SIGNALING PATHWAYS THAT REGULATE DEVELOPMENT

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Members of the SWI2/SNF2 family of "helicase-like", DNA-dependent ATPases modulate transcription of chromatin templates. Proteins in the CHD branch of this family contain a chromodomain and a DNA-binding motif, in addition to the central helicase-like domain, and studies implicate CHD members in control of gene expression. We report the characterization of a presumptive CHD1 ortholog in Dictyostelium . Expression of CHD1 is induced as cells become competent to enter the multicellular stage of the Dictyostelium life-cycle, coincident with de-repression of the earliest developmentally regulated genes. Disruption of CHD1 leads to severe developmental defects including aberrant aggregation, reduction in prespore-specific gene expression, inappropriate patterning of both prespore and prestalk cells, and developmental arrest prior to the formation of terminally differentiated fruiting bodies. During aggregation, cells expressing prestalk genes normally sort to the top of the aggregate and form the extending tip, an organizer for further differentiation and for prestalk and prespore patterning. In the absence of CHD1, prestalk cells remain dispersed through the aggregate, even as extensions project from the mound. We propose that a primary defect in cells lacking CHD1 is the inability of prestalk cells to establish a functional tip. Consequently, prespore-specific gene expression is not maximally activated and cellular differentiation cannot proceed. These studies suggest that CHD1 is essential for differentiation and patterning of the major cell types in Dictyostelium. We are now extending our studies on developmental cellular signaling in Dictyostelium to the g-secretase/Presenilin (PS) pathway. The g-secretase complex Presenilin (PS), Nicastrin (Nct), Aph1 and Pen2 cleaves single-pass transmembrane proteins to release intracellular domain moieties that regulate a variety of signaling pathways. Proteolytic activity appears to reside within PS. In metazoa, g-secretase is responsible for processing of Notch, resulting in Notch-dependent gene expression and subsequent cell fate specification; loss-of-function mutations in PS leads to Notch-like embryonic lethality. In humans, mutations in PS alter specificity in cleavage of Amyoid Precursor Protein (APP) and ultimately cause Alzheimers disease. Dictyostelium has 2 genes that encode PS-like proteins and one each for Nct, Aph1, and Pen2. Loss of the two PS genes of Dictyostelium does not cause lethality, making it ideal to study g-secretase signaling. We have screened for novel functions of PS in Dictyostelium and for distinct roles of the different complex components. Although the genes in Dictyostelium display significant similarity to their mammalian counterparts, they are nonetheless highly diverged. To assess a definitive functional association with g-secretase activity, we expressed the mammalian substrate APP in WT and mutant variants of Dictyostelium. g-secretase processing of APP was observed in WT cells, but not in strains lacking PS, Nct, and Aph1, or in multiply mutated stains. Analysis of single and double mutations of PS, Nct and Aph1 genes, indicates that all the components are required for proper prespore/spore differentiation by a cell-autonomous pathway. We have tentatively identified several endogenous proteins in Dictyostelium that are candidate substrates for g-secretase processing in vivo and are testing if these proteins are indeed subject to PS cleavage and if their processing is essential to regulate Dictyostelium development.