Transcriptional Control of Monocyte Development

摘要 非经典巡逻单核细胞（在小鼠中鉴定为Ly 6Clo，在人类中鉴定为CD 14 dimCD 16+）是不同的 来自经典单核细胞（小鼠中的Ly 6Chi和人中的CD 14 + CD 16-），并表现出独特的主动调节 在稳态和炎症条件下巡视血管内皮。非经典单核细胞 其功能是从脉管系统中清除病原体、氧化脂蛋白和碎片。的重点 本申请的目的是鉴定控制非经典单核细胞发育的新转录因子。靶向 这些转录因子可以让我们增加这些抗炎因子的数量或功能， 体内单核细胞。增强子调节基因表达的细胞特异性模式，并且可以被量化。 使用针对组蛋白修饰H3 K4 me 2和H3 K27 ac的ChIP-Seq进行全基因组测序。使用 为了标记活性增强子区域，我们鉴定了H3 K27 ac上游的一个大的增强子区域。 在Ly 6Clo非经典单核细胞中显示选择性活性增加的转录因子Nr 4a 1。我们 在该增强子中选择了3个约1.5kb的活性区域，这些区域在人类中也是保守的 CD 14 dim单核细胞，并且我们使用CRISPR/Cas9方法产生了这3种增强子的敲除小鼠 in vivo.在小鼠体内缺失这些增强子区域中的一个导致产生小鼠， 血液中完全缺乏非经典Ly 6Clo单核细胞。在具体目标1中，我们将研究这个增强子区域 鉴定新的转录因子，其通过调节 Nr4a1。特异性目标2将测试这些非经典单核细胞是否具有动脉粥样硬化保护作用，我们将 机械地鉴定该单核细胞亚群在早期动脉粥样硬化形成中的功能。的结果予以 这些研究将增强我们对非经典单核细胞在动脉粥样硬化中作用的理解， 鉴定控制这种抗炎单核细胞亚群发育的新转录因子。我们 预期增加Nr 4a 1表达和非经典单核细胞数量的疗法可能有助于 人类在慢性疾病，包括动脉粥样硬化炎症的目标分辨率。

Abstract Nonclassical patrolling monocytes (identified as Ly6Clo in mice, and CD14dimCD16+ in humans) are distinct from classical monocytes (Ly6Chi in mice, and CD14+CD16– in humans), and exhibit a unique ability to actively patrol the vascular endothelium under both homeostatic and inflammatory conditions. Nonclassical monocytes function to remove pathogens, oxidized lipoproteins, and debris from the vasculature. The focus of this application is to identify new transcription factors that control nonclassical monocyte development. Targeting such transcription factors could allow us to increase numbers or functions of these anti-inflammatory monocytes in vivo. Enhancers regulate cell-specific patterns of gene expression and can be quantified genome-wide using ChIP-Seq directed against the histone modifications H3K4me2 and H3K27ac. Using antibodies to H3K27ac to mark active enhancer regions, we identified a large enhancer region upstream of the transcription factor Nr4a1 that shows increased activity selectively in Ly6Clo nonclassical monocytes. We chose 3 active ~1.5kb regions that were located within this large enhancer that were also conserved in human CD14dim monocytes, and we generated knockout mice for these 3 enhancers using CRISPR/Cas9 approaches in vivo. Deletion of one of these enhancer regions in mice in vivo resulted in generation of a mouse that completely lacks nonclassical Ly6Clo monocytes in blood. In Specific Aim 1, we will study this enhancer region to identify new transcription factors that control nonclassical monocyte development through regulation of Nr4a1. Specific Aim 2 will test whether these nonclassical monocytes are atheroprotective, and we will mechanistically identify the functions of this monocyte subset in early atherogenesis. The results of these studies will enhance our understanding of the roles of nonclassical monocytes in atherosclerosis, and will identify new transcription factors that control development of this anti-inflammatory monocyte subset. We anticipate that therapies that increase Nr4a1 expression and nonclassical monocyte numbers may be useful in humans to target resolution of inflammation in chronic diseases, including atherosclerosis.