Developmental regulation of cytochrome P-450 (b+e) and glutathione transferase (Ya+Yc) gene expression in rat liver

The expression of cytochrome P-450 (b+e) and glutathione transferase (Ya+Yc) genes has been studied as a function of development in rat liver. The levels of cytochrome P-450 (b+e) mRNAs and their transcription rates are too low for detection in the 19-day old fetal liver before or after phenobarbitone treatment. However, glutathione transferase (Ya+Yc) mRNAs can be detected in the fetal liver as well as their induction after phenobarbitone treatment can be demonstrated. These mRNAs contents as well as their inducibility with phenobarbitone are lower in maternal liver than that of adult nonpregnant female rat liver. Steroid hormone administration to immature rats blocks substantially the phenobarbitone mediated induction of the two mRNA families as well as their transcription. It is suggested that steroid hormones constitute one of the factors responsible for the repression of the cytochrome P-450 (b+e) and glutathione transferase (Ya+Yc) genes in fetal liver.

Regulation of Growth, Cell Shape, Cell Division, and Gene Expression by Second Messengers (p)ppGpp and Cyclic Di-GMP in Mycobacterium smegmatis

The alarmone (p)ppGpp regulates transcription, translation, replication, virulence, lipid synthesis, antibiotic sensitivity, biofilm formation, and other functions in bacteria. Signaling nucleotide cyclic di-GMP (c-di-GMP) regulates biofilm formation, motility, virulence, the cell cycle, and other functions. In Mycobacterium smegmatis, both (p) ppGpp and c-di-GMP are synthesized and degraded by bifunctional proteins Rel(Msm) and DcpA, encoded by rel(Msm) and dcpA genes, respectively. We have previously shown that the Delta rel(Msm) and Delta dcpA knockout strains are antibiotic resistant and defective in biofilm formation, show altered cell surface properties, and have reduced levels of glycopeptidolipids and polar lipids in their cell wall (K. R. Gupta, S. Kasetty, and D. Chatterji, Appl Environ Microbiol 81:2571-2578, 2015, http://dx.doi.org/10.1128/AEM.03999-14). In this work, we have explored the phenotypes that are affected by both (p) ppGpp and c-di-GMP in mycobacteria. We have shown that both (p) ppGpp and c-di-GMP are needed to maintain the proper growth rate under stress conditions such as carbon deprivation and cold shock. Scanning electron microscopy showed that low levels of these second messengers result in elongated cells, while high levels reduce the cell length and embed the cells in a biofilm-like matrix. Fluorescence microscopy revealed that the elongated Delta rel(Msm) and Delta dcpA cells are multinucleate, while transmission electron microscopy showed that the elongated cells are multiseptate. Gene expression analysis also showed that genes belonging to functional categories such as virulence, detoxification, lipid metabolism, and cell-wall-related processes were differentially expressed. Our results suggests that both (p) ppGpp and c-di-GMP affect some common phenotypes in M. smegmatis, thus raising a possibility of cross talk between these two second messengers in mycobacteria. IMPORTANCE Our work has expanded the horizon of (p) ppGpp and c-di-GMP signaling in Gram-positive bacteria. We have come across a novel observation that M. smegmatis needs (p) ppGpp and c-di-GMP for cold tolerance. We had previously shown that the Delta rel(Msm) and Delta dcpA strains are defective in biofilm formation. In this work, the overproduction of (p) ppGpp and c-di-GMP encased M. smegmatis in a biofilm-like matrix, which shows that both (p) ppGpp and c-di-GMP are needed for biofilm formation. The regulation of cell length and cell division by (p) ppGpp was known in mycobacteria, but our work shows that c-di-GMP also affects the cell size and cell division in mycobacteria. This is perhaps the first report of c-di-GMP regulating cell division in mycobacteria.

Engineering nucleic acid mechanisms for regulation and readout of gene expression : conditional Dicer substrate formation and sensitive multiplexed northern blots

Nucleic acids are most commonly associated with the genetic code, transcription and gene expression. Recently, interest has grown in engineering nucleic acids for biological applications such as controlling or detecting gene expression. The natural presence and functionality of nucleic acids within living organisms coupled with their thermodynamic properties of base-pairing make them ideal for interfacing (and possibly altering) biological systems. We use engineered small conditional RNA or DNA (scRNA, scDNA, respectively) molecules to control and detect gene expression. Three novel systems are presented: two for conditional down-regulation of gene expression via RNA interference (RNAi) and a third system for simultaneous sensitive detection of multiple RNAs using labeled scRNAs.

RNAi is a powerful tool to study genetic circuits by knocking down a gene of interest. RNAi executes the logic: If gene Y is detected, silence gene Y. The fact that detection and silencing are restricted to the same gene means that RNAi is constitutively on. This poses a significant limitation when spatiotemporal control is needed. In this work, we engineered small nucleic acid molecules that execute the logic: If mRNA X is detected, form a Dicer substrate that targets independent mRNA Y for silencing. This is a step towards implementing the logic of conditional RNAi: If gene X is detected, silence gene Y. We use scRNAs and scDNAs to engineer signal transduction cascades that produce an RNAi effector molecule in response to hybridization to a nucleic acid target X. The first mechanism is solely based on hybridization cascades and uses scRNAs to produce a double-stranded RNA (dsRNA) Dicer substrate against target gene Y. The second mechanism is based on hybridization of scDNAs to detect a nucleic acid target and produce a template for transcription of a short hairpin RNA (shRNA) Dicer substrate against target gene Y. Test-tube studies for both mechanisms demonstrate that the output Dicer substrate is produced predominantly in the presence of a correct input target and is cleaved by Dicer to produce a small interfering RNA (siRNA). Both output products can lead to gene knockdown in tissue culture. To date, signal transduction is not observed in cells; possible reasons are explored.

Signal transduction cascades are composed of multiple scRNAs (or scDNAs). The need to study multiple molecules simultaneously has motivated the development of a highly sensitive method for multiplexed northern blots. The core technology of our system is the utilization of a hybridization chain reaction (HCR) of scRNAs as the detection signal for a northern blot. To achieve multiplexing (simultaneous detection of multiple genes), we use fluorescently tagged scRNAs. Moreover, by using radioactive labeling of scRNAs, the system exhibits a five-fold increase, compared to the literature, in detection sensitivity. Sensitive multiplexed northern blot detection provides an avenue for exploring the fate of scRNAs and scDNAs in tissue culture.

Cell-targeted regulation of gene expression through synthetic RNA devices

The ability to interface with and program cellular function remains a challenging research frontier in biotechnology. Although the emerging field of synthetic biology has recently generated a variety of gene-regulatory strategies based on synthetic RNA molecules, few strategies exist through which to control such regulatory effects in response to specific exogenous or endogenous molecular signals. Here, we present the development of an engineered RNA-based device platform to detect and act on endogenous protein signals, linking these signals to the regulation of genes and thus cellular function.

We describe efforts to develop an RNA-based device framework for regulating endogenous genes in human cells. Previously developed RNA control devices have demonstrated programmable ligand-responsive genetic regulation in diverse cell types, and we attempted to adapt this class of cis-acting control elements to function in trans. We divided the device into two strands that reconstitute activity upon hybridization. Device function was optimized using an in vivo model system, and we found that device sequence is not as flexible as previously reported. After verifying the in vitro activity of our optimized design, we attempted to establish gene regulation in a human cell line using additional elements to direct device stability, structure, and localization. The significant limitations of our platform prevented endogenous gene regulation.

We next describe the development of a protein-responsive RNA-based regulatory platform. Employing various design strategies, we demonstrated functional devices that both up- and downregulate gene expression in response to a heterologous protein in a human cell line. The activity of our platform exceeded that of a similar, small-molecule-responsive platform. We demonstrated the ability of our devices to respond to both cytoplasmic- and nuclear-localized protein, providing insight into the mechanism of action and distinguishing our platform from previously described devices with more restrictive ligand localization requirements. Finally, we demonstrated the versatility of our device platform by developing a regulatory device that responds to an endogenous signaling protein.

The foundational tool we present here possesses unique advantages over previously described RNA-based gene-regulatory platforms. This genetically encoded technology may find future applications in the development of more effective diagnostic tools and targeted molecular therapy strategies.

MicroRNA regulation and the variability of human cortical gene expression

Understanding the driving forces of gene expression variation within human populations will provide important insights into the molecular basis of human phenotypic variation. In the genome, the gene expression variability differs among genes, and at prese

Regulation of zygotic gene activation in the preimplantation mouse embryo: Global activation and repression of gene expression

Superimposed on the activation of the embryonic genome in the preimplantation mouse embryo is the formation of a transcriptionally repressive state during the two-cell stage. This repression appears mediated at the level of chromatin structure, because it is reversed by inducing histone hyperacetylation or inhibiting the second round of DNA replication. We report that of more than 200 amplicons analyzed by mRNA differential display, about 45% of them are repressed between the two-cell and four-cell stages. This repression is scored as either a decrease in amplicon expression that occurs between the two-cell and four-cell stages or on the ability of either trichostatin A tan inhibitor of histone deacetylases) or aphidicolin tan inhibitor of replicative DNA polymerases) to increase the level of amplicon expression. Results of this study also indicate that about 16% of the amplicons analyzed likely are novel genes whose sequence doesn't correspond to sequences in the current databases, whereas about 20% of the sequences expressed during this transition likely are repetitive sequences. Lastly, inducing histone hyperacetylation in the two-cell embryos inhibits cleavage to the four-cell stage. These results suggest that genome activation is global and relatively promiscuous and that a function of the transcriptionally repressive state is to dictate the appropriate profile of gene expression that is compatible with further development.

Expression regulation and functional characterization of a novel interferon inducible gene Gig2 and its promoter

Grass carp hemorrhagic virus (GCHV)-induced gene 2 (Gig2) is a novel gene previously identified from UV-inactivated GCHV-treated Carassius auratus blastulae embryonic (CAB) cells, suggesting that it should play a pivotal role in the interferon (IFN) antiviral response. In this study, a polyclonal anti-Gig2 antiserum was generated and used to study the inductive expression pattern by Western blot analysis, showing no basal expression in normal CAB cells but a significant up-regulation upon UV-inactivated GCHV, polyinosinic:polycytidylic acid (Poly I:Q and recombinant IFN (rIFN). However, constitutive expression of Gig2 is observed in all tested tissues from grass carp (Ctenopharyngodon idellus), and Poly I:C injection increases the relative amount of Gig2 protein in skin, spleen, trunk kidney, gill, hindgut and thymus. Moreover, the genomic sequence covering the whole Gig2 ORF and the upstream promoter region were amplified by genomic walking. Significantly, the Gig2 promoter contains three IFN-stimulated response elements (ISREs), nine GAAA/TfTC motifs and five gamma-IFN activating sites (GAS), which are the characteristics of genes responsive to both type I IFN and type 11 IFN. Subsequently, the complete Gig2 promoter sequence was cloned into pGL3-Basic vector, and its activity was measured by luciferase assays in the transfected CAB cells. The Gig2 promoter-driven construct is highly induced in CAB cells after treatment with Poly I:C or rIFN, and the functional capability is dependent on IFN regulatory factor 7 (IRF7), because its activity can be stimulated by IRF7. Collectively, the data provide strong evidence that Gig2 is indeed a novel IFN inducible gene and its expression is likely dependent on IRF7 upon Poly I:C or IFN. (C) 2009 Elsevier Ltd. All rights reserved.

Molecular coordinated regulation of gene expression during ovarian development in the penaeid shrimp

To understand the molecular events of ovarian development in penaeid shrimp, RNA arbitrarily primed polymerase chain reaction (RAP-PCR) was used to identify differentially expressed genes during ovarian maturation in Metapenaeus ensis. From a screening of 700 clones in a cDNA library of the shrimp ovary by the products of RAP-PCR of different maturation stages, 91 fragments with differentially expressed pattern as revealed by dot-blot hybridization were isolated and sequenced. Forty-two of these fragments show significant sequence similarity to known gene products and the differentially expressed pattern of 10 putative genes were further characterized via Northern hybridization. Putative glyceraldehyde-3-phosphate dehydrogenase and arginine kinase are related to provision of energy for active cellular function in oocyte development. Translationally controlled tumor protein, actin, and keratin are related to the organization of cytoskeleton to accomplish growth and development of oocytes. High mobility group protein DSP1, heat shock protein 70, and nucleoside diphosphate kinase may act as repressors before the onset of ovarian maturation. Peptidyl-prolyl cis-trans isomerase and glutathione peroxidase are related to the stabilization of proteins and oocytes. This study provides new insights on the molecular events in the ovarian development in the shrimp.

Regulation and consequences of differential gene expression in diabetic kidney disease

DIN (diabetic nephropathy) is the leading cause of end-stage renal disease worldwide and develops in 25-40% of patients with Type 1 or Type 2 diabetes mellitus. Elevated blood glucose over long periods together with glomerular hypertension leads to progressive glomerulosclerosis and tubulointerstitial fibrosis in susceptible individuals. Central to the pathology of DIN are cytokines and growth factors such as TGF-beta (transforming growth factor beta) superfamily members, including BMPs (bone morphogenetic protein) and TGF-beta 1, which play key roles in fibrogenic responses of the kidney, including podocyte loss, mesangial cell hypertrophy, matrix accumulation and tubulointerstitial fibrosis. Many of these responses can be mimicked in in vitro models of cells cultured in high glucose. We have applied differential gene expression technologies to identify novel genes expressed in in vitro and in vivo models of DN and, importantly, in human renal tissue. By mining these datasets and probing the regulation of expression and actions of specific molecules, we have identified novel roles for molecules such as Gremlin, IHG-1 (induced in high glucose-1) and CTGF (connective tissue growth factor) in DIN and potential regulators of their bioactions.

Global down-regulation of HOX gene expression in PML-RAR alpha(+) acute promyelocytic leukemia identified by small-array real-time PCR

Acute promyelocytic leukemia (APL) is associated with a reciprocal and balanced translocation involving the retinoic acid receptor-alpha (RARalpha). All-trans retinoic acid (ATRA) is used to treat APL and is a potent morphogen that regulates HOX gene expression in embryogenesis and organogenesis. HOX genes are also involved in hematopoiesis and leukemogenesis. Thirty-nine mammalian HOX genes have been identified and classified into 13 paralogous groups clustered on 4 chromosomes. They encode a complex net-Work of transcription regulatory proteins whose precise targets remain poorly understood. The overall function of the network appears to be dictated by gene dosage. To investigate the mechanisms involved in HOX gene regulation in hematopoiesis and leukemogenesis by precise measurement of individual HOX genes, a small-array real-time HOX (SMART-HOX) quantitative polymerase chain reaction (PCR) platform was designed and validated. Application of SMART-HOX to 16 APL bone marrow samples revealed a global down-regulation of 26 HOX genes compared with normal controls. HOX gene expression was also altered during differentiation induced by ATRA in the PML-RARalpha(+) NB4 cell line. PML-RARalpha, fusion proteins have been reported to act as part of a repressor complex during myelold cell differentiation, and a model linking HOX gene expression to this PML-RARalpha repressor complex is now proposed.

Regulation of erythropoietin gene expression depends on two different oxygen-sensing mechanisms

Erythropoietin (Epo), a glycoprotein hormone produced principally in the fetal kidney and in the adult liver in response to hypoxia, is the prime regulator of growth and differentiation in erythroid progenitor cells. The regulation of Epo gene expression is not fully understood, but two mechanisms have been proposed. One involves the participation of a heme protein capable of reversible oxygenation and the other depends on the intracellular concentration of reactive oxygen species (ROS), assumed to be a function of pO2. We have investigated the production of Epo in response to three stimuli, hypoxia, cobalt chloride, and the iron chelator desferrioxamine, in Hep3B cells. As expected, hypoxia caused a marked rise in Epo production. When the cells were exposed to the paired stimuli of hypoxia and cobalt no further increase was found. In contrast, chelation of iron under hypoxic conditions markedly enhanced Epo production, suggesting that the two stimuli act by separate pathways. The addition of carbon monoxide inhibited hypoxia-induced Epo production, independent of desferrioxamine concentration. Taken together these data support the concept that pO2 and ROS are sensed independently.