Translational independence between overlapping genes for a restriction endonuclease and its transcriptional regulator

<p>Abstract</p> <p>Background</p> <p>Most type II restriction-modification (RM) systems have two independent enzymes that act on the same DNA sequence: a modification methyltransferase that protects target sites, and a restriction endonuclease that cleaves unmethylated target sites. When RM genes enter a new cell, methylation must occur before restriction activity appears, or the host's chromosome is digested. Transcriptional mechanisms that delay endonuclease expression have been identified in some RM systems. A substantial subset of those systems is controlled by a family of small transcription activators called C proteins. In the PvuII system, C.PvuII activates transcription of its own gene, along with that of the downstream endonuclease gene. This regulation results in very low R.PvuII mRNA levels early after gene entry, followed by rapid increase due to positive feedback. However, given the lethal consequences of premature REase accumulation, transcriptional control alone might be insufficient. In C-controlled RM systems, there is a ± 20 nt overlap between the C termination codon and the R (endonuclease) initiation codon, suggesting possible translational coupling, and in many cases predicted RNA hairpins could occlude the ribosome binding site for the endonuclease gene.</p> <p>Results</p> <p>Expression levels of <it>lacZ </it>translational fusions to <it>pvuIIR </it>or <it>pvuIIC </it>were determined, with the native <it>pvuII </it>promoter having been replaced by one not controlled by C.PvuII. In-frame <it>pvuIIC </it>insertions did not substantially decrease either <it>pvuIIC-lacZ </it>or <it>pvuIIR-lacZ </it>expression (with or without C.PvuII provided <it>in trans</it>). In contrast, a frameshift mutation in <it>pvuIIC </it>decreased expression markedly in both fusions, but mRNA measurements indicated that this decrease could be explained by transcriptional polarity. Expression of <it>pvuIIR-lacZ </it>was unaffected when the <it>pvuIIC </it>stop codon was moved 21 nt downstream from its WT location, or 25 or 40 bp upstream of the <it>pvuIIR </it>initiation codon. Disrupting the putative hairpins had no significant effects.</p> <p>Conclusions</p> <p>The initiation of translation of <it>pvuIIR </it>appears to be independent of that for <it>pvuIIC</it>. Direct tests failed to detect regulatory rules for either gene overlap or the putative hairpins. Thus, at least during balanced growth, transcriptional control appears to be sufficiently robust for proper regulation of this RM system.</p

Forced Hepatic Overexpression of CEACAM1 Curtails Diet-Induced Insulin Resistance

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) regulates insulin sensitivity by promoting hepatic insulin clearance. Liver-specific inactivation or global null-mutation of Ceacam1 impairs hepatic insulin extraction to cause chronic hyperinsulinemia, resulting in insulin resistance and visceral obesity. In this study we investigated whether diet-induced insulin resistance implicates changes in hepatic CEACAM1. We report that feeding C57/BL6J mice a high-fat diet reduced hepatic CEACAM1 levels by >50% beginning at 21 days, causing hyperinsulinemia, insulin resistance, and elevation in hepatic triacylglycerol content. Conversely, liver-specific inducible CEACAM1 expression prevented hyperinsulinemia and markedly limited insulin resistance and hepatic lipid accumulation that were induced by prolonged high-fat intake. This was partly mediated by increased hepatic β-fatty acid oxidation and energy expenditure. The data demonstrate that the high-fat diet reduced hepatic CEACAM1 expression and that overexpressing CEACAM1 in liver curtailed diet-induced metabolic abnormalities by protecting hepatic insulin clearance

A Polymorphism in Intron I of the Human Angiotensinogen Gene (hAGT) Affects Binding by HNF3 and hAGT Expression and Increases Blood Pressure in Mice

Angiotensinogen (AGT) is the precursor of one of the most potent vasoconstrictors, peptide angiotensin-II. Genome-wide association studies (GWAS) have shown that two A/G polymorphisms (rs2493134 and rs2004776) located at +507 and +1164 in intron I of human AGT (hAGT) gene are associated with hypertension. Polymorphisms of the AGT gene result in two main haplotypes. Hap-I contains the variants -217A, -6A, +507G, and +1164A and is pro-hypertensive, whereas Hap-II contains the variants -217G, -6G, +507A, and +1164G and does not affect blood pressure. The nucleotide sequence of intron I of the hAGT gene containing the +1164A variant has a stronger homology with hepatocyte nuclear factor 3 (HNF3)-binding site than does +1164G. Here, we found that (a) an oligonucleotide containing +1164A binds HNF3beta more strongly than does +1164G, and (b) Hap I-containing reporter gene constructs have increased basal and HNF3- and glucocorticoid-induced promoter activity in transiently transfected liver and kidney cells. Using a knock-in approach at the HPRT locus, we generated transgenic mouse model containing the human renin (hREN) gene and either Hap-I or Hap-II. We show that transgenic animals containing Hap-I have increased blood pressure compared with those containing Hap-II. Moreover, the transcription factors glucocorticoid receptor (GR), CCAAT enhancer-binding protein beta (C/EBPbeta), and HNF3beta bound more strongly to chromatin obtained from the liver of transgenic animals containing Hap-I than to liver chromatin from Hap-II-containing animals. These findings suggest that unlike Hap-II variants, Hap-I variants of the hAGT gene have increased transcription rates, resulting in elevated blood pressure

High-calorie diet exacerbates prostate neoplasia in mice with haploinsufficiency of Pten tumor suppressor gene

Objective: Association between prostate cancer and obesity remains controversial. Allelic deletions of PTEN, a tumor suppressor gene, are common in prostate cancer in men. Monoallelic Pten deletion in mice causes low prostatic intraepithelial neoplasia (mPIN). This study tested the effect of a hypercaloric diet on prostate cancer in Pten+/− mice. Methods: 1-month old mice were fed a high-calorie diet deriving 45% calories from fat for 3 and 6 months before prostate was analyzed histologically and biochemically for mPIN progression. Because Pten+/− mice are protected against diet-induced insulin resistance, we tested the role of insulin on cell growth in RWPE-1 normal human prostatic epithelial cells with siRNA knockdown of PTEN. Results: In addition to activating PI3 kinase/Akt and Ras/MAPkinase pathways, high-calorie diet causes neoplastic progression, angiogenesis, inflammation and epithelial–mesenchymal transition. It also elevates the expression of fatty acid synthase (FAS), a lipogenic gene commonly elevated in progressive cancer. SiRNA-mediated downregulation of PTEN demonstrates increased cell growth and motility, and soft agar clonicity in addition to elevation in FAS in response to insulin in RWPE-1 normal human prostatic cells. Downregulating FAS in addition to PTEN, blunted the proliferative effect of insulin (and IL-6) in RWPE-1 cells. Conclusion: High-calorie diet promotes prostate cancer progression in the genetically susceptible Pten haploinsufficient mouse while preserving insulin sensitivity. This appears to be partly due to increased inflammatory response to high-caloric intake in addition to increased ability of insulin to promote lipogenesis

Estrogen receptor activation reduces lipid synthesis in pancreatic islets and prevents β cell failure in rodent models of type 2 diabetes

The failure of pancreatic β cells to adapt to an increasing demand for insulin is the major mechanism by which patients progress from insulin resistance to type 2 diabetes (T2D) and is thought to be related to dysfunctional lipid homeostasis within those cells. In multiple animal models of diabetes, females demonstrate relative protection from β cell failure. We previously found that the hormone 17β-estradiol (E2) in part mediates this benefit. Here, we show that treating male Zucker diabetic fatty (ZDF) rats with E2 suppressed synthesis and accumulation of fatty acids and glycerolipids in islets and protected against β cell failure. The antilipogenic actions of E2 were recapitulated by pharmacological activation of estrogen receptor α (ERα) or ERβ in a rat β cell line and in cultured ZDF rat, mouse, and human islets. Pancreas-specific null deletion of ERα in mice (PERα–/–) prevented reduction of lipid synthesis by E2 via a direct action in islets, and PERα–/– mice were predisposed to islet lipid accumulation and β cell dysfunction in response to feeding with a high-fat diet. ER activation inhibited β cell lipid synthesis by suppressing the expression (and activity) of fatty acid synthase via a nonclassical pathway dependent on activated Stat3. Accordingly, pancreas-specific deletion of Stat3 in mice curtailed ER-mediated suppression of lipid synthesis. These data suggest that extranuclear ERs may be promising therapeutic targets to prevent β cell failure in T2D

Caloric Restriction Reverses Hepatic Insulin Resistance and Steatosis in Rats with Low Aerobic Capacity

Rats selectively bred for low aerobic running capacity exhibit the metabolic syndrome, including hyperinsulinemia, insulin resistance, visceral obesity, and dyslipidemia. They also exhibit features of nonalcoholic steatohepatitis, including chicken-wire fibrosis, inflammation, and oxidative stress. Hyperinsulinemia in these rats is associated with impaired hepatic insulin clearance. The current studies aimed to determine whether these metabolic abnormalities could be reversed by caloric restriction (CR). CR by 30% over a period of 2–3 months improved insulin clearance in parallel to inducing the protein content and activation of the carcinoembryonic antigen-related cell adhesion molecule 1, a main player in hepatic insulin extraction. It also reduced glucose and insulin intolerance and serum and tissue (liver and muscle) triglyceride levels. Additionally, CR reversed inflammation, oxidative stress, and fibrosis in liver. The data support a significant role of CR in the normalization of insulin and lipid metabolism in liver