Determinants of Human Growth Hormone Expression and Pituitary Cell Differentiation

摘要 垂体前叶的促生长激素和促乳素谱系的分化构成了一个高度的 哺乳动物细胞分化的信息量大、医学上相关、特征明确的模型。 值得注意的是，这两个谱系的分化依赖于相同的脑下垂体特异的活性。 转录因子Pit-1(POU1-F1)。定义两个谱系中每一个的标志性基因，如生长 激素(GH)和催乳素(PRL)受Pit-1依赖的顺式调控元件的直接控制。损失 Pit-1的表达导致小鼠和人的垂体前叶缺乏这两种类型的细胞 随之而来的是综合激素缺乏综合征。尽管Pit-1对发展具有核心重要性 以及垂体前叶的功能，它激活的驱动分化的机制和途径 生长抑素和乳促乳素谱系的维持尚不清楚。我们假设Pit-1， 与差异表达的转录因子共同作用，控制 通过与血统定义的顺式调节元件结合来调节生长和乳激素谱系，介导长- 范围转录相互作用，组织核小体结构，并定义血统依赖的三个 三维(3D)染色质网络。在目标1中，我们检验以下假设：Pit-1在主要 人类生长激素基因座控制区(HSI)中的增强子元件激活了一系列时间上定义的 对垂体hGH-N转录的强健和选择性激活至关重要的功能 生长激素及其在乳酸菌谱系中的相互抑制。在目标2中，我们测试假设 Pit-1驱动谱系分化的能力取决于它与协同转录因子(S)的相互作用。 候选因子通过初级流动分选生长激素的比较转录组分析来确定 和乳酸菌，并通过一套令人信服的功能分析进行验证。在目标3中，我们检验这一假设 3D染色质相互作用的不同网络在整个促生长激素和嗜乳素中建立 基因组来整合和协调特定血统的基因激活和抑制程序，这些 3-D架构依赖于特定血统的Pit-1动作。所有这三个目标都是基于对 从生理完整的野生型或转基因小鼠系的脑垂体中分离出的原代细胞。这些 研究将扩大我们对垂体功能的理解，并将使我们能够预测和定义 激素表达的表型变异，确定遗传和获得性内分泌的致病突变 疾病，并强调新的治疗干预措施的目标。此外，该计划将建立一个 具有里程碑意义的模式中的哺乳动物发育范例，并作为研究 在广泛的实验环境中的分化和基因组调控。

Abstract The divergence of the somatotrope and lactotrope lineages in the anterior pituitary constitutes a highly informative, medically relevant, and well-characterized model of mammalian cellular differentiation. Remarkably, the differentiation of both lineages is dependent on the activity of the same pituitary-specific transcription factor, Pit-1 (POU1-F1). Landmark genes defining each of the two lineages, such as Growth Hormone (GH) and Prolactin (Prl), are under direct control of Pit-1 dependent cis-regulatory elements. Loss of Pit-1 expression results in absence of both cell types from the anterior pituitary in mice and humans with consequent combined hormone deficiency syndromes. Despite the central importance of Pit-1 to development and function of the anterior pituitary, the mechanisms and pathways that it activates to drive the differentiation and maintenance of the somatotrope and lactotrope lineages remain unclear. We hypothesize that Pit-1, acting in conjunction with differentially expressed transcription factors, controls the divergence of the somatotrope and lactotrope lineages by binding to lineage-defined cis-regulatory elements, mediating long- range transcriptional interactions, organizing nucleosomal architectures, and defining lineage-dependent three dimensional (3D) chromatin networks. In Aim 1, we test the hypothesis that Pit-1 occupancy at the major enhancer element within the human GH locus control region (HSI) activates a series of temporally-defined functions that are critical to the robust and selective activation of hGH-N transcription in the pituitary somatotrope and its reciprocal repression in the lactotrope lineage. In Aim 2 we test the hypothesis that the ability of Pit-1 to drive lineage divergence depends on its interactions with cooperating transcriptional factor(s). Candidate factors are identified by comparative transcriptome analyses of primary flow-sorted somatotropes and lactotropes and are validated by a set of compelling functional assays. In Aim 3 we test the hypothesis that distinct networks of 3D chromatin interactions are established throughout the somatotrope and lactotrope genomes to integrate and coordinate lineage-specific gene activation and repression programs and that these 3-D architectures are dependent on lineage-specific Pit-1 actions. All three Aims are based on analyses of primary cells isolated from the pituitaries of physiologically intact wild type or transgenic mouse lines. These studies will extend our understanding of pituitary function and will allow us to predict and define the basis for phenotypic variations in hormone expression, identify causative mutations in inherited and acquired endocrine disorders, and highlight targets for novel therapeutic interventions. Furthermore, this program will establish a paradigm of mammalian development in a landmark model and serve as a template for investigations of differentiation and genome regulation in a broad spectrum of experimental settings.