Antigen-specific stimulation of T helper (Th) cells initiates signaling cascades that ultimately result in the activation of the transcription factors NF-jB, NFAT, and AP-1 which regulate, together with other factors, many T-cell functions such as cytokine production, proliferation, and differentiation. Ordered assembly and different phosphorylation events, along with subcellular translocation of the CARMA1/Bcl-10/MALT1 complex, determine NF-jB activation after T-cell receptor (TCR) triggering. We now provide evidence that inhibition of the Ser/Thr phosphatase calcineurin (CaN) prevents dephosphorylation of Bcl-10. CaN, in constant interaction with the Bcl-10/MALT1 complex, is able to dephosphorylate Bcl-10. The CaN inhibitor cyclosporine A (CsA) converts a transient phosphorylation of Bcl-10 Ser138 during the immediate early phase of T-cell activation into a persistent state. Thus, subsequent processes such as IKKb phosphorylation, IjBa degradation, p65 nuclear translocation, and DNA binding are diminished. Consistently, CsA treatment does not affect the phosphorylation pattern of the upstream kinase PKCh. Together, our findings demonstrate that CaN functions as a critical signaling molecule during Th cell activation, regulating Bcl-10 phosphorylation and thereby NF-jB activation.Keywords: Calcineurin . Cell activation . T cell . TCR signaling . Transcription factors Supporting Information available online IntroductionAntigen-specific stimulation of T helper (Th) cells induces activation of the transcription factors nuclear factor of activated T cells (NFAT) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) via a complex signaling network. The cooperative action of these and other transcription factors results in gene expression profiles leading to the activation of Th cells, production of growth factors, in particular cytokines, and proliferation [1][2][3]. T-cell receptor (TCR) engagement triggers phosphorylation of membrane-associated tyrosine kinases that activate PLCg, which in turn generates the second messengers IP 3 and DAG. The IP 3 -induced Ca 21 influx triggers the Ser/Thr phosphatase calcineurin (CaN) which dephosphorylates NFAT. This dephosphorylation step is crucial for the nuclear translocation of NFAT and binding to promoters of target genes. The second messenger, DAG, activates the NF-kB signaling cascade by binding to the protein kinase C-y (PKCy) leading to a change in PKCy conformation into an open, active state. The precise nature of these changes is not entirely clear, but phosphorylation at different sites, subcellular translocation, and assembly of assorted molecules contributes to this process (reviewed in [4,5] [6,7]. The importance of Bcl-10 for NF-kB activation is underlined by studies in Bcl-10 knockout mice showing that Bcl-10 is essential for NF-kB-driven cytokine production, cell proliferation, and B-as well as T-cell development [8,9]. Furthermore, Bcl-10 overexpression in MALT B-cell lymphomas is characterized by constant NF-kB activation [10,11]. Bcl-1...

The cell biology of circadian clocks is still in its infancy. Here, we describe an efficient strategy for generating knock-in reporter cell lines using CRISPR technology that is particularly useful for genes expressed transiently or at low levels, such as those coding for circadian clock proteins. We generated single and double knock-in cells with endogenously expressed PER2 and CRY1 fused to fluorescent proteins allowing us to simultaneously monitor the dynamics of CRY1 and PER2 proteins in live single cells. Both proteins are highly rhythmic in the nucleus of human cells with PER2 showing a much higher amplitude than CRY1. Surprisingly, CRY1 protein is nuclear at all circadian times indicating the absence of circadian gating of nuclear import. Furthermore, in the nucleus of individual cells CRY1 abundance rhythms are phase-delayed (~5 hours), and CRY1 levels are much higher (>5 times) compared to PER2 questioning the current model of the circadian oscillator.

Transcription factors of the nuclear factor of activated T cell (NFAT) family are essential for antigen-specific T cell activation and differentiation. Their cooperative DNA binding with other transcription factors, such as AP1 proteins (FOS, JUN, and JUNB), FOXP3, IRFs, and EGR1, dictates the gene regulatory action of NFATs. To identify as yet unknown interaction partners of NFAT, we purified biotin-tagged NFATc1/αA, NFATc1/βC, and NFATc2/C protein complexes and analyzed their components by stable isotope labeling by amino acids in cell culture-based mass spectrometry. We revealed more than 170 NFAT-associated proteins, half of which are involved in transcriptional regulation. Among them are many hitherto unknown interaction partners of NFATc1 and NFATc2 in T cells, such as Raptor, CHEK1, CREB1, RUNX1, SATB1, Ikaros, and Helios. The association of NFATc2 with several other transcription factors is DNA-dependent, indicating cooperative DNA binding. Moreover, our computational analysis discovered that binding motifs for RUNX and CREB1 are found preferentially in the direct vicinity of NFAT-binding motifs and in a distinct orientation to them. Furthermore, we provide evidence that mTOR and CHEK1 kinase activity influence NFAT's transcriptional potency. Finally, our dataset of NFAT-associated proteins provides a good basis to further study NFAT's diverse functions and how these are modulated due to the interplay of multiple interaction partners.

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Special attention has recently been drawn to the molecular network of different genes that are responsible for the development of erythroid cells. The aim of the present study was to establish in detail the immunophenotype of early erythroid cells and to compare the gene expression profile of freshly isolated early erythroid precursors with that of the CD34-positive (CD34+) compartment. Multiparameter flow cytometric analyses of human bone marrow mononuclear cell fractions (n=20) defined three distinct early erythroid stages. The gene expression profile of sorted early erythroid cells was analyzed by Affymetrix array technology. For 4524 genes, a differential regulation was found in CD105-positive erythroid cells as compared with the CD34+ progenitor compartment (2362 upregulated genes). A highly significant difference was observed in the expression level of genes involved in transcription, heme synthesis, iron and mitochondrial metabolism and transforming growth factor-β signaling. A comparison with recently published data showed over 1000 genes that as yet have not been reported to be upregulated in the early erythroid lineage. The gene expression level within distinct pathways could be illustrated directly by applying the Ingenuity software program. The results of gene expression analyses can be seen at the Gene Expression Omnibus repository.

Nonsense-mediated messenger RNA (mRNA) decay (NMD) has been intensively studied as a surveillance pathway that degrades erroneous transcripts arising from mutations or RNA processing errors. While additional roles in physiological control of mRNA stability have emerged, possible functions in mammalian physiology in vivo remain unclear. Here, we created a conditional mouse allele that allows converting the NMD effector nuclease SMG6 from wild-type to nuclease domain-mutant protein. We find that NMD down-regulation affects the function of the circadian clock, a system known to require rapid mRNA turnover. Specifically, we uncover strong lengthening of free-running circadian periods for liver and fibroblast clocks and direct NMD regulation of Cry2 mRNA, encoding a key transcriptional repressor within the rhythm-generating feedback loop. Transcriptome-wide changes in daily mRNA accumulation patterns in the entrained liver, as well as an altered response to food entrainment, expand the known scope of NMD regulation in mammalian gene expression and physiology.

Circadian clocks are endogenous oscillators essential for orchestrating daily rhythms in physiology, metabolism and behavior. While mouse models have been instrumental to elucidate the molecular mechanism of circadian rhythm generation, our knowledge about the molecular makeup of circadian oscillators in humans is still limited. Here, we used duplex CRISPR/Cas9 technology to generate three cellular models for studying human circadian clocks: CRY1 knockout cells, CRY2 knockout cells as well as CRY1 / CRY2 double knockout cells. Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes by using two guide RNAs targeting exon-flanking intron regions of human osteosarcoma cells (U-2 OS). Resulting cell clones did not express CRY proteins and showed short period, low-amplitude rhythms (for CRY1 knockout), long period rhythms (for CRY2 knockout) or were arrhythmic (for CRY1 / CRY2 double knockout) similar to circadian phenotypes of cells derived from classical knockout mouse models.

The current model of the mammalian circadian oscillator is predominantly based on data from genetics and biochemistry experiments, while the cell biology of circadian clocks is still in its infancy. Here, we describe a new strategy for the efficient generation of knock-in reporter cell lines using CRISPR technology that is particularly useful for lowly or transiently expressed genes, such as those coding for circadian clock proteins. We generated single and double knock-in cells with endogenously expressed PER2 and CRY1 fused to fluorescent proteins, which allowed to simultaneously monitor the dynamics of CRY1 and PER2 proteins in live single cells. Both proteins are highly rhythmic in the nucleus of human cells with PER2 showing a much higher amplitude than CRY1. Surprisingly, CRY1 protein is nuclear at all circadian times indicating the absence of circadian gating of nuclear import. Furthermore, in the nucleus of individual cells CRY1 abundance rhythms are phase-delayed (~5 hours), and CRY1 levels are much higher (>6 times) compared to PER2 questioning the current model of the circadian oscillator.Our knock-in strategy will allow the generation of additional single, double or triple knock-in cells for circadian clock proteins, which should greatly advance our understanding about the cell biology of circadian clocks.Recent biochemical studies with mouse liver lysates suggest that during the repressive phase, essentially all nuclear PER and CRY proteins are coordinated together in one large repressive complex, with only a minor amount of CRY1 monomers detectable (17). Again, double knock-out of either Per1/2 or Cry1/2 completely prevented the formation of this repressive complex.Most of the current knowledge of PER and CRY protein dynamics resulted either from biochemical data with mixed lysates of many thousand cells, or from single-cell imaging of over-expressed fluorescent tagged fusion proteins (12,13,17,18). Both approaches have clear limitations: Population samplinge.g. cell fractionation followed by Western Blot, chromatography or immunoprecipitation -not only conceals spatial information but also suffers from much reduced temporal resolution. Most importantly, however, population sampling averages signals from thousands of cells thereby masking individual cell properties (e.g. regarding circadian period, phase and amplitude) and degree of noise.While fluorescent tagged proteins constitute an outstanding tool to monitor protein expression and localization in individual living cells, overexpression of PER-and CRY-proteins in most cases disrupts the circadian oscillator and data from such experiments have to be conceived with caution (19,20).Such limitations can be overcome by incorporating a fluorescent tag directly into the proteins' genomic locus. In this case, expression dynamics and level of the resulting fusion protein often remain similar to the wild-type protein and the clock stays intact. Indeed, the PER2-Luciferase and the PER2-Venus knock-in mice -in which PER2 is tagged at the genomic level with ...