Mechanism of c-MYC repression by IRF8 in myeloid lineages

IRF8 在骨髓谱系中抑制 c-MYC 的机制

• 批准号: 10493389
• 负责人: Kenneth M Murphy
• 金额: $ 23.63万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-22 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10493389
• 关键词: Age; Binding; Binding Sites; CD8-Positive T-Lymphocytes; CRISPR/Cas technology; Cell Lineage; Cells; Chromatin; Code; Common Lymphoid Progenitor; Data; Defect; Dendritic Cells; Development; Enhancers; Failure; Family; Family member; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Germ-Line Mutation; Growth; Helix-Turn-Helix Motifs; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Homologous Gene; Human; Hyperplasia; Immune; Immunoprecipitation; Knock-out; Left; Lymphocyte; Lymphoid; MYC Family Genes; MYC gene; Mediating; Methods; Modeling; Molecular; Mus; Myelogenous; Myeloid Cells; Pattern; Population; Publishing; Regulation; Regulatory Element; Reporting; Repression; Response Elements; Site; Solid; Structure; Supporting Cell; T-Lymphocyte; Testing; Transcriptional Activation; Type I Epithelial Receptor Cell; Untranslated RNA; Weight; activating transcription factor; base; c-myc Genes; gene repression; genetic element; genomic locus; in vivo; macrophage; member; metabolic fitness; metabolic phenotype; monocyte; mouse model; novel; prevent; progenitor; prototype; self-renewal; stem cell population; stem cells; transcription factor; tumorigenesis

ABSTRACT Growth, differentiation and survival of immune cells are regulated members of MYC gene family. This family is a member of the basic helix-loop-helix (bHLH) family of transcription factors, and contains three members, the prototype c-MYC (encoded by Myc), N-MYC (Mycn) and L-Myc (Mycl, Mycl1). c-MYC is the most widely used among the many types of immune lineages, but N-MYC is expressed in early hematopoietic stem cells (HSCs), while L-MYC, we discovered several years ago, is expressed in the myeloid subsets of dendritic cells (DCs). We reported that the switch to expression of L-MYC occurs at the stage of the common dendritic cell progeni- tor (CDP), when c-MYC is shut off and L-MYC is induced. We also discovered that L-MYC serves a function in DCs of supporting a robust metabolic phenotype and is required for optimal T cell priming by dendritic cells. The regulation of these various MYC family members is under tight control, but the mechanisms underlying the coordination of their expression is not known. In particular, the mechanism by which c-MYC is repressed is unknown but obviously important at a basic level. In our studies, we have uncovered the fact that the repres- sion of c-MYC at the CDP stage is dependent on the transcription factor IRF8 and that IRF8-deficient mice fail to repress c-MYC and continue to express it in myeloid lineages including classical DCs and plasmacytoid DCs (pDCs). This observation is puzzling because the weight of evidence indicates that IRF8 is an activating tran- scription factor, and no example of direct molecular repression of gene expression is known. To understand how IRF8 can repress c-MYC, we examined the Myc gene locus for IRF8 binding sites by chromatin immune precipitation (ChIP) in a set of progenitor stages of myeloid and DC lineages. We identified several specific regions of IRF8 binding that suggest a concrete hypothesis to explain suppression of c-MYC. These binding sites are located between the c-MYC coding locus and the known Myc enhancer, called BENC, that is located nearly 2 megabases downstream of the Myc gene. IRF8-mediated activation transcription of non-coding RNA that are located between the Myc gene and its enhancer BENC have the potential to alter the chromosomal loop structure of the locus and create a functional blockade preventing access of the Myc gene with its en- hancer, thus causing loss of expression. This R21 application will test this hypothesis directly by deleting the specific IRF8 binding sites specific in primary cells and in vivo using CRISPR/Cas9 methods that we have al- ready established and for which we have a number of published results.

抽象的 免疫细胞的生长、分化和存活是由MYC基因家族成员调控的。这家人是 转录因子基本螺旋-环-螺旋 (bHLH) 家族的成员，包含三个成员： 原型c-MYC（由Myc编码）、N-MYC（Mycn）和L-Myc（Mycl、Mycl1）。 c-MYC是应用最广泛的 在多种类型的免疫谱系中，但 N-MYC 在早期造血干细胞 (HSC) 中表达， 而我们几年前发现的 L-MYC 在树突状细胞 (DC) 的骨髓亚群中表达。 我们报道，L-MYC 表达的转变发生在共同树突状细胞祖细胞阶段。 tor (CDP)，当 c-MYC 关闭并且 L-MYC 被诱导时。我们还发现 L-MYC 具有以下功能： DC 支持强大的代谢表型，是树突状细胞最佳 T 细胞启动所必需的。 这些不同 MYC 家族成员的调控受到严格控制，但其背后的机制 他们的表达协调性尚不清楚。特别是，c-MYC 被抑制的机制是 未知但在基础层面上显然很重要。在我们的研究中，我们发现了这样一个事实： c-MYC 在 CDP 阶段的表达依赖于转录因子 IRF8，IRF8 缺陷的小鼠会失败 抑制 c-MYC 并继续在髓系谱系（包括经典 DC 和浆细胞样 DC）中表达它 （pDC）。这一观察结果令人费解，因为大量证据表明 IRF8 是一种激活转录因子。 转录因子，并且没有直接分子抑制基因表达的例子。要了解 IRF8如何抑制c-MYC，我们通过染色质免疫检查了Myc基因座的IRF8结合位点 骨髓和 DC 谱系的一组祖细胞阶段中的沉淀 (ChIP)。我们确定了几个具体的 IRF8 结合区域提出了解释 c-MYC 抑制的具体假设。这些绑定 位点位于 c-MYC 编码位点和已知的 Myc 增强子（称为 BENC）之间，该增强子位于 Myc 基因下游近 2 兆碱基。 IRF8介导的非编码RNA转录激活 位于 Myc 基因及其增强子 BENC 之间的基因有可能改变染色体 基因座的环结构并创建功能封锁，防止 Myc 基因及其 en- hancer，从而导致表达丧失。这个 R21 应用程序将通过删除直接测试这个假设 使用我们已经掌握的 CRISPR/Cas9 方法，在原代细胞和体内具有特定的 IRF8 结合位点 准备就绪，我们已经发布了许多结果。