Reconstitution of Pituitary Specific Transcription

垂体特异性转录的重建

• 批准号: 7570594
• 负责人: ARTHUR GUTIERREZ-HARTMANN
• 金额: $ 29.68万
• 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 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7570594
• 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; Modeling; Molecular; Molecular Biology; Mus; Nude Mice; PRL gene; Phenotype; Pituitary Gland; 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

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-IB isoform with regards to Pit-1-dependent pituitary cell ontogeny and gene expression, and to define the mechanism that mediates B-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-IB proteins. Pit-IB 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-1and Pit-IB share identical structures otherwise. Naturally occurring Pit-1mutations in mice and humans resulting in heritable dwarfism map to regions common to both Pit-1 and Pit-IB. However, the precise biological contributions of Pit-IB to pituitary cell-specific ontogeny and gene expression remain unknown. AlthoughPit- 1B 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 B-domain functions as a transcription switch motif resulting in B-isoform-specific transcriptional responses. We found that Pit-IB consistently inhibits PRL, GH and TSHB promoter activities in all pituitary cell lines tested, whereas Pit-IB consistently activates these same promoters in all nonpituitary cell lines tested. Moreover, Pit-IB inhibited basal, cAMP- and Ras- stimulated rPRL promoter activity in GH4 pituitary cells. We elucidated mechanism, demonstrating that the B-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 B-domain can function as an autonomous, modular, active and HDAC-dependent represser motif when fused to the Gal4 DBD. Finally, adenoviral encoded HA-Pit-1B 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-IB pair provides a prototypical model to study transcription factor isoform-specific functions. We hypothesize that a pituitary- restricted represser complex that associates with the B-domain dictates Pit-IB isoform-specific transcriptional responses. A corollary hypothesis is that pituitary cells expressing increased Pit-IB- will have a distinct phenotype. To address this hypothesis, we propose four Specific Aims: (1) To determine whether HA-Pit-1B- 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-IB in GH4 cells. (3) To use RNAi to determine the biological role of Pit-IB and defined co-repressors in mediating B-specific responses. (4) To identify and functionally validate the represser complex associated with the B-domain. Insights gained from these studies will not only provide a better understanding of Pit-IB 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.

这项建议的广泛授权是使用转基因、分子和生化方法来确定在 Pit-IB亚型对依赖Pit-1的垂体细胞个体发育和基因的体内生物学效应 表达，并确定在脑垂体细胞中介导B-异构体特异性转录反应的机制。 POU同源结构域转录因子Pit-1控制着促生长激素、催乳素和促甲状腺激素的发育 垂体细胞类型，并调节GH、PRL和TSHB基因的细胞特异性表达。单个Pit-1基因是 在Pit-1谱系中表达为两个选择性剪接的mRNAs，导致Pit-1和Pit-IB蛋白。PIT-IB包含 在Pit-1的Aa48位插入了一个独特的26个氨基酸的S结构域，恰好位于转录激活的中间 域(TAD)。然而，Pit-1和Pit-IB在其他方面具有相同的结构。自然发生的Pit-1突变 导致可遗传侏儒症的小鼠和人类映射到Pit-1和Pit-IB共同的区域。然而，准确的 Pit-IB在垂体细胞特异性个体发育和基因表达中的生物学作用尚不清楚。尽管Pit- 在最初的描述之后，1B被忽视了，在之前的资助期内，我们使其变得重要、新颖和 对我们理解Pit-1 B结构域功能的精确分子机制的独特贡献 作为转录开关基序，导致B-异构体特异的转录反应。我们发现Pit-IB 在所有被测试的垂体细胞系中，Pit-IB一致地抑制PRL、GH和TSHB启动子的活性 在所有测试的非垂体细胞系中激活这些相同的启动子。此外，Pit-IB抑制基础、cAMP-和RAS- 刺激GH4细胞的rPRL启动子活性。我们阐明了机制，证明了B-结构域 作为脑垂体特异性阻遏基序，通过五种疏水氨基酸招募HDAC活性 改变rPRL近端启动子的组蛋白乙酰化状态。重要的是，B-结构域可以起到 当与Gal4 DBD融合时，自主、模块化、主动和依赖HDAC的抑制器基序。最后，腺病毒 编码HA-Pit-1B抑制内源性催乳素和细胞周期蛋白DI，但激活Rb基因表达；抑制GH4细胞 裸鼠体内的增殖和肿瘤生长，为其生物学效应提供了证据。因此，Pit-1/Pit-IB对 提供了一个研究转录因子异构体特异性功能的原型模型。我们假设一个脑下垂体- 与B结构域相关的限制性抑制物复合体决定Pit-IB异构体特异性转录 回应。一个必然的假设是，表达增加的Pit-IB-的垂体细胞将具有不同的表型。至 针对这一假设，我们提出了四个具体的目标：(1)确定HA-Pit-1B-转基因是否表达 控制转基因小鼠的个体发育和/或Pit-1谱系的扩展。(2)生物测定 Pit-IB诱导的GH4细胞的特异性反应。(3)利用RNAi技术确定Pit-IB的生物学功能，并对Pit-IB和Pit-IB的功能进行比较 在调节B-特异性反应中定义的共抑制物。(4)识别和功能验证抑制子复合体 与B-结构域相关联。从这些研究中获得的见解不仅将提供对Pit-IB的更好理解 但也将提供一个概念和实验框架来研究其他高度相关的 与重叠的DMA位点结合的转录因子。