Deleting an Nr4a1 Super-Enhancer Subdomain Ablates Ly6C low Monocytes while Preserving Macrophage Gene Function

Abstract

Summary Mononuclear phagocytes are a heterogeneous family that occupy all tissues and assume numerous roles to support tissue function and systemic homeostasis. Our ability to dissect the roles of individual subsets is limited by a lack of technologies that ablate gene function within specific mononuclear phagocyte sub-populations. Using Nr4a1-dependent Ly6Clow monocytes we present a proof-of-principle approach that addresses these limitations. Combining ChIP-Seq and molecular approaches we identified a single, conserved, sub-domain within the Nr4a1 enhancer that was essential for Ly6Clow monocyte development. Mice lacking this enhancer lacked Ly6Clow monocytes but retained Nr4a1 gene expression in macrophages during steady state and in response to LPS. As Nr4a1 regulates inflammatory gene expression and differentiation of Ly6Clow monocytes, decoupling these processes allows Ly6Clow monocytes to be studied independently.

Scavenger Receptor CD36 Directs Nonclassical Monocyte Patrolling Along the Endothelium During Early Atherogenesis

“…We generated mice selectively lacking Ly6C − monocytes (E2-def) with no observable changes in any other immune cell type in vivo 31 , and transplanted bone marrow from either WT or E2-def mice into Ldlr −/− recipients. After checking for successful bone marrow reconstitution (Supplemental Figure IIIB) and feeding a high cholesterol diet for 15 weeks, we assessed plaque formation by Oil Red O staining in WT-Ldlr −/− and E2-def-Ldlr −/− mice.…”

Endothelial Protective Monocyte Patrolling in Large Arteries Intensified by Western Diet and Atherosclerosis

“…Third, a local imbalance of monocyte subsets or other leukocytes in situ could adversely affect endothelial fitness of Nr4a1-deficient mice. Future monocyte studies will be greatly improved by using a new enhancer-knockout mouse with specific effects on patrollers but not macrophages 36 .…”

Monocyte heterogeneity and functions in cancer

“…Nr4a1 is also expressed in T cells and Mϕs, motivating us to use epigenetic approaches to search for more specific regulatory factors specifically active during Ly6C lo monocyte generation. We identified a super‐enhancer region upstream of the Nr4a1 gene that binds the transcription factor KLF2 and exclusively regulates Ly6C lo monocyte development . In addition to KLF2, a similar epigenetic approach identified the transcription factor C/EBPβ as a regulator of the Nr4a1 gene and generation of Ly6C lo monocytes .…”

“…We identified a super-enhancer region upstream of the Nr4a1 gene that binds the transcription factor KLF2 and exclusively regulates Ly6C lo monocyte development. 30 In addition to KLF2, a similar epigenetic approach identified the transcription factor C/EBP as a regulator of the Nr4a1 gene and generation of Ly6C lo monocytes. 11 In agreement with these findings, Ly6C lo monocytes are absent in C/EBP -deficient mice.…”