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Activin induces the expression of different genes in a concentration-dependent manner. In this paper, we show that the initial response of cells to activin, whether assayed in dispersed cells or in a bead-implantation regime in intact animal caps, is to activate expression of both Xbra and goosecoid. However, differential expression of the two genes, with downregulation of Xbra, occurs very rapidly and certainly within 3 h of the initial phase of expression. This rapid refinement of gene expression can occur in dispersed cells and thus does not require cell-cell interactions. Refinement of gene expression does, however, require protein synthesis but not goosecoid function. Together, our results place the burden of threshold formation not on the initial induction of different genes but on regulatory interactions between the genes once they have been activated.

Vertebrate neural development is initiated by factors released by the Spemann organizer that, by inhibiting BMP signaling, induce dorsal ectodermal cells to adopt a neural fate. We have isolated a gene, XSIP1, that might play a role downstream of neural inducers in early neural development. XSIP1 is the Xenopus homologue to the mouse Smad-interacting zinc finger/homeodomain repressor SIP1 (Verschueren et al., 1999, JBC 16, 274: 20489-98). By whole-mount in situ hybridization, high XSIP1 expression is first detected in the prospective neurectoderm at the beginning of gastrulation. At late gastrula and neurula stages, XSIP1 is widely expressed in the neural plate with the exception of the dorsal midline. After neural plate closure, XSIP1 expression is detected throughout the brain, in the neural tube, in the eye and cephalic neural crest cells. At larval stages, while XSIP1 expression decreases in the neural tube, strong expression is detected in trunk neural crest cells contiguous to the dorsal neural tube. As expected for a bona fide neural gene, expression of XSIP1 is induced in animal caps by attenuation of BMP signals. Preliminary overexpression experiments using mouse SIP1 in Xenopus embryos indicate that it is effective in blocking epidermal gene expression. The function and mode of action of XSIP1 during neural and neural crest development is currently investigated.

Histone variants are key epigenetic players, but their functional and physiological roles remain poorly understood. Here, we show that depletion of the histone variant H2A.Z in mouse skeletal muscle causes oxidative stress, oxidation of proteins, accumulation of DNA damages, and both neuromuscular junction and mitochondria lesions that consequently lead to premature muscle aging and reduced life span. Investigation of the molecular mechanisms involved shows that H2A.Z is required to initiate DNA double strand break repair by recruiting Ku80 at DNA lesions. This is achieved via specific interactions of Ku80 vWA domain with H2A.Z. Taken as a whole, our data reveal that H2A.Z containing nucleosomes act as a molecular platform to bring together the proteins required to initiate and process DNA double strand break repair., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Inherited defects in the base-excision repair gene MBD4 predispose individuals to adenomatous polyposis and colorectal cancer, which is characterized by an accumulation of C > T transitions resulting from spontaneous deamination of 5'-methylcytosine., Methods: Here, we have investigated the potential role of MBD4 in regulating DNA methylation levels using genome-wide transcriptome and methylome analyses. Additionally, we have elucidated its function through a series of in vitro experiments., Results: Here we show that the protein MBD4 is required for DNA methylation maintenance and G/T mismatch repair. Transcriptome and methylome analyses reveal a genome-wide hypomethylation of promoters, gene bodies and repetitive elements in the absence of MBD4 in vivo. Methylation mark loss is accompanied by a broad transcriptional derepression phenotype affecting promoters and retroelements with low methylated CpG density. MBD4 in vivo forms a complex with the mismatch repair proteins (MMR), which exhibits high bi-functional glycosylase/AP-lyase endonuclease specific activity towards methylated DNA substrates containing a G/T mismatch. Experiments using recombinant proteins reveal that the association of MBD4 with the MMR protein MLH1 is required for this activity., Conclusions: Our data identify MBD4 as an enzyme specifically designed to repair deaminated 5-methylcytosines and underscores its critical role in safeguarding against methylation damage. Furthermore, it illustrates how MBD4 functions in normal and pathological conditions., (© 2023. The Author(s).)

CENP-A is an essential histone variant that replaces the canonical H3 at the centromeres and marks these regions epigenetically. The CENP-A nucleosome is the specific building block of centromeric chromatin, and it is recognized by CENP-C and CENP-N, two components of the constitutive centromere-associated network (CCAN), the first protein layer of the kinetochore. Recent proposals of the yeast and human (h)CCAN structures position the assembly on exposed DNA, suggesting an elusive spatiotemporal recognition. We summarize the data on the structural organization of the CENP-A nucleosome and the binding of CENP-C and CENP-N. The latter posits an apparent contradiction in engaging the CENP-A nucleosome versus the CCAN. We propose a reconciliatory model for the assembly of CCAN on centromeric chromatin., Competing Interests: Declaration of interests None are declared by the author., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Cholesterol 24-hydroxylase (CH24H, CYP46A1) is a cytochrome P450 family enzyme that maintains the homeostasis of brain cholesterol. Soticlestat, a potent and selective CH24H inhibitor, is in development as a therapeutic agent for Dravet syndrome and Lennox-Gastaut syndrome. Herein, we report the discovery of aryl-piperidine derivatives as potent and selective CH24H positron emission tomography (PET) tracers which can be used for dose guidance of a clinical CH24H inhibitor and as a diagnostic tool for CH24H-related pathology. Starting from compound 1 (IC 50  = 16 nM, logD = 1.7), which was reported as a CH24H inhibitor with lower lipophilicity, a 18 F-labeling site (3-fluoroazetidine) was incorporated by structure-based drug design (SBDD) utilizing the co-crystal structure of a compound 1 analog. Subsequent optimization to adjust key parameters for PET tracers, such as potency, lipophilicity, brain penetration, and unbound plasma protein binding, enabled compounds 3f (IC 50  = 8.8 nM) and 3g (IC 50  = 8.7 nM) as PET imaging candidates. Selectivity of these compounds for CH24H was validated by a brain distribution study using CH24H-WT and KO mice. In non-human primate PET imaging, [ 18 F]3f and [ 18 F]3g showed similar regional uptake in the brain, indicating that these tracers were specific to the CH24H-expressed regions and validated the expression of CH24H in the living brain by different tracers., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The histone variant H3.3 is encoded by two distinct genes, H3f3a and H3f3b, exhibiting identical amino-acid sequence. H3.3 is required for spermatogenesis, but the molecular mechanism of its spermatogenic function remains obscure. Here, we have studied the role of each one of H3.3A and H3.3B proteins in spermatogenesis. We have generated transgenic conditional knock-out/knock-in (cKO/KI) epitope-tagged FLAG-FLAG-HA-H3.3B (H3.3BHA) and FLAG-FLAG-HA-H3.3A (H3.3AHA) mouse lines. We show that H3.3B, but not H3.3A, is required for spermatogenesis and male fertility. Analysis of the molecular mechanism unveils that the absence of H3.3B led to alterations in the meiotic/post-meiotic transition. Genome-wide RNA-seq reveals that the depletion of H3.3B in meiotic cells is associated with increased expression of the whole sex X and Y chromosomes as well as of both RLTR10B and RLTR10B2 retrotransposons. In contrast, the absence of H3.3B resulted in down-regulation of the expression of piRNA clusters. ChIP-seq experiments uncover that RLTR10B and RLTR10B2 retrotransposons, the whole sex chromosomes and the piRNA clusters are markedly enriched of H3.3. Taken together, our data dissect the molecular mechanism of H3.3B functions during spermatogenesis and demonstrate that H3.3B, depending on its chromatin localization, is involved in either up-regulation or down-regulation of expression of defined large chromatin regions., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Purpose: Cholesterol 24-hydroxylase (CH24H) is a brain-specific enzyme that plays a major role in brain cholesterol homeostasis by converting cholesterol into 24S-hydroxycholesterol. The selective CH24H inhibitor soticlestat (TAK-935) is being pursued as a drug for treatment of seizures in developmental and epileptic encephalopathies. Herein, we describe the successful discovery and the preclinical validation of the novel radiolabeled CH24H ligand (3-[ 18 F]fluoroazetidin-1-yl){1-[4-(4-fluorophenyl)pyrimidin-5-yl]piperidin-4-yl}methanone ([ 18 F]T-008) and its tritiated analog, [ 3 H]T-008., Methods: In vitro autoradiography (ARG) studies in the CH24H wild-type (WT) and knockout (KO) mouse brain sections were conducted using [ 3 H]T-008. PET imaging was conducted in two adult rhesus macaques using [ 18 F]T-008. Each macaque received two test-retest baseline scans and a series of two blocking doses of soticlestat administered prior to [ 18 F]T-008 to determine the CH24H enzyme occupancy. PET data were analyzed with Logan graphical analysis using plasma input. A Lassen plot was applied to estimate CH24H enzyme occupancy by soticlestat., Results: In ARG studies, binding of [ 3 H]T-008 was specific to CH24H in the mouse brain sections, which was not observed in CH24H KO or in wild-type mice after pretreatment with soticlestat. In rhesus PET studies, the rank order of [ 18 F]T-008 uptake was striatum > cortical regions > cerebellum, which was consistent with CH24H distribution in the brain. Pre-blocking with soticlestat reduced the maximum uptake and increased the washout in all brain regions in a dose-dependent manner. Calculated global occupancy values for soticlestat at a dose of 0.89 mg/kg were 97-98%, indicating maximum occupancy., Conclusion: The preclinical in vitro and in vivo evaluation of labeled T-008 demonstrates that [ 18 F]T-008 is suitable for imaging CH24H in the brain and warrants further studies in humans., (© 2021. The Author(s).)

Non-catechol-based high-affinity selective dopamine D 1 receptor (D1R) agonists were recently described, and candidate PET ligands were selected on the basis of favorable properties. The objective of this study was to characterize in vivo in nonhuman primates 2 novel D1R agonist PET radiotracers, racemic 18 F-MNI-800 and its more active atropisomeric (-)-enantiomer, 18 F-MNI-968. Methods: Ten brain PET experiments were conducted with 18 F-MNI-800 on 2 adult rhesus macaques and 2 adult cynomolgus macaques, and 8 brain PET experiments were conducted with 18 F-MNI-968 on 2 adult rhesus macaques and 2 adult cynomolgus macaques. PET data were analyzed with both plasma-input-based methods and reference-region-based methods. Whole-body PET images were acquired with 18 F-MNI-800 from 2 adult rhesus macaques for radiation dosimetry estimates. Results: 18 F-MNI-800 and 18 F-MNI-968 exhibited regional uptake consistent with D1R distribution. Specificity and selectivity were demonstrated by dose-dependent blocking with the D 1 antagonist SCH-23390. 18 F-MNI-968 showed a 30% higher specific signal than 18 F-MNI-800, with a nondisplaceable binding potential of approximately 0.3 in the cortex and approximately 1.1 in the striatum. Dosimetry radiation exposure was favorable, with an effective dose of about 0.023 mSv/MBq. Conclusion: 18 F-MNI-968 has significant potential as a D1R agonist PET radiotracer, and further characterization in human subjects is warranted., (© 2021 by the Society of Nuclear Medicine and Molecular Imaging.)

The Rett syndrome protein MeCP2 was described as a methyl-CpG-binding protein, but its exact function remains unknown. Here we show that mouse MeCP2 is a microsatellite binding protein that specifically recognizes hydroxymethylated CA repeats. Depletion of MeCP2 alters chromatin organization of CA repeats and lamina-associated domains and results in nucleosome accumulation on CA repeats and genome-wide transcriptional dysregulation. The structure of MeCP2 in complex with a hydroxymethylated CA repeat reveals a characteristic DNA shape, with considerably modified geometry at the 5-hydroxymethylcytosine, which is recognized specifically by Arg 133 , a key residue whose mutation causes Rett syndrome. Our work identifies MeCP2 as a microsatellite DNA binding protein that targets the 5hmC-modified CA-rich strand and maintains genome regions nucleosome-free, suggesting a role for MeCP2 dysfunction in Rett syndrome., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Dysregulation of histone H3 lysine 4 (H3K4) methylation is implicated in the pathogenesis of neurodevelopmental disorders. Lysine-specific demethylase 1 (LSD1) determines the methylation status of H3K4 through flavin adenine dinucleotide (FAD)-mediated histone demethylation. Therefore, LSD1 inhibition in the brain can be a novel therapeutic option for treating these disorders. Positron emission tomography (PET) imaging of LSD1 allows for investigating LSD1 expression levels under normal and disease conditions and validating target engagement of therapeutic LSD1 inhibitors. This study designed and synthesized (2-aminocyclopropyl)phenyl derivatives with irreversible binding to LSD1 as PET imaging agents for LSD1 in the brain. We optimized lipophilicity of the lead compound to minimize the risk of nonspecific binding and identified 1e with high selectivity over monoamine oxidase A and B, which are a family of FAD-dependent enzymes homologous to LSD1. PET imaging in a monkey showed a high uptake of [ 18 F] 1e to regions enriched with LSD1, indicating its specific binding to LSD1.

Epigenetic modifications and nucleosome positioning play an important role in modulating gene expression. However, how the patterns of epigenetic modifications and nucleosome positioning are established around promoters is not well understood. Here, we have addressed these questions in a series of genome-wide experiments coupled to a novel bioinformatic analysis approach. Our data reveal a clear correlation between CpG density, promoter activity and accumulation of active or repressive histone marks. CGI boundaries define the chromatin promoter regions that will be epigenetically modified. CpG-rich promoters are targeted by histone modifications and histone variants, while CpG-poor promoters are regulated by DNA methylation. CGIs boundaries, but not transcriptional activity, are essential determinants of H2A.Z positioning in vicinity of the promoters, suggesting that the presence of H2A.Z is not related to transcriptional control. Accordingly, H2A.Z depletion has no impact on gene expression of arrested mouse embryonic fibroblasts. Therefore, the underlying DNA sequence, the promoter CpG density and, to a lesser extent, transcriptional activity, are key factors implicated in promoter chromatin architecture., Competing Interests: Declarations of Interest None., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

18 F-PI-2620 is a PET tracer with high binding affinity for aggregated tau, a key pathologic feature of Alzheimer disease (AD) and other neurodegenerative disorders. Preclinically, 18 F-PI-2620 binds to both 3-repeat and 4-repeat tau isoforms. The purpose of this first-in-humans study was to evaluate the ability of 18 F-PI-2620 to detect tau pathology in AD patients using PET imaging, as well as to assess the safety and tolerability of this new tau PET tracer. Methods: Participants with a clinical diagnosis of probable AD and healthy controls (HCs) underwent dynamic 18 F-PI-2620 PET imaging for 180 min. 18 F-PI-2620 binding was assessed visually and quantitatively using distribution volume ratios (DVR) estimated from noninvasive tracer kinetics and SUV ratio (SUVR) measured at different time points after injection, with the cerebellar cortex as the reference region. Time-activity curves and SUVR were assessed in AD and HC subjects, as well as DVR and SUVR correlations and effect size (Cohen's d) over time. Results: 18 F-PI-2620 showed peak brain uptake around 5 min after injection and fast washout from nontarget regions. In AD subjects, focal asymmetric uptake was evident in temporal and parietal lobes, precuneus, and posterior cingulate cortex. DVR and SUVR in these regions were significantly higher in AD subjects than in HCs. Very low background signal was observed in HCs. 18 F-PI-2620 administration was safe and well tolerated. SUVR time-activity curves in most regions and subjects achieved a secular equilibrium after 40 min after injection. A strong correlation ( R 2 > 0.93) was found between noninvasive DVR and SUVR for all imaging windows starting at more than 30 min after injection. Similar effect sizes between AD and HC groups were obtained across the different imaging windows. 18 F-PI-2620 uptake in neocortical regions significantly correlated with the degree of cognitive impairment. Conclusion: Initial clinical data obtained in AD and HC subjects demonstrated a high image quality and excellent signal-to-noise ratio of 18 F-PI-2620 PET for imaging tau deposition in AD subjects. Noninvasive quantification using DVR and SUVR for 30-min imaging windows between 30 and 90 min after injection-for example, 45-75 min-provides robust and significant discrimination between AD and HC subjects. 18 F-PI-2620 uptake in expected regions correlates strongly with neurocognitive performance., (© 2020 by the Society of Nuclear Medicine and Molecular Imaging.)

18 F-PI-2620 is a next-generation tau PET tracer that has demonstrated ability to image the spatial distribution of suspected tau pathology. The objective of this study was to assess the tracer biodistribution, dosimetry, and quantitative methods of 18 F-PI-2620 in the human brain. Full kinetic modeling to quantify tau load was investigated. Noninvasive kinetic modeling and semiquantitative methods were evaluated against the full tracer kinetics. Finally, the reproducibility of PET measurements from test and retest scans was assessed. Methods: Three healthy controls (HCs) and 4 Alzheimer disease (AD) subjects underwent 2 dynamic PET scans, including arterial sampling. Distribution volume ratio (DVR) was estimated using full tracer kinetics (reversible 2-tissue-compartment [2TC] model and Logan graphical analysis [LGA]) and noninvasive kinetic models (noninvasive LGA [NI-LGA] and the multilinear reference tissue model [MRTM2]). SUV ratio (SUVR) was determined at different imaging windows after injection. The correlation between DVR and SUVR, effect size (Cohen's d), and test-retest variability (TRV) were evaluated. Additionally, 6 HCs received 1 tracer administration and underwent whole-body PET for dosimetry calculation. Organ doses and the whole-body effective dose were calculated using OLINDA 2.0. Results: A strong correlation was found across different kinetic models ( R 2 > 0.97) and between DVR(2TC) and SUVR between 30 and 90 min, with an R 2 of more than 0.95. Secular equilibrium was reached at around 40 min after injection in most regions and subjects. TRV and effect size for SUVR across different regions were similar at 30-60 min (TRV, 3.8%; Cohen's d, 3.80), 45-75 min (TRV, 4.3%; Cohen's d, 3.77) and 60-90 min (TRV, 4.9%; Cohen's d, 3.73) and increased at later time points. Elimination was via the hepatobiliary and urinary systems. The whole-body effective dose was 33.3 ± 2.1 μSv/MBq for an adult female and 33.1 ± 1.4 μSv/MBq for an adult male, with a 1.5-h urinary bladder voiding interval. Conclusion: 18 F-PI-2620 exhibits fast kinetics, suitable dosimetry, and low TRV. DVR measured using the 2TC model with arterial sampling correlated strongly with DVR measured by NI-LGA, MRTM2, and SUVR. SUVR can be used for 18 F-PI-2620 PET quantification of tau deposits, avoiding arterial blood sampling. Static 18 F-PI-2620 PET scans between 45 and 75 min after injection provide excellent quantification accuracy, a large effect size, and low TRV., (© 2020 by the Society of Nuclear Medicine and Molecular Imaging.)

While the histone variant H2A.Z is known to be required for mitosis, it is also enriched in nucleosomes surrounding the transcription start site of active promoters, implicating H2A.Z in transcription. However, evidence obtained so far mainly rely on correlational data generated in actively dividing cells. We have exploited a paradigm in which transcription is uncoupled from the cell cycle by developing an in vivo system to inactivate H2A.Z in terminally differentiated post-mitotic muscle cells. ChIP-seq, RNA-seq and ATAC-seq experiments performed on H2A.Z KO post-mitotic muscle cells show that this histone variant is neither required to maintain nor to activate transcription. Altogether, this study provides in vivo evidence that in the absence of mitosis H2A.Z is dispensable for transcription and that the enrichment of H2A.Z on active promoters is a marker but not an active driver of transcription., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The brain tumor microenvironment has recently become a major challenge in all pediatric cancers, but especially in brain tumors like high-grade gliomas. Hypoxia is one of the extrinsic tumor features that interacts with tumor cells, but also with the blood-brain barrier and all normal brain cells. It is the result of a dramatic proliferation and expansion of tumor cells that deprive the tissues of oxygen inflow. However, cancer cells, especially tumor stem cells, can endure extreme hypoxic conditions by rescheduling various genes' expression involved in cell proliferation, metabolism and angiogenesis and thus, promote tumor expansion, therapeutic resistance and metabolic adaptation. This cellular adaptation implies Hypoxia-Inducible Factors (HIF), namely HIF-1α and HIF-2α. In pediatric high-grade gliomas (pHGGs), several questions remained open on hypoxia-specific role in normal brain during gliomagenesis and pHGG progression, as well how to model it in preclinical studies and how it might be counteracted with targeted therapies. Therefore, this review aims to gather various data about this key extrinsic tumor factor in pHGGs.

Purpose: Synaptic vesicle protein 2A (SV2A) serves as a biomarker of synaptic density and positron emission tomography (PET) imaging of SV2A could provide a tool to assess progression of neurodegenerative diseases. Two tracers have primarily been reported and characterized in vivo: [ 11 C]UCB-J and [ 18 F]UCB-H. In early human studies, [ 11 C]UCB-J showed promising results, while its F-18-labeled analogue [ 18 F]UCB-H showed suboptimal specific signal in comparison to [ 11 C]UCB-J. Considering the limited use of [ 11 C]UCB-J to facilities with a cyclotron, having a F-18 variant would facilitate large, multicenter imaging trials. We have screened several F-18 derivatives of UCB-J in non-human primates and identified a promising F-18 PET candidate, [ 18 F]MNI-1126, with additional investigations of the racemate [ 18 F]MNI-1038, affording a signal comparable to [ 11 C]UCB-J., Procedures: F-18 derivatives of UCB-J and UCB-H were synthesized and administered to non-human primates for microPET imaging. Following screenings, [ 18 F]MNI-1038 (racemate) and [ 18 F]MNI-1126 (R-enantiomer) were identified with the highest signal and favorable kinetics and were selected for further imaging. Kinetic modeling with one- and two-tissue compartmental models, and linear methods were applied to PET data using metabolite-corrected arterial input function. Pre-block scans with levetiracetam (LEV, 10, 30 mg/kg, iv) were performed to determine the tracers' in vivo specificity for SV2A. Two whole-body PET studies were performed with [ 18 F]MNI-1038 in one male and one female rhesus, and radiation absorbed dose estimates and effective dose (ED, ICRP-103) were estimated with OLINDA/EXM 2.0., Results: All compounds screened displayed very good brain penetration, with a plasma-free fraction of ~ 40 %. [ 18 F]MNI-1126 and [ 18 F]MNI-1038 showed uptake and distribution the most consistent with UCB-J, while the other derivatives showed suboptimal results, with similar or lower uptake than [ 18 F]UCB-H. V T of [ 18 F]MNI-1126 and [ 18 F]MNI-1038 was high in all gray matter regions (within animal averages ~ 30 ml/cm 3 ) and highly correlated with [ 11 C]UCB-J (r > 0.99). Pre-blocking of [ 18 F]MNI-1126 or [ 18 F]MNI-1038 with LEV showed robust occupancy across all gray matter regions, similar to that reported with [ 11 C]UCB-J (~ 85 % at 30 mg/kg, ~ 65 % at 10 mg/kg). Using the centrum semiovale as a reference region, BP ND of [ 18 F]MNI-1126 reached values of up to ~ 30 to 40 % higher than those reported for [ 11 C]UCB-J. From whole-body imaging average ED of [ 18 F]MNI-1038 was estimated to be 22.3 μSv/MBq, with tracer being eliminated via both urinary and hepatobiliary pathways., Conclusions: We have identified a F-18-labeled tracer ([ 18 F]MNI-1126) that exhibits comparable in vivo characteristics and specificity for SV2A to [ 11 C]UCB-J in non-human primates, which makes [ 18 F]MNI-1126 a promising PET radiotracer for imaging SV2A in human trials.

The Drosophila circadian oscillator relies on a negative transcriptional feedback loop, in which the PERIOD (PER) and TIMELESS (TIM) proteins repress the expression of their own gene by inhibiting the activity of the CLOCK (CLK) and CYCLE (CYC) transcription factors. A series of posttranslational modifications contribute to the oscillations of the PER and TIM proteins but few posttranscriptional mechanisms have been described that affect mRNA stability. Here we report that down-regulation of the POP2 deadenylase, a key component of the CCR4-NOT deadenylation complex, alters behavioral rhythms. Down-regulating POP2 specifically increases TIM protein and tim mRNA but not tim pre-mRNA, supporting a posttranscriptional role. Indeed, reduced POP2 levels induce a lengthening of tim mRNA poly(A) tail. Surprisingly, such effects are lost in per 0 mutants, supporting a PER-dependent inhibition of tim mRNA deadenylation by POP2. We report a deadenylation mechanism that controls the oscillations of a core clock gene transcript., Competing Interests: The authors declare no conflict of interest.

Down-regulation of UHRF1 (Ubiquitin-like containing PHD and Ring Finger 1) in Jurkat cells, induced by natural anticancer compounds such as thymoquinone, allows re-expression of tumor suppressor genes such as p73 and p16 INK4A . In order to decipher the mechanisms of UHRF1 down-regulation, we investigated the kinetic of expression of HAUSP (herpes virus-associated ubiquitin-specific protease), UHRF1, cleaved caspase-3 and p73 in Jurkat cells treated with thymoquinone. We found that thymoquinone induced degradation of UHRF1, correlated with a sharp decrease in HAUSP and an increase in cleaved caspase-3 and p73. UHRF1 concomitantly underwent a rapid ubiquitination in response to thymoquinone and this effect was not observed in the cells expressing mutant UHRF1 RING domain, suggesting that UHRF1 commits an auto-ubiquitination through its RING domain in response to thymoquinone treatment. Exposure of cells to Z-DEVD, an inhibitor of caspase-3 markedly reduced the thymoquinone-induced down-regulation of UHRF1, while proteosomal inhibitor MG132 had no such effect. The present findings indicate that thymoquinone induces in cancer cells a fast UHRF1 auto-ubiquitination through its RING domain associated with HAUSP down-regulation. They further suggest that thymoquinone-induced UHRF1 auto-ubiquitination followed by its degradation is a key event in inducing apoptosis through a proteasome-independent mechanism., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no financial conflicts of interest.

Circadian clocks have evolved as time-measuring molecular devices to help organisms adapt their physiology to daily changes in light and temperature. Transcriptional oscillations account for a large fraction of rhythmic protein abundance. However, cycling of various posttranslational modifications, such as ubiquitylation, also contributes to shape the rhythmic protein landscape. In this study, we used an in vivo ubiquitin labeling assay to investigate the circadian ubiquitylated proteome of Drosophila melanogaster. We find that cyclic ubiquitylation affects MEGATOR (MTOR), a chromatin-associated nucleoporin that, in turn, feeds back to regulate the core molecular oscillator. Furthermore, we show that the ubiquitin ligase subunits CULLIN-3 (CUL-3) and SUPERNUMERARY LIMBS (SLMB) cooperate for ubiquitylating the TIMELESS protein. These findings stress the importance of ubiquitylation pathways in the Drosophila circadian clock and reveal a key component of this system., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cytoplasmic polyadenylation is a widespread mechanism to regulate mRNA translation. In vertebrates, this process requires two sequence elements in target 3' UTRs: the U-rich cytoplasmic polyadenylation element and the AAUAAA hexanucleotide. In Drosophila melanogaster , cytoplasmic polyadenylation of Toll mRNA occurs independently of these canonical elements and requires a machinery that remains to be characterized. Here we identify Dicer-2 as a component of this machinery. Dicer-2, a factor previously involved in RNA interference (RNAi), interacts with the cytoplasmic poly(A) polymerase Wispy. Depletion of Dicer-2 from polyadenylation-competent embryo extracts and analysis of wispy mutants indicate that both factors are necessary for polyadenylation and translation of Toll mRNA. We further identify r2d2 mRNA, encoding a Dicer-2 partner in RNAi, as a Dicer-2 polyadenylation target. Our results uncover a novel function of Dicer-2 in activation of mRNA translation through cytoplasmic polyadenylation., (© 2018 Coll et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Piwi-interacting RNAs (piRNAs) and PIWI proteins play a crucial role in germ cells by repressing transposable elements and regulating gene expression. In Drosophila, maternal piRNAs are loaded into the embryo mostly bound to the PIWI protein Aubergine (Aub). Aub targets maternal mRNAs through incomplete base-pairing with piRNAs and can induce their destabilization in the somatic part of the embryo. Paradoxically, these Aub-dependent unstable mRNAs encode germ cell determinants that are selectively stabilized in the germ plasm. Here we show that piRNAs and Aub actively protect germ cell mRNAs in the germ plasm. Aub directly interacts with the germline-specific poly(A) polymerase Wispy, thus leading to mRNA polyadenylation and stabilization in the germ plasm. These results reveal a role for piRNAs in mRNA stabilization and identify Aub as an interactor of Wispy for mRNA polyadenylation. They further highlight the role of Aub and piRNAs in embryonic patterning through two opposite functions.

Fbw7 is a tumor suppressor often deleted or mutated in human cancers. It serves as the substrate-recruiting subunit of a SCF ubiquitin ligase that targets numerous critical proteins for degradation, including oncoproteins and master transcription factors. Cyclin E was the first identified substrate of the SCFFbw7 ubiquitin ligase. In human cancers bearing FBXW7-gene mutations, deregulation of cyclin E turnover leads to its aberrant expression in mitosis. We investigated Fbw7 regulation in Xenopus eggs, which, although arrested in a mitotic-like phase, naturally express high levels of cyclin E. Here, we report that Fbw7α, the only Fbw7 isoform detected in eggs, is phosphorylated by PKC (protein kinase C) at a key residue (S18) in a manner coincident with Fbw7α inactivation. We show that this PKC-dependent phosphorylation and inactivation of Fbw7α also occurs in mitosis during human somatic cell cycles, and importantly is critical for Fbw7α stabilization itself upon nuclear envelope breakdown. Finally, we provide evidence that S18 phosphorylation, which lies within the intrinsically disordered N-terminal region specific to the α-isoform reduces the capacity of Fbw7α to dimerize and to bind cyclin E. Together, these findings implicate PKC in an evolutionarily-conserved pathway that aims to protect Fbw7α from degradation by keeping it transiently in a resting, inactive state.