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: 10379675
• 负责人: Kenneth M Murphy
• 金额: $ 19.69万
• 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/10379675
• 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.

摘要 免疫细胞的生长、分化和存活是MYC基因家族的重要成员。这个家庭是 转录因子碱性螺旋-环-螺旋（bHLH）家族的一员，包含三个成员， 原型c-MYC（由Myc编码）、N-MYC（Mycn）和L-Myc（Mycl，Mycl 1）。c-MYC是最广泛使用的 在许多类型的免疫谱系中，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）的髓样亚群中表达。 我们报道了L-MYC表达的转换发生在共同树突状细胞祖细胞的阶段， tor（CDP），当c-MYC关闭而L-MYC被诱导时。我们还发现L-MYC在以下方面发挥作用： DC支持稳健的代谢表型，并且是树突状细胞的最佳T细胞引发所需的。 这些不同的MYC家族成员的调节受到严格控制，但MYC家族成员的潜在机制是不稳定的。 其表达的协调性尚不清楚。特别是，c-MYC被抑制的机制是 不为人知，但在基本层面上显然很重要。在我们的研究中，我们发现了一个事实，即代表- c-MYC在CDP阶段的锡永依赖于转录因子IRF 8，IRF 8缺陷小鼠不能表达c-MYC。 抑制c-MYC并继续在包括经典DC和浆细胞样DC在内的髓系中表达 （pDC）。这一观察结果令人困惑，因为大量证据表明IRF 8是一种激活的跨膜转运蛋白。 转录因子，并且没有已知的基因表达的直接分子阻遏的实例。了解 IRF 8如何抑制c-MYC，我们通过染色质免疫检查了Myc基因位点的IRF 8结合位点 在一组髓系和DC谱系的祖细胞阶段中使用ChIP沉淀（ChIP）。我们发现了几个 IRF 8结合区域，提出了一个具体的假设来解释c-MYC的抑制。这些结合 位点位于c-MYC编码基因座和已知的Myc增强子（称为BENC）之间， 在Myc基因下游近2兆碱基处。IRF 8介导的非编码RNA的激活转录 位于Myc基因和其增强子BENC之间的基因具有改变染色体的潜力。 环结构的基因座，并建立一个功能性封锁，防止访问的Myc基因与其en- 因此，失去表达能力。此R21应用程序将通过删除 在原代细胞和体内使用CRISPR/Cas9方法特异性的特异性IRF 8结合位点， 已经建立，并且我们有一些已发表的结果。