Functional Interaction between the Coactivator Drosophila CREB-Binding Protein and ASH1, a Member of the Trithorax Group of Chromatin Modifiers

CREB-binding protein (CBP) is a coactivator for multiple transcription factors that transduce a variety of signaling pathways. Current models propose that CBP enhances gene expression by bridging the signal-responsive transcription factors with components of the basal transcriptional machinery and by augmenting the access of transcription factors to DNA through the acetylation of histones. To define the pathways and proteins that require CBP function in a living organism, we have begun a genetic analysis of CBP in flies. We have overproduced Drosophila melanogaster CBP (dCBP) in a variety of cell types and obtained distinct adult phenotypes. We used an uninflated-wing phenotype, caused by the overexpression of dCBP in specific central nervous system cells, to screen for suppressors of dCBP overactivity. Two genes with mutant versions that act as dominant suppressors of the wing phenotype were identified: the PKA-C1/DCO gene, encoding the catalytic subunit of cyclic AMP protein kinase, and ash1, a member of the trithorax group (trxG) of chromatin modifiers. Using immunocolocalization, we showed that the ASH1 protein is specifically expressed in the majority of the dCBP-overexpressing cells, suggesting that these proteins have the potential to interact biochemically. This model was confirmed by the findings that the proteins interact strongly in vitro and colocalize at specific sites on polytene chromosomes. The trxG proteins are thought to maintain gene expression during development by creating domains of open chromatin structure. Our results thus implicate a second class of chromatin-associated proteins in mediating dCBP function and imply that dCBP might be involved in the regulation of higher-order chromatin structure.

CREB结合蛋白（CREB-binding protein, CBP）是一类可转导多种信号通路的转录因子的共激活因子。现有模型认为，CBP通过两种途径增强基因表达：其一，将信号响应转录因子与基础转录机器的组分进行桥接；其二，通过组蛋白乙酰化作用提升转录因子对DNA的可及性。为明确活生物体中依赖CBP功能的通路与蛋白质，我们启动了果蝇体内CBP的遗传分析研究。我们在多种细胞类型中过量表达黑腹果蝇（Drosophila melanogaster）CBP（简称dCBP），并获得了多种不同的成虫表型。我们利用在特定中枢神经系统细胞中过量表达dCBP所诱导的翅膀未展开表型，筛选dCBP过度活跃的抑制因子。最终鉴定出两个携带突变形式的基因可作为该翅膀表型的显性抑制因子：分别是编码环腺苷酸蛋白激酶催化亚基的PKA-C1/DCO基因，以及染色质修饰因子trithorax家族（trithorax group, trxG）成员ash1。通过免疫共定位实验，我们证实ASH1蛋白在大多数dCBP过量表达的细胞中特异性表达，提示二者具备发生生化相互作用的潜力。这一假说通过后续实验结果得到验证：两种蛋白质在体外存在强烈相互作用，且可在多线染色体的特定位点共定位。目前学界认为，trxG蛋白可通过形成开放染色质结构域，维持发育过程中的基因表达。综上，我们的研究结果将第二类染色质相关蛋白纳入介导dCBP功能的范畴，并暗示dCBP可能参与调控高级染色质结构。