Epigenetic control of the vasopressin promoter explains physiological ability to regulate vasopressin transcription in dehydration and salt loading states in the rat

The synthesis of arginine vasopressin (AVP) in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus is sensitive to increased plasma osmolality and a decreased blood volume, and thus is robustly increased by both dehydration (increased plasma osmolality and decreased blood volume) and salt loading (increased plasma osmolality). Both stimuli result in functional remodelling of the SON and PVN, a process referred to as functional‐related plasticity. Such plastic changes in the brain have recently been associated with altered patterns of DNA methylation at CpG (cytosine‐phosphate‐guanine) residues, a process considered to be important for the regulation of gene transcription. In this regard, the proximal Avp promoter contains a number of CpG sites and is recognised as one of four CpG islands for the Avp gene, suggesting that methylation may be regulating Avp transcription. In the present study, we show that, in an immortalised hypothalamic cell line 4B, the proximal Avp promoter is highly methylated, and treatment of these cells with the DNA methyltransferase inhibitor 5‐Aza‐2′‐deoxycytidine to demethylate DNA dramatically increases basal and stimulated Avp biosynthesis. We report no changes in the expression of DNA methyltransferases, Dnmt1 and Dnmt3a, whereas there is decreased expression of the demethylating enzyme ten‐eleven‐translocation 2, Tet2, in the SON by dehydration and salt loading. We found higher methylation of the SON Avp promoter in dehydrated but not salt‐loaded rats. By analysis of individual CpG sites, we observed hypomethylation, hypermethylation and no change in methylation of specific CpGs in the SON Avp promoter of the dehydrated rat. Using reporter gene assays, we show that mutation of individual CpGs can result in altered Avp promoter activity. We propose that methylation of the SON Avp promoter is necessary to co‐ordinate the duel inputs of increased plasma osmolality and decreased blood volume on Avp transcription in the chronically dehydrated rat

Transcription factor Creb3l1 maintains proteostasis in neuroendocrine cells

OBJECTIVES: Dynamic changes to neuropeptide hormone synthesis and secretion by hypothalamic neuroendocrine cells is essential to ensure metabolic homeostasis. The specialised molecular mechanisms that allow neuroendocrine cells to synthesise and secrete vast quantities of neuropeptides remain ill defined. The objective of this study was to identify novel genes and pathways controlled by transcription factor and endoplasmic reticulum stress sensor Creb3l1 which is robustly activated in hypothalamic magnocellular neurones in response to increased demand for protein synthesis. METHODS: We adopted a multiomic strategy to investigate specific roles of Creb3l1 in rat magnocellular neurones. We first performed chromatin immunoprecipitation followed by genome sequencing (ChIP-seq) to identify Creb3l1 genomic targets and then integrated this data with RNA sequencing data from physiologically stimulated and Creb3l1 knockdown magnocellular neurones. RESULTS: The data converged on Creb3l1 targets that code for ribosomal proteins and endoplasmic reticulum proteins crucial for the maintenance of cellular proteostasis. We validated genes that compose the PERK arm of the unfolded protein response pathway including Eif2ak3, Eif2s1, Atf4 and Ddit3 as direct Creb3l1 targets. Importantly, knockdown of Creb3l1 in the hypothalamus led to a dramatic depletion in neuropeptide synthesis and secretion. The physiological outcomes from studies of paraventricular and supraoptic nuclei Creb3l1 knockdown animals were changes to food and water consumption. CONCLUSION: Collectively, our data identify Creb3l1 as a comprehensive controller of the PERK signalling pathway in magnocellular neurones in response to physiological stimulation. The broad regulation of neuropeptide synthesis and secretion by Creb3l1 presents a new therapeutic strategy for metabolic diseases

Untangling the Roles of Anti-Apoptosis in Regulating Programmed Cell Death using Humanized Yeast Cells

Genetically programmed cell death (PCD) mechanisms, including apoptosis, are important for the survival of metazoans since it allows, among things, the removal of damaged cells that interfere with normal function. Cell death due to PCD is observed in normal processes such as aging and in a number of pathophysiologies including hypoxia (common causes of heart attacks and strokes) and subsequent tissue reperfusion. Conversely, the loss of normal apoptotic responses is associated with the development of tumors. So far, limited success in preventing unwanted PCD has been reported with current therapeutic approaches despite the fact that inhibitors of key apoptotic inducers such as caspases have been developed. Alternative approaches have focused on mimicking anti-apoptotic processes observed in cells displaying increased resistance to apoptotic stimuli. Hormesis and pre-conditioning are commonly observed cellular strategies where sub-lethal levels of pro-apoptotic stimuli lead to increased resistance to higher or lethal levels of stress. Increased expression of anti-apoptotic sequences is a common mechanism mediating these protective effects. The relevance of the latter observation is exemplified by the observation that transgenic mice overexpressing anti-apoptotic genes show significant reductions in tissue damage following ischemia. Thus strategies aimed at increasing the levels of anti-apoptotic proteins, using gene therapy or cell penetrating recombinant proteins are being evaluated as novel therapeutics to decrease cell death following acute periods of cell death inducing stress. In spite of its functional and therapeutic importance, more is known regarding the processes involved in apoptosis than anti-apoptosis. The genetically tractable yeast Saccharomyces cerevisiae has emerged as an exceptional model to study multiple aspects of PCD including the mitochondrial mediated apoptosis observed in metazoans. To increase our knowledge of the process of anti-apoptosis, we screened a human heart cDNA expression library in yeast cells undergoing PCD due to the conditional expression of a mammalian pro-apoptotic Bax cDNA. Analysis of the multiple Bax suppressors identified revealed several previously known as well as a large number of clones representing potential novel anti-apoptotic sequences. The focus of this review is to report on recent achievements in the use of humanized yeast in genetic screens to identify novel stress-induced PCD suppressors, supporting the use of yeast as a unicellular model organism to elucidate anti-apoptotic and cell survival mechanisms

Chromosome-breakage genomic instability and chromothripsis in breast cancer

Background: Chromosomal breakage followed by faulty DNA repair leads to gene amplifications and deletions in cancers. However, the mere assessment of the extent of genomic changes, amplifications and deletions may reduce the complexity of genomic data observed by array comparative genomic hybridization (array CGH). We present here a novel approach to array CGH data analysis, which focuses on putative breakpoints responsible for rearrangements within the genome. Results: We performed array comparative genomic hybridization in 29 primary tumors from high risk patients with breast cancer. The specimens were flow sorted according to ploidy to increase tumor cell purity prior to array CGH. We describe the number of chromosomal breaks as well as the patterns of breaks on individual chromosomes in each tumor. There were differences in chromosomal breakage patterns between the 3 clinical subtypes of breast cancers, although the highest density of breaks occurred at chromosome 17 in all subtypes, suggesting a particular proclivity of this chromosome for breaks. We also observed chromothripsis affecting various chromosomes in 41% of high risk breast cancers. Conclusions: Our results provide a new insight into the genomic complexity of breast cancer. Genomic instability dependent on chromosomal breakage events is not stochastic, targeting some chromosomes clearly more than others. We report a much higher percentage of chromothripsis than described previously in other cancers and this suggests that massive genomic rearrangements occurring in a single catastrophic event may shape many breast cancer genomes

Regulation of cAMP Responsive Element Binding Protein 3-Like 1 (Creb3l1) Expression by Orphan Nuclear Receptor Nr4a1

Cyclic AMP (cAMP) inducible transcription factor cAMP responsive element binding protein 3 like 1 (Creb3l1) is strongly activated in the hypothalamus in response to hyperosmotic cues such as dehydration (DH). We have recently shown that Creb3l1 expression is upregulated by cAMP pathways in vitro, however the exact mechanisms are not known. Here we show that increasing Creb3l1 transcription by raising cAMP levels in mouse pituitary AtT20 cells automatically initiates cleavage of Creb3l1, leading to a greater abundance of the transcriptionally active N-terminal portion. Inhibiting protein synthesis indicated that de novo protein synthesis of an intermediary transcription factor was required for Creb3l1 induction. Strategic mining of our microarray data from dehydrated rodent hypothalamus revealed four candidates, reduced to two by analysis of acute hyperosmotic-induced transcriptional activation profiles in the hypothalamus, and one, orphan nuclear receptor Nr4a1, by direct shRNA mediated silencing in AtT20 cells. We show that activation of Creb3l1 transcription by Nr4a1 involves interaction with a single NBRE site in the promoter region. The ability to activate Creb3l1 transcription by this pathway in vitro is dictated by the level of methylation of a CpG island within the proximal promoter/5′UTR of this gene. We thus identify a novel cAMP-Nr4a1-Creb3l1 transcriptional pathway in AtT20 cells and also, our evidence would suggest, in the hypothalamus

Practice characteristics of Emergency Department extracorporeal cardiopulmonary resuscitation (eCPR) programs in the United States: The current state of the art of Emergency Department extracorporeal membrane oxygenation (ED ECMO).

PURPOSE: To characterize the current scope and practices of centers performing extracorporeal cardiopulmonary resuscitation (eCPR) on the undifferentiated patient with cardiac arrest in the emergency department. METHODS: We contacted all US centers in January 2016 that had submitted adult eCPR cases to the Extracorporeal Life Support Organization (ELSO) registry and surveyed them, querying for programs that had performed eCPR in the Emergency Department (ED ECMO). Our objective was to characterize the following domains of ED ECMO practice: program characteristics, patient selection, devices and techniques, and personnel. RESULTS: Among 99 centers queried, 70 responded. Among these, 36 centers performed ED ECMO. Nearly 93% of programs are based at academic/teaching hospitals. 65% of programs are less than 5 years old, and 60% of programs perform ≤3 cases per year. Most programs (90%) had inpatient eCPR or salvage ECMO programs prior to starting ED ECMO programs. The majority of programs do not have formal inclusion and exclusion criteria. Most programs preferentially obtain vascular access via the percutaneous route (70%) and many (40%) use mechanical CPR during cannulation. The most commonly used console is the Maquet Rotaflow(®). Cannulation is most often performed by cardiothoracic (CT) surgery, and nearly all programs (\u3e85%) involve CT surgeons, perfusionists, and pharmacists. CONCLUSIONS: Over a third of centers that submitted adult eCPR cases to ELSO have performed ED ECMO. These programs are largely based at academic hospitals, new, and have low volumes. They do not have many formal inclusion or exclusion criteria, and devices and techniques are variable