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MYC基因家族的调节成员。这个家庭是 基本螺旋 - 环螺旋（BHLH）的转录因子家族的成员，包含三个成员， 原型C-MYC（由MYC编码），N-MYC（MYCN）和L-MYC（MYCL，MYCL1）。 C-MYC是使用最广泛的 在多种类型的免疫谱系中，但是N-MYC在早期造血干细胞（HSC）中表达， 我们几年前发现的L-MYC在树突状细胞（DC）的髓样子群中表达。 我们报道说，转向L-MYC的表达发生在公共树突状细胞的阶段 当C-Myc关闭并诱导L-MYC时，TOR（CDP）。我们还发现L-MYC在 支持强大的代谢表型的DC，是树突状细胞最佳T细胞启动所必需的。 这些各种MYC家庭成员的调节受到严格控制，但是 其表达的协调尚不清楚。特别是，C-MYC被抑制的机制是 在基本层面上未知但显然很重要。在我们的研究中，我们发现了一个事实，即代表 CDP阶段C-MYC的SION取决于转录因子IRF8，而IRF8缺陷小鼠失败 抑制C-MYC并继续在包括经典DC和浆细胞样DC在内的髓样谱系中表达它 （PDC）。该观察结果令人困惑，因为证据的重量表明IRF8是一种激活的tran- 术语因子，也不知道基因表达的直接分子抑制。理解 IRF8如何抑制C-MYC，我们通过染色质免疫检查了IRF8结合位点的MYC基因座 在一组髓样和直流谱系的祖细胞阶段的降水（芯片）。我们确定了几个特定的 IRF8结合的区域，提出了一种具体的假设来解释抑制C-Myc的抑制作用。这些结合 站点位于C-MYC编码基因座和已知的MYC增强器（称为Benc）之间 MYC基因下游的近2个巨蛋。非编码RNA的IRF8介导的激活转录 位于MYC基因及其增强子BENC之间的潜力可以改变染色体 基因座的循环结构并创建功能阻滞，以防止MYC基因访问其en- Hancer，从而导致表达丧失。该R21应用程序将通过删除该假设直接检验该假设 特定的IRF8结合位点在原代细胞和体内特异 准备就绪，并为此我们有许多已发表的结果。