![]() ![]() The tree was plotted as an organic tree and nodes were colored according to the z-score in the flow cytometry-based screen. The phylogenetic tree was build in Matlab and imported into Cytoscape (2.6.3) with the PhyloTree (v.0.1) plug-in. A similarity matrix was calculated and the data were clustered using the Matlab Bioinformatics Toolbox. The MHC class II related parameters were replicated four times and the Golgi related parameters were replicated two times to increase their weight in the consideration. All these features were z-score normalized in Excel (Table S4). After cross-validation accuracy calculation (Figure S4B) six MHC class II parameters, two for EEA1 and five for Golgi were selected. During supervised machine learning seven bins for MHC class II, three for early endosome and five for Golgi features were designed. The CP Analyst 2 program was used to determine the minimal number of parameters distinguishing and describing phenotypes of interest. The membrane area was defined as the area between the cell perimeter expanded or shrunken by 10 pixels. EEA1 and MHC class II vesicles and the Golgi were detected as primary objects and then assigned to a cell containing this object (their parent). From these nuclei the cell was detected based on cytosolic background staining. Briefly, nuclei were detected as primary objects. Divecha (The University of Manchester, UK) ( GFP-PIP5K1A GFP was a generous gift from Dr. D'Souza-Schorey (University of Notre Dame, FR). Double PH-domains of PSD4 (aa776-aa892) were amplified and cloned into pRP265 and pEGFP-N1 using BglII, EcoRI and BamH1 for lipid binding. SEC7 domains of PSD4 (aa555-aa738) and CYTH1 (aa73-aa202) were amplified from IMAGE clone 5757431 (PSD4) and IMAGE clone 4755203 (CYTH1) and cloned into pMAL-c2X using BamHI and HinDIII restriction sites. Those constructs were used for protein production, co-immune precipitation and colocalization experiments. ![]() ARF7EP was amplified form IMAGE clone 6062049 and cloned into pRP265 and p2HA-C1 via BglII and EcoRI (p2HA-C1 was retrieved from pEGFP-C1 (Clontech) where GFP was exchanged for HA-HA using Nhe1 and BglII cloning sites). ARL14/ARF7 was cloned, using restriction sites EcoRI and BamHI, into pRP265 and mCherry-N1 via EcoRI and BamHI. Cluster 2 has two areas where the genes cohered ( Figure 5A ) one was enriched for GO terms like “MHC-II protein complex” ( Figure 5B) and another for “cytosolic ribosome” terms (cells with reduced intracellular MHC-II ).ĪRL14/ARF7 Q68L missing the sequence coding for the myristoylation site (first two N-terminal amino acids) was amplified from IMAGE: 4747382 and cloned into pGBT9 via EcoRI and BamH1 restriction sites to use for Yeast Two-Hybrid assay. When we combined this information with the microscopy phenotypes, we noted that the genes in cluster 4 did not affect MHC-II distribution, whereas those in cluster 2 showed MHC-II redistribution to the cell surface that resembles an mDC phenotype ( Figure 6A). Subsequent GO analysis of the expanded groups indicated that clusters 2 and 4 ( Figure 4C) were enriched for “MHC-II protein complex” ( Table S5). These increased the number of proteins involved in the same functional pathway. Neighbors with a log-likelihood score ≥ 1 according to Humannet and a |z| ≥ 1.645 (p < 0.1) in our original flow cytometry screen were also included in this analysis ( Table S6). The AUC of 0.6175 indicates that many connected genes (neighbors) also genuinely interact. We first measured the degree of connectivity between our candidates by the area under the receiver operating characteristic (ROC) curve (AUC) ( Figure S6). ), which combines information from several expression and protein-protein interaction databases. These genome-wide systems analyses have thus identified factors and pathways controlling MHC-II transcription and transport, defining targets for manipulation of MHC-II antigen presentation in infection and autoimmunity. This complex controls movement of MHC-II vesicles along the actin cytoskeleton in human dendritic cells (DCs). MHC-II transport was controlled by the GTPase ARL14/ARF7, which recruits the motor myosin 1E via an effector protein ARF7EP. All data sets were integrated to answer two fundamental questions: what regulates tissue-specific MHC-II transcription, and what controls MHC-II transport in dendritic cells? MHC-II transcription was controlled by nine regulators acting in feedback networks with higher-order control by signaling pathways, including TGFβ. To unravel processes controlling MHC-II antigen presentation, we performed a genome-wide flow cytometry-based RNAi screen detecting MHC-II expression and peptide loading followed by additional high-throughput assays. MHC class II molecules (MHC-II) present peptides to T helper cells to facilitate immune responses and are strongly linked to autoimmune diseases.
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