Regulation of proto-oncogene expression in adult and developing lungs.

Activation of immediate-early gene expression has been associated with mitogenesis, differentiation, nerve cell depolarization, and recently, terminal differentiation processes and programmed cell death. Previous evidence also suggested that immediate-early genes play a role in the physiology of the lungs (J. I. Morgan, D. R. Cohen, J. L. Hempstead, and T. Curran, Science 237:192-197, 1987). Therefore, we analyzed c-fos expression in adult and developing lung tissues. Seizures elicited by chemoconvulsants induced expression of mRNA for c-fos, c-jun, and junB and Fos-like immunoreactivity in lung tissue. The use of pharmacological antagonists and adrenalectomy indicated that this increased expression was neurogenic. Interestingly, by using a fos-lacZ transgenic mouse, it was shown that Fos-LacZ expression in response to seizure occurred preferentially in clusters of epithelial cells at the poles of the bronchioles. This was the same location of Fos-LacZ expression detected during early lung development. These data imply that pharmacological induction of immediate-early gene expression in adult mice recapitulates an embryological program of gene expression.     

Hippocampal gene expression analysis highlights Ly6a/Sca-1 as candidate gene for previously mapped novelty induced behaviors in mice

In this study, we show that the covariance between behavior and gene expression in the brain can help further unravel the determinants of neurobehavioral traits. Previously, a QTL for novelty induced motor activity levels was identified on murine chromosome 15 using consomic strains. With the goal of narrowing down the linked region and possibly identifying the gene underlying the quantitative trait, gene expression data from this F(2)-population was collected and used for expression QTL analysis. While genetic variation in these mice was limited to chromosome 15, eQTL analysis of gene expression showed strong cis-effects as well as trans-effects elsewhere in the genome. Using weighted gene co-expression network analysis, we were able to identify modules of co-expressed genes related to novelty induced motor activity levels. In eQTL analyses, the expression of Ly6a (a.k.a. Sca-1) was found to be cis-regulated by chromosome 15. Ly6a also surfaced in a group of genes resulting from the network analysis that was correlated with behavior. Behavioral analysis of Ly6a knock-out mice revealed reduced novelty induced motor activity levels when compared to wild type controls, confirming functional importance of Ly6a in this behavior, possibly through regulating other genes in a pathway. This study shows that gene expression profiling can be used to narrow down a previously identified behavioral QTL in mice, providing support for Ly6a as a candidate gene for functional involvement in novelty responsiveness.     

Dexamethasone induction of hypertension and diabetes is PPAR-alpha dependent in LDL receptor-null mice

Hypertension and diabetes are common side effects of glucocorticoid treatment. To determine whether peroxisome proliferator-activated receptor-alpha (PPAR-alpha) mediates these sequelae, mice deficient in low-density lipoprotein receptor (Ldlr-/-), with (Ppara+/+) or without (Ppara-/-) PPAR-alpha, were treated chronically with dexamethasone. Ppara+/+, but not Ppara-/-, mice developed hyperglycemia, hyperinsulinemia and hypertension. Similar effects on glucose metabolism were seen in a different model using C57BL/6 mice. Hepatic gluconeogenic gene expression was increased and insulin-mediated suppression of endogenous glucose production was less effective in dexamethasone-treated Ppara+/+ mice. Adenoviral reconstitution of PPAR-alpha in the livers of nondiabetic, normotensive, dexamethasone-treated Ppara-/- mice induced hyperglycemia, hyperinsulinemia and increased gluconeogenic gene expression. It also increased blood pressure, renin activity, sympathetic nervous activity and renal sodium retention. Human hepatocytes treated with dexamethasone and the PPAR-alpha agonist Wy14,643 induced PPARA and gluconeogenic gene expression. These results identify hepatic activation of PPAR-alpha as a mechanism underlying glucocorticoid-induced insulin resistance.     

Restricted feeding induces daily expression of clock genes and Pai-1 mRNA in the heart of Clock mutant mice

Plasminogen activator inhibitor-1 (PAI-1) is a key factor of fibrinolytic activity. The activity and mRNA abundance show a daily rhythm. To elucidate the mechanism of daily Pai-1 gene expression, the expression of Pai-1 and several clock genes was examined in the heart of homozygous Clock mutant (Clock/Clock) mice. Damping of the daily oscillation of Pai-1 gene expression in Clock/Clock mice was accompanied with damped or attenuated oscillations of mPer1, mPer2, mBmal1, and mNpas2 mRNA. Daily restricted feeding induced a daily mRNA rhythm of all clock genes and Pai-1 mRNA in Clock/Clock mice as well as wild-type mice. The peaks of clock genes and Pai-1 mRNA were phase-advanced in the heart of both genotypes after 6 days of restricted feeding. The present results demonstrate that daily Pai-1 gene expression depends on clock gene expression in the heart and that a functional Clock gene is not required for restricted feeding-induced resetting of the peripheral clock.     

Effects of catechin on homocysteine metabolism in hyperhomocysteinemic mice

We have recently focused on the interaction between hyperhomocysteinemia, defined by high plasma homocysteine levels, and paraoxonase-1 expression and found a reduced activity of paraoxonase-1 associated with a reduced gene expression in the liver of cystathionine beta synthase (CBS) deficient mice, a murine model of hyperhomocysteinemia. As it has been demonstrated that polyphenolic compounds could modulate the expression level of the paraoxonase-1 gene in vitro, we have investigated the possible effect of flavonoid supplementation on the impaired paraoxonase-1 gene expression and activity induced by hyperhomocysteinemia and have evaluated the link with homocysteine metabolism. High-methionine diet significantly increased serum homocysteine levels, decreased hepatic CBS activity, and down-regulated paraoxonase-1 mRNA and its activity. However, chronic administration of catechin but not quercetin significantly reduced plasma homocysteine levels, attenuated the reduction of the hepatic CBS activity, and restored the decreased paraoxonase-1 gene expression and activity induced by chronic hyperhomocysteinemia. These data suggest that catechin could act on the homocysteine levels by increasing the rate of catabolism of homocysteine.     

Tyrosine hydroxylase activity and expression of its gene in mice with contrasting capacity to dominate in social stress]

Changes of tyrosine hydroxylase (TH) activity and level of mRNA of TH gene in PT and CBA/Lac mouse strains, which are contrast by ability to dominate in heterogenous populations, were investigated. It was established, that the activity of TH both in dominate PT and subordinate CBA/Lac mice in hypothalamus, hippocampus and brain stem elevated in one hour after forming of micropopulations. But the appearance of this increase was different: activation of TH in hypothalamus and brain stem of PT mice was stronger then one in CBA/Lac mice. Moreover, the beginning of the reaction in brain stem of PT mice was earlier then that of CBA/Lac mice. MRNA level of TH gene in hypothalamus and brain stem in one hour was elevated only in PT mice for 50% and 200%, respectively. No changing in expression TH gene was found in hippocampus. In conclusion, it was suggested that the activation of catecholamine biosynthesis under social stress in hypothalamus and brain stem of male mice was due to the TH activation and increase of its gene expression.     

Efficient inducible Pan-neuronal cre-mediated recombination in SLICK-H transgenic mice

Large-scale functional genomics in mice is becoming feasible through projects to develop conditional knockout alleles for every gene. Inducible neuron-specific gene knockout in such mice will permit the analysis of neuronal phenotypes while circumventing developmental defects or embryonic lethality. Here we describe a transgenic line, termed SLICK-H, that facilitates widespread inducible conditional genetic manipulation within most populations of projection neurons. In SLICK-H mice, the Thy1 promoter drives robust and relatively uniform expression of a drug-inducible form of cre recombinase throughout the peripheral and central nervous system. This permits efficient induction of cre-mediated genetic manipulation upon tamoxifen administration in adult mice. Importantly, cre activity in the absence of tamoxifen is minimal, permitting tight control of recombination. In the present study, we catalog in detail the transgene expression patterns and recombination efficiencies in SLICK-H mice. Our results highlight the utility of SLICK-H mice for functional genomics in the nervous system.     

Improvement of DNA vaccine immunogenicity by a dual antigen expression system

This study examined whether increased antigen expression resulted in enhanced antigen-specific immune responses in the context of DNA vaccines. To increase antigen expression, two copies of antigen expression cassettes were arranged in a plasmid pDX. BALB/c mice were intramuscularly immunized with various constructs that express influenza antigens and analysed for DNA-raised immunity. The plasmid pDX that expresses two copies of the antigen gene induced stronger antigen-specific immune responses than the plasmid pGA which expresses single antigen gene. To explore the in vivo transgene expression by pDX and pGA, luciferase activity was measured in the muscles transduced with luciferase expression plasmids. The pDX expressing two copies of luciferase induced the highest luciferase activity, which corresponded to the results from vaccination. We concluded that increasing the number of antigen expression cassettes in a vaccine construct improved antigen expression in the transduced tissue, which induced stronger DNA-raised immune responses.     

Activation of antioxidative enzymes induced by low-dose-rate whole-body gamma irradiation: adaptive response in terms of initial DNA damage

An adaptive response induced by long-term low-dose-rate irradiation in mice was evaluated in terms of the amount of DNA damage in the spleen analyzed by a comet assay. C57BL/ 6N female mice were irradiated with 0.5 Gy of (137)Cs gamma rays at 1.2 mGy/h; thereafter, a challenge dose (0.4, 0.8 or 1.6 Gy) at a high dose rate was given. Less DNA damage was observed in the spleen cells of preirradiated mice than in those of mice that received the challenge dose only; an adaptive response in terms of DNA damage was induced by long-term low-dose-rate irradiation in mice. The gene expression of catalase and Mn-SOD was significantly increased in the spleen after 23 days of the low-dose-rate radiation (0.5 Gy). In addition, the enzymatic activity of catalase corresponded to the gene expression level; the increase in the activity was observed at day 23 (0.5 Gy). These results suggested that an enhancement of the antioxidative capacities played an important role in the reduction of initial DNA damage by low-dose-rate radiation.     

Stimulation of leukocyte lysophospholipase activity by noninfectious agents

Mouse peritoneal leukocyte lysophospholipase (LPL) activity was studied to determine whether or not noninfectious agents cause increased enzyme activity and whether neutrophils have LPL activity. In the first study, mice infected with Ascaris suum, a known inducer of LPL activity, were given intraperitoneal injections of proteose peptone, thioglycolate, bovine albumin, paraffin, glycogen, or A. suum whole worm extract (WWE). Cell populations collected from mice injected with A. suum WWE, proteose peptone, thioglycolate, or bovine albumin contained increased numbers of neutrophils and eosinophils. These cell populations had increased LPL activity when treated, in vitro, with either A. suum WWE, zymosan-activated complement, or with the agent they were induced with. However, the LPL activity of the different cell populations did not respond to all treatments in the same way. In a second study, A. suum-infected or noninfected mice were given intraperitoneal injections of paraffin, thioglycolate, glycogen, or A. suum WWE. Enriched cell populations containing either lymphocytes or macrophages, from infected or noninfected mice, did not have increased LPL activity following in vitro stimulation with A. suum WWE, zymosan-activated complement, or with the agent they were induced with. Enriched neutrophil populations from infected or noninfected mice had increased LPL activity following in vitro treatment with A. suum WWE or zymosan-activated complement. Results demonstrate that the LPL activity of peritoneal leukocytes can be induced by noninfectious agents and that neutrophils have increased LPL activity following in vitro stimulation.     

The subunit 1 of the COP9 signalosome suppresses gene expression through its N-terminal domain and incorporates into the complex through the PCI domain

The COP9 signalosome is a conserved protein complex composed of eight subunits. Individual subunits of the complex have been linked to various signal transduction pathways leading to gene expression and cell cycle control. However, it is not understood how each subunit executes these activities as part of a large protein complex. In this study, we dissected structure and function of the subunit 1 (CSN1 or GPS1) of the COP9 signalosome relative to the complex. We demonstrated that the C-terminal half of CSN1 encompassing the PCI domain is responsible for interaction with CSN2, CSN3, and CSN4 subunits and is required for incorporation of the subunit into the complex. The N-terminal fragment of CSN1 cannot stably associate with the complex but can translocate to the nucleus on its own. We further show that CSN1 or the N-terminal fragment of CSN1 (CSN1-N) can inhibit c-fos expression from either a transfected template or a chromosomal transgene ( fos-lacZ). Moreover, CSN1 as well as CSN1-N can potently suppress signal activation of a AP-1 promoter and moderately suppress serum activation of a SRE promoter, but is unable to inhibit PKA-induced CRE promoter activity. We conclude that the N-terminal half of CSN1 harbors the activity domain that confers most of the repression functions of CSN1 while the C-terminal half allows integration of the protein into the COP9 signalosome.     

Overexpression of calmodulin induces cardiac hypertrophy by a calcineurin-dependent pathway

The possible role of calcineurin in cardiac hypertrophy induced by calmodulin (CaM) overexpression in the heart was investigated. CaM transgenic (CaM-TG) mice developed marked cardiac hypertrophy and exhibited up-regulation of atrial natriuretic factor (ANF) and beta-myosin heavy chain gene expression in the heart during the first 2 weeks after birth. The activity of calcineurin in the heart was also significantly increased in CaM-TG mice compared with wild-type littermates. Treatment of CaM-TG mice with the calcineurin inhibitor FK506 (1mg/kg per day) prevented the increase in the heart-to-body weight ratio as well as that in cardiomyocyte width. FK506 also inhibited the induction of fetal-type cardiac gene expression in CaM-TG mice. Overexpression of CaM in cultured rat cardiomyocytes activated the ANF gene promoter in a manner sensitive to FK506. Activation of a calcineurin-dependent pathway thus contributes to the development of cardiac hypertrophy induced by CaM overexpression in the heart.     

The mouse L-histidine decarboxylase gene: structure and transcriptional regulation by CpG methylation in the promoter region

To investigate the regulation of mouse L-histidine decarboxylase (HDC) gene expression, we isolated genomic DNA clones encoding HDC. Structural analysis revealed that the mouse HDC gene was composed of 12 exons, spanning approximately 24 kb. Northern blotting analysis indicated that, among the cell lines examined, a high level of HDC gene expression was restricted to mature mast cell lines and an erythroblastic cell line. The gene was induced strongly in the mouse immature mast cell line P815 after incubation in the peritoneal cavity of BDF1 mice. We observed that the promoter region was demethylated in the HDC-expressing cell lines and in induced P815 cells. Interestingly, forced demethylation by 5-azacytidine (5-azaC) treatment induced high expression of HDC mRNA in P815 cells. The activity of a mouse HDC promoter-reporter construct stably transfected in P815 cells was repressed by in vitro patch-methylation. This low promoter activity of the patch-methylated reporter construct was restored after 5-azaC treatment, which demethylated the patch-methylated promoter. These results indicate that DNA methylation state of the promoter region controls HDC gene expression.     

PiggyBac转座酶转基因小鼠模型的建立

目的 建立表达PiggyBac转座酶转基因小鼠模型,为研究PiggyBac转座子介导基因修饰在小鼠中的应用提供工具.方法 利用Cytomegalovirus( CMV)启动子驱动PiggyBac转座酶基因的表达,经显微注射法建立C57BL/6J表达PiggyBac转座酶的转基因小鼠.PCR鉴定转基因小鼠的基因型,RT-PCR检测PiggyBac转座酶在小鼠生殖系睾丸中的表达情况.PiggyBac转座酶转基因小鼠活性的检测,是通过与转座子供体转基因小鼠杂交检测供体位置变化来确定的.结果 显微注射产生7只转基因小鼠并能传代,经RT-PCR筛选出一株在睾丸中相对高表达PiggyBac转座酶的转基因小鼠.随后与转座子供体转基因小鼠杂交,子代双阳小鼠与野生型小鼠杂交基因型分离,产生的子代转座子供体单阳性小鼠中具有转座子供体片段的转座反应.结论 成功建立了表达PiggyBac转座酶转基因小鼠动物模型,该模型为PiggyBac转座子技术在小鼠中的应用提供了有价值的工具动物.     

Variation in the large-scale organization of gene expression levels in the hippocampus relates to stable epigenetic variability in behavior

Background

Despite sharing the same genes, identical twins demonstrate substantial variability in behavioral traits and in their risk for disease. Epigenetic factors–DNA and chromatin modifications that affect levels of gene expression without affecting the DNA sequence–are thought to be important in establishing this variability. Epigenetically-mediated differences in the levels of gene expression that are associated with individual variability traditionally are thought to occur only in a gene-specific manner. We challenge this idea by exploring the large-scale organizational patterns of gene expression in an epigenetic model of behavioral variability.

Methodology/Findings

To study the effects of epigenetic influences on behavioral variability, we examine gene expression in genetically identical mice. Using a novel approach to microarray analysis, we show that variability in the large-scale organization of gene expression levels, rather than differences in the expression levels of specific genes, is associated with individual differences in behavior. Specifically, increased activity in the open field is associated with increased variance of log-transformed measures of gene expression in the hippocampus, a brain region involved in open field activity. Early life experience that increases adult activity in the open field also similarly modifies the variance of gene expression levels. The same association of the variance of gene expression levels with behavioral variability is found with levels of gene expression in the hippocampus of genetically heterogeneous outbred populations of mice, suggesting that variation in the large-scale organization of gene expression levels may also be relevant to phenotypic differences in outbred populations such as humans. We find that the increased variance in gene expression levels is attributable to an increasing separation of several large, log-normally distributed families of gene expression levels. We also show that the presence of these multiple log-normal distributions of gene expression levels is a universal characteristic of gene expression in eurkaryotes. We use data from the MicroArray Quality Control Project (MAQC) to demonstrate that our method is robust and that it reliably detects biological differences in the large-scale organization of gene expression levels.

Conclusions

Our results contrast with the traditional belief that epigenetic effects on gene expression occur only at the level of specific genes and suggest instead that the large-scale organization of gene expression levels provides important insights into the relationship of gene expression with behavioral variability. Understanding the epigenetic, genetic, and environmental factors that regulate the large-scale organization of gene expression levels, and how changes in this large-scale organization influences brain development and behavior will be a major future challenge in the field of behavioral genomics.