CHD1 Remodels Chromatin and Influences Transient DNA Methylation at the Clock Gene frequency

Circadian-regulated gene expression is predominantly controlled by a transcriptional negative feedback loop, and it is evident that chromatin modifications and chromatin remodeling are integral to this process in eukaryotes. We previously determined that multiple ATP–dependent chromatin-remodeling enzymes function at frequency (frq). In this report, we demonstrate that the Neurospora homologue of chd1 is required for normal remodeling of chromatin at frq and is required for normal frq expression and sustained rhythmicity. Surprisingly, our studies of CHD1 also revealed that DNA sequences within the frq promoter are methylated, and deletion of chd1 results in expansion of this methylated domain. DNA methylation of the frq locus is altered in strains bearing mutations in a variety of circadian clock genes, including frq, frh, wc-1, and the gene encoding the frq antisense transcript (qrf). Furthermore, frq methylation depends on the DNA methyltransferase, DIM-2. Phenotypic characterization of Δdim-2 strains revealed an approximate WT period length and a phase advance of approximately 2 hours, indicating that methylation plays only an ancillary role in clock-regulated gene expression. This suggests that DNA methylation, like the antisense transcript, is necessary to establish proper clock phasing but does not control overt rhythmicity. These data demonstrate that the epigenetic state of clock genes is dependent on normal regulation of clock components

Systems Biology of the Clock in Neurospora crassa

A model-driven discovery process, Computing Life, is used to identify an ensemble of genetic networks that describe the biological clock. A clock mechanism involving the genes white-collar-1 and white-collar-2 (wc-1 and wc-2) that encode a transcriptional activator (as well as a blue-light receptor) and an oscillator frequency (frq) that encodes a cyclin that deactivates the activator is used to guide this discovery process through three cycles of microarray experiments. Central to this discovery process is a new methodology for the rational design of a Maximally Informative Next Experiment (MINE), based on the genetic network ensemble. In each experimentation cycle, the MINE approach is used to select the most informative new experiment in order to mine for clock-controlled genes, the outputs of the clock. As much as 25% of the N. crassa transcriptome appears to be under clock-control. Clock outputs include genes with products in DNA metabolism, ribosome biogenesis in RNA metabolism, cell cycle, protein metabolism, transport, carbon metabolism, isoprenoid (including carotenoid) biosynthesis, development, and varied signaling processes. Genes under the transcription factor complex WCC ( = WC-1/WC-2) control were resolved into four classes, circadian only (612 genes), light-responsive only (396), both circadian and light-responsive (328), and neither circadian nor light-responsive (987). In each of three cycles of microarray experiments data support that wc-1 and wc-2 are auto-regulated by WCC. Among 11,000 N. crassa genes a total of 295 genes, including a large fraction of phosphatases/kinases, appear to be under the immediate control of the FRQ oscillator as validated by 4 independent microarray experiments. Ribosomal RNA processing and assembly rather than its transcription appears to be under clock control, suggesting a new mechanism for the post-transcriptional control of clock-controlled genes

Cross-Talk between the Cellular Redox State and the Circadian System in Neurospora

The circadian system is composed of a number of feedback loops, and multiple feedback loops in the form of oscillators help to maintain stable rhythms. The filamentous fungus Neurospora crassa exhibits a circadian rhythm during asexual spore formation (conidiation banding) and has a major feedback loop that includes the FREQUENCY (FRQ)/WHITE COLLAR (WC) -1 and -2 oscillator (FWO). A mutation in superoxide dismutase (sod)-1, an antioxidant gene, causes a robust and stable circadian rhythm compared with that of wild-type (Wt). However, the mechanisms underlying the functions of reactive oxygen species (ROS) remain unknown. Here, we show that cellular ROS concentrations change in a circadian manner (ROS oscillation), and the amplitudes of ROS oscillation increase with each cycle and then become steady (ROS homeostasis). The ROS oscillation and homeostasis are produced by the ROS-destroying catalases (CATs) and ROS-generating NADPH oxidase (NOX). cat-1 is also induced by illumination, and it reduces ROS levels. Although ROS oscillation persists in the absence of frq, wc-1 or wc-2, its homeostasis is altered. Furthermore, genetic and biochemical evidence reveals that ROS concentration regulates the transcriptional function of WCC and a higher ROS concentration enhances conidiation banding. These findings suggest that the circadian system engages in cross-talk with the cellular redox state via ROS-regulatory factors

Morphological and Pathological Evolution of the Brain Microcirculation in Aging and Alzheimer’s Disease

Key pathological hallmarks of Alzheimer’s disease (AD), including amyloid plaques, cerebral amyloid angiopathy (CAA) and neurofibrillary tangles do not completely account for cognitive impairment, therefore other factors such as cardiovascular and cerebrovascular pathologies, may contribute to AD. In order to elucidate the microvascular changes that contribute to aging and disease, direct neuropathological staining and immunohistochemistry, were used to quantify the structural integrity of the microvasculature and its innervation in three oldest-old cohorts: 1) nonagenarians with AD and a high amyloid plaque load; 2) nonagenarians with no dementia and a high amyloid plaque load; 3) nonagenarians without dementia or amyloid plaques. In addition, a non-demented (ND) group (average age 71 years) with no amyloid plaques was included for comparison. While gray matter thickness and overall brain mass were reduced in AD compared to ND control groups, overall capillary density was not different. However, degenerated string capillaries were elevated in AD, potentially suggesting greater microvascular “dysfunction” compared to ND groups. Intriguingly, apolipoprotein ε4 carriers had significantly higher string vessel counts relative to non-ε4 carriers. Taken together, these data suggest a concomitant loss of functional capillaries and brain volume in AD subjects. We also demonstrated a trend of decreasing vesicular acetylcholine transporter staining, a marker of cortical cholinergic afferents that contribute to arteriolar vasoregulation, in AD compared to ND control groups, suggesting impaired control of vasodilation in AD subjects. In addition, tyrosine hydroxylase, a marker of noradrenergic vascular innervation, was reduced which may also contribute to a loss of control of vasoconstriction. The data highlight the importance of the brain microcirculation in the pathogenesis and evolution of AD

Interplay of Substrate Retention and Export Signals in Endoplasmic Reticulum Quality Control

BACKGROUND: Endoplasmic reticulum (ER) quality control mechanisms are part of a comprehensive system to manage cell stress. The flux of molecules is monitored to retain folding intermediates and target misfolded molecules to ER-associated degradation (ERAD) pathways. The mechanisms of sorting remain unclear. While some proteins are retained statically, the classical model substrate CPY* is found in COPII transport vesicles, suggesting a retrieval mechanism for retention. However, its management can be even more dynamic. If ERAD is saturated under stress, excess CPY* traffics to the vacuole for degradation. These observations suggest that misfolded proteins might display different signals for their management. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report the existence of a functional ER exit signal in the pro-domain of CPY*. Compromising its integrity causes ER retention through exclusion from COPII vesicles. The signal co-exists with other signals used for retention and degradation. Physiologically, the export signal is important for stress tolerance. Disabling it converts a benign protein into one that is intrinsically cytotoxic. CONCLUSIONS/SIGNIFICANCE: These data reveal the remarkable interplay between opposing signals embedded within ERAD substrate molecules and the mechanisms that decipher them. Our findings demonstrate the diversity of mechanisms deployed for protein quality control and maintenance of protein homeostasis

The Intracellular Transport and Secretion of Calumenin-1/2 in Living Cells

Calumenin isoforms 1 and 2 (calu-1/2), encoded by the CALU gene, belong to the CREC protein family. Calu-1/2 proteins are secreted into the extracellular space, but the secretory process and regulatory mechanism are largely unknown. Here, using a time-lapse imaging system, we visualized the intracellular transport and secretory process of calu-1/2-EGFP after their translocation into the ER lumen. Interestingly, we observed that an abundance of calu-1/2-EGFP accumulated in cellular processes before being released into the extracellular space, while only part of calu-1/2-EGFP proteins were secreted directly after attaching to the cell periphery. Moreover, we found the secretion of calu-1/2-EGFP required microtubule integrity, and that calu-1/2-EGFP-containing vesicles were transported by the motor proteins Kif5b and cytoplasmic dynein. Finally, we determined the export signal of calu-1/2-EGFP (amino acid positions 20–46) and provided evidence that the asparagine at site 131 was indispensable for calu-1/2-EGFP stabilization. Taken together, we provide a detailed picture of the intracellular transport of calu-1/2-EGFP, which facilitates our understanding of the secretory mechanism of calu-1/2

Comparative Proteomic Analysis of the PhoP Regulon in Salmonella enterica Serovar Typhi Versus Typhimurium

Background: S. Typhi, a human-restricted Salmonella enterica serovar, causes a systemic intracellular infection in humans (typhoid fever). In comparison, S. Typhimurium causes gastroenteritis in humans, but causes a systemic typhoidal illness in mice. The PhoP regulon is a well studied two component (PhoP/Q) coordinately regulated network of genes whose expression is required for intracellular survival of S. enterica. Methodology/Principal Findings: Using high performance liquid chromatography mass spectrometry (HPLC-MS/MS), we examined the protein expression profiles of three sequenced S. enterica strains: S. Typhimurium LT2, S. Typhi CT18, and S. Typhi Ty2 in PhoP-inducing and non-inducing conditions in vitro and compared these results to profiles of $phoP^−/Q^−$ mutants derived from S. Typhimurium LT2 and S. Typhi Ty2. Our analysis identified 53 proteins in S. Typhimurium LT2 and 56 proteins in S. Typhi that were regulated in a PhoP-dependent manner. As expected, many proteins identified in S. Typhi demonstrated concordant differential expression with a homologous protein in S. Typhimurium. However, three proteins (HlyE, STY1499, and CdtB) had no homolog in S. Typhimurium. HlyE is a pore-forming toxin. STY1499 encodes a stably expressed protein of unknown function transcribed in the same operon as HlyE. CdtB is a cytolethal distending toxin associated with DNA damage, cell cycle arrest, and cellular distension. Gene expression studies confirmed up-regulation of mRNA of HlyE, STY1499, and CdtB in S. Typhi in PhoP-inducing conditions. Conclusions/Significance: This study is the first protein expression study of the PhoP virulence associated regulon using strains of Salmonella mutant in PhoP, has identified three Typhi-unique proteins (CdtB, HlyE and STY1499) that are not present in the genome of the wide host-range Typhimurium, and includes the first protein expression profiling of a live attenuated bacterial vaccine studied in humans (Ty800)

A Gain-of-Function Germline Mutation in Drosophila ras1 Affects Apoptosis and Cell Fate during Development

The RAS/MAPK signal transduction pathway is an intracellular signaling cascade that transmits environmental signals from activated receptor tyrosine kinases (RTKs) on the cell surface and other endomembranes to transcription factors in the nucleus, thereby linking extracellular stimuli to changes in gene expression. Largely as a consequence of its role in oncogenesis, RAS signaling has been the subject of intense research efforts for many years. More recently, it has been shown that milder perturbations in Ras signaling during embryogenesis also contribute to the etiology of a group of human diseases. Here we report the identification and characterization of the first gain-of-function germline mutation in Drosophila ras1 (ras85D), the Drosophila homolog of human K-ras, N-ras and H-ras. A single amino acid substitution (R68Q) in the highly conserved switch II region of Ras causes a defective protein with reduced intrinsic GTPase activity, but with normal sensitivity to GAP stimulation. The ras1R68Q mutant is homozygous viable but causes various developmental defects associated with elevated Ras signaling, including cell fate changes and ectopic survival of cells in the nervous system. These biochemical and functional properties are reminiscent of germline Ras mutants found in patients afflicted with Noonan, Costello or cardio-facio-cutaneous syndromes. Finally, we used ras1R68Q to identify novel genes that interact with Ras and suppress cell death