NR4A1 and NR4A3 restrict HSC proliferation via reciprocal regulation of C/EBPα and inflammatory signaling

Abstract

Members of the NR4A subfamily of nuclear receptors have complex, overlapping roles during hematopoietic cell development and also function as tumor suppressors of hematologic malignancies. We previously identified NR4A1 and NR4A3 (NR4A1/3) as functionally redundant suppressors of acute myeloid leukemia (AML) development. However, their role in hematopoietic stem cell (HSC) homeostasis remains to be disclosed. Using a conditional / knockout mouse (CDKO), we show that codepletion of NR4A1/3 promotes acute changes in HSC homeostasis including loss of HSC quiescence, accumulation of oxidative stress, and DNA damage while maintaining stem cell regenerative and differentiation capacity. Molecular profiling of CDKO HSCs revealed widespread upregulation of genetic programs governing cell cycle and inflammation and an aberrant activation of the interferon and NF-κB signaling pathways in the absence of stimuli. Mechanistically, we demonstrate that NR4A1/3 restrict HSC proliferation in part through activation of a C/EBPα-driven antiproliferative network by directly binding to a hematopoietic-specific enhancer and activating transcription. In addition, NR4A1/3 occupy the regulatory regions of NF-κB-regulated inflammatory cytokines, antagonizing the activation of NF-κB signaling. Taken together, our results reveal a novel coordinate control of HSC quiescence by NR4A1/3 through direct activation of C/EBPα and suppression of activation of NF-κB-driven proliferative inflammatory responses.

Steroid resistance in Diamond Blackfan anemia associates with p57Kip2 dysregulation in erythroid progenitors

“…One of the proteins whose abundance was upregulated by dexamethasone in PB-derived CFU-Es, but not CB-derived CFU-Es, was NR4A1 ( Figure 3, C and D), a negative cell-cycle regulator. NR4A1 is an interesting target, given its role as a regulator of the cell cycle (22) and of T cell differentiation (33). NR4A1 also binds to HIF1α (34), an important regulator of erythroid differentiation (35).…”

“…Dexamethasone did not affect BFU-Es, but using a new set of cell-surface markers, we identified a unique, PB progenitor-generated subset of transitional CD34 + CD36 + CD71 hi CD105 med CFU-Es that were dexamethasone responsive. Mass spectrometry-based quantitative proteomics analyses revealed substantial differences in the effects of dexamethasone on PB and CB progenitors, with upregulation of nuclear receptor subfamily 4 group A member 1 (NR4A1), a negative cell-cycle regulator (22), in the former cell type. Furthermore, we found that the p57 Kip2 Cip/Kip CKI was specifically upregulated by dexamethasone in PB-derived CFU-Es and that its downregulation significantly attenuated the effects of this glucocorticoid.…”

Lrp5 and Lrp6 are required for maintaining self‐renewal and differentiation of hematopoietic stem cells

“…Nr4a1, Gata2, Junb , and Btg2 as up‐regulated genes in dKO mice may also contribute to the decelerated cell cycle of HSCs. Nr4a1 and Nr4a2 are preferentially expressed in HSCs and control HSC quiescence by acting upstream of key HSC transcriptional networks (42). GATA2 is essential for HSC survival (43).…”

PU.1 enforces quiescence and limits hematopoietic stem cell expansion during inflammatory stress

“…Cells were then washed and either stored at 4 o C or resuspended in SM containing 2 µg/ml DAPI and analyzed on a BD LSRII analyzer equipped with a UV laser (Becton-Dickenson). Myc staining was performed as previously described (Freire and Conneely, 2018). 10 7 BM cells were stained for 30 minutes on ice with SM containing the following antibodies: PE-Cy5-conjugated anti-CD3, CD4, CD5, CD8, Gr-1 and Ter119 as a lineage exclusion stain, Flk2biotin, CD34-FITC, EPCR-PE, Mac-1-PE/Cy7, CD48-A700, and cKit-APC/Cy7, washed in SM and stained for 30 min in 1:4 Brilliant buffer:SM containing Sca-1-BV421, SA-BV605 and CD150-BV785.…”