The circadian clock drives daily changes of physiology, including sleep-wake cycles, through regulation of transcription, protein abundance, and function. Circadian phosphorylation controls cellular ...processes in peripheral organs, but little is known about its role in brain function and synaptic activity. We applied advanced quantitative phosphoproteomics to mouse forebrain synaptoneurosomes isolated across 24 hours, accurately quantifying almost 8000 phosphopeptides. Half of the synaptic phosphoproteins, including numerous kinases, had large-amplitude rhythms peaking at rest-activity and activity-rest transitions. Bioinformatic analyses revealed global temporal control of synaptic function through phosphorylation, including synaptic transmission, cytoskeleton reorganization, and excitatory/inhibitory balance. Sleep deprivation abolished 98% of all phosphorylation cycles in synaptoneurosomes, indicating that sleep-wake cycles rather than circadian signals are main drivers of synaptic phosphorylation, responding to both sleep and wake pressures.
SNAIL is a key transcriptional regulator in embryonic development and cancer. Its effects in physiology and disease are believed to be linked to its role as a master regulator of ...epithelial-to-mesenchymal transition (EMT). Here, we report EMT-independent oncogenic SNAIL functions in cancer. Using genetic models, we systematically interrogated SNAIL effects in various oncogenic backgrounds and tissue types. SNAIL-related phenotypes displayed remarkable tissue- and genetic context-dependencies, ranging from protective effects as observed in KRAS- or WNT-driven intestinal cancers, to dramatic acceleration of tumorigenesis, as shown in KRAS-induced pancreatic cancer. Unexpectedly, SNAIL-driven oncogenesis was not associated with E-cadherin downregulation or induction of an overt EMT program. Instead, we show that SNAIL induces bypass of senescence and cell cycle progression through p16
-independent inactivation of the Retinoblastoma (RB)-restriction checkpoint. Collectively, our work identifies non-canonical EMT-independent functions of SNAIL and unravel its complex context-dependent role in cancer.
Abbreviations 7-AAD 7-Aminoactinomycin D AKT protein kinase B ANOVA analysis of variance BET bromodomain and extra-terminal motif BETi BET inhibitor BrdU 5-bromo-2'-deoxyuridine CI Combination Index ...CNV Copy Number Variation E2F E2 factor (E2F) family of transcription factors EGFR epidermal growth factor receptor ERBB Erb-B receptor tyrosine kinases (EGFR family) FDR False Discovery Rate GI50 Half-maximal growth inhibitory (GI50) concentration GSEA gene set enrichment analysis KEGG Kyoto Encyclopedia of Genes and Genomes MAPK Mitogen-activated protein kinase MEK MAPK/ERK kinase mRNA messenger ribonucleic acid mTOR mechanistic target of rapamycin mTORi mTOR inhibitor MYC myelocytomatosis oncogene PDAC pancreatic ductal adenocarcinoma PI3K Phosphoinositide 3-kinase PLK1 Serine/threonine-protein kinase PLK1 qPCR quantitative Polymerase Chain Reaction RNA ribonucleic acid RNA-seq RNA-sequencing SNP single-nucleotide polymorphism ZIP Zero interaction potency Dear Editor, Pancreatic ductal adenocarcinoma (PDAC) remains a significant health problem with an increase in the incidence and a five-year survival rate of only 10% 1. ...we aimed to understand the molecular underpinnings of mTORi resistance and treated 20 well-characterized (transcriptomics, single nucleotide polymorphisms SNPs, copy number variations CNVs) murine PDAC cell lines 7 with the potent mTORi INK128 (Sapanisertib) to determine the half-maximal growth inhibitory concentration (GI50) (Figure 1A). In PPT-9091MYCER cells, a conditional MYC gain-of-function model 8, activation of MYC led to a doubling of the INK128 GI50 value (Figure 1C), corroborating that MYC confers mTORi resistance, which is consistent with a recent report 9. Downstream signaling, as measured by investigating the phosphorylation of the mTOR target Eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), was distinctly reduced in the deficient clones (Supplementary Figure S2B).
A defining property of circadian clocks is temperature compensation, characterized by the resilience of their near 24-hour free-running periods against changes in environmental temperature within the ...physiological range. While temperature compensation is evolutionary conserved across different taxa of life and has been studied within many model organisms, its molecular underpinnings remain elusive. Posttranscriptional regulations such as temperature-sensitive alternative splicing or phosphorylation have been described as underlying reactions. Here, we show that knockdown of cleavage and polyadenylation specificity factor subunit 6 (CPSF6), a key regulator of 3'-end cleavage and polyadenylation, significantly alters circadian temperature compensation in human U-2 OS cells. We apply a combination of 3'-end-RNA-seq and mass spectrometry-based proteomics to globally quantify changes in 3' UTR length as well as gene and protein expression between wild-type and CPSF6 knockdown cells and their dependency on temperature. Since changes in temperature compensation behavior should be reflected in alterations of temperature responses within one or all of the 3 regulatory layers, we statistically assess differential responses upon changes in ambient temperature between wild-type and CPSF6 knockdown cells. By this means, we reveal candidate genes underlying circadian temperature compensation, including eukaryotic translation initiation factor 2 subunit 1 (EIF2S1).
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