chromatin-immunoprecipitation-chip-in-mouse-t-cell-lines

Chromatin Immunoprecipitation (ChIP) in Mouse T-cell Lines

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational ...

University of Giessen

Several methods have been developed for measurement of plasma total homocysteine (tHcy), a proven graded risk factor for cardiovascular diseases. The aim of the study was to compare 3 commonly used clinical methods (enzyme immunoassay, Axis Biochemicals, Oslo, Norway; and fluorescence polarization immunoassay [FPIA], Axis, on the Abbott IMx System, Abbott Laboratories, Abbott Park, IL; and BioRad high-performance liquid chromatography [HPLC], Laboratories, Munich, Germany) for tHcy determination in samples from cardiovascular patients also undergoing methionine load measurement. Only HPLC reached the required coefficient of variation (<6%) for desirable performance for tHcy concentrations ranging between 0.68 and 5.41 mg/L (5 and 40 micromol/L). A higher variability was observed for postmethionine load vs fasting samples, which reached statistical significance for FPIA (P = .0013). The closest agreement between methods was observed between HPLC and FPIA for fasting tHcy concentrations (-0.23 mg/L [-1.69 micromol/L], -30% to 8%).Both immunoassays could be suitable alternatives for laboratories with high workloads when HPLC is not available. Differences among tHcy values must be taken into account when these methods will be used interchangeably for diagnosis and monitoring of treatment.

Performance comparison of three assay methods used in fasting and postmethionine load plasma homocysteine determinations from patients with vascular disease.

Maternal care (MC) and dopamine modulate brain activity during emotion processing in inferior frontal gyrus (IFG), striatum and amygdala. Reuptake of dopamine from the synapse is performed by the dopamine transporter (DAT), whose abundance is predicted by variation in its gene (DAT 3'VNTR; 10 > 9-repeat alleles). Here, we investigated the interaction between perceived MC and DAT 3'VNTR genotype on brain activity during processing of aversive facial emotional stimuli.

DAT by perceived MC interaction on human prefrontal activity and connectivity during emotion processing.

Physiologically, Notch signal transduction plays a pivotal role in differentiation; pathologically, Notch signaling contributes to the development of cancer. Transcriptional activation of Notch target genes involves cleavage of the Notch receptor in response to ligand binding, production of the Notch intracellular domain (NICD), and NICD migration into the nucleus and assembly of a coactivator complex. Posttranslational modifications of the NICD are important for its transcriptional activity and protein turnover. Deregulation of Notch signaling and stabilizing mutations of Notch1 have been linked to leukemia development. We found that the methyltransferase CARM1 (coactivator-associated arginine methyltransferase 1; also known as PRMT4) methylated NICD at five conserved arginine residues within the C-terminal transactivation domain. CARM1 physically and functionally interacted with the NICD-coactivator complex and was found at gene enhancers in a Notch-dependent manner. Although a methylation-defective NICD mutant was biochemically more stable, this mutant was biologically less active as measured with Notch assays in embryos of Xenopus laevis and Danio rerio. Mathematical modeling indicated that full but short and transient Notch signaling required methylation of NICD.

Site-specific methylation of Notch1 controls the amplitude and duration of the Notch1 response.

The transcriptional shift from repression to activation of target genes is crucial for the fidelity of Notch responses through incompletely understood mechanisms that likely involve chromatin-based control. To activate silenced genes, repressive chromatin marks are removed and active marks must be acquired. Histone H3 lysine-4 (H3K4) demethylases are key chromatin modifiers that establish the repressive chromatin state at Notch target genes. However, the counteracting histone methyltransferase required for the active chromatin state remained elusive. Here, we show that the RBP-J interacting factor SHARP is not only able to interact with the NCoR corepressor complex, but also with the H3K4 methyltransferase KMT2D coactivator complex. KMT2D and NCoR compete for the C-terminal SPOC-domain of SHARP. We reveal that the SPOC-domain exclusively binds to phosphorylated NCoR. The balance between NCoR and KMT2D binding is shifted upon mutating the phosphorylation sites of NCoR or upon inhibition of the NCoR kinase CK2β. Furthermore, we show that the homologs of SHARP and KMT2D in Drosophila also physically interact and control Notch-mediated functions in vivo Together, our findings reveal how signaling can fine-tune a committed chromatin state by phosphorylation of a pivotal chromatin-modifier.

Francesca Ferrante

Institute of Biochemistry

University of Giessen

Chromatin Immunoprecipitation (ChIP) in Mouse T-cell Lines

Francesca Ferrante

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Institute of Biochemistry

University of Giessen

Chromatin Immunoprecipitation (ChIP) in Mouse T-cell Lines

Institute of Biochemistry