Dictyostelium discoideum is an exceptionally powerful eukaryotic model to study many aspects of growth, development, and fundamental cellular processes. Its small-sized, haploid genome allows highly efficient targeted homologous recombination for gene disruption and knock-in epitope tagging. We had developed a robust system for the generation of multiple gene mutations in Dictyostelium by recycling the Blasticidin S selectable marker after transient expression of the Cre recombinase. We have now further optimized the system for higher efficiency and, additionally, coupled it to both knock-out and knock-in gene targeting, allowing the characterization of multiple and cooperative gene functions in a single cell line. Micrococcal nuclease (MNase) is an endonuclease that cleaves native DNA at high frequency, but is blocked in chromatin by sites of intimate DNA-protein interaction, including nucleosomal regions. Protection from MNase cleavage has often been used to map transcription factor binding sites and nucleosomal positions on a single gene basis; however, by combining MNase digestion with high-throughput, paired-end DNA sequencing, we map protein-DNA interaction regions across the entire genome. Biochemical and bioinformatic protocols have been optimized for global mono-nucleosome positioning at 160 bp spacing coverage, but are applicable to mapping more broadly or for site-specific binding of transcription factors at 50 bp resolution. Control of chromatin structure is critical for multicellular development and regulation of cell differentiation. The CHD (Chromodomain-Helicase-DNA binding) protein family is one of the major ATP-dependent, chromatin remodeling factors that regulate nucleosome positioning and access of transcription factors and RNA polymerase to the eukaryotic genome. There are 3 mammalian CHD subfamilies and their impaired functions are associated with several human diseases. We have identified three CHD orthologs (ChdA, ChdB, and ChdC) in Dictyostelium discoideum. These CHDs are expressed throughout development, but with unique patterns. Null mutants lacking each CHD have distinct, non-redundant phenotypes that reflect their expression patterns and suggest functional specificity. Accordingly, utilizing genome-wide (RNA-seq) transcriptome profiling for each null strain, we showed that the different CHDs regulate independent gene sets during both growth and development. ChdC is an apparent ortholog of the mammalian Class III CHD group that is associated with the human CHARGE syndrome, and GO analyses of aberrant gene expression in chdC-nulls suggest defects in both cell-autonomous and non-autonomous signaling, which have been confirmed through analyses of chdC-nulls developed in pure populations or with low levels of WT cells. We have provided novel insight into the broad function of CHDs in the regulation development and disease, through chromatin-mediated changes in directed gene expression. Migratory cells, like mammalian leukocytes and Dictyostelium, utilize G protein coupled receptor(GPCR) signaling to regulate MAPK/ERK, PI3K, TORC2/AKT, adenylyl cyclase, and actin polymerization, which collectively direct chemotaxis. Upon ligand binding, mammalian GPCRs are phosphorylated at cytoplasmic residues, uncoupling G protein pathways, but activating others. Still, connections between GPCR phosphorylation and chemotaxis are unclear. In developing Dictyostelium, secreted cAMP serves as a chemoattractant, with extracellular cAMP propagated as oscillating waves to ensure directional migratory signals. cAMP oscillations derive from transient excitatory responses of adenylyl cyclase, which then rapidly adapts. We have studied chemotactic signaling in Dictyostelium that express non-phosphorylatable cAMP receptors and show through chemotaxis modeling, single-cell FRET imaging, pure and chimeric population wavelet quantification, biochemical analyses, and TIRF microscopy, that receptor phosphorylation is required to regulate adenylyl cyclase adaptation, long-range oscillatory cAMP wave production, and cytoskeletal actin response. Phosphorylation defects, thus, promote hyperactive actin polymerization at the cell periphery, misdirected pseudopodia, and the loss of directional chemotaxis. Our data indicate that chemoattractant receptor phosphorylation is required to co-regulate essential pathways for migratory cell polarization and chemotaxis. Our results significantly extend the understanding of GPCR phosphorylation function, providing strong evidence that this evolutionarily conserved mechanism is required in a signal attenuation pathway that is necessary to maintain persistent directional movement of Dictyostelium, neutrophils, and other migratory cells. Global stimulation of Dictyostelium with different chemoattractants elicits multiple transient signaling responses, including synthesis of cAMP and cGMP, actin polymerization, activation of kinases ERK2, TORC2, and PI3K, and Ras-GTP accumulation; mechanisms that down-regulate these responses are poorly understood. We examined transient activation of TORC2 in response to chemically distinct chemoattractants, cAMP and folate, and suggest that TORC2 is regulated by adaptive, de-sensitizing responses to stimulatory ligands that are independent of downstream, feedback or feed-forward circuits. Cells with acquired insensitivity to either folate or cAMP remain fully responsive to TORC2 activation if stimulated with the other ligand. Thus, TORC2 responses to cAMP or folate are not cross-inhibitory. Using a series of signaling mutants, we have shown that folate and cAMP activate TORC2 through an identical GEF/Ras pathway, but separate receptors and G protein couplings. Since the common GEF/Ras pathway also remains fully responsive to one chemoattractant after de-sensitization to the other, GEF/Ras must act downstream and independently of adaptation to persistent ligand stimulation. When initial chemoattractant concentrations are immediately diluted, cells rapidly regain full responsiveness. We suggest that ligand adaptation functions in upstream inhibitory pathways that involve chemoattractant-specific receptor/G protein complexes and regulate multiple response pathways.
NIH ExPorter
NIH ExPorter