NR4A act upstream of a C/EBPα transcriptional network in HSCs. (A) RT-qPCR confirmation of C/EBPα downregulation in CDKO relative to control HSCs after acute NR4A1/3 codepletion. (B) Experimental design for overexpression of NR4A1 and C/EBPα in bone marrow Lin⁻ cells. (C) Frequency of LSKs and (D) 2 hours in vitro BrdU incorporation after 48 hours in culture after retroviral transduction in Lin⁻ cells. (E) Pairwise alignment of mouse and human +37/42 Kb CEBPA enhancer containing the NR4A binding element. Modified from Cooper et al.³⁵  (F) ChIP-qPCR analysis of NR4A1 and NR4A1^CEAA (DNA-binding mutant) recruitment to the CEBPA +42 Kb enhancer in expanded CD34⁺ bone marrow cells from healthy donors. (G) ChIP-qPCR analysis of NR4A3 binding to the Cebpa +37 Kb enhancer in murine 32Dcl3 cell line. (H) Luciferase reporter assay of the activation of the Cebpa +37 Kb enhancer by NR4A1/3 in 32Dcl3 cells. Vector schemes are shown on the left, and luciferase measurements on the right. RLU, relative light units. Results expressed as mean ± SD, n = 3. *P ≤ .05; **P ≤ .01; ***P ≤ .001. ChIP, chromatin immunoprecipitation.