SIGNALING PATHWAYS THAT REGULATE DEVELOPMENT

SWI 2/SNF 2家族的“解旋酶样”DNA依赖性ATP酶的成员调节染色质模板的转录。该家族的CHD分支中的蛋白质除了中心解旋酶样结构域之外还含有一个染色体结构域和一个DNA结合基序，并且研究暗示CHD成员控制基因表达。我们报告的一个假定的CHD 1直系同源在Dictyosteopathy的表征。CHD 1的表达被诱导为细胞变得有能力进入网骨藻生命周期的多细胞阶段，与最早的发育调控基因的去阻遏一致。CHD 1的破坏导致严重的发育缺陷，包括异常聚集、前孢子特异性基因表达减少、前孢子和前柄细胞的不适当图案化以及在形成终末分化的子实体之前的发育停滞。在聚集过程中，表达前茎基因的细胞通常分选到聚集体的顶部并形成延伸的尖端，这是进一步分化以及前茎和前孢子图案化的组织者。在没有CHD 1的情况下，前柄细胞仍然分散在聚集体中，即使是从丘延伸出来。我们提出，缺乏CHD 1的细胞的主要缺陷是前柄细胞无法建立功能性尖端。因此，前孢子特异性基因表达没有被最大限度地激活，细胞分化不能进行。这些研究表明，CHD 1是必不可少的分化和图案的主要细胞类型的网骨藻。 我们现在正在扩大我们的研究在Dictyosteoblasts的g-分泌酶/早老素（PS）通路的发育细胞信号。G-分泌酶复合物Presenilin（PS）、Nicastrin（Nct）、Aph 1和Pen 2切割单程跨膜蛋白以释放调节多种信号传导途径的细胞内结构域部分。蛋白水解活性似乎存在于PS内。在后生动物中，G-分泌酶负责Notch的加工，导致Notch依赖的基因表达和随后的细胞命运特化; PS中的功能缺失突变导致Notch样胚胎致死。在人类中，PS突变改变淀粉样前体蛋白（APP）切割的特异性，并最终导致阿尔茨海默病。Dictyosteoprotein有2个编码PS样蛋白的基因，Nct、Aph 1和Pen 2各有一个。两个PS基因的丢失不会导致Dictyosteoblasts的致死性，使其成为理想的研究g-分泌酶信号。我们已经筛选了新的功能PS在Dictyosteopathy和不同的复杂的组件的不同作用。虽然在Dictyosteroid中的基因显示出与它们的哺乳动物对应物的显著相似性，但它们仍然高度分化。为了评估与g-分泌酶活性的确定的功能关联，我们表达了哺乳动物底物APP在野生型和突变体的Dictyosteroid。在WT细胞中观察到APP的g-分泌酶加工，但在缺乏PS、Nct和Aph 1的菌株或多重突变菌株中未观察到。PS，Nct和Aph 1基因的单突变和双突变的分析表明，所有的组件都需要适当的前孢子/孢子分化的细胞自主途径。我们已经初步确定了几种内源性蛋白质在Dictyosteoprotein的候选底物的g-分泌酶在体内加工，并测试这些蛋白质是否确实受到PS裂解，如果他们的加工是必不可少的调节Dictyosteoprotein的发展。

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.