Project description:This project investigated the effect of Cln3-deficiency on protein secretion in the social amoeba Dictyostelium discoideum by performing LC-MS/MS on conditioned media harvested from starved WT and cln3- cells.
Project description:The Unfolded Protein Response (UPR) is an adaptive pathway that restores cellular homeostasis after endoplasmic reticulum (ER) stress caused by an impairment of its protein folding capacity. The ER-resident kinase/ribonuclease Ire1 is the only UPR sensor that has been conserved during evolution from yeast to mammals; in these organisms, Ire1 transmits information from the ER to the nucleus trough the non-conventional splicing of Hac1 (yeast)/Xbp1 (metazoans) mRNA. We described the Dictyostelium discoideum ER-stress response and characterized its single bonafide Ire1 orthologue, IreA. We found that tunicamycin (TN) triggers a gene-expression program that increases the protein folding capacity of the ER and that alleviates ER protein load. Further, IreA resulted essential not only for cell-survival after TN-induced ER-stress, but also to accomplish about nearly 40% of the transcriptional changes induced upon a TN treatment. In addition, we described that autophagy is activated in Dictyostelium cells after a TN treatment and that autophagy-defective mutants exhibited increased sensitivity to this drug. The response of Dictyostelium cells to ER-stress involves the combined activation of an IreA-dependent gene expression program and the autophagy pathway. Overall design: Poly A RNA profile of wild-type (WT) and ireA- mutant Dictyostelium cells treated and non-treated with tunicamycin (TN) were generated by deep sequencing, in duplicate, using illumina MiSeq. The assembly of the data was performed using TopHat and Cufflinks software. Onthology enrichment analysis were performed with PANTHER and DAVID web tools
Project description:In many developmental systems, morphogenesis is coupled with dramatic changes in spatiotemporal gene expression, often orchestrated by the coordinated action of transcription factors. Development of the social soil amoebae Dictyostelium discoideum proceeds through a sequence of morphological and transcriptional changes, but the role of transcription factors in development is not well understood. GtaC, a GATA-type zinc-finger transcription factor, is essential for Dictyostelium development. It decodes pulsatile extracellular cAMP signals during early development and mediates cell-type differentiation at later stages. Here, we studied the developmental regulatory roles of GtaC through the concerted analysis of temporal ChIP- and RNA-sequencing data from strains that carry different alleles of gtaC. We show that GtaC exhibits temporally distinctive DNA-binding patterns throughout early development, accompanied by largely cotemporaneous expression of its target genes. We also show that GtaC binds DNA in two modes. One of these modes exhibits binding preferences for canonical GATA-like sequences, the regulatory consequences accompanying which is predominantly up-regulation of target gene expression. The other binding mode is mostly associated with down-regulation. Among its targets we find transcription factors that are essential for development as well as genes involved in cAMP signaling and cell-type specification. Our results suggest that GtaC is a master regulator that regulates multiple physiological processes during early development, when Dictyostelium transitions from a group of unicellular amoebae to an integrated multicellular organism. Cotemporaneous transcriptional profiling and ChIP sequencing during early Dictyostelium development