Abstract 408: Identifying the Molecular Basis of Monocyte Development Using Enhancer Profiling

Abstract

Intro: Monocytes (Mo) are key drivers of atherosclerosis. In mouse the two main monocyte populations are distinguished based on Ly6C expression. Classical Ly6Chi Mo drive atherosclerosis while Ly6Clow Mo help to maintain vascular integrity. Indirect evidence also suggests that Ly6Clow Mo protect against atherosclerosis. Mo develop in the bone marrow (BM) from the macrophage dendritic cell precursor (MDP) and common monocyte progenitor (cMoP), although precise ontological relationships are unclear. We have recently described an obligate cell-intrinsic role for the transcription factor Nr4a1 in Ly6Clow monocyte development. Hypothesis: Enhancers drive transcription factor-dependent programs of gene expression in a cell-specific manner. We hypothesize that a functional analysis of Nr4a1-associated enhancers will provide insight into mechanisms regulating Ly6Clow Mo development. Furthermore, a genome-wide analysis of Mo and their progenitor enhancers will consolidate our understanding of lineage relationships between these cells. Methods: Using H3K4me2 and H3K27ac to define enhancers and enhancer activity respectively we have performed ChIP-Seq on primary mouse MDP, cMoP, Ly6Chi and Ly6Clow Mo. Results: Of 114,755 enhancers 10,070 were differentially regulated. Hierarchical cluster analysis of these regions supports the consensus view that Ly6Clow Mo arise from Ly6Chi Mo. A 20kb ‘super-enhancer’ spanning Nr4a1 (Nr4a1se) is selectively induced in Ly6Clo Mo. We have begun to dissect Nr4a1se to identify regulators of Ly6Clow monocyte development. Three Nr4a1se regions have in vitro enhancer activity and show high H3K27ac in CD14dimCD16+ Mo, the proposed orthologue of mouse Ly6Clow Mo. This is evidence that Nr4a1 expression is conserved between humans and mice. Conclusion: We have identified candidate enhancers regulating Ly6Clow monocyte development. We are currently knocking out these regions using the CRISPR-Cas9 system to test their role in Ly6Clow Mo development in vivo. We aim to present preliminary data from these experiments at this meeting.