Reconstitution of Pituitary Specific Transcription

垂体特异性转录的重建

• 批准号: 7172312
• 负责人: ARTHUR GUTIERREZ-HARTMANN
• 金额: $ 30.33万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7172312
• 关键词: Activins; Address; Alternative Splicing; Amino Acids; Binding; Biochemical; Biological; Cell Line; Cell Ontogeny; Cell Proliferation; Cells; Complex; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; DNA; DNA Binding; DNA Sequence; Data; Development; Dwarfism; Event; Family; Funding; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Goals; Growth; Hela Cells; Histone Acetylation; Histone Deacetylase; Human; Maps; Mediating; Mitolactol; Modeling; Molecular; Molecular Biology; Mus; Mutation; Nude Mice; Numbers; PRL gene; Phenotype; Pituitary Gland; Prolactin; Protein Isoforms; Proteins; RNA Interference; RNA Splicing; Recruitment Activity; Research Personnel; Role; Set protein; Site; Specificity; Structure; Testing; Transcriptional Activation; Transgenic Mice; Transgenic Organisms; cell type; homeodomain; in vivo; insight; lactotroph; mutant; novel; programs; promoter; reconstitution; response; transcription factor; transcription factor Pit-1; transgene expression; tumor growth

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.

描述（由申请人提供）：本提案的广泛任务是使用转基因、分子和生物化学方法来确定 Pit-1 异构体在依赖 Pit-1 的垂体细胞个体发育和基因表达方面特异的体内生物效应，并定义介导垂体细胞中 ¿ 异构体特异性转录反应的机制。 POU 同源域转录因子 Pit-1 控制生长激素细胞、催乳细胞和促甲状腺激素垂体细胞类型的发育，并调节 GH、PRL 和 TSHB 基因的细胞特异性表达。单个 Pit-1 基因在 Pit-1 谱系中表达为两个选择性剪​​接的 mRNA，从而产生 Pit-1 和 Pit-1¿ 蛋白。 Pit-1¿ 包含一个独特的 26 个氨基酸 (AA) S 结构域，插入 Pit-1 的 AA 48，恰好位于转录激活结构域 (TAD) 的中间。然而，Pit-1 和 Pit-1¿ 在其他方面具有相同的结构。小鼠和人类中自然发生的 Pit-1 突变导致遗传性侏儒症，该突变映射到 Pit-1 和 Pit-1¿ 共有的区域。然而，Pit-1对垂体细胞特异性个体发育和基因表达的精确生物学贡献仍然未知。尽管 Pit-1¿ 在最初的描述后被忽视，但在之前的资助期间，我们为理解精确的分子机制做出了重要、新颖和独特的贡献，通过该机制，Pit-1 ¿ 结构域作为转录开关基序发挥作用，从而导致 ¿ 亚型特异性转录反应。我们发现 Pit-1¿ 在所有测试的垂体细胞系中始终抑制 PRL、GH 和 TSHB 启动子活性，而 Pit-1¿ 在所有测试的非垂体细胞系中始终激活这些相同的启动子。此外，Pit-1¿ 抑制 GH4 垂体细胞中基础、cAMP 和 Ras 刺激的 rPRL 启动子活性。我们阐明了机制，证明 ¿ 结构域作为垂体特异性阻遏基序发挥作用，通过五个疏水性氨基酸发挥作用，募集 HDAC 活性以改变近端 rPRL 启动子的组蛋白乙酰化状态。重要的是，当与 Gal4 DBD 融合时，¿ 结构域可以作为自主、模块化、活性和 HDAC 依赖性阻遏基序发挥作用。最后，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；并抑制裸鼠GH4细胞增殖和肿瘤生长，为其生物学效应提供了证据。因此，Pit-1/Pit-1¿对提供了研究转录因子亚型特异性功能的原型模型。我们假设与 ¿ 结构域相关的垂体限制性阻遏物复合物决定了 Pit-1 异构体特异性转录反应。一个推论假设是，表达增加的 Pit-1¿ 的垂体细胞将具有独特的表型。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中 Pit-1 谱系的个体发育和/或扩张。 (2) 确定 Pit-1¿ 在 GH4 细胞中特异性诱导的生物反应。 (3) 使用 RNAi 来确定 Pit-1¿ 和确定的辅阻遏物在介导 ¿ 特异性反应中的生物学作用。 (4) 鉴定和功能验证与 ¿ 结构域相关的阻遏物复合物。从这些研究中获得的见解不仅可以更好地理解 Pit-1¿ 同工型特异性功能，而且还将提供一个概念和实验框架来研究与重叠 DMA 位点结合的其他高度相关的转录因子。