Matthew J. Schipma

Pancreatic β cell enhancers regulate rhythmic transcription of genes controlling insulin secretion.

The clockwork of insulin release In healthy people, blood glucose levels are maintained within a narrow range by several physiological mechanisms. Key among them is the release of the hormone insulin by pancreatic β cells, which occurs when glucose levels rise after a meal. In response to insulin, blood glucose is taken up by tissues that need fuel, such as muscle. β cells can anticipate the bodys varying demand for insulin throughout the 24-hour day because they have their own circadian clock. How this clock controls insulin release has been unclear. Perelis et al. now show that the activity of transcriptional enhancers specific to β cells regulates the rhythmic expression of genes involved in the assembly and trafficking of insulin secretory vesicles (see the Perspective by Dibner and Schibler). Science, this issue p. 10.1126/science.aac4250; see also p. 628 Circadian control of insulin release is mediated by transcriptional enhancers active specifically in pancreatic β cells. [Also see Perspective by Dibner and Schibler] INTRODUCTION The circadian clock is a molecular oscillator that coordinates behavior and physiology in anticipation of the daily light cycle. Desynchrony of circadian cycles, through genetic or environmental perturbation, contributes to metabolic disorders such as cardiovascular disease, obesity, and type 2 diabetes. We previously showed that disruption of the clock transcription factors CLOCK and BMAL1 in the pancreas causes hypoinsulinemic diabetes in mice. The mechanism(s) linking clock dysfunction to pancreatic β cell failure and the means by which CLOCK and BMAL1 affect glucose metabolism in the whole organism are not well understood. RATIONALE The circadian system helps to maintain glucose homeostasis across the sleep-wake cycle. This system requires cross-talk between the master clock in the central nervous system, which coordinates feeding and sleep, and peripheral tissue clocks, which synchronize behavior with the storage, mobilization, and synthesis of glucose. Although it is clear that clocks within distinct organs participate in glucose turnover, the molecular basis for time-of-day variation in organismal glucose responsiveness is still not understood. Here, we combined genome-wide analyses with gene targeting in mice to study the impact of the cell-autonomous clock on β cell function. RESULTS We found that cell-autonomous expression of CLOCK and BMAL1 in pancreatic islets isolated from wild-type mice generates robust 24-hour rhythms of glucose- and potassium chloride–stimulated insulin secretion ex vivo. About 27% of the β cell transcriptome exhibited circadian oscillation. Many of these transcripts correspond to genes coding for proteins that are involved in the assembly, trafficking, and membrane fusion of vesicles that participate in insulin secretion. Chromatin immunoprecipitation sequencing revealed that CLOCK and BMAL1 regulate cycling genes in β cells by binding at distal regulatory elements distinct from those controlling the circadian transcription of metabolic gene networks within the liver. The regulatory sites of cycling genes in the β cell resided primarily within transcriptionally active enhancers that were also bound by the pancreatic transcription factor PDX1. Finally, we found that in islets from adult mice, Bmal1 ablation either in vivo or ex vivo abrogates nutrient-responsive insulin secretion, demonstrating clock control of pancreatic β cell function throughout adult life. CONCLUSION Our results show that local clock-driven genomic rhythms program cell function across the light-dark cycle, including the priming of insulin secretion within limited time windows each day. Cell type–specific transcriptional regulation by the clock localizes to rhythmic enhancers that are unique to the β cell. Thus, our findings uncover a transcriptional process through which the core clock aligns physiology with the light cycle, revealing pathways that are important in both health and disease states such as type 2 diabetes. β cell–specific enhancers control the rhythmic transcription of genes linked to insulin secretion. Peripheral clocks maintain glucose homeostasis across the sleep-wake cycle by gating β cell insulin secretion through genome-wide transcriptional control of the assembly and trafficking of insulin secretory vesicles. Clock transcription factors bind within cell type–specific enhancer neighborhoods of cycling genes, revealing the mechanisms that synchronize rhythmic metabolism at transcriptional and physiologic levels across the light-dark cycle. The mammalian transcription factors CLOCK and BMAL1 are essential components of the molecular clock that coordinate behavior and metabolism with the solar cycle. Genetic or environmental perturbation of circadian cycles contributes to metabolic disorders including type 2 diabetes. To study the impact of the cell-autonomous clock on pancreatic β cell function, we examined pancreatic islets from mice with either intact or disrupted BMAL1 expression both throughout life and limited to adulthood. We found pronounced oscillation of insulin secretion that was synchronized with the expression of genes encoding secretory machinery and signaling factors that regulate insulin release. CLOCK/BMAL1 colocalized with the pancreatic transcription factor PDX1 within active enhancers distinct from those controlling rhythmic metabolic gene networks in liver. We also found that β cell clock ablation in adult mice caused severe glucose intolerance. Thus, cell type–specific enhancers underlie the circadian control of peripheral metabolism throughout life and may help to explain its dysregulation in diabetes.

Inhibition of amyloid-β-induced neurotoxicity and apoptosis by moderate ethanol preconditioning

Consumers of moderate amounts of ethanol have a lower risk of Alzheimers dementia than do abstainers. In Alzheimers disease the brain contains many extracellular plaques composed of amyloid-&bgr; (A&bgr;), a neurotoxic protein linked to pathogenesis of the disease. Here we report that moderate ethanol preconditioning (20–30 mM for 6 days) of organotypic hippocampal-entorhinal slice cultures prevents A&bgr;-induced neurotoxicity and apoptosis as measured by media lactate dehydrogenase levels and staining with propidium iodide and Hoechst 33342. With A&bgr;, as with our previous studies of the neurotoxic HIV-1 protein gp120, moderate ethanol preconditioning may interfere with various glial-mediated neurotoxic responses in the slices to A&bgr;. In addition, we found that moderate ethanol preconditioning causes an almost 3-fold increase in brain levels of heat shock protein 70 (hsp70), a protective molecular chaperone. Our results suggest possible molecular mechanisms underlying the protective effect of moderate drinking against Alzheimers dementia.

Genome Sequence of the Fish Pathogen Renibacterium salmoninarum Suggests Reductive Evolution away from an Environmental Arthrobacter Ancestor

Renibacterium salmoninarum is the causative agent of bacterial kidney disease and a significant threat to healthy and sustainable production of salmonid fish worldwide. This pathogen is difficult to culture in vitro, genetic manipulation is challenging, and current therapies and preventative strategies are only marginally effective in preventing disease. The complete genome of R. salmoninarum ATCC 33209 was sequenced and shown to be a 3,155,250-bp circular chromosome that is predicted to contain 3,507 open-reading frames (ORFs). A total of 80 copies of three different insertion sequence elements are interspersed throughout the genome. Approximately 21% of the predicted ORFs have been inactivated via frameshifts, point mutations, insertion sequences, and putative deletions. The R. salmoninarum genome has extended regions of synteny to the Arthrobacter sp. strain FB24 and Arthrobacter aurescens TC1 genomes, but it is approximately 1.9 Mb smaller than both Arthrobacter genomes and has a lower G+C content, suggesting that significant genome reduction has occurred since divergence from the last common ancestor. A limited set of putative virulence factors appear to have been acquired via horizontal transmission after divergence of the species; these factors include capsular polysaccharides, heme sequestration molecules, and the major secreted cell surface antigen p57 (also known as major soluble antigen). Examination of the genome revealed a number of ORFs homologous to antibiotic resistance genes, including genes encoding beta-lactamases, efflux proteins, macrolide glycosyltransferases, and rRNA methyltransferases. The genome sequence provides new insights into R. salmoninarum evolution and may facilitate identification of chemotherapeutic targets and vaccine candidates that can be used for prevention and treatment of infections in cultured salmonids.

Suspension array analysis of 16S rRNA from Fe- and SO(4)2- reducing bacteria in uranium-contaminated sediments undergoing bioremediation.

ABSTRACT A 16S rRNA-targeted tunable bead array was developed and used in a retrospective analysis of metal- and sulfate-reducing bacteria in contaminated subsurface sediments undergoing in situ U(VI) bioremediation. Total RNA was extracted from subsurface sediments and interrogated directly, without a PCR step. Bead array validation studies with total RNA derived from 24 isolates indicated that the behavior and response of the 16S rRNA-targeted oligonucleotide probes could not be predicted based on the primary nucleic acid sequence. Likewise, signal intensity (absolute or normalized) could not be used to assess the abundance of one organism (or rRNA) relative to the abundance of another organism (or rRNA). Nevertheless, the microbial community structure and dynamics through time and space and as measured by the rRNA-targeted bead array were consistent with previous data acquired at the site, where indigenous sulfate- and iron-reducing bacteria and near neighbors of Desulfotomaculum were the organisms that were most responsive to a change in injected acetate concentrations. Bead array data were best interpreted by analyzing the relative changes in the probe responses for spatially and temporally related samples and by considering only the response of one probe to itself in relation to a background (reference) environmental sample. By limiting the interpretation of the data in this manner and placing it in the context of supporting geochemical and microbiological analyses, we concluded that ecologically relevant and meaningful information can be derived from direct microarray analysis of rRNA in uncharacterized environmental samples, even with the current analytical uncertainty surrounding the behavior of individual probes on tunable bead arrays.

Genome-Wide Analysis of Small RNAs Expressed by Yersinia pestis Identifies a Regulator of the Yop-Ysc Type III Secretion System

Small noncoding RNA (sRNA) molecules are integral components of the regulatory machinery for many bacterial species and are known to posttranscriptionally regulate metabolic and stress-response pathways, quorum sensing, virulence factors, and more. The Yop-Ysc type III secretion system (T3SS) is a critical virulence component for the pathogenic Yersinia species, and the regulation of this system is tightly controlled at each step from transcription to translocation of effectors into host cells. The contribution of sRNAs to the regulation of the T3SS in Yersinia has been largely unstudied, however. Previously, our lab identified a role for the sRNA chaperone protein Hfq in the regulation of components of the T3SS in the gastrointestinal pathogen Yersinia pseudotuberculosis. Here we present data demonstrating a similar requirement for Hfq in the closely related species Yersinia pestis. Through deep sequencing analysis of the Y. pestis sRNA-ome, we found 63 previously unidentified putative sRNAs in this species. We identified a Yersinia-specific sRNA, Ysr141, carried by the T3SS plasmid pCD1 that is required for the production of multiple T3SS proteins. In addition, we show that Ysr141 targets an untranslated region upstream of yopJ to posttranscriptionally activate the synthesis of the YopJ protein. Furthermore, Ysr141 may be an unstable and/or processed sRNA, which could contribute to its function in the regulation of the T3SS. The discovery of an sRNA that influences the synthesis of the T3SS adds an additional layer of regulation to this tightly controlled virulence determinant of Y. pestis.

Nuclear Condensation during Mouse Erythropoiesis Requires Caspase-3-Mediated Nuclear Opening.

Mammalian erythropoiesis involves chromatin condensation that is initiated in the early stage of terminal differentiation. The mechanisms of chromatin condensation during erythropoiesis are unclear. Here, we show that the mouse erythroblast forms large, transient, and recurrent nuclear openings that coincide with the condensation process. The opening lacks nuclear lamina, nuclear pore complexes, and nuclear membrane, but it is distinct from nuclear envelope changes that occur during apoptosis and mitosis. A fraction of the major histones are released from the nuclear opening and degraded in the cytoplasm. We demonstrate that caspase-3 is required for the nuclear opening formation throughout terminal erythropoiesis. Loss of caspase-3 or ectopic expression of a caspase-3 non-cleavable lamin B mutant blocks nuclear opening formation, histone release, chromatin condensation, and terminal erythroid differentiation. We conclude that caspase-3-mediated nuclear opening formation accompanied by histone release from the opening is a critical step toward chromatin condensation during erythropoiesis in mice.

Oligonucleotide microarray identification of Bacillus anthracis strains using support vector machines

The capability of a custom microarray to discriminate between closely related DNA samples is demonstrated using a set of Bacillus anthracis strains. The microarray was developed as a universal fingerprint device consisting of 390 genome-independent 9mer probes. The genomes of B. anthracis strains are monomorphic and therefore, typically difficult to distinguish using conventional molecular biology tools or microarray data clustering techniques. Using support vector machines (SVMs) as a supervised learning technique, we show that a low-density fingerprint microarray contains enough information to discriminate between B. anthracis strains with 90% sensitivity using a reference library constructed from six replicate arrays and three replicates for new isolates.

PBRM1 Regulates the Expression of Genes Involved in Metabolism and Cell Adhesion in Renal Clear Cell Carcinoma.

Polybromo-1 (PBRM1) is a component of the PBAF (Polybromo-associated-BRG1- or BRM-associated factors) chromatin remodeling complex and is the second most frequently mutated gene in clear-cell renal cell Carcinoma (ccRCC). Mutation of PBRM1 is believed to be an early event in carcinogenesis, however its function as a tumor suppressor is not understood. In this study, we have employed Next Generation Sequencing to profile the differentially expressed genes upon PBRM1 re-expression in a cellular model of ccRCC. PBRM1 re-expression led to upregulation of genes involved in cellular adhesion, carbohydrate metabolism, apoptotic process and response to hypoxia, and a downregulation of genes involved in different stages of cell division. The decrease in cellular proliferation upon PBRM1 re-expression was confirmed, validating the functional role of PBRM1 as a tumor suppressor in a cell-based model. In addition, we identified a role for PBRM1 in regulating metabolic pathways known to be important for driving ccRCC, including the regulation of hypoxia response genes, PI3K signaling, glucose uptake, and cholesterol homeostasis. Of particular novelty is the identification of cell adhesion as a major downstream process uniquely regulated by PBRM1 expression. Cytoskeletal reorganization was induced upon PBRM1 reexpression as evidenced from the increase in the number of cells displaying cortical actin, a hallmark of epithelial cells. Genes involved in cell adhesion featured prominently in our transcriptional dataset and overlapped with genes uniquely regulated by PBRM1 in clinical specimens of ccRCC. Genes involved in cell adhesion serve as tumor suppressor and maybe involved in inhibiting cell migration. Here we report for the first time genes linked to cell adhesion serve as downstream targets of PBRM1, and hope to lay the foundation of future studies focusing on the role of chromatin remodelers in bringing about these alterations during malignancies.

Diagnostic Oligonucleotide Microarray Fingerprinting of Bacillus Isolates

ABSTRACT A genome-independent microarray and new statistical techniques were used to genotype Bacillus strains and quantitatively compare DNA fingerprints with the known taxonomy of the genus. A synthetic DNA standard was used to understand process level variability and lead to recommended standard operating procedures for microbial forensics and clinical diagnostics.

Aberrant overexpression of CD14 on granulocytes sensitizes the innate immune response in mDia1 heterozygous del(5q) MDS

The myelodysplastic syndromes (MDSs) include a spectrum of stem cell malignancies characterized by an increased risk of developing acute myeloid leukemia. Heterozygous loss of chromosome 5q (del[5q]) is the most common cytogenetic abnormality in MDS. DIAPH1 is localized to 5q31 and encodes one of the formin proteins, mDia1, which is involved in linear actin polymerization. Mice with mDia1 deficiency develop hematologic features with age mimicking human myeloid neoplasm, but its role in the pathogenesis of MDS is unclear. Here we report that mDia1 heterozygous and knockout mice develop MDS phenotypes with age. In these mice, CD14 was aberrantly overexpressed on granulocytes in a cell-autonomous manner, leading to a hypersensitive innate immune response to lipopolysaccharide (LPS) stimuli through CD14/Toll-like receptor 4 signaling. Chronic stimulation with LPS accelerated the development of MDS in mDia1 heterozygous and knockout mice that can be rescued by lenalidomide. Similar findings of CD14 overexpression were observed on the bone marrow granulocytes of del(5q) MDS patients. Mechanistically, mDia1 deficiency led to a downregulation of membrane-associated genes and a specific upregulation of CD14 messenger RNA in granulocytes, but not in other lineages. These results underscore the significance of mDia1 heterozygosity in deregulated innate immune responses in del(5q) MDS.