The ATF4-RPS19BP1 Axis Modulates Ribosome Biogenesis to Promote Erythropoiesis
Authors: Zheng Zhaofeng , Yang Shangda , Gou Fanglin , Tang Chao , Zhang Zhaoru , Jiang Penglei , Qian Pengxu , Zhu Ping , Cheng Hui , Cheng Tao 
Published: December 2023

The function of hematopoietic stem cells (HSCs) and the process of hematopoiesis are intricately governed by a complex interplay of intrinsic programs and extrinsic signals from the microenvironment (Crane et al., Nat Rev Immunol, 2017). Previous work reported by our group and others has demonstrated that ATF4, a basic region-leucine zipper transcription factor, plays pivotal roles in fetal liver hematopoiesis, HSC maintenance, bone formation and tumorigenesis (Zhao et al., Blood, 2015; Sun et al., Sci Adv, 2021; Yang et al., Cell, 2004). However, the precise function of ATF4 in the bone marrow niche and how ATF4 regulates adult hematopoiesis remain largely unknown.

Here, we employ four cell-type-specific mouse Cre lines to conditionally knock out Atf4 in Cdh5 + endothelial cells (ECs), Prx1 + bone marrow stromal cells (BMSCs), Osx + osteo-progenitor cells, and Mx1 + hematopoietic cells, and uncover the role of Atf4 in niche cells and hematopoiesis. We found that Atf4 deletion in BMSCs reduced BMSC numbers and impaired their CFU-F activity as well as differentiation toward osteoblasts, but spares hematopoiesis and HSC function in adult mice. Furthermore, Atf4 depletion in the ECs or osteo-progenitors of adult mice has a very mild effect on the BM hematopoiesis and HSC maintenance.

Intriguingly, Atf4 depletion in the hematopoietic system resulted in severe anemia. More than 70% of the Mx1cre; Atf4 fl/fl (Δ/Δ) mice gradually died from anemia. Atf4 deficiency markedly reduced the frequency of erythroid progenitor cells (EPCs) (including MEP, PreCFU-E, and CFU-E cells) and impaired their CFU-E and BFU-E colony-forming ability. Atf4 depletion severely impaired the repopulation ability of BM cells and the self-renewal capacity of HSCs. Single cell RNA-seq (scRNA-seq) analysis of BM Lin -cKit + cells confirmed the defects of erythropoiesis after Atf4 depletion. The absence of ATF4 compelled early erythroid progenitors to enter the S phase, which led to replication stress and in turn activated both the DNA damage response and apoptosis.

Subsequently, we conducted an integrative analysis of the genes that were identified as downregulated in MEPs by RNA-seq, ATAC-seq, and H3K4me3 Cut&Tag and genes that were identified as downregulated in erythroid progenitors (i.e., PreMegE, Ery1, and Ery2) by scRNA-seq, which predicted Rps19bp1 as the top target gene of Atf4. Luciferase reporter and quantitative chromatin immunoprecipitation (qChIP) assays confirmed that ATF4 was a transcriptional activator of Rps19bp1. Downregulation of Rps19bp1 caused by Atf4 deletion led to decreased assembles of 40S proteins, such as RPS3, RPS6 and RPS19. O-propargyl-puromycin (OP-Puro) incorporation and SUnSET assay further confirmed the global protein synthesis defect in Atf4-deficient MEP cells. The protein synthesis defects and impaired erythropoiesis were rescued upon RPS19BP1 overexpression in Atf4 knockdown MEL cell line or BM cKit + cells from Atf4-deficient mice. Ribosome profiling of CMP cells from Δ/Δ and fl/fl mice further demonstrated that the expression of proteins involved in ribosome biogenesis was significantly lower in Atf4-depleted than in wildtype CMP cells. The expression of gene sets linked to PreCFU-E signature was also lower in Atf4-depleted than in wildtype CMPs, suggesting that the reduction in ribosome biogenesis significantly affected the translation efficiency of erythroid-related pathway components, thereby impeding erythroid lineage commitment. Finally, we demonstrate that under conditions of 5-fluorouracil-induced stress, Atf4 depletion impedes the recovery of hematopoietic lineages, which requires efficient ribosome biogenesis.

Taken together, we demonstrated that, unlike in the fetal liver, ATF4 governs adult HSC function and erythropoiesis in a cell-intrinsic manner. We revealed a novel role for ATF4 in erythropoiesis, which links it to RPS19BP1, ribosome biogenesis and protein translation. Our findings have highlighted the crucial importance of protein synthesis regulation during erythroid lineage commitment. This discovery will likely have extended implications for understanding and treating ribosomopathy-associated erythroid failure.