and responses to environmental challenges. As a well-known medicinal and edible plant, the harsh natural growth habitat endows Lycium plants with ideal materials for stress biology research. However, the details of their molecular switch for water transport remain unclear. In the present work, we first identified and characterized AQP family genes from Lycium (L.) barbarum at the genome scale and conducted systemic bioinformatics and expression analyses. The results showed that there were 38 Lycium barbarum AQPs (LbAQPs) in L. barbarum, which were classified into four subfamilies, including 17 LbPIP, 9 LbTIP, 10 LbNIP, and 2 LbXIP. Their encoded genes were unevenly distributed on all 12 chromosomes, except chromosome 10. Three of these genes encoded truncated proteins and three genes underwent clear gene duplication events. Cis-acting element analysis indicated that the expression of LbAQPs may be mainly regulated by biotic/abiotic stress, phytohormones and light. The qRT-PCR assay indicated that this family of genes presented a clear tissue-specific expression pattern, in which most of the genes had maximal transcript levels in roots, stems, and leaves, while there were relatively lower levels in flowers and fruits. Most of the LbAQP genes were downregulated during L. barbarum fruit ripening and presented a negative correlation with the fruit relative water content (RWC). Most of their transcripts presented a quick and sharp upregulation response to heat stress following exposure of the 2-month-old seedlings to a 42 °C temperature for 0, 1, 3, 12, or 24 h. Our results proposed that LbAQPs were involved in L. barbarum key development events and abiotic stress responses, which may lay a foundation for further studying the molecular mechanism of the water relationship of Lycium plants, especially in harsh environments.

Anemia-Activated Cis Regulatory Requirements for Erythroid Regeneration

Acute anemia elicits broad transcriptional changes in erythroid progenitors and precursors. Whether anemia-activated transcription and underlying usage of cis-regulatory elements to control gene expression are unique to anemia or overlap with physiological erythropoiesis is an unresolved question regarding anemia response mechanisms. We previously discovered that a cis-regulatory transcriptional enhancer at the Samd14 locus (S14E), defined by a CANNTG-spacer-AGATAA (named E-box-GATA) composite motif and occupied by GATA1 and TAL1 transcription factors, is required for survival in severe anemia. Our working model proposes that the S14E is part of an ensemble of similar anemia-responsive cis-elements promoting erythroid precursor activities. To determine whether loci containing a cohort of S14E-like cis-elements are transcriptionally upregulated in anemia, we conducted single cell RNA-sequencing of Kit+ cells isolated from mouse spleen during the recovery phase of acute anemia. Single-cell regulatory network inference and clustering (SCENIC) analysis - used to predict transcription factors involved in regulatory mechanisms - revealed that anemia-activated transcripts co-expressed with Gata1 and Tal1 were localized in specific populations of erythroid cells. We identified 65 anemia-activated genes associated with GATA1/TAL1-occupied S14E-like cis-elements including Samd14, suggesting common transcriptional mechanisms are involved in restoring homeostasis. Using Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE), we found the chromatin accessibility of S14E-like cis-elements associated with synovial sarcoma X-2 interacting protein (Ssx2ip) and tubulin tyrosine ligase-like12 (Ttll12) was significantly increased in anemia. To test S14E-like function in transcriptional activation, we targeted these sequences using sgRNA-guided Cas9 and dCas9-KRAB in mouse and human erythroid cell lines (G1E, K562 and HUDEP-2). Both SSX2IP and TTLL12 S14E-like enhancers were required for maximal gene transcription. These data demonstrate that a cohort of S14E-like cis-elements promote anemia-activated gene expression. Ssx2ip is a poorly studied gene that is upregulated by anemia in erythroid precursors (19.2-fold, p<0.0001). To connect cis-regulatory activation in anemia with erythroid regeneration, we next tested Ssx2ip function in erythroid precursor cells isolated from a mouse model of acute anemia. shRNA mediated knockdown of Ssx2ip decreased the percentage of early stage (CD71lowTer119-) erythroid precursors 4.4-fold (p=0.001). Ssx2ip knockdown decreased burst-forming unit-erythroid (BFU-E) and colony forming unit-erythroid (CFU-E) colony forming potential of erythroid precursors by 1.3-fold (p=0.008) and 1.6-fold (p<0.0001), respectively. To investigate a possible role for Ssx2ip in cell proliferation, we conducted BrdU incorporation and cell proliferation assays. These experiments revealed that Ssx2ip is an important determinant of erythroid progenitor cell cycle progression, and proliferation in anemia. Together, we provide rigorous evidence that S14E-like cis-elements associate with anemia-activated genes, activate gene expression, and that the anemia-activated Ssx2ip gene plays important roles in erythroid precursor cells. Our results highlight a common mechanism of gene activation that regulates erythroid regeneration via S14E-like enhancers. Ongoing efforts will establish mechanisms of Ssx2ip activities in erythropoiesis and functional requirements of additional cohorts of S14E-like enhancers involved in gene regulation during anemia recovery. These findings provide a framework to understand anemia-specific transcriptional mechanisms, ineffective erythropoiesis, and anemia recovery. Given the role of Ssx2ip in erythroid regeneration, it is worth considering the pathological implications of this mechanism in acute/chronic anemia and leukemia.

Protective Responses at the Biochemical and Molecular Level Differ between a Coffea arabica L. Hybrid and Its Parental Genotypes to Supra-Optimal Temperatures and Elevated Air [CO2

Climate changes with global warming associated with rising atmospheric [CO2] can strongly impact crop performance, including coffee, which is one of the most world’s traded agricultural commodities. Therefore, it is of utmost importance to understand the mechanisms of heat tolerance and the potential role of elevated air CO2 (eCO2) in the coffee plant response, particularly regarding the antioxidant and other protective mechanisms, which are crucial for coffee plant acclimation. For that, plants of Coffea arabica cv. Geisha 3, cv. Marsellesa and their hybrid (Geisha 3 × Marsellesa) were grown for 2 years at 25/20 °C (day/night), under 400 (ambient CO2, aCO2) or 700 µL (elevated CO2, eCO2) CO2 L−1, and then gradually submitted to a temperature increase up to 42/30 °C, followed by recovery periods of 4 (Rec4) and 14 days (Rec14). Heat (37/28 °C and/or 42/30 °C) was the major driver of the response of the studied protective molecules and associated genes in all genotypes. That was the case for carotenoids (mostly neoxanthin and lutein), but the maximal (α + β) carotenes pool was found at 37/28 °C only in Marsellesa. All genes (except VDE) encoding for antioxidative enzymes (catalase, CAT; superoxide dismutases, CuSODs; ascorbate peroxidases, APX) or other protective proteins (HSP70, ELIP, Chape20, Chape60) were strongly up-regulated at 37/28 °C, and, especially, at 42/30 °C, in all genotypes, but with maximal transcription in Hybrid plants. Accordingly, heat greatly stimulated the activity of APX and CAT (all genotypes) and glutathione reductase (Geisha3, Hybrid) but not of SOD. Notably, CAT activity increased even at 42/30 °C, concomitantly with a strongly declined APX activity. Therefore, increased thermotolerance might arise through the reinforcement of some ROS-scavenging enzymes and other protective molecules (HSP70, ELIP, Chape20, Chape60). Plants showed low responsiveness to single eCO2 under unstressed conditions, while heat promoted changes in aCO2 plants. Only eCO2 Marsellesa plants showed greater contents of lutein, the pool of the xanthophyll cycle components (V + A + Z), and β-carotene, compared to aCO2 plants at 42/30 °C. This, together with a lower CAT activity, suggests a lower presence of H2O2, likely also associated with the higher photochemical use of energy under eCO2. An incomplete heat stress recovery seemed evident, especially in aCO2 plants, as judged by the maintenance of the greater expression of all genes in all genotypes and increased levels of zeaxanthin (Marsellesa and Hybrid) relative to their initial controls. Altogether, heat was the main response driver of the addressed protective molecules and genes, whereas eCO2 usually attenuated the heat response and promoted a better recovery. Hybrid plants showed stronger gene expression responses, especially at the highest temperature, when compared to their parental genotypes, but altogether, Marsellesa showed a greater acclimation potential. The reinforcement of antioxidative and other protective molecules are, therefore, useful biomarkers to be included in breeding and selection programs to obtain coffee genotypes to thrive under global warming conditions, thus contributing to improved crop sustainability.

The ABCISIC ACID INSENSITIVE (ABI) 4 Transcription Factor Is Stabilized by Stress, ABA and Phosphorylation.

The Arabidopsis transcription factor ABSCISIC ACID INSENSITIVE 4 (ABI4) is a key player in the plant hormone abscisic acid (ABA) signaling pathway and is involved in plant response to abiotic stress and development. Expression of the ABI4 gene is tightly regulated, with low basal expression. Maximal transcript levels occur during the seed maturation and early seed germination stages. Moreover, ABI4 is an unstable, lowly expressed protein. Here, we studied factors affecting the stability of the ABI4 protein using transgenic Arabidopsis plants expressing 35S::HA-FLAG-ABI4-eGFP. Despite the expression of eGFP-tagged ABI4 being driven by the highly active 35S CaMV promoter, low steady-state levels of ABI4 were detected in the roots of seedlings grown under optimal conditions. These levels were markedly enhanced upon exposure of the seedlings to abiotic stress and ABA. ABI4 is degraded rapidly by the 26S proteasome, and we report on the role of phosphorylation of ABI4-serine 114 in regulating ABI4 stability. Our results indicate that ABI4 is tightly regulated both post-transcriptionally and post-translationally. Moreover, abiotic factors and plant hormones have similar effects on ABI4 transcripts and ABI4 protein levels. This double-check mechanism for controlling ABI4 reflects its central role in plant development and cellular metabolism.

Effect of the Carotenoid Content and Activity of the Carotene cis-trans Isomerase CRTISO on Tomato Fruit Color

The tomato Solanum lycopersicum L. is a convenient model for studying carotenoid metabolism due to the wide variety of carotenoid-mediated pigmentation of the ripe fruit. Carotene cis-trans isomerase CRTISO catalyzes cis-trans isomerization of double bonds leading to the formation of all-trans-lycopene, which is the most powerful antioxidant among carotenoids and a substrate for subsequent synthesis of α-, β-carotenes, and xanthophylls. In this work, 18 accessions of tomato cultivars and lines differing in the color of ripe fruit have been analyzed. The obtained biochemical data showed a dependence of fruit color on the content and composition of carotenoids and the presence or absence of chlorophylls. Expression analysis of three CRTISO homologous genes performed in silico has shown that the highest expression level in the fruit is only characteristic of gene CRTISO that has the maximal transcription at the stages of color change (from green to red) and biological ripeness of the fruit. Quantitative real-time PCR did not reveal any strong correlation between the level of CRTISO gene expression and total carotenoids, which may be explained by a different quantity of metabolites preceding prolycopene.

Enhanced replication fitness of MERS-CoV clade B over clade A strains in camelids explains the dominance of clade B strains in the Arabian Peninsula

ABSTRACT Middle East respiratory syndrome coronavirus (MERS-CoV) continues infecting humans and dromedary camels. While MERS-CoV strains from the Middle East region are subdivided into two clades (A and B), all the contemporary epidemic viruses belong to clade B. Thus, MERS-CoV clade B strains may display adaptive advantages over clade A in humans and/or reservoir hosts. To test this hypothesis in vivo, we compared an early epidemic clade A strain (EMC/2012) with a clade B strain (Jordan-1/2015) in an alpaca model monitoring virological and immunological parameters. Further, the Jordan-1/2015 strain has a partial amino acid (aa) deletion in the double-stranded (ds) RNA binding motif of the open reading frame ORF4a protein. Animals inoculated with the Jordan-1/2015 variant had higher MERS-CoV replicative capabilities in the respiratory tract and larger nasal viral shedding. In the nasal mucosa, the Jordan-1/2015 strain caused an early IFN response, suggesting a role for ORF4a as a moderate IFN antagonist in vivo. However, both strains elicited maximal transcription of antiviral interferon-stimulated genes (ISGs) at the peak of infection on 2 days post inoculation, correlating with subsequent decreases in tissular viral loads. Genome alignment analysis revealed several clade B-specific amino acid substitutions occurring in the replicase and the S proteins, which could explain a better adaptation of clade B strains in camelid hosts. Differences in replication and shedding reported herein indicate a better fitness and transmission capability of MERS-CoV clade B strains than their clade A counterparts.

An optimal growth law for RNA composition and its partial implementation through ribosomal and tRNA gene locations in bacterial genomes.

The distribution of cellular resources across bacterial proteins has been quantified through phenomenological growth laws. Here, we describe a complementary bacterial growth law for RNA composition, emerging from optimal cellular resource allocation into ribosomes and ternary complexes. The predicted decline of the tRNA/rRNA ratio with growth rate agrees quantitatively with experimental data. Its regulation appears to be implemented in part through chromosomal localization, as rRNA genes are typically closer to the origin of replication than tRNA genes and thus have increasingly higher gene dosage at faster growth. At the highest growth rates in E. coli, the tRNA/rRNA gene dosage ratio based on chromosomal positions is almost identical to the observed and theoretically optimal tRNA/rRNA expression ratio, indicating that the chromosomal arrangement has evolved to favor maximal transcription of both types of genes at this condition.

Fusarium verticillioides and Aspergillus flavus Co-Occurrence Influences Plant and Fungal Transcriptional Profiles in Maize Kernels and In Vitro.

Climate change will increase the co-occurrence of Fusarium verticillioides and Aspergillus flavus, along with their mycotoxins, in European maize. In this study, the expression profiles of two pathogenesis-related (PR) genes and four mycotoxin biosynthetic genes, FUM1 and FUM13, fumonisin pathway, and aflR and aflD, aflatoxin pathway, as well as mycotoxin production, were examined in kernels and in artificial medium after a single inoculation with F. verticillioides or A. flavus or with the two fungi in combination. Different temperature regimes (20, 25 and 30 °C) over a time-course of 21 days were also considered. In maize kernels, PR genes showed the strongest induction at 25 °C in the earlier days post inoculation (dpi)with both fungi inoculated singularly. A similar behaviour was maintained with fungi co-occurrence, but with enhanced defence response at 9 dpi under 20 °C. Regarding FUM genes, in the kernels inoculated with F. verticillioides the maximal transcript levels occurred at 6 dpi at 25 °C. At this temperature regime, expression values decreased with the co-occurrence of A. flavus, where the highest gene induction was detected at 20 °C. Similar results were observed in fungi grown in vitro, whilst A. flavus presence determined lower levels of expression along the entire time-course. As concerns afl genes, considering both A. flavus alone and in combination, the most elevated transcript accumulation occurred at 30 °C during all time-course both in infected kernels and in fungi grown in vitro. Regarding mycotoxin production, no significant differences were found among temperatures for kernel contamination, whereas in vitro the highest production was registered at 25 °C for aflatoxin B1 and at 20 °C for fumonisins in the case of single inoculation. In fungal co-occurrence, both mycotoxins resulted reduced at all the temperatures considered compared to the amount produced with single inoculation.

A Hill type equation can predict target gene expression driven by p53 pulsing.

Many factors determine target gene expression dynamics under p53 pulsing. In this study, I sought to determine the mechanism by which duration, frequency, binding affinity and maximal transcription rate affect the expression dynamics of target genes. Using an analytical method to solve a simple model, I found that the fold change of target gene expression increases relative to the number of p53 pulses, and the optimal frequency, 0.18 h−1, from two real p53 pulses drives the maximal fold change with a decay rate of 0.18 h−1. Moreover, p53 pulses may also lead to a higher fold change than sustained p53. Finally, I discovered that a Hill‐type equation, including these effect factors, can characterise target gene expression. The average error between the theoretical predictions and experiments was 23%. Collectively, this equation advances the understanding of transcription factor dynamics, where duration and frequency play a significant role in the fine regulation of target gene expression with higher binding affinity.

Gene Transcription as a Limiting Factor in Protein Production and Cell Growth.

Cell growth is driven by the synthesis of proteins, genes, and other cellular components. Defining processes that limit biosynthesis rates is fundamental for understanding the determinants of cell physiology. Here, we analyze the consequences of engineering cells to express extremely high levels of mCherry proteins, as a tool to define limiting processes that fail to adapt upon increasing biosynthetic demands. Protein-burdened cells were transcriptionally and phenotypically similar to mutants of the Mediator, a transcription coactivator complex. However, our binding data suggest that the Mediator was not depleted from endogenous promoters. Burdened cells showed an overall increase in the abundance of the majority of endogenous transcripts, except for highly expressed genes. Our results, supported by mathematical modeling, suggest that wild-type cells transcribe highly expressed genes at the maximal possible rate, as defined by the transcription machinery’s physical properties. We discuss the possible cellular benefit of maximal transcription rates to allow a coordinated optimization of cell size and cell growth.

Expression of a hypomorphic Pomc allele alters leptin dynamics during late pregnancy.

Proopiomelanocortin (POMC) neurons in the hypothalamic arcuate nucleus (ARC) are essential for normal energy homeostasis. Maximal ARC Pomc transcription is dependent on neuronal Pomc enhancer 1 (nPE1), located 12 kb upstream from the promoter. Selective deletion of nPE1 in mice decreases ARC Pomc expression by 70%, sufficient to induce mild obesity. Because nPE1 is located exclusively in the genomes of placental mammals, we questioned whether its hypomorphic mutation would also alter placental Pomc expression and the metabolic adaptations associated with pregnancy and lactation. We assessed placental development, pup growth, circulating leptin and expression of Pomc, Agrp and alternatively spliced leptin receptor (LepR) isoforms in the ARC and placenta of Pomc∆1/∆1 and Pomc+/+ dams. Despite indistinguishable body weights, lean mass, food intake, placental histology and Pomc expression and overall pregnancy outcomes between the genotypes, Pomc ∆1/∆1 females had increased pre-pregnancy fat mass that paradoxically decreased to control levels by parturition. However, Pomc∆1/∆1 dams had exaggerated increases in circulating leptin, up to twice of that of the typically elevated levels in Pomc+/+ mice at the end of pregnancy, despite their equivalent fat mass. Pomc∆1/∆1dams also had increased placental expression of soluble leptin receptor (LepRe), although the protein levels of LEPRE in circulation were the same as Pomc+/+ controls. Together, these data suggest that the hypomorphic Pomc∆1/∆1 allele is responsible for the perinatal super hyperleptinemia of Pomc∆1/∆1 dams, possibly due to upregulated leptin secretion from individual adipocytes.

The HIV-Tat protein interacts with Sp3 transcription factor and inhibits its binding to a distal site of the sod2 promoter in human pulmonary artery endothelial cells

Redox imbalance results in damage to cellular macromolecules and interferes with signaling pathways, leading to an inflammatory cellular and tissue environment. As such, the cellular oxidative environment is tightly regulated by several redox-modulating pathways. Many viruses have evolved intricate mechanisms to manipulate these pathways for their benefit, including HIV-1, which requires a pro-oxidant cellular environment for optimal replication. One such virulence factor responsible for modulating the redox environment is the HIV Transactivator of transcription (Tat). Tat is of particular interest as it is actively secreted by infected cells and internalized by uninfected bystander cells where it can elicit pro-oxidant effects resulting in inflammation and damage. Previously, we demonstrated that Tat regulates basal expression of Superoxide Dismutase 2 (sod2) by altering the binding of the Sp-transcription factors at regions relatively near (approx. −210 nucleotides) upstream of the transcriptional start site. Now, using in silico analysis and a series of sod2 promoter reporter constructs, we have identified putative clusters of Sp-binding sites located further upstream of the proximal sod2 promoter, between nucleotides −3400 to −210, and tested their effect on basal transcription and for their sensitivity to HIV-1 Tat. In this report, we demonstrate that under basal conditions, maximal transcription requires a cluster of Sp-binding sites in the −584 nucleotide region, which is extremely sensitive to Tat. Using chromatin immunoprecipitation (ChIP) we demonstrate that Tat results in altered occupancy of Sp1 and Sp3 at this distal Tat-sensitive regulatory element and strongly stimulated endogenous expression of SOD2 in human pulmonary artery endothelial cells (HPAEC). We also report altered expression of Sp1 and Sp3 in Tat-expressing HPAEC as well as in the lungs of HIV-1 infected humanized mice. Lastly, Tat co-immunoprecipitated with endogenous Sp3 but not Sp1 and did not alter the acetylation state of Sp3. Thus, here, we have defined a novel and important cis-acting factor in HIV-1 Tat-mediated regulation of SOD2, demonstrated that modulation of Sp1 and Sp3 activity by Tat promotes SOD2 expression in primary human pulmonary artery endothelial cells and determined that pulmonary levels of Sp3 as well as SOD2 are increased in the lungs of a mouse model of HIV infection.

Non-canonical transcriptional regulation of heme oxygenase in Aedes aegypti

Heme oxygenase (HO) is a ubiquitous enzyme responsible for heme breakdown, which yields carbon monoxide (CO), biliverdin (BV) and ferrous ion. Here we show that the Aedes aegypti heme oxygenase gene (AeHO – AAEL008136) is expressed in different developmental stages and tissues. AeHO expression increases after a blood meal in the midgut, and its maximal transcription levels overlaps with the maximal rate of the further modified A. aegypti biglutaminyl-biliverdin (AeBV) pigment production. HO is a classical component of stress response in eukaryotic cells, being activated under oxidative stress or increased heme levels. Indeed, the final product of HO activity in the mosquito midgut, AeBV, exerts a protective antioxidant activity. AeHO, however, does not seem to be under a classical redox-sensitive transcriptional regulation, being unresponsive to heme itself, and even down regulated when insects face a pro-oxidant insult. In contrast, AeHO gene expression responds to nutrient sensing mechanisms, through the target of rapamycin (TOR) pathway. This unusual transcriptional control of AeHO, together with the antioxidant properties of AeBV, suggests that heme degradation by HO, in addition to its important role in protection of Aedes aegypti against heme exposure, also acts as a digestive feature, being an essential adaptation to blood feeding.

3H-Imidazo[4,5-b]pyridine-6-carboxylic acid derivatives as rexinoids with reduced teratogenicity.

Several retinoid X receptor (RXR) ligands (rexinoids), such as bexarotene (1), exhibit teratogenicity, which is a serious impediment to their clinical application. We considered that rexinoids with a lower level of maximal RXR transcription activation (i.e., partial agonists) and lower lipid solubility might show weaker adverse side effects. Based on this idea, we modified our previously reported pentamethyltetralin-type RXR partial agonists 5 and 6 to reduce their lipophilicity. Here, we report a new RXR partial agonist, 3-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)-2-(trifluoromethyl)-3H-imidazo[4,5-b]pyridine-6-carboxylic acid (8, CATF-PMN), which showed greatly reduced teratogenicity in zebrafish embryos.

Entrainment of a cellular circadian oscillator by light in the presence of molecular noise.

In this paper, we consider a stochastic molecular circadian oscillator described by a sequence of biological reactions and its deterministic kinetics governed by a system of ordinary differential equations in the limit of large numbers of molecules. The oscillations in the model are generated by negative feedback regulation of a gene. The focus of this paper is the entrainment of the oscillator by a periodic light signal that affects the maximal transcription rate of the gene. We introduce two scalings of the model parameters that provide independent control over the natural frequency of the oscillator and the relative noise level. We study entrainment in two ways: by visualizing the stochastic limit cycle in various projections of the discrete phase space of the system and by evaluating the maximum of the normalized cross correlation of the light signal with the number of protein molecules in the cell. The visualization method ignores the phase of the oscillator, and we find in this way that entrainment has a subtle organizing effect on the limit cycle as a whole. The cross correlation results reveal an interval of natural frequencies of the oscillator surrounding the frequency of the light signal within which maximal entrainment occurs with rather sharp drops in entrainment at the edges of this interval. The width of the interval of maximal entrainment increases with the amplitude of the light signal. These statements are applicable both to the stochastic oscillator and to its deterministic limit, but the results are most clear-cut in the deterministic case and degrade from there as the relative noise level increases.

SWI/SNF and RSC cooperate to reposition and evict promoter nucleosomes at highly expressed genes in yeast.

The nucleosome remodeling complex RSC functions throughout the yeast genome to set the positions of -1 and +1 nucleosomes and thereby determines the widths of nucleosome-depleted regions (NDRs). The related complex SWI/SNF participates in nucleosome remodeling/eviction and promoter activation at certain yeast genes, including those activated by transcription factor Gcn4, but did not appear to function broadly in establishing NDRs. By analyzing the large cohort of Gcn4-induced genes in mutants lacking the catalytic subunits of SWI/SNF or RSC, we uncovered cooperation between these remodelers in evicting nucleosomes from different locations in the promoter and repositioning the +1 nucleosome downstream to produce wider NDRs-highly depleted of nucleosomes-during transcriptional activation. SWI/SNF also functions on a par with RSC at the most highly transcribed constitutively expressed genes, suggesting general cooperation by these remodelers for maximal transcription. SWI/SNF and RSC occupancies are greatest at the most highly expressed genes, consistent with their cooperative functions in nucleosome remodeling and transcriptional activation. Thus, SWI/SNF acts comparably with RSC in forming wide nucleosome-free NDRs to achieve high-level transcription but only at the most highly expressed genes exhibiting the greatest SWI/SNF occupancies.

B-cell differentiation is pressuromodulated as determined by pressuromodulation mapping: Part I, cell differentiation

BackgroundThe episodic sub-episode block sums split-integrated weighted average-averaged gene overexpression tropy quotient (esebssiwaagoTQ) is a measure of the 5′ → 3′ reading direction intergene distance tropy that needs to be overcome for horizontal alignment of a gene for maximal transcription; and it is also an arbitrary unit measure of the intracellular pressure needed for maximal gene expression.In this study, B-cell differentiation is studied by esebssiwaagoTQ-based pressuromodulation mapping of B-cell stage marker genes.MethodsLocations of 25 B-cell differentiation stage genes, and locations of downstream and upstream genes were mined at GeneCards and at LNCipedia, pseudogenes included and enhancers excluded. The esebssiwaagoTQs for each gene were determined. A pressuromodulation map was generated by arranging overexpressed B-cell stage marker genes in descending and ascending order by esebssiwaagoTQ in reference to periods of B-cell polarization.ResultsThe gene esebssiwaagoTQs are CD34 0.65 (0.648), PRDM1 0.36 (0.356), PTPRC 0.35 (0.345), MKI67 0.33 (0.329), ENPP1 0.31 (0.308), RAG2 0.31 (0.306), MS4A1 0.30 (0.299), PCNA 0.28 (0.285), ESPL1 0.28 (0.275), CD79B 0.27 (0.271), AICDA 0.27 (0.266), CD40 0.26 (0.257), APOBEC3A/-B 0.22 (0.216), CD38 0.21 (0.212), CD27 0.19 (0.194), APOBEC3C/−D/-F/−G 0.17 (0.173), CD19 0.15 (0.153), RAG1 0.14 (0.139), CD79A 0.14 (0.137), CR2 0.11 (0.109), and APOBEC3H 0.10 (0.102); these are pressuromodulation mapped in reference to B-cell polarization state and differentiation stage.ConclusionsThe esebssiwaagoTQ-based pressuromodulation map of B-cell differentiation simulates the in vivo B-cell maturation process for the classical pathway (T-cell mediated pressuromodulation effect pathway) and applies to the parallel non-classical pathway (T-cell independent antigen-mediated pressuromodulation effect pathway). Henceforth the B-cell pressuromodulation map can be utilized as the template for the study of specific B-cell events including bi-allelic V(D)J gene recombination, IGHM internal consensus recognition sequence, IGHD homologous recombination or initial allelic exclusion, further consensus recognition sequence isotype switchings, and somatic hypermutation, as in Part II.

B-cell antibody class switchings are pressuromodulated events: Part II, gene recombination

BackgroundThe esebssiwaagoTQ method is applicable for the of study cell gene recombination events as the esebssiwaagoTQ is a measure of the intracellular pressure required to establish a horizontal reading frame for alignment of a gene and its intergene bases for maximal transcription and recombination enzyme activity.B-cell differentiation stages have recently been studied by gene esebssiwaagoTQ-based pressuromodulation mapping. In this study, the B-cell differentiation stage pressuromodulation map is utilized as a template to simulate B-cell immunoglobulin locus recombination events that take place in the pressuromodulated state in vivo.MethodsChromosome 14 (−) strand location 105,566,277 and 106,879,844 germline genes were recombined after determination of gene esebssiwaagoTQs with respect to the germline, and then recombined genes were recombined further after determination of gene esebssiwaagoTQs with respect to rearranged configurations. For both alleles, first, IGHD_-_ to IGHJ_ was performed, and then IGHV_-_ to IGHD_-_-IGHJ_ was performed. For Allele 1 (IGHM), internal consensus recognition sequence (iCSR) and further CSR isotype switchings were performed; and for Allele 2 (IGHD), homologous recombination was performed and initial allelic exclusion determined.ResultsFirst, the esebssiwaagoTQ of a joining (J_) and diversity (D_-_) gene in its native germline configuration is the basis for predictable subsequent gene rearrangement. Second, D_-_ to J_ gene recombination events are bi-allelic and mutually exclusive. Third, the entire process from beginning to end depends on the grade of the pressuromodulation effect, and as per the classical pathway it is an antigen presenting cell (APC)-dependent CD4R+ T-cell-mediated B-cell polarization process. Fourth, CD4R+ T-cells are positively pressuromodulated, while B-cells are subject to the effect of both positive and negative forms of antigen pressuromodulation. And fifth, B-cell to plasma cell transformation and the extra-nodal periphery/tissue nidus phase take place in the presence of antigen load and either positive or negative pressuromodulation of the cell to its recombined antibody gene expression intracellular pressure.ConclusionsB-cell gene recombination rearrangement events can be predicted with a reasonable degree of certainty.It is envisioned that further esebssiwaagoTQ-based study of the remaining B-cell variability gene recombinations isotype switching events will further our understanding of pressuromodulated basis for antigen selection including the evolutionary underpinnings of.