Translational Regulation of the DOUBLETIME/CKIδ/ε Kinase by LARK Contributes to Circadian Period Modulation

The Drosophila homolog of Casein Kinase I δ/ε, DOUBLETIME (DBT), is required for Wnt, Hedgehog, Fat and Hippo signaling as well as circadian clock function. Extensive studies have established a critical role of DBT in circadian period determination. However, how DBT expression is regulated remains largely unexplored. In this study, we show that translation of dbt transcripts are directly regulated by a rhythmic RNA-binding protein (RBP) called LARK (known as RBM4 in mammals). LARK promotes translation of specific alternative dbt transcripts in clock cells, in particular the dbt-RC transcript. Translation of dbt-RC exhibits circadian changes under free-running conditions, indicative of clock regulation. Translation of a newly identified transcript, dbt-RE, is induced by light in a LARK-dependent manner and oscillates under light/dark conditions. Altered LARK abundance affects circadian period length, and this phenotype can be modified by different dbt alleles. Increased LARK delays nuclear degradation of the PERIOD (PER) clock protein at the beginning of subjective day, consistent with the known role of DBT in PER dynamics. Taken together, these data support the idea that LARK influences circadian period and perhaps responses of the clock to light via the regulated translation of DBT. Our study is the first to investigate translational control of the DBT kinase, revealing its regulation by LARK and a novel role of this RBP in Drosophila circadian period modulation

Psychology and aggression

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/68264/2/10.1177_002200275900300301.pd

The James Webb Space Telescope Mission

Twenty-six years ago a small committee report, building on earlier studies, expounded a compelling and poetic vision for the future of astronomy, calling for an infrared-optimized space telescope with an aperture of at least $4m$. With the support of their governments in the US, Europe, and Canada, 20,000 people realized that vision as the $6.5m$ James Webb Space Telescope. A generation of astronomers will celebrate their accomplishments for the life of the mission, potentially as long as 20 years, and beyond. This report and the scientific discoveries that follow are extended thank-you notes to the 20,000 team members. The telescope is working perfectly, with much better image quality than expected. In this and accompanying papers, we give a brief history, describe the observatory, outline its objectives and current observing program, and discuss the inventions and people who made it possible. We cite detailed reports on the design and the measured performance on orbit.Comment: Accepted by PASP for the special issue on The James Webb Space Telescope Overview, 29 pages, 4 figure

Drosophila Muller F Elements Maintain a Distinct Set of Genomic Properties Over 40 Million Years of Evolution

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25–50%) than euchromatic reference regions (3–11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11–27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4–3.6 vs. 8.4–8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu

Development and Validation of a Risk Score for Chronic Kidney Disease in HIV Infection Using Prospective Cohort Data from the D:A:D Study

Ristola M. on työryhmien DAD Study Grp ; Royal Free Hosp Clin Cohort ; INSIGHT Study Grp ; SMART Study Grp ; ESPRIT Study Grp jäsen.Background Chronic kidney disease (CKD) is a major health issue for HIV-positive individuals, associated with increased morbidity and mortality. Development and implementation of a risk score model for CKD would allow comparison of the risks and benefits of adding potentially nephrotoxic antiretrovirals to a treatment regimen and would identify those at greatest risk of CKD. The aims of this study were to develop a simple, externally validated, and widely applicable long-term risk score model for CKD in HIV-positive individuals that can guide decision making in clinical practice. Methods and Findings A total of 17,954 HIV-positive individuals from the Data Collection on Adverse Events of Anti-HIV Drugs (D:A:D) study with >= 3 estimated glomerular filtration rate (eGFR) values after 1 January 2004 were included. Baseline was defined as the first eGFR > 60 ml/min/1.73 m2 after 1 January 2004; individuals with exposure to tenofovir, atazanavir, atazanavir/ritonavir, lopinavir/ritonavir, other boosted protease inhibitors before baseline were excluded. CKD was defined as confirmed (>3 mo apart) eGFR In the D:A:D study, 641 individuals developed CKD during 103,185 person-years of follow-up (PYFU; incidence 6.2/1,000 PYFU, 95% CI 5.7-6.7; median follow-up 6.1 y, range 0.3-9.1 y). Older age, intravenous drug use, hepatitis C coinfection, lower baseline eGFR, female gender, lower CD4 count nadir, hypertension, diabetes, and cardiovascular disease (CVD) predicted CKD. The adjusted incidence rate ratios of these nine categorical variables were scaled and summed to create the risk score. The median risk score at baseline was -2 (interquartile range -4 to 2). There was a 1: 393 chance of developing CKD in the next 5 y in the low risk group (risk score = 5, 505 events), respectively. Number needed to harm (NNTH) at 5 y when starting unboosted atazanavir or lopinavir/ritonavir among those with a low risk score was 1,702 (95% CI 1,166-3,367); NNTH was 202 (95% CI 159-278) and 21 (95% CI 19-23), respectively, for those with a medium and high risk score. NNTH was 739 (95% CI 506-1462), 88 (95% CI 69-121), and 9 (95% CI 8-10) for those with a low, medium, and high risk score, respectively, starting tenofovir, atazanavir/ritonavir, or another boosted protease inhibitor. The Royal Free Hospital Clinic Cohort included 2,548 individuals, of whom 94 individuals developed CKD (3.7%) during 18,376 PYFU (median follow-up 7.4 y, range 0.3-12.7 y). Of 2,013 individuals included from the SMART/ESPRIT control arms, 32 individuals developed CKD (1.6%) during 8,452 PYFU (median follow-up 4.1 y, range 0.6-8.1 y). External validation showed that the risk score predicted well in these cohorts. Limitations of this study included limited data on race and no information on proteinuria. Conclusions Both traditional and HIV-related risk factors were predictive of CKD. These factors were used to develop a risk score for CKD in HIV infection, externally validated, that has direct clinical relevance for patients and clinicians to weigh the benefits of certain antiretrovirals against the risk of CKD and to identify those at greatest risk of CKD.Peer reviewe

Characterization of DNA-Protein Interactions at the NT/N Promoter: Proles for AP-1 and ATF Proteins

The focus of experiments presented in this dissertation is to determine how signals created by exposure to environmental stimuli are integrated at the level of transcription, resulting in the generation of specific patterns of gene expression. The model system used was expression of the neurotensinl neuromedin N (NT/N) neuropeptide gene in the neuroendocrine PC12 cell line. This gene is synergistically activated in PC12 cells in response to nerve growth factor, lithium, glucocorticoids, and activators of adenylate cyclase. Several cis-regulatory elements were identified within a 200 bp regulatory region, including AP-1, CRE, and GRE-like elements. Mutational analysis confirmed the importance of these elements for responses to inducer combinations. The primary objective was to identify proteins that interact with NT/N promoter sequences and determine if they are important in mediating responses to inducer combinations. The first set of experiments was designed to investigate changes in AP-1 binding activity. Previous analysis had shown that mutation of the AP-1 site severely curtails responses to all inducer combinations indicating that AP-1 plays a pivotal role in NT/N gene activation. DNA binding studies using in vitro synthesized AP-1 proteins revealed that all heterodimeric combinations could bind both the AP-1 and JARE sites; however, these complexes displayed a higher affinity for the AP-1 site. c-Jun homodimers were also found to bind both these sites albeit with a lower affinity and with a preference for the JARE site. These studies revealed that specificity is probably not at the level of DNA binding. Therefore, it was possible that only a subset of AP-1 proteins were activated upon stimulation. DNase I footprint analysis using nuclear extracts from PC12 cells showed changes in protection at the consensus AP-1 site upon treatment with inducers suggesting changes in AP-1 binding activity. It was found that AP-1 binding activity was increased upon stimulation, with the major component being Jun B. However, substantial levels of c-Fos and c-Jun were also detected at some time points. These results coupled with transfection data demonstrating that forced expression of c-Jun and c-Fos result in potent synergistic activation of the NT/N promoter support the hypothesis that c-Jun and c-Fos are also involved in NT/N gene activation. DNase I footprinting studies using PC12 nuclear extracts also revealed substantial areas of protection surrounding the CRE element. This result, along with the high degree of conservation of these sequences between human and rat, suggested they play a role in the regulation of the NT/N gene in PC12 cells. Mutational analysis of this region showed that sequences upstream of the CRE were important for full activation of the NT/N promoter. Specific mutation of the CRE resulted in a 75% decrease in activity upon induction, a level similar to that observed previously with less precise linker scanner mutations. This site had also been shown to be critical for c-Jun mediated NT/N activation, even though c-Jun homodimers do not bind this site in vitro. Therefore, nuclear extracts from PC12 cells were tested for the presence of proteins which could bind this site. Complexes composed of both c-Jun and ATF-2 were found in extracts from both uninduced and induced PC12 cells. ATF-2 could mediate both the recruitment of c-Jun to this site as well as mediate the effect of activators of adenylate cyclase, since ATF-2 has been shown to be a target for protein kinase A in vitro. Expression of ATF-2 in PC12 cells resulted in a modest increase in NT/N promoter activation. The significant levels of endogenous ATF-2 protein in PC12 cells most likely accounts for the relatively small magnitude of this effect. Experiments with the closely related protein, ATF-a2, revealed that it potently antagonizes c-Jun activation while forced expression of ATF-2 did not affect c-Jun activation under the conditions analyzed. Therefore, ATF proteins could be involved in both activation and repression of the NT/N gene. Both c-Jun and ATF-2 have been shown to be activated by c-Jun N-terminal kinase (JNK) in response to environmental stress or cytokine activation. Therefore, the ability of inducers to activate the previously described N-terminal ATF-2 activation domain was investigated using a GAL4-ATF-2 (1-109) chimer construct. This construct was not significantly activated by inducer combinations that result in high level NT/N gene expression, indicating that activation of ATF-2 through this pathway is not involved in NT/N gene activation. Also activation of JNK, a MAPK which activates both c-Jun and ATF-2, only partially substituted for NGF indicating that NGF activates an additional pathway. The data presented here support a model involving synergistic transcriptional activation of the NT/N promoter by c-Jun/c-Fos, ATF-2, ATF-2/c-Jun and the GR. ATF-2 was found to enhance NT/N promoter activation while a splice variant (ATF-2 195) lacking a central portion of ATF-2 that is rich in Ser/Thr residues had no effect suggesting that this region could be important for ATF-2 activation in PC12 cells. The identification of the signaling pathways that mediate the effects of inducer combinations on NT/N gene activation will be an important future goal and should provide insights into the control of neuronal gene expression

Synergistic activation of neurotensin/neuromedin N gene expression by c-Jun and glucocorticoids: novel effects of Fos family proteins

The cis-regulatory region of the neurotensin/neuromedin N (NT/N) gene integrates diverse environmental signals in the neuroendocrine PC12 cell line, resulting in remarkable synergistic regulation. An AP-1 site appears to play a pivotal role in cooperative NT/N gene activation, as mutations in this site decrease responses to all inducer combinations by at least an order of magnitude. Here we report that c-Jun acts synergistically with glucocorticoids to activate the NT/N promoter, and that Fos family proteins have novel regulatory effects on this interaction. Cotransfection of individual pCMV-AP-1 expression plasmids revealed that c-Jun most potently activates the NT/N promoter and that costimulation with dexamethasone results in a further 6- to 12-fold increase in expression. Unlike its general inhibitory effects on glucocorticoid regulation in other systems, c-Fos potentiated activation by glucocorticoids when coexpressed with c-Jun, and Fos B had a similar, but more limited, positive effect. In contrast, Fra-1 reversed the direction of glucocorticoid regulation, and Fra-2 abolished synergism. AP-1, cAMP response element, and glucocorticoid response element motifs are required for full cooperative activation by either c-Jun or c-Jun/c-Fos and glucocorticoids. These results indicate that NT/N promoter activation involves synergistic interactions between specific AP-1 complexes and ligand-activated glucocorticoid receptor, and similar mechanisms may regulate NT/N gene expression in central neurons

Synergistic induction of neurotensin gene transcription in PC12 cells parallels changes in AP-1 activity

A consensus AP-1 site in the promoter of the rat neurotensin/neuromedin N (NT/N) gene is a critical regulatory element required for synergistic regulation by combinations of nerve growth factor (NGF), lithium, glucocorticoids, and adenylate cyclase activators. A rapid RNase protection assay was developed to examine the kinetics of NT/N gene activation and to determine whether activation requires newly synthesized proteins. Either NGF or lithium in combination with dexamethasone and forskolin transiently activated NT/N gene expression, but with distinct kinetics. Protein synthesis was not required for activation when NGF was used as the permissive inducer, but was required for the rapid down-regulation of the response. In contrast, lithium responses were attenuated in the absence of protein synthesis, consistent with a requirement for newly synthesized AP-1 complexes in activation. In all cases, increases in NT/N gene expression closely paralleled increases in AP-1 binding activity. Lithium in combination with other inducers caused delayed increases in both AP-1 binding activity and c-jun, c-fos and fra-1 gene expression. These results indicate that NGF and lithium exert their effects on NT/N gene expression through distinct pathways. The lithium pathway is active in neuronally-differentiated PC12 cells and could potentially be involved in the regulation of NT/N gene expression in the nervous system

Translational Regulation of the DOUBLETIME/CKIδ/ε Kinase by LARK Contributes to Circadian Period Modulation

<div><p>The Drosophila homolog of Casein Kinase I δ/ε, DOUBLETIME (DBT), is required for Wnt, Hedgehog, Fat and Hippo signaling as well as circadian clock function. Extensive studies have established a critical role of DBT in circadian period determination. However, how DBT expression is regulated remains largely unexplored. In this study, we show that translation of <i>dbt</i> transcripts are directly regulated by a rhythmic RNA-binding protein (RBP) called LARK (known as RBM4 in mammals). LARK promotes translation of specific alternative <i>dbt</i> transcripts in clock cells, in particular the <i>dbt</i>-<i>RC</i> transcript. Translation of <i>dbt</i>-<i>RC</i> exhibits circadian changes under free-running conditions, indicative of clock regulation. Translation of a newly identified transcript, <i>dbt</i>-<i>RE</i>, is induced by light in a LARK-dependent manner and oscillates under light/dark conditions. Altered LARK abundance affects circadian period length, and this phenotype can be modified by different <i>dbt</i> alleles. Increased LARK delays nuclear degradation of the PERIOD (PER) clock protein at the beginning of subjective day, consistent with the known role of DBT in PER dynamics. Taken together, these data support the idea that LARK influences circadian period and perhaps responses of the clock to light via the regulated translation of DBT. Our study is the first to investigate translational control of the DBT kinase, revealing its regulation by LARK and a novel role of this RBP in Drosophila circadian period modulation.</p></div