Identification of Elf5 targets in pregnant mammary gland progenitor cells

 描述（由申请人提供）：确定调节乳腺细胞分化层次的分子机制对于更好地了解此类细胞命运决定如何影响乳腺发育、成熟以及各种乳腺癌亚型的发生和进展至关重要。然而，控制乳腺细胞谱系选择的转录和基因调控机制还不清楚，特别是在乳腺癌反应性妊娠乳腺的体内背景下。因此，我们的长期目标是全面描绘怀孕期间乳腺的转录网络。Elf 5是一个关键的转录因子，在催乳素受体下游发挥作用，对乳腺小叶肺泡发育至关重要。使用Elf 5条件性敲除动物，我们已经表明，Elf 5-null乳腺完全不能启动肺泡在怀孕期间。此外，Elf 5缺失的乳腺具有扩大的干细胞和管腔祖细胞库，并表现出上皮间充质转化（EMT）的特征-这些发现进一步突出了Elf 5在驱动细胞命运选择中的重要性。虽然Elf 5是乳腺上皮发育，特别是管腔祖细胞分化的重要调节因子，但富含Elf 5的管腔祖细胞的分子性质的几个关键方面仍然未知。具体地，存在未满足的需要来定义和更好地机械地理解Elf 5如何在妊娠期间在管腔祖细胞中介导其下游转录活性。为了解决这些知识的差距，我们将利用一种新的转基因小鼠模型，选择性地表达绿色荧光蛋白（GFP）报告基因的Elf 5丰富的管腔祖细胞。我们将使用抗Elf 5抗体和深度测序进行ChIP-exo研究，以在从小鼠妊娠乳腺获得的纯化管腔祖细胞中以高分辨率和深度覆盖鉴定全局Elf 5靶位点。这些研究将通过生物信息学方法进行补充，以确定富集的其他转录因子结合位点。 在调控区中与Elf 5结合位点共同代表。总的来说，这些实验将揭示Elf 5调控的转录控制机制和信号通路。这项工作具有高度创新性和重要意义，因为我们提议使用尖端技术来研究基因调节的基本转录机制，这将为乳腺的发育决策带来新的发现。这些信息不仅是至关重要的，在怀孕期间乳腺生理的正常控制过程中提供重要的见解，但也将泄露的病理条件，如基底乳腺癌，这是由管腔祖细胞产生的重要线索。

 DESCRIPTION (provided by applicant): Identifying the molecular mechanisms that regulate the mammary cell differentiation hierarchy is critically important to better understand how such cell fate decisions impact mammary gland development, maturation and the initiation and progression of various subtypes of breast cancer. However, the transcriptional and gene-regulatory mechanisms that control cell lineage choices in mammary gland is not well understood, particularly in the in vivo context of the hormone-responsive pregnant mammary glands. Hence our long-term goal is to comprehensively delineate the transcriptional network of mammary glands during pregnancy. Elf5 is a key transcription factor that functions downstream of the prolactin receptor and is essential for lobuloalveolar development of the mammary gland. Using Elf5 conditional knockout animals, we have shown that Elf5-null mammary glands completely fail to initiate alveologenesis during pregnancy. In addition, Elf5-null mammary glands harbor an expanded pool of stem and luminal progenitor cells and exhibit features of Epithelial Mesenchymal Transition (EMT) - these findings further highlight the importance of Elf5 in driving cell fate choices. Although Elf5 is an essential regulator of mammary epithelial development and in particular of luminal progenitor differentiation, several key aspects of the molecular nature of the Elf5 enriched luminal progenitors remain unknown. Specifically, there is unmet need to define and mechanistically better understand how Elf5 mediates its downstream transcriptional activities in luminal progenitor cells during pregnancy. To address these knowledge gaps, we will utilize a novel transgenic mouse model that selectively expresses the Green Fluorescent Protein (GFP) reporter in the Elf5-enriched luminal progenitor cells. We will perform ChIP-exo studies using anti-Elf5 antibodies and deep sequencing to identify global Elf5 targets sites at high resolution and deep coverage in the purified luminal progenitor cells obtained from mouse pregnant mammary glands. These studies will be complemented by a bioinformatics approach to identify additional transcription factor binding sites that are enriched for and co-represented with Elf5 binding sites in regulatory regions. Collectively, these experiments will shed light on Elf5-governed transcriptional control mechanisms and signaling pathways. This work is highly innovative and significant because our proposed use of cutting edge technologies to examine fundamental transcriptional mechanisms of gene regulation will lead to new discoveries into developmental decisions of the mammary gland. Such information is not only crucial in providing important insights into the process of normal controls of mammary gland physiology during pregnancy, but will also divulge important clues on pathological conditions such as basal breast cancers, which arise from luminal progenitor cells.