Project 1: Mitotic Gene Bookmarking as an Epigenetic Mechanism to Maintain the Mammary Epithelial Phenotype

项目 1：有丝分裂基因书签作为维持乳腺上皮表型的表观遗传机制

• 批准号: 10608053
• 负责人: Gary S. Stein
• 金额: $ 40.49万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10608053
• 关键词: Biological; Breast Cancer Cell; Breast Cancer Patient; Breast Cancer cell line; Breast Epithelial Cells; CRISPR screen; Cause of Death; Cell Line; Cell Proliferation; Cell division; Cells; ChIP-seq; Chromatin; Chromosomes; Clinical; Collaborations; DNA Binding; Data; Development; Developmental Process; Dimensions; Epigenetic Process; Epithelial Cell Proliferation; Epithelial Cells; Epithelium; Equilibrium; Estrogen Receptors; Estrogen receptor positive; Estrogens; Event; Gene Expression; Genes; Genetic Transcription; Genome; Genomic Segment; Genomic approach; Genomics; Growth; Hi-C; Histones; Knowledge; Link; Maintenance; Malignant Neoplasms; Mammary Neoplasms; Maps; Mediating; Mitosis; Mitotic; Mitotic Chromosome; Modeling; Molecular; Mutation; Neighborhoods; Outcome; Phenotype; Physiological; Play; Property; RUNX1 gene; Receptor Signaling; Regulation; Research Personnel; Resolution; Role; Signal Transduction; Specimen; Structure; Supporting Cell; Testing; Transcriptional Regulation; United States; Validation; Woman; acute myeloid leukemia 1 protein; breast cancer progression; cancer cell; cancer initiation; clinically relevant; epigenome; epithelial to mesenchymal transition; histone modification; in vivo; innovation; live cell microscopy; loss of function; malignant breast neoplasm; mammary; mammary epithelium; novel; novel strategies; nuclease; pharmacologic; preservation; prevent; programs; therapeutic target; transcription factor; transcriptome; tumor; tumor progression

PROJECT 1 | SUMMARY Mechanisms underlying the epithelial to mesenchymal transition (EMT)—a normal developmental process and an early event at the onset of epithelium-derived cancers—are minimally understood. Our findings, corroborated by others in multiple biologically relevant models, have established mitotic gene bookmarking—occupancy of target genes by transcription factors during mitosis—as a novel dimension to epigenetic control of lineage commitment and cell identity. Because EMT results from loss of the epithelial phenotype, Project 1 will address whether disruption of mitotic gene bookmarking constitutes a key mechanistic component of EMT. Recently, we have shown that depletion of the RUNX1 transcription factor in normal mammary epithelial (NME) cells results in EMT in both in vivo and cell-based models, indicating that RUNX1 stabilizes the normal ME phenotype. Our preliminary data show that in ME cells undergoing mitosis, RUNX1 bookmarks genes involved in cell proliferation, growth and maintenance of the epithelial phenotype. These findings provide the rationale for our hypothesis that mitotic bookmarking of target genes by RUNX1 is a key epigenetic mechanism for maintenance of the normal mammary epithelial phenotype, and perturbation of this mechanism leads to EMT. Innovative molecular and cellular experimental strategies will be combined with state-of-the-art genomics approaches to investigate how the RUNX1 mitotic epigenome contributes to initiation of EMT, an early event that leads to development of breast cancer. Aim 1 will use candidate and unbiased CRISPR screens to determine how RUNX1 bookmarked genes are physiologically linked to the mammary epithelial phenotype and whether estrogen receptor signaling—a key determinant of NME phenotype—plays a role in regulation of RUNX1- bookmarked genes. Aim 2 will employ Hi-ChIP and HIPMap to establish whether RUNX1 bookmarked genes reside in same genomic neighborhoods for coordinate control. We will assess whether disruption of these genomic regions confers EMT. Aim 3 will take advantage of degron-based RUNX1 depletion to investigate the regulatory impact of RUNX1 mitotic bookmarking on target gene expression and maintenance of the NME phenotype. Project 1 studies will address significant gaps in current knowledge of the epigenetic mechanisms that regulate EMT. Our findings will establish RUNX1 mitotic bookmarking as a unique dimension to epigenetic control of the normal mammary epithelial phenotype, which when disrupted, results in EMT, a key early event in the development and progression of breast cancer.

项目1|总结 上皮细胞向间质细胞转化（EMT）的机制-一个正常的发育过程， 上皮源性癌症发病的早期事件--很少被了解。我们的发现，证实了 其他人在多种生物学相关模型中，已经建立了有丝分裂基因书签占有率， 有丝分裂过程中转录因子靶向基因--作为谱系表观遗传控制新维度 承诺和细胞身份。由于EMT是上皮表型丧失的结果，项目1将解决 是否有丝分裂基因书签的破坏构成了EMT的关键机制组成部分。 最近，我们发现在正常乳腺上皮细胞（NME）中RUNX 1转录因子的缺失， 细胞在体内和基于细胞的模型中均导致EMT，表明RUNX 1稳定正常ME 表型我们的初步数据表明，在ME细胞进行有丝分裂，RUNX 1书签基因参与 在细胞增殖、生长和维持上皮表型方面。这些发现提供了理由， 我们假设RUNX 1对靶基因的有丝分裂书签是一种关键的表观遗传机制， 维持正常的乳腺上皮表型，并且该机制的扰动导致EMT。 创新的分子和细胞实验策略将与最先进的基因组学相结合 研究RUNX 1有丝分裂表观基因组如何促进EMT启动的方法，EMT是一种早期事件， 导致乳腺癌的发展。Aim 1将使用候选和无偏见的CRISPR筛选来确定 RUNX 1书签标记基因在生理学上如何与乳腺上皮表型相关，以及是否 雌激素受体信号传导-NME表型的关键决定因素-在RUNX 1- 书签基因目标2将使用Hi-ChIP和HIPMap来确定RUNX 1书签标记的基因是否 存在于相同的基因组邻域中以进行协调控制。我们将评估是否破坏这些 基因组区域赋予EMT。目标3将利用基于degron的RUNX 1耗尽来研究 RUNX 1有丝分裂书签对靶基因表达和NME维持的调控影响 表型 项目1的研究将解决目前对表观遗传机制的认识存在的重大空白， 急救员我们的发现将建立RUNX 1有丝分裂书签作为表观遗传控制的一个独特的方面， 正常的乳腺上皮表型，当被破坏时，导致EMT，这是乳腺癌的关键早期事件。 乳腺癌的发生和发展。