Increase In Rate Of Transcription Research Articles

MicroRNA-based single-gene circuits buffer protein synthesis rates against perturbations.

Achieving precise control of mammalian transgene expression has remained a long-standing, and increasingly urgent, challenge in biomedical science. Despite much work, single-cell methods have consistently revealed that mammalian gene expression levels remain susceptible to fluctuations (noise) and external perturbations. Here, we show that precise control of protein synthesis can be realized using a single-gene microRNA (miRNA)-based feed-forward loop (sgFFL). This minimal autoregulatory gene circuit consists of an intronic miRNA that targets its own transcript. In response to a step-like increase in transcription rate, the network generated a transient protein expression pulse before returning to a lower steady state level, thus exhibiting adaptation. Critically, the steady state protein levels were independent of the size of the stimulus, demonstrating that this simple network architecture effectively buffered protein production against changes in transcription. The single-gene network architecture was also effective in buffering against transcriptional noise, leading to reduced cell-to-cell variability in protein synthesis. Noise was up to 5-fold lower for a sgFFL than for an unregulated control gene with equal mean protein levels. The noise buffering capability varied predictably with the strength of the miRNA-target interaction. Together, these results suggest that the sgFFL single-gene motif provides a general and broadly applicable platform for robust gene expression in synthetic and natural gene circuits.

Direct measurement of transcription rates reveals multiple mechanisms for configuration of the Arabidopsisambient temperature response

BackgroundSensing and responding to ambient temperature is important for controlling growth and development of many organisms, in part by regulating mRNA levels. mRNA abundance can change with temperature, but it is unclear whether this results from changes in transcription or decay rates, and whether passive or active temperature regulation is involved.ResultsUsing a base analog labelling method, we directly measured the temperature coefficient, Q10, of mRNA synthesis and degradation rates of the Arabidopsis transcriptome. We show that for most genes, transcript levels are buffered against passive increases in transcription rates by balancing passive increases in the rate of decay. Strikingly, for temperature-responsive transcripts, increasing temperature raises transcript abundance primarily by promoting faster transcription relative to decay and not vice versa, suggesting a global transcriptional process exists that controls mRNA abundance by temperature. This is partly accounted for by gene body H2A.Z which is associated with low transcription rate Q10, but is also influenced by other marks and transcription factor activities.ConclusionsOur data show that less frequent chromatin states can produce temperature responses simply by virtue of their rarity and the difference between their thermal properties and those of the most common states, and underline the advantages of directly measuring transcription rate changes in dynamic systems, rather than inferring rates from changes in mRNA abundance.

Stimulus-induced modulation of transcriptional bursting in a single mammalian gene

Mammalian genes are often transcribed discontinuously as short bursts of RNA synthesis followed by longer silent periods. However, how these "on" and "off" transitions, together with the burst sizes, are modulated in single cells to increase gene expression upon stimulation is poorly characterized. By combining single-cell time-lapse luminescence imaging with stochastic modeling of the time traces, we quantified the transcriptional responses of the endogenous connective tissue growth factor gene to different physiological stimuli: serum and TGF-β1. Both stimuli caused a rapid and acute increase in burst sizes. Whereas TGF-β1 showed prolonged transcriptional activation mediated by an increase of transcription rate, serum stimulation resulted in a large and temporally tight first transcriptional burst, followed by a refractory period in the range of hours. Our study thus reveals how different physiological stimuli can trigger kinetically distinct transcriptional responses of the same gene.

Hypoxia induces a HIF-1α dependent signaling cascade to make a complex metabolic switch in SGBS-adipocytes

To elucidate the complex impact of hypoxia on adipose tissue, resulting in biased metabolism, insulin resistance and finally diabetes we used mature adipocytes derived from a Simpson-Golabi-Behmel syndrome patient for microarray analysis. We found a significantly increased transcription rate of genes involved in glycolysis and a striking association between the pattern of upregulated genes and disease biomarkers for diabetes mellitus and insulin resistance. Although their upregulation turned out to be HIF-1α-dependent, we identified further transcription factors mainly AP-1 components to play also an important role in hypoxia response. Analyzing the regulatory network of mentioned transcription factors and glycolysis targets we revealed a clear hint for directing glycolysis to glutathione and glycogen synthesis. This metabolic switch in adipocytes enables the cell to prevent oxidative damage in the short term but might induce lipogenesis and establish systemic metabolic disorders in the long run.

A Meta-Analysis Of Hodgkin Lymphoma Reveals 19p13.3 (TCF3) As a Novel Susceptibility Loc

BackgroundRecent genome wide association studies (GWAS) of Hodgkin lymphoma (HL) have identified several associations at both HLA and non-HLA loci. However, much of HL heritability remains unexplained. MethodsTo identify novel risk loci, we performed a meta-analysis of 3 HL GWAS including a total of 1,810 cases and 7,879 controls. Results were replicated in an independent set of 1,163 cases and 2,580 controls, for a total of 3,097 and 11,097 cases and controls combined, respectively. participants in discovery and replication stages were of European descent. quality control and imputation we conducted a meta-analysis addressing 1,004,829 variants (λ= 1.10, λ1000= 1.03).Associations between SNP genotypes and HL risk were evaluated under a log-additive model of inheritance adjusting for sex, study center and significant principal components to control for population stratification. We performed an analysis with all HL cases and then conducted stratified analyses by histological subtype (classical, nodular sclerosis and mixed cellularity), age at diagnosis (nodular sclerosis among those diagnosed at 15- 35 years in all studies, and those diagnosed at 35 and older years in the European Study only) and EBV tumor status (negative and positive). We then used a bioinformatic approach (FunciSNP) to identify potential functional variants associated with HD risk correlated with risk loci of interest. We extracted publically available ENCODE data on biofeatures to identify potential functional motifs associated with the index SNP or correlated SNPs. Finally, we measured expression levels of the two alternative mRNA transcripts in lymphoblastoid cell lines (LCLs) from 49 post-therapy HL patients and 25 unaffected controls. RT-PCR was carried out in triplicate. Relative expression levels were calculated relative to TBP as housekeeping gene. Linear models were used to assess correlation between genotype and TCF3expression levels. ResultsThe meta-analysis identified a novel susceptibility variant at chromosome 19p13.3 (rs1860661) associated with HL risk (Odds Ratio [OR]= 0.78, P=2.0*10-8, I2=0%). variant is located in intron 2 of TCF3 (also known as E2A), a regulator of B- and T-cell lineage commitment. was also significantly associated with HL (OR= 0.85, P=0.002) in the replication series of 1,281 cases and 3,218 controls. the combined analysis consisting of the discovery and replication sets, rs1860661was strongly associated with HL (OR=0.81,=3.5*10-10), with no evidence of heterogeneity between contributing studies (Phom=0.41, I2=0%).The number of G alleles defined by rs1860661 was significantly associated with a reduced risk of each HL subtype except EBV positive HL. rs1860661 and two correlated SNPs, rs10413888 (r2=0.90) and rs8103453 (r2=0.89) identified by FunciSNP analysis map in or near marks of open chromatin and in DNAse hypersensitivity sites in TCF3 in CD20+ B cell lines., the protective minor alleles of these SNPs as defined by the G-G-G haplotype map to the binding sites for ZBTB7a (rs10413888 and rs1860661) and (rs8103453) transcription factors, likely improving the binding efficiency to the sites which may result in increased transcription rates of TCF3. TCF3 is encoded by two alternative transcripts (E12 and E47). Higher expression levels of TCF3-E47, whose transcription start site is located close to rs1860661, was associated with the rs1860661-G allele in controls (P=0.02), but not in HL patients (P=0.22). Conclusion/DiscussionTCF3 is essential for the commitment of lymphoid progenitors to both B-cell and T-cell lineage development. A molecular and phenotypic hallmark of classical HL is the loss of the B-cell phenotype in HRS cells, including lack of demonstrable B-cell receptor and most B-cell specific markers such as CD19 or CD20. HRS cells have a low level of TCF3, particularly homodimers of the isoform E47, due to expression of the ABF-1 and ID2 inhibitors that bind to TCF3. Thus, higher TCF3 levels in HRS precursor cells may lead to enhanced retention of the B cell phenotype, thereby conferring a protective effect. These data suggest a link between the 19p13.3 locus including TCF3 and HL risk, indicating that TCF3 could be relevant to HL etiology and pathogenesis. Disclosures:Link:Genentech: Consultancy; Millenium: Consultancy; Pharmacyclics: Consultancy; Spectrum: Consultancy.

Regulatory Polymorphisms in β-Tubulin IIa Are Associated with Paclitaxel-Induced Peripheral Neuropathy

Peripheral neuropathy is the dose-limiting toxicity of paclitaxel, a chemotherapeutic drug widely used to treat several solid tumors such as breast, lung, and ovary. The cytotoxic effect of paclitaxel is mediated through β-tubulin binding in the cellular microtubules. In this study, we investigated the association between paclitaxel neurotoxicity risk and regulatory genetic variants in β-tubulin genes. We measured variation in gene expression of three β-tubulin isotypes (I, IVb, and IIa) in lymphocytes from 100 healthy volunteers, sequenced the promoter region to identify polymorphisms putatively influencing gene expression and assessed the transcription rate of the identified variants using luciferase assays. To determine whether the identified regulatory polymorphisms were associated with paclitaxel neurotoxicity, we genotyped them in 214 patients treated with paclitaxel. In addition, paclitaxel-induced cytotoxicity in lymphoblastoid cell lines was compared with β-tubulin expression as measured by Affymetrix exon array. We found a 63-fold variation in β-tubulin IIa gene (TUBB2A) mRNA content and three polymorphisms located at -101, -112, and -157 in TUBB2A promoter correlated with increased mRNA levels. The -101 and -112 variants, in total linkage disequilibrium, conferred TUBB2A increased transcription rate. Furthermore, these variants protected from paclitaxel-induced peripheral neuropathy [HR, 0.62; 95% confidence interval (CI), 0.42-0.93; P = 0.021, multivariable analysis]. In addition, an inverse correlation between TUBB2A and paclitaxel-induced apoptosis (P = 0.001) in lymphoblastoid cell lines further supported that higher TUBB2A gene expression conferred lower paclitaxel sensitivity. This is the first study showing that paclitaxel neuropathy risk is influenced by polymorphisms regulating the expression of a β-tubulin gene.

Gestational high fat diet programs hepatic phosphoenolpyruvate carboxykinase gene expression and histone modification in neonatal offspring rats

In insulin resistance and type II diabetes, there is an elevation of hepatic gluconeogenesis, which contributes to hyperglycaemia. Studies in experimental animals have provided evidence that consumption of high fat (HF) diets by female rats programs the progeny for glucose intolerance in adulthood, but the mechanisms behind the in utero programming remain poorly understood. The present study analysed the effect of a maternal HF diet on fetal gluconeogenic gene expression and potential regulation mechanism related to histone modifications. Dams were fed either a Control (C, 16% kcal fat) or a high-fat (HF, 45% kcal fat) diet throughout gestation. Livers of the offspring were collected on gestational day 21 and analysed to determine the consequences of a maternal HF diet on molecular markers of fetal liver gluconeogenesis. We demonstrated that offspring of HF-fed dams were significantly heavier and had significantly higher blood glucose levels at the time of delivery than offspring of dams fed the C diet. While maternal gluconeogenesis and plasma glucose were not affected by the HF diet, offspring of HF-fed dams had significantly higher mRNA contents of gluconeogenic genes in addition to the elevated plasma glucose. In addition to increased transcription rate, a gestational HF diet resulted in modifications of the Pck1 histone code in livers of offspring. Our results demonstrate that in utero exposure to HF diet has the potential to program the gluconeogenic capacity of offspring through epigenetic modifications, which could potentially lead to excessive glucose production and altered insulin sensitivity in adulthood.

Gestational High Fat Diet Programs Hepatic Gluconeogenic Gene Expression And Histone Modification In Offspring Rats

According to current CDC figures, diabetes rates continue to rise, but the mechanisms behind the in-utero programming of diabetes development are unclear. It has been reported that a maternal high-fat (HF) diet affects fetal liver development, which can lead to altered glucose metabolism in adulthood. In the current study, Obesity Resistant (OR) dams were fed a control (C) or a high-fat (HF) diet during gestation. After cesarean delivery, offspring livers were analyzed to determine the consequences of a maternal HF diet on molecular markers of gluconeogenesis. We demonstrated that while HF-fed dams were not heavier at the end of gestation and did not gain more gestational weight than C dams, HF offspring were heavier and had higher blood glucose at birth than C offspring. Liver mRNA analysis demonstrated that while maternal gluconeogenic genes were not affected by the HF diet, offspring of HF-fed dams had significantly higher expression of hepatic G6Pase, Srebp1c, Cebpα, Pgc1a, and Pck1, which also had an increased transcription rate. Pck1 ChIP analysis showed that a gestational HF diet resulted in epigenetic modifications of the histone code in offspring livers. Our results demonstrate that in-utero exposure to HF diet has the potential to program the gluconeogenic capacity of offspring through epigenetic modifications, which could inevitably lead to altered insulin sensitivity in adulthood. Grant Funding Source: USDA

Over-expression of functional Saccharomyces cerevisiae GUP1, induces proliferation of intracellular membranes containing ER and Golgi resident proteins

High-level expression of the GUP1 gene in Saccharomyces cerevisiae resulted in the formation of proliferated structures, which hosted endoplasmic reticulum (ER), Golgi and itinerant proteins. The GUP1 over-expression enhanced ER biogenesis, as shown by the coordinated increased transcription rate of genes involved in both ER and Golgi metabolism and in phospholipids biosynthesis. The formation of Gup1-induced proliferation revealed that it depended on an intact unfolded protein response, because their assembly was reported to be lethal to yeast strains unable to initiate the UPR (Unfolded Protein Response) pathway. GUP1 over-expression affected global ER and Golgi structure and resulted in the biogenesis of novel membrane arrays with Golgi and ER hybrid composition. In fact, a number of ER and Golgi resident proteins together with itinerant proteins that normally cycle between ER and Golgi, were localized in the proliferated stacked membranes. The described assembling of novel membrane structures was affected by the functionality of the Gup1 O-acyltransferase domain, which regulates the Gup1 protein role as remodelase in the glycosylphosphatidylinositol (GPI) anchored proteins biosynthesis. To our knowledge, we presented the first evidence of sub cellular modifications in response over-expression of a GPI-anchor remodelase in S. cerevisiae.

Polymorphisms in the human glutathione transferase Kappa ( GSTK1) promoter alter gene expression

The level of glutathione transferase Kappa (GSTK1-1) has been correlated with obesity (Liu et.al. 2008 PNAS 105: 18302-7) and a polymorphism in the hGSTK1 promoter has been associated with insulin secretion and fat deposition (Gao et al 2009 Endocr J 56: 487-94). We searched for additional polymorphisms that may influence GSTK1-1 function or expression. Two SNPs were identified in the 5’ non-coding region. A SNP at -1308 that occurs in Chinese subjects is predicted to eliminate a FXR/RXR transcription factor-binding site and causes a 55% increase in transcription rate in HepG2 cells and a 59% decrease in HEK293 cells. These data suggest that the impact of this polymorphism is complex and tissue specific. A SNP at -1032 alters a methylation site and represses transcription by 38%. These observations provide the first functional insight into genetic factors that regulate hGSTK1 expression and may directly influence insulin secretion and fat deposition.

High Expression of the PAX3-FKHR Oncoprotein Is Required to Promote Tumorigenesis of Human Myoblasts

PAX3-FKHR is a fusion oncoprotein generated by the 2;13 chromosomal translocation in alveolar rhabdomyosarcoma (ARMS), a cancer associated with the skeletal muscle lineage. Previous studies determined that high-level PAX3-FKHR expression is a consistent feature in ARMS tumors. To investigate the relationship between expression and phenotype in human myogenic cells, PAX3-FKHR was introduced into immortalized human myoblasts to produce a low overall PAX3-FKHR expression level. Although PAX3-FKHR alone failed to exert transforming activity, a combination of PAX3-FKHR and MYCN induced transforming activity in cell culture assays. Furthermore, myoblasts expressing PAX3-FKHR with or without MYCN formed tumors in SCID mice. These tumors demonstrated invasive features and expressed myogenic markers, consistent with rhabdomyosarcoma. Comparisons of tumor and parental cells revealed that only a subset of parental cells developed into tumors and that tumor cells expressed high PAX3-FKHR levels compared with transduced parental cells. Subcloning of parental PAX3-FKHR/MYCN-transduced myoblasts identified rare high PAX3-FKHR-expressing subclones with high transforming and tumorigenic activity; however, most subclones expressed low PAX3-FKHR and showed neither transforming nor tumorigenic activity. Finally, RNA interference experiments in myoblast-derived tumor and ARMS cells revealed that high PAX3-FKHR expression plays a crucial role in regulating proliferation, transformation, and differentiation. These findings support the premise that high PAX3-FKHR-expressing cells are selected during tumorigenesis.

Carcinoembryonic cell adhesion molecule 6 in human lung: regulated expression of a multifunctional type II cell protein

Carcinoembryonic cell adhesion molecule 6 (CEACAM6) is a glycosylated, glycosylphosphatidylinositol (GPI)-anchored protein expressed in epithelial cells of various human tissues. It binds gram-negative bacteria and is overexpressed in cancers, where it is antiapoptotic and promotes metastases. To characterize CEACAM6 expression in developing lung, we cultured human fetal lung epithelial cells and examined responses to differentiation-promoting hormones, adenovirus expressing thyroid transcription factor-1 (TTF-1), and silencing of TTF-1 with small inhibitory RNA. Glucocorticoid and cAMP had additive stimulatory effects on CEACAM6 content, and combined treatment maximally increased transcription rate, mRNA, and protein approximately 10-fold. Knockdown of TTF-1 reduced hormone induction of CEACAM6 by 80%, and expression of recombinant TTF-1 increased CEACAM6 in a dose-dependent fashion. CEACAM6 content of lung tissue increased during the third trimester and postnatally. By immunostaining, CEACAM6 was present in fetal type II cells, but not mesenchymal cells, and localized to both the plasma membrane and within surfactant-containing lamellar bodies. CEACAM6 was secreted from cultured type II cells and was present in both surfactant and supernatant fractions of infant tracheal aspirates. In functional studies, CEACAM6 reduced inhibition of surfactant surface properties by proteins in vitro and blocked apoptosis of electroporated cultured cells. We conclude that CEACAM6 in fetal lung epithelial cells is developmentally and hormonally regulated and a target protein for TTF-1. Because CEACAM6 acts as an antiapoptotic factor and stabilizes surfactant function, in addition to a putative role in innate defense against bacteria, we propose that it is a multifunctional alveolar protein.

Characterization of the ubiquitin–proteasome system in bortezomib-adapted cells

Resistance towards the proteasome inhibitor bortezomib is poorly understood. We adapted the HL-60, ARH-77 and AMO-1 cell lines (myeloid leukemia, plasmocytoid lymphoma, myeloma) to bortezomib exceeding therapeutic plasma levels, and compared characteristics of the ubiquitin-proteasome system, alternative proteases and the unfolded protein response (UPR) between adapted cells and parental lines. Adapted cells showed increased transcription rates, activities and polypeptide levels of the bortezomib-sensitive beta5, but also of the beta2 proteasome subunit and consistently retained elevated levels of active beta1/beta5-type proteasome subunits in the presence of therapeutic levels of bortezomib. Bortezomib-adapted HL-60 cells showed increased expression and proteasome association of the 11S proteasome activator, and did not accumulate poly-ubiquitinated protein, activate the UPR or UPR-mediated apoptosis in response to bortezomib. The rate of protein biosynthesis was reduced, and the transcription of chaperone genes downmodulated. We did not observe major changes in the activities of TPPII, cathepsins or deubiquitinating proteases. We conclude that different types of bortezomib-adapted cell lines, including myeloma, show similar patterns of changes in the proteasomal machinery which result in residual proteasome activity in the presence of bortezomib and a quantitative balance between protein biosynthesis and destruction.

Gene plasticity in colonic circular smooth muscle cells underlies motility dysfunction in a model of postinfective IBS

The cellular mechanisms of motility dysfunction in postinfectious irritable bowel syndrome (PI-IBS) are not known. We used a rat model of neonatal inflammation to test the hypothesis that gene plasticity in colonic circular smooth muscle cells underlies motility dysfunction in PI-IBS. Mild/moderate or severe inflammation was induced in neonatal and adult rats. Experiments were performed in tissues obtained at 7 days (short term) and 6-8 wk (long term) after the induction of inflammation. Severe inflammation in neonatal rats induced persistent long-term smooth muscle hyperreactivity to acetylcholine (ACh), whereas that in adult rat caused smooth muscle hyporeactivity that showed partial recovery in the long term. Mild/moderate inflammation had no effect in neonatal rats, but it induced smooth muscle hyporeactivity to ACh in adult rats, which recovered fully in the long term. Smooth muscle hyperreactivity to ACh resulted in accelerated colonic transit and increase in defecation rate, whereas hyporeactivity had opposite effects. Smooth muscle hyperreactivity to ACh was associated with increase in transcription rate of key cell-signaling proteins of the excitation-contraction coupling alpha1C subunit of Cav1.2 (L-type) calcium channels, Galphaq, and 20-kDa myosin light chain (MLC20), whereas hyporeactivity was associated with their suppression. Inflammation in adult rats induced classical inflammatory response, which was absent in neonatal rats. Severe neonatal inflammation enhanced plasma norepinephrine and muscularis propria vasoactive intestinal polypeptide in the long term. We conclude that severe, but not mild/moderate, inflammation in a state of immature or impaired stress and immune response systems alters the transcription rate of key cell-signaling proteins of excitation-contraction coupling in colonic circular smooth muscle cells to enhance their contractility and accelerate colonic transit and defecation rate.

The combined immunodetection of AP-2α and YY1 transcription factors is associated with ERBB2 gene overexpression in primary breast tumors

IntroductionOverexpression of the ERBB2 oncogene is observed in about 20% of human breast tumors and is the consequence of increased transcription rates frequently associated with gene amplification. Several studies have shown a link between activator protein 2 (AP-2) transcription factors and ERBB2 gene expression in breast cancer cell lines. Moreover, the Yin Yang 1 (YY1) transcription factor has been shown to stimulate AP-2 transcriptional activity on the ERBB2 promoter in vitro. In this report, we examined the relationships between ERBB2, AP-2α, and YY1 both in breast cancer tissue specimens and in a mammary cancer cell line.MethodsERBB2, AP-2α, and YY1 protein levels were analyzed by immunohistochemistry in a panel of 55 primary breast tumors. ERBB2 gene amplification status was determined by fluorescent in situ hybridization. Correlations were evaluated by a χ2 test at a p value of less than 0.05. The functional role of AP-2α and YY1 on ERBB2 gene expression was analyzed by small interfering RNA (siRNA) transfection in the BT-474 mammary cancer cell line followed by real-time reverse transcription-polymerase chain reaction and Western blotting.ResultsWe observed a statistically significant correlation between ERBB2 and AP-2α levels in the tumors (p < 0.01). Moreover, associations were found between ERBB2 protein level and the combined high expression of AP-2α and YY1 (p < 0.02) as well as between the expression of AP-2α and YY1 (p < 0.001). Furthermore, the levels of both AP-2α and YY1 proteins were inversely correlated to ERBB2 gene amplification status in the tumors (p < 0.01). Transfection of siRNAs targeting AP-2α and AP-2γ mRNAs in the BT-474 breast cancer cell line repressed the expression of the endogenous ERBB2 gene at both the mRNA and protein levels. Moreover, the additional transfection of an siRNA directed against the YY1 transcript further reduced the ERBB2 protein level, suggesting that AP-2 and YY1 transcription factors cooperate to stimulate the transcription of the ERBB2 gene.ConclusionThis study highlights the role of both AP-2α and YY1 transcription factors in ERBB2 oncogene overexpression in breast tumors. Our results also suggest that high ERBB2 expression may result either from gene amplification or from increased transcription factor levels.

Interleukin-3 and erythropoietin cooperate in the regulation of the expression of erythroid-specific transcription factors during erythroid differentiation

To characterize how interleukin-3 and erythropoietin regulate cell fate by modulating the expression of lineage-specific transcription factors. This study analyzed mRNA and protein levels, gene transcription rates, and mRNA and protein stabilities of erythroid-specific transcription factors in lineage-restricted cells derived from the 32D cell line cultured either in interleukin-3 or erythropoietin. Erythroid 32D subclones expressed levels of Idl, Gata-2, and Scl comparable and levels of Eklf and Gata-1 higher than those expressed by myeloid subclones. While maintained in interleukin-3, erythroid cells remained immature despite their high expression of Gata-1, Gata-2, Scl, Eklf, and Idl. Switching the erythroid cells to erythropoietin induced cell maturation (within 48 hours) and reduced expression of Gata-2 and Idl (in 24 hours) but did not alter the expression of Gata-1. The effects of interleukin-3 were mostly mediated by increases in transcription rates (Scl and Gata-2), and that of erythropoietin was apparently due to increased mRNA and protein (Gata-1, Scl, and Eklf) stability. In particular, erythropoietin increased the stability of the processed and transcriptionally more active form of GATA-1 protein. These results suggest that interleukin-3 and erythropoietin cooperate to establish the lineage-specific transcription factor milieu of erythroid cells: interleukin-3 regulates mainly gene transcription and erythropoietin consistently increases mRNA and protein stability.

Angiotensin II Type 1 Receptor Gene Regulation

Angiotensin II (Ang II), the major bioactive peptide of the renin–angiotensin system, was originally described as a potent vasoconstrictor. It is now recognized as a multifunctional hormone influencing many cellular processes, including cell growth, apoptosis, migration, inflammation, and fibrosis.1,2 Under pathological conditions, the vasoconstrictor, mitogenic, proinflammatory, and profibrotic actions of Ang II contribute to altered vascular tone, structural remodeling, and endothelial dysfunction.3–6 Thus, Ang II plays a key role in the pathogenesis of cardiovascular diseases. The biological responses of Ang II are mediated by its interaction with 2 distinct high-affinity G protein–coupled receptors now designated Ang II type 1 receptor (AT1R) and Ang II type 2 receptor.7 Most of the known physiological and pathophysiological effects of Ang II are mediated via the AT1R.1–7 This receptor subtype is expressed in cardiovascular-relevant cell types including vascular smooth muscle cells (VSMCs), endothelial cells, cardiac myocytes, cardiac fibroblasts, and renal mesangial cells.7 The multiple actions of Ang II, mediated through the AT1R, are a result of complex intracellular signaling pathways including stimulation of the phospholipase C/inositol 1,4,5-trisphosphate/diacylglycerol cascade, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases, tyrosine kinases, and RhoA/Rho kinase.1,2,4,6,7 In addition, AT1Rs mediate many of their pathophysiological effects by stimulating reactive oxygen species generation via an reduced nicotinamide-adenine dinucleotide/reduced nicotinamide-adenine dinucleotide phosphate oxidase-dependent mechanism.2,8 Reactive oxygen species, in turn, influences downstream signaling molecules, including transcription factors, tyrosine kinases/phosphatases, Ca2+ channels, and MAPKs.2,8 The expression level of the AT1R defines the biological efficacy of Ang II; hence, overexpression of the AT1R is one potential mechanism by which Ang II can contribute to cardiovascular disease. Importantly, aberrant expression of the AT1R has been shown to have pathophysiological relevance in cell culture, animal studies, …

Matrix Metalloproteinase-2 Expression by Vascular Smooth Muscle Cells Is Mediated by Both Stimulatory and Inhibitory Signals in Response to Growth Factors

In response to growth factors, vascular smooth muscle cells (VSMCs) undergo a phenotypic modulation from a contractile, non-proliferative state to an activated, migratory state. This transition is characterized by changes in their gene expression profile, particularly by a significant down-regulation of contractile proteins. Platelet-derived growth factor (PDGF)-BB has long been known to initiate VSMC de-differentiation and mitogenesis. Insulin-like growth factor (IGF)-I, on the other hand, has differing effects depending on the model studied. Here, we report that both IGF-I and PDGF-BB stimulated VSMC de-differentiation of rat heart-derived SMCs in culture, although only PDGF-BB was capable of inducing proliferation. Although both PDGF-BB and IGF-I stimulation resulted in decreased smooth muscle alpha-actin expression and increased matrix metalloproteinase (MMP)-2 expression, the response to IGF-I was significantly more rapid. The increased MMP-2 expression in response to both growth factors was due to increased transcription rates and was dependent on the action of phosphatidylinositol 3-kinase (PI3K) and its downstream effector, Akt. Both PDGF-BB and IGF-I activated PI3K/Akt to similar degrees; however, only PDGF-BB concomitantly stimulated an inhibitory signaling pathway that antagonized the effects of Akt but did not alter the extent or duration of Akt activation. Together, these findings suggest that changes in MMP-2 expression are part of the program of VSMC phenotypic modulation and that both PDGF-BB and IGF-I, despite their different abilities to induce proliferation in this model, are capable of inducing VSMC activation.

Mitogenic Effects of the Up-Regulation of Minichromosome Maintenance Proteins in Anaplastic Thyroid Carcinoma

Anaplastic thyroid carcinomas (ATC) are among the most aggressive human malignancies and are characterized by high mitotic activity. Minichromosome maintenance proteins (MCM) 2-7 are required to initiate eukaryotic DNA replication, and their overexpression has been associated with dysplasia and malignancy. In an attempt to cast light on the mechanisms governing ATC, we evaluated MCM5 and MCM7 expression in human normal, papillary (PTC), and anaplastic thyroid samples, as well as in primary culture cells and transgenic mouse models. MCM5 and MCM7 expression was high in 65% of ATC and negligible in normal thyroid tissue and papillary thyroid carcinomas. In ATC, high MCM5 and MCM7 expression was paralleled by high levels of MCM2 and MCM6. An analysis of human ATC primary cell cultures and of a transgenic mouse model of ATC confirmed these findings. An increased transcription rate accounted for MCM7 up-regulation, because the activity of the MCM7 promoter was more than 10-fold higher in ATC cells compared with normal thyroid cells. Adoptive overexpression of wild-type p53, but not of its inactive (R248W and R273H) mutants, strongly down-regulated transcription from the MCM7 promoter, suggesting that p53 knock-out contributes to MCM7 up-regulation in ATC. Treatment with small inhibitory duplex RNAs, which decrease MCM7 protein levels, reduced the rate of DNA synthesis in ATC cells. MCM proteins are overexpressed in ATC and sustain the high proliferative capacity of ATC cells.

Chromatin remodeling in plant cell culture: patterns of DNA methylation and histone H3 and H4 acetylation vary during growth of asynchronous potato cell suspensions

Changes in DNA cytosine methylation and core histone multi-acetylation were determined in cell suspension cultures of potato ( Solanum tuberosum L. cv. Russet Burbank) during 15 days of in vitro culture. Cell subculture induced a transient 33% decrease in genome-wide 5-methylcytosine (5mC) content and a transient threefold increase in transcription rates that were most evident at 6 and 9 days after subculture, respectively. In contrast to the global reduction in 5mC content, subculture resulted in a transient twofold increase in 5mC levels within 5′-CCGG-3′ sequences and no detectable change in 5′-CG-3′ methylation. Multi-acetylation of histones H3.1, H3.2 and H4 rose 2-, 1.5- and 3-fold by 9, 9 and 12 days after subculture, respectively. All observed epigenetic changes were reset during aging of cell cultures. Inclusion of the histone deacetylase inhibitor trichostatin A (TSA) and/or the cytosine methylation inhibitor 5-azacytidine (5AC) in culture sequentially decreased genome-wide 5mC levels by ~25% at day 9, then decreased 5′- mC mCGG-3′ by 30–50% and increased H3 and H4 multi-acetylation by 30–60% at day 15, compared to controls. Treatment with 5AC or TSA alone or in combination had no effect on RNA synthesis at day 9. At day 15, 5AC treatment remained ineffective, while de novo RNA synthesis was approximately twofold higher in cells grown in both inhibitors or in TSA alone. Collectively, these results demonstrate that in potato suspension cultures, rapid, reversible changes in 5mC levels precede regulatory post-translational acetylation of core histones, and suggest that interactions between these epigenetic processes appear to be necessary to power transcription and growth induction in potato cells.