The frequency natural antisense transcript first promotes, then represses, frequency gene expression via facultative heterochromatin.

The circadian clock is controlled by a network of interconnected feedback loops that require histone modifications and chromatin remodeling. Long noncoding natural antisense transcripts (NATs) originate from Period in mammals and frequency (frq) in Neurospora. To understand the role of NATs in the clock, we put the frq antisense transcript qrf (frq spelled backwards) under the control of an inducible promoter. Replacing the endogenous qrf promoter altered heterochromatin formation and DNA methylation at frq. In addition, constitutive, low-level induction of qrf caused a dramatic effect on the endogenous rhythm and elevated circadian output. Surprisingly, even though qrf is needed for heterochromatic silencing, induction of qrf initially promoted frq gene expression by creating a more permissible local chromatin environment. The observation that antisense expression can initially promote sense gene expression before silencing via heterochromatin formation at convergent loci is also found when a NAT to hygromycin resistance gene is driven off the endogenous vivid (vvd) promoter in the Δvvd strain. Facultative heterochromatin silencing at frq functions in a parallel pathway to previously characterized VVD-dependent silencing and is needed to establish the appropriate circadian phase. Thus, repression via dicer-independent siRNA-mediated facultative heterochromatin is largely independent of, and occurs alongside, other feedback processes.

The histone H3 lysine 9 methyltransferase DIM-5 modifies chromatin at frequency and represses light-activated gene expression.

The transcriptional program controlling the circadian rhythm requires coordinated regulation of chromatin. Characterization of the chromodomain helicase DNA-binding enzyme CHD1 revealed DNA methylation in the promoter of the central clock gene frequency (frq) in Neurospora crassa. In this report, we show that the DNA methylation at frq is not only dependent on the DNA methyltransferase DIM-2 but also on the H3K9 methyltransferase DIM-5 and HP1. Histone H3 lysine 9 trimethylation (H3K9me3) occurs at frq and is most prominent 30 min after light-activated expression. Strains lacking dim-5 have an increase in light-induced transcription, and more White Collar-2 is found associated with the frq promoter. Consistent with the notion that DNA methylation assists in establishing the proper circadian phase, loss of H3K9 methylation results in a phase advance suggesting it delays the onset of frq expression. The dim-5 deletion strain displays an increase in circadian-regulated conidia formation on race tubes and there is a synthetic genetic interaction between dim-5 and ras-1(bd). These results indicate DIM-5 has a regulatory role in muting circadian output. Overall, the data support a model where facultative heterochromatic at frq serves to establish the appropriate phase, mute the light response, and repress circadian output.

Regulated DNA methylation and the circadian clock: implications in cancer.

Since the cloning and discovery of DNA methyltransferases (DNMT), there has been a growing interest in DNA methylation, its role as an epigenetic modification, how it is established and removed, along with the implications in development and disease. In recent years, it has become evident that dynamic DNA methylation accompanies the circadian clock and is found at clock genes in Neurospora, mice and cancer cells. The relationship among the circadian clock, cancer and DNA methylation at clock genes suggests a correlative indication that improper DNA methylation may influence clock gene expression, contributing to the etiology of cancer. The molecular mechanism underlying DNA methylation at clock loci is best studied in the filamentous fungi, Neurospora crassa, and recent data indicate a mechanism analogous to the RNA-dependent DNA methylation (RdDM) or RNAi-mediated facultative heterochromatin. Although it is still unclear, DNA methylation at clock genes may function as a terminal modification that serves to prevent the regulated removal of histone modifications. In this capacity, aberrant DNA methylation may serve as a readout of misregulated clock genes and not as the causative agent. This review explores the implications of DNA methylation at clock loci and describes what is currently known regarding the molecular mechanism underlying DNA methylation at circadian clock genes.

A universal cloning method based on yeast homologous recombination that is simple, efficient, and versatile.

Cloning by homologous recombination (HR) in Saccharomyces cerevisiae is an extremely efficient and cost-effective alternative to other methods of recombinant DNA technologies. Unfortunately, it is incompatible with all the various specialized plasmids currently used in microbiology and biomedical research laboratories, and is therefore, not widely adopted. In an effort to dramatically improve the versatility of yeast gap-repair cloning and make it compatible with any DNA plasmid, we demonstrate that by simply including a yeast-cloning cassette (YCC) that contains the 2-micron origin of replication (2µm ori) and the ura3 gene for selection, multiple DNA fragments can be assembled into any DNA vector. We show this has almost unlimited potential by building a variety of plasmid for different uses including: recombinant protein production, epitope tagging, site-directed mutagenesis, and expression of fluorescent fusion proteins. We demonstrate the use in a variety of plasmids for use in microbial systems and even demonstrate it can be used in a vertebrate model. This method is remarkably simple and extremely efficient, plus it provides a significant cost saving over commercially available kits.

Synthetic and crystallographic studies of a new inhibitor series targeting Bacillus anthracis dihydrofolate reductase.

Bacillus anthracis, the causative agent of anthrax, poses a significant biodefense danger. Serious limitations in approved therapeutics and the generation of resistance have produced a compelling need for new therapeutic agents against this organism. Bacillus anthracis is known to be insensitive to the clinically used antifolate, trimethoprim, because of a lack of potency against the dihydrofolate reductase enzyme. Herein, we describe a novel lead series of B. anthracis dihydrofolate reductase inhibitors characterized by an extended trimethoprim-like scaffold. The best lead compound adds only 22 Da to the molecular weight and is 82-fold more potent than trimethoprim. An X-ray crystal structure of this lead compound bound to B. anthracis dihydrofolate reductase in the presence of NADPH was determined to 2.25 A resolution. The structure reveals several features that can be exploited for further development of this lead series.

Highly efficient ligands for dihydrofolate reductase from Cryptosporidium hominis and Toxoplasma gondii inspired by structural analysis.

The search for effective therapeutics for cryptosporidiosis and toxoplasmosis has led to the discovery of novel inhibitors of dihydrofolate reductase (DHFR) that possess high ligand efficiency: compounds with high potency and low molecular weight. Detailed analysis of the crystal structure of dihydrofolate reductase-thymidylate synthase from Cryptosporidium hominis and a homology model of DHFR from Toxoplasma gondii inspired the synthesis of a new series of compounds with a propargyl-based linker between a substituted 2,4-diaminopyrimidine and a trimethoxyphenyl ring. An enantiomerically pure compound in this series exhibits IC50 values of 38 and 1 nM against C. hominis and T. gondii DHFR, respectively. Improvements of 368-fold or 5714-fold (C. hominis and T. gondii) relative to trimethoprim were generated by synthesizing just 14 new analogues and by adding only a total of 52 Da to the mass of the parent compound, creating an efficient ligand as an excellent candidate for further study.

Structure-activity relationships of Bacillus cereus and Bacillus anthracis dihydrofolate reductase: toward the identification of new potent drug leads.

New and improved therapeutics are needed for Bacillus anthracis, the etiological agent of anthrax. To date, antimicrobial agents have not been developed against the well-validated target dihydrofolate reductase (DHFR). In order to address whether DHFR inhibitors could have potential use as clinical agents against Bacillus, 27 compounds were screened against this enzyme from Bacillus cereus, which is identical to the enzyme from B. anthracis at the active site. Several 2,4-diamino-5-deazapteridine compounds exhibit submicromolar 50% inhibitory concentrations (IC(50)s). Four of the inhibitors displaying potency in vitro were tested in vivo and showed a marked growth inhibition of B. cereus; the most potent of these has MIC(50) and minimum bactericidal concentrations at which 50% are killed of 1.6 mug/ml and 0.09 mug/ml, respectively. In order to illustrate structure-activity relationships for the classes of inhibitors tested, each of the 27 inhibitors was docked into homology models of the B. cereus and B. anthracis DHFR proteins, allowing the development of a rationale for the inhibition profiles. A combination of favorable interactions with the diaminopyrimidine and substituted phenyl rings explains the low IC(50) values of potent inhibitors; steric interactions explain higher IC(50) values. These experiments show that DHFR is a reasonable antimicrobial target for Bacillus anthracis and that there is a class of inhibitors that possess sufficient potency and antibacterial activity to suggest further development.

Doxorubicin cardiotoxicity: prevention of congestive heart failure with serial cardiac function monitoring with equilibrium radionuclide angiocardiography in the current era.

BACKGROUND: Congestive heart failure (CHF) is among the most serious toxicities of doxorubicin, a potent cancer chemotherapeutic agent. Serial left ventricular ejection fraction (LVEF) monitoring during doxorubicin therapy for preventing CHF was proposed over 20 years ago. The current utility and cost-effectiveness of this approach in the present era are not known. METHODS AND RESULTS: Clinical and follow-up data of 265 patients with cancer (age, 53 +/- 14 years; 76% women) undergoing doxorubicin chemotherapy with serial equilibrium radionuclide angiocardiography (ERNA) monitoring (> or =2 studies) were analyzed retrospectively. Patients with a normal baseline LVEF (> or =50%) and a 10% or greater point fall in LVEF to a final value of less than 50% during doxorubicin therapy were considered "at risk" for CHF (n = 41). Over 679 +/- 426 days of follow-up, 7 patients (2.6%) had CHF develop and 90 (34%) died (all cancer-related deaths, with none due to CHF). A comparison of "at-risk" (n = 41 [15%]) and "low-risk" (n = 224 [85%]) groups showed a higher incidence of CHF (12% vs 0.9%, P <.0001), lower baseline LVEF (58% +/- 8% vs 64% +/- 8%, P <.0001), lower value for the lowest LVEF (42% +/- 8% vs 57% +/- 7%, P <.0001), and higher rate of cancer-related deaths (59% vs 29%, P =.0003) in the former despite similar cumulative doxorubicin dose (304 +/- 124 mg/m(2) vs 284 +/- 110 mg/m(2), P = not significant). There were no differences in age, gender, cancer type, and co-morbidity. Cost analysis showed the overall cost of ERNA studies to be lower than the 1-year cost of caring for additional cases of CHF that would potentially be prevented by routine LVEF monitoring. CONCLUSIONS: An incipient fall in LVEF detected on serial ERNA during doxorubicin therapy provides an appropriate and cost-effective approach for predicting and preventing impending CHF. Use of this approach was associated with a low incidence of CHF (2.6%) and no CHF-related mortality in this study.

Pharmacologic stress perfusion imaging with adenosine: role of simultaneous low-level treadmill exercise.

BACKGROUND: Adenosine is commonly used for pharmacologic stress myocardial perfusion imaging (MPI). However, it frequently results in adverse effects, and the subdiaphragmatic tracer uptake may interfere with the image interpretation. Our aim was to determine the feasibility of combining low-level treadmill exercise with adenosine MPI and its impact on adverse effects, image quality, and myocardial ischemia. METHODS AND RESULTS: Forty-one patients underwent technetium 99m sestamibi single photon emission computed tomography following adenosine and adenosine with low-level exercise (adenosine-Ex) on separate occasions and rest MPI. A comparison was made of symptoms, hemodynamic response, electrocardiographic changes, image quality, and image interpretation between the 2 protocols. With adenosine-Ex, fewer patients had one or more adverse effects (61% vs 90%; P =.006), more patients had ischemic electrocardiographic changes (34% vs 15%; P =.03), a higher percentage had excellent- or fair-quality images (88% vs 61%; P =.003), and they had higher heart-liver ratios (1.0 +/- 0.37 vs 0.84 +/- 0.29; P =.002) compared with adenosine alone. Four adenosine MPI studies, but only 2 adenosine-Ex studies, were uninterpretable because of excessive subdiaphragmatic radiotracer activity. Of the 39 patients with at least 1 interpretable stress study, interpretation was discordant in 11 (28%): 7 showed greater ischemia with adenosine-Ex, 2 uninterpretable adenosine studies were interpretable with adenosine-Ex, and 2 studies interpreted as abnormal with adenosine were normal by adenosine-Ex (both had normal coronary angiograms). CONCLUSIONS: Simultaneous low-level treadmill exercise with adenosine Tc-99m sestamibi imaging is safe and feasible, significantly reduces unfavorable side effects, enhances image quality, and may result in greater ischemia detection compared with adenosine alone.