SIGNALING PATHWAYS THAT REGULATE DEVELOPMENT

SWI2/SNF2“解旋酶样”家族成员，dna依赖性atp酶调节染色质模板的转录。该家族的冠心病分支蛋白除了中央解旋酶样结构域外，还包含一个色域和一个dna结合基元，研究表明冠心病成员控制基因表达。我们报道了Dictyostelium中推测的CHD1同源物的特征。CHD1的表达随着细胞进入盘基骨柱生命周期的多细胞阶段而被诱导，与早期发育调节基因的去抑制相一致。CHD1的破坏会导致严重的发育缺陷，包括异常聚集、孢子前特异性基因表达减少、孢子前和孢子前细胞的不适当模式以及在最终分化的子实体形成之前发育停滞。在聚集过程中，表达前柄基因的细胞通常排序到聚集体的顶部并形成延伸尖端，这是进一步分化的组织者，也是前柄和前孢子模式的组织者。在缺乏CHD1的情况下，即使从丘状突起延伸出来，预柄细胞仍然分散在聚集体中。我们认为缺乏CHD1的细胞的主要缺陷是前柄细胞无法建立功能性尖端。因此，孢子前特异性基因表达不能被最大限度地激活，细胞分化不能进行。这些研究表明，CHD1对盘基骨柱中主要细胞类型的分化和模式形成至关重要。

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.