Context matters: role of ATF4 in hematopoiesis

In this issue of Blood, Zheng et al have identified new roles for activating transcription factor 4 (ATF4) in regulating ribosome protein S19 binding protein 1 (RPS19BP1) transcription and ribosome biogenesis to promote erythropoiesis in mouse bone marrow.¹ 

The production of functional hematopoietic stem cells (HSCs) during development is governed by the intricate interplay between cell-intrinsic programs and extrinsic signals from the microenvironment or “niche.” Ontogeny-driven changes in HSC function and HSC-niche interactions have important implications for human disease. However, our knowledge of the mechanisms that control developmental stage-specific changes in HSC maintenance and lineage differentiation remains incomplete.

ATF4 is a basic leucine zipper transcription factor that plays crucial roles in various cellular processes, particularly in response to different forms of cellular stresses, such as the unfolded protein response, oxidative stress, and nutrient deprivation. In the hematopoietic system, ATF4 has been shown to regulate HSC expansion and maintenance in the murine fetal liver² and HSC aging in the bone marrow.³ Additionally, the ATF4-mediated signaling pathway is activated by the erythroid-specific eukaryotic translation initiation factor 2 (eIF2α) kinase, called heme-regulated inhibitor (HRI), to control terminal erythroid cell maturation in response to iron or heme deficiency.⁴ The HRI-ATF4 regulatory axis also modulates MYB and BCL11A transcription to impact the expression of fetal hemoglobin genes,^5,6 highlighting a pleiotropic role of ATF4 in erythropoiesis. Despite these findings, the functional roles of ATF4 in adult HSCs and the bone marrow microenvironment have not been systematically analyzed.

In this study, the authors investigated whether and how ATF4 regulates steady-state HSC function and erythropoiesis in mouse bone marrow by generating conditional ATF4 inactivation in Prx1⁺ stromal cells, Cdh5⁺ endothelial cells, Osx⁺ osteoprogenitor cells, and Mx1⁺ hematopoietic cells.¹ ATF4 deletion in stromal cells reduced the number of bone marrow mesenchymal stromal cells (MSCs) and impaired MSC differentiation, leading to shortened limbs and reduced body size, whereas hematopoiesis and HSC function were minimally affected. The knockout of ATF4 in endothelial cells and osteoprogenitors also had little effects on HSC maintenance and hematopoiesis in mouse bone marrow. In contrast, hematopoietic-selective deletion of ATF4 by inducible Mx1-cyclic recombinase (Cre) increased the frequencies of HSCs, multipotent progenitors, and myeloid progenitor subsets, whereas HSC repopulation activity was significantly impaired (see figure). A notable phenotype caused by ATF4 deficiency was the markedly decreased erythroid progenitor cells, including megakaryocyte-erythroid progenitor cells, and defective erythropoiesis, resulting in macrocytic anemia and mortality.¹ 

The results in the current report are particularly intriguing, as previous studies of ATF4^–/– conventional knockout mice have showed that ATF4 deletion does not affect HSC generation in the aorta-gonad-mesonephros region but markedly impairs the expansion of functional HSCs in the developing fetal liver.² ATF4 regulates fetal HSC development, at least in part, by acting through the transcriptional regulation of genes encoding cytokines, such as angiopoietin protein-like 3, in the fetal liver microenvironment.² Moreover, ATF4 deficiency in adult HSCs is associated with aging-like phenotypes through the downregulation of hypoxia-inducible factor 1α and the upregulation of mitochondrial reactive oxygen species.³ Therefore, ATF4 regulates HSC maintenance and lineage differentiation in a highly context-dependent manner, wherein ATF4-dependent cell-intrinsic and cell-extrinsic pathways contribute to hematopoiesis at distinct oncogenic locations. The current study also raises several interesting questions to further our understanding of the multifaceted roles of ATF4 in hematopoiesis. For example, how does ATF4 deficiency cause the expansion of defective HSCs in the bone marrow? How does ATF4 loss lead to biased HSC lineage differentiation? Does ATF4 regulate fetal HSCs and erythropoiesis through similar or different mechanisms compared with those in adult bone marrow? Addressing these questions using the genetic mouse models developed in the current study should continue to provide novel insights into understanding the context-dependent function of a major stress-responsive factor in development and disease.

The observed defects in erythropoiesis caused by ATF4 loss prompted the authors to delve deeper into the underlying mechanisms. Through integrative analyses of single-cell and bulk RNA-sequencing profiles, chromatin accessibility via the assay for transposase-accessible chromatin with sequencing (ATAC-seq), and histone H3 lysine 4 trimethylation (H3K4me3) histone modification via cleavage under targets and tagmentation (CUT&Tag), the authors described several interesting observations. ATF4-deficient erythroid progenitor cells exhibited altered cell cycles and expression of erythroid transcriptional regulators, leading to replication stress and impaired differentiation. Most importantly, the gene encoding RPS19BP1 (alias AROS) was markedly downregulated on ATF4 loss, whereas ectopic expression of RPS19BP1 rescued ATF4 deficiency-induced erythroid defects.¹ RPS19BP1 interacts with RPS19 to regulate ribosome biogenesis,^7,8 consistent with the observed decreases in 40S ribosomal subunits and protein synthesis in ATF4-deficient erythroid progenitor cells (see figure). Together, these studies establish a previously unrecognized regulatory axis involving ATF4 and RPS19BP1 in ribosome biogenesis required for erythropoiesis and hematopoietic recovery from stresses.

The specification of the erythroid lineage from hematopoietic stem cells is characterized by increased ribosome synthesis, protein translation, and mitochondrial biogenesis.⁹ Ribosomopathies, such as Diamond-Blackfan anemia and Shwachman-Diamond syndrome, comprise a diverse group of genetic disorders characterized by defective ribosome biogenesis or function, which can cause congenital anemia and bone marrow failure.¹⁰ As ribosomopathies typically manifest in specific cell and tissue types, a key question remains regarding the principles governing the sensitivity of specific cell types to defective ribosome homeostasis. The results presented in this work provide new evidence that erythroid progenitors display exquisite sensitivity to impaired ribosome biogenesis, potentially due to the increased demand for protein synthesis required for erythroid cell maturation.⁹ Therefore, this study represents an important step toward a better understanding of the multifaceted roles of ATF4 in normal and disordered erythropoiesis. Further elucidation of the molecular pathways underlying context-dependent regulation and susceptibility to ribosome biogenesis may uncover new strategies to mitigate the deleterious effects of ribosomal defects in human disorders.

Conflict-of-interest disclosure: The authors declare no competing financial interests.