Identification of Elf5 targets in pregnant mammary gland progenitor cells

 描述(由申请人提供)：确定调控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、成熟以及不同亚型乳腺癌的发生和发展至关重要。然而，控制乳腺细胞谱系选择的转录和基因调控机制还不是很清楚，特别是在荷尔蒙反应的怀孕乳腺的活体环境中。因此，我们的长期目标是全面描述怀孕期间乳腺的转录网络。ELF5是一个关键的转录因子，作用于催乳素受体下游，对乳腺小叶肺泡发育至关重要。使用ELF5条件性基因敲除动物，我们已经证明ELF5缺失的乳腺在怀孕期间完全无法启动肺泡形成。此外，ELF5缺失的乳腺拥有扩大的干细胞库和腔前体细胞池，并表现出上皮间充质转化(EMT)的特征--这些发现进一步强调了ELF5在驱动细胞命运选择方面的重要性。虽然ELF5是乳腺上皮发育的重要调节因子，尤其是腔祖细胞的分化，但ELF5丰富的腔祖细胞分子性质的几个关键方面仍不清楚。具体地说，还没有得到满足的需求是定义并从机械上更好地了解ELF5如何在怀孕期间调节其在腔前体细胞中的下游转录活动。为了解决这些知识空白，我们将利用一种新的转基因小鼠模型，在ELF5富含的腔前体细胞中选择性地表达绿色荧光蛋白(GFP)报告。我们将使用抗ELF5抗体和深度测序进行芯片外显研究，以高分辨率和深度覆盖从小鼠怀孕乳腺获得的纯化腔前体细胞中确定全球ELF5靶点。这些研究将得到生物信息学方法的补充，以确定更多的富含转录因子结合位点 与调控区域中的ELF5结合位点结合并共同代表。总的来说，这些实验将阐明ELF5控制的转录控制机制和信号通路。这项工作具有很高的创新性和重要意义，因为我们建议使用尖端技术来研究基因调控的基本转录机制，这将导致对乳腺发育决策的新发现。这些信息不仅对了解妊娠期间正常对照的乳腺生理过程至关重要，而且还将揭示基础乳腺癌等病理情况的重要线索，这些疾病是由腔祖细胞引起的。

 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.