Reconstitution of Pituitary Specific Transcription

描述（由申请人提供）：本提案的广泛任务是使用转基因、分子和生化方法来确定Pit-1异构体在Pit-1依赖性垂体细胞个体发育和基因表达方面的体内生物学效应，并定义垂体细胞中介导-异构体特异性转录反应的机制。ppo同源结构域转录因子Pit-1控制生长营养型、乳营养型和甲状腺营养型垂体细胞的发育，调控GH、PRL和TSHB基因的细胞特异性表达。单个Pit-1基因在Pit-1谱系中作为两个可选剪接的mrna表达，产生Pit-1和Pit-1¿蛋白。Pit-1¿含有一个独特的26个氨基酸（AA）的s结构域，插入在Pit-1的AA 48上，正好位于转录激活域（TAD）的中间。然而，Pit-1和Pit-1在其他方面具有相同的结构。在小鼠和人类中自然发生的Pit-1突变导致遗传性侏儒症映射到Pit-1和Pit-1共同的区域。然而，Pit-1¿对垂体细胞特异性个体发育和基因表达的确切生物学贡献尚不清楚。虽然Pit-1¿在其最初的描述之后被忽视了，但在之前的资助期间，我们对Pit-1¿-结构域作为转录开关基序导致-异构体特异性转录反应的精确分子机制的理解做出了重要，新颖和独特的贡献。我们发现，在所有垂体细胞系中，Pit-1¿始终抑制PRL、GH和TSHB启动子的活性，而在所有非垂体细胞系中，Pit-1¿始终激活这些启动子。此外，Pit-1¿抑制了GH4垂体细胞中基底、cAMP-和Ras-刺激的rPRL启动子活性。我们阐明了机制，证明了¿-结构域作为垂体特异性抑制基序通过五个疏水氨基酸募集HDAC活性来改变近端rPRL启动子的组蛋白乙酰化状态。重要的是，当与Gal4 DBD融合时，¿-结构域可以作为一个自主的、模块化的、主动的和hdac依赖的抑制基序发挥作用。腺病毒编码的HA-Pit-1¿抑制内源性PRL和cyclinDI，但激活RB基因表达；抑制裸小鼠GH4细胞增殖和肿瘤生长，为其生物学效应提供证据。因此，Pit-1/Pit-1¿对为研究转录因子同种异构体特异性功能提供了一个原型模型。我们假设与-结构域相关的垂体限制性抑制复合物决定了Pit-1 -异构体特异性转录反应。一个必然的假设是，垂体细胞表达增加的Pit-1¿-将有一个独特的表型。为了解决这一假设，我们提出了四个具体目标：(1)确定HA-Pit-1¿-转基因表达是否控制转基因小鼠的个体发育和/或Pit-1谱系的扩增。(2)确定Pit-1¿在GH4细胞中特异性诱导的生物学反应。(3)利用RNAi确定Pit-1和定义的共抑制因子在介导特异性反应中的生物学作用。(4)鉴定和功能验证与¿-结构域相关的抑制复合体。从这些研究中获得的见解不仅将提供对Pit-1异构体特异性功能的更好理解，而且还将为研究与重叠DMA位点结合的其他高度相关的转录因子提供概念和实验框架。

DESCRIPTION (provided by applicant): The broad mandate of this proposal is to use transgenic, molecular and biochemical approaches to determine the in vivo biological effects specific to the Pit-1¿ isoform with regards to Pit-1-dependent pituitary cell ontogeny and gene expression, and to define the mechanism that mediates ¿ -isoform-specific transcriptional responses in pituitary cells. The POU-homeodomain transcription factor, Pit-1, controls the development of somatotroph, lactotroph and thyrotroph pituitary cell-types, and regulates the cell-specific expression of GH, PRL and TSHB genes. The single Pit-1 gene is expressed in the Pit-1 lineage as two alternatively-spliced mRNAs, resulting in Pit-1 and Pit-1¿ proteins. Pit-1¿ contains a unique 26 amino-acid (AA) S-domain inserted at AA 48 of Pit-1, precisely in the middle of the transcription activation domain (TAD). However, Pit-1 and Pit-1¿ share identical structures otherwise. Naturally occurring Pit-1 mutations in mice and humans resulting in heritable dwarfism map to regions common to both Pit-1 and Pit-1¿. However, the precise biological contributions of Pit-1¿ to pituitary cell-specific ontogeny and gene expression remain unknown. Although Pit- 1¿ has been overlooked after its initial description, during the previous funding period we made important, novel and unique contributions to our understanding of the precise molecular mechanisms by which the Pit-1 ¿ -domain functions as a transcription switch motif resulting in ¿ -isoform-specific transcriptional responses. We found that Pit-1¿ consistently inhibits PRL, GH and TSHB promoter activities in all pituitary cell lines tested, whereas Pit-1¿ consistently activates these same promoters in all nonpituitary cell lines tested. Moreover, Pit-1¿ inhibited basal, cAMP- and Ras- stimulated rPRL promoter activity in GH4 pituitary cells. We elucidated mechanism, demonstrating that the ¿ -domain functions as a pituitary-specific represser motif acting via five hydrophobic amino acids that recruit HDAC activity to alter the histone acetylation state of the proximal rPRL promoter. Importantly, the ¿ -domain can function as an autonomous, modular, active and HDAC-dependent represser motif when fused to the Gal4 DBD. Finally, adenoviral encoded HA-Pit-1¿ inhibits endogenous PRL and cyclinDI, but activates RB gene expression; and inhibits GH4 cell proliferation and tumor growth in nude mice, providing evidence of its biological effects. Thus, the Pit-1/Pit-1¿ pair provides a prototypical model to study transcription factor isoform-specific functions. We hypothesize that a pituitary- restricted represser complex that associates with the ¿ -domain dictates Pit-1¿ isoform-specific transcriptional responses. A corollary hypothesis is that pituitary cells expressing increased Pit-1¿ - will have a distinct phenotype. To address this hypothesis, we propose four Specific Aims: (1) To determine whether HA-Pit-1¿ - transgene expression governs the ontogeny and/or expansion of the Pit-1 lineage in transgenic mice. (2) To determine the biological responses specifically induced by Pit-1¿ in GH4 cells. (3) To use RNAi to determine the biological role of Pit-1¿ and defined co-repressors in mediating ¿ -specific responses. (4) To identify and functionally validate the represser complex associated with the ¿ -domain. Insights gained from these studies will not only provide a better understanding of Pit-1¿ isoform-specific functions, but will also provide a conceptual and experimental framework to study other highly-related transcription factors that bind to overlapping DMA sites.