Reconstitution of Pituitary Specific Transcription

这项建议的主要任务是利用转基因、分子和生物化学方法来确定基因组中的 Pit-1B亚型对Pit-1依赖性垂体细胞个体发育和基因的体内生物学效应 表达，并确定在垂体细胞中介导B-亚型特异性转录反应的机制。 POU同源域转录因子Pit-1控制着促生长素、促乳素和促甲状腺素的发育 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谱系中表达为两种选择性剪接的mRNA，产生Pit-1和Pit-1B蛋白。Pit-IB包含 一个独特的26个氨基酸（AA）的S-结构域插入在AA 48的Pit-1，正是在中间的转录激活 域（domain）。然而，Pit-1和Pit-1B在其他方面具有相同的结构。天然发生的Pit-1突变， 小鼠和人导致遗传性侏儒症，映射到Pit-1和Pit-1B共同的区域。然而，精确 Pit-1B对垂体细胞特异性个体发育和基因表达的生物学贡献仍然未知。虽然坑- 1B在最初的描述之后被忽视了，在上一个资助期，我们提出了重要的，新颖的， 对我们理解Pit-1 B结构域功能的精确分子机制做出了独特的贡献 作为转录开关基序，导致B亚型特异性转录反应。我们发现Pit-IB 在所有测试的垂体细胞系中，Pit-IB一致地抑制PRL、GH和TSHB启动子活性，而Pit-IB一致地抑制PRL、GH和TSHB启动子活性。 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-1B抑制基础，cAMP-和Ras- 刺激GH 4垂体细胞rPRL启动子活性。我们阐明了机制，表明B结构域 作为垂体特异性阻遏物基序发挥作用，通过5个疏水氨基酸募集HDAC活性， 改变近端rPRL启动子的组蛋白乙酰化状态。重要的是，B结构域可以作为一个 当与Gal 4 DBD融合时，具有自主、模块化、活性和HDAC依赖性阻遏基序。最后，腺病毒 编码的HA-Pit-1B抑制内源性PRL和cyclinDI，但激活RB基因表达，并抑制GH4细胞 增殖和裸鼠肿瘤生长，提供其生物学效应的证据。因此，Pit-1/Pit-1B对 为研究转录因子亚型特异性功能提供了原型模型。我们假设脑垂体- 与B结构域相关的限制性阻遏物复合物决定Pit-1B亚型特异性转录 应答一个必然的假设是，垂体细胞表达增加Pit-IB-将有一个独特的表型。到 针对这一假设，我们提出了四个具体的目的：（1）确定HA-Pit-1B-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/或扩增。(2)为了确定 Pit-1B在GH4细胞中特异性诱导的应答。(3)为了使用RNAi来确定Pit-1B的生物学作用， 在介导B特异性反应中定义的共阻遏物。(4)为了鉴定和功能验证阻遏复合物， 与B域相关。从这些研究中获得的见解不仅可以更好地了解Pit-IB 异构体特异性功能，但也将提供一个概念和实验框架，以研究其他高度相关的 与重叠的DNA位点结合的转录因子。

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.