Zfp521 is essential for the quiescence and maintenance of adult hematopoietic stem cells under stress

Summary

Zinc finger protein 521 (Zfp521), a quiescent hematopoietic stem cell (HSC)-enriched transcription factor, is involved in the self-renewal and differentiation of fetal liver HSC. However, its role in adult hematopoiesis remains elusive. Here, we found that Zfp521 deletion did not inhibit adult hematopoiesis under homeostatic conditions. In contrast, Zfp521-null chimeric mice showed significantly reduced pool size of HSC and hematopoietic progenitor cells associated with increased apoptosis and loss of quiescence. Competitive serial transplantation assays revealed that Zfp521 regulates HSC self-renewal and differentiation under regenerative stress. Mechanistically, Zfp521 transcriptionally repressed Rela expression by increasing H3K9ac and decreasing H3K9me3 levels in its promoter. Knockdown of Rela inhibited the hyper-activated NF-κB pathway and reversed the loss of quiescence in Zfp521-null HSC under stress. Thus, our results reveal a previously unrecognized role for Zfp521 as critical regulator of quiescence and self-renewal of HSC in adult hematopoiesis mediated at least partly by controlling Rela expression.

Graphical abstract

Highlights

• •Zfp521 deletion does not inhibit adult hematopoiesis under homeostatic conditions
• •Zfp521 regulates the quiescence and maintenance of HSC under stress
• •Zfp521 deficiency affects the NF-κB pathway in HSC
• •Knockdown of Rela reverses the loss of quiescence in Zfp521-null HSC under stress

Molecular Biology; Cell Biology; Stem Cell Research; Transcriptomics

Introduction

Mammalian definitive hematopoiesis is a complex and finely regulated process in which hematopoietic stem cells (HSCs) give rise to all blood lineages of the organism including fetal liver and adult hematopoiesis during ontogeny (Dzierzak and Speck, 2008). The corresponding fetal liver and adult HSC are regulated by distinct intrinsic and extrinsic mechanisms (Wilkinson et al., 2020). In adult hematopoiesis, HSC mainly resides in the bone marrow (BM) niche and remains quiescent in a steady state. However, HSC can exit quiescence, and rapidly expand and differentiate to reconstitute the hematopoietic system under stress such as blood loss (Enver et al., 1998) and myelotoxic insults (Cheshier et al., 1999). The balance between quiescence and proliferation is tightly regulated by both intrinsic and extrinsic mechanisms in HSC under stress but the precise mechanisms remain to be fully elucidated. A better understanding of how these processes are regulated could lead to improved strategies for the clinical utility of HSC and provide insight into the basis of hematopoietic malignancy.

Zinc finger protein 521 (human, ZNF521; mouse, Zfp521) is a C2H2-type zinc finger transcription factor containing a conserved amino terminal motif that binds to the nucleosome remodeling and histone deacetylase complex (NuRD), which is associated with transcriptional repression (Lin et al., 2004). ZNF521 was initially identified as having restricted expression in human CD34⁺ progenitor cells (Bond et al., 2004); however, it is widely expressed in many tissues where it regulates self-renewal and differentiation. Zfp521 negatively modulates differentiation by repressing ZFP432, EBF1, and RUNX2 (Addison et al., 2014; Kang et al., 2012b; Wu et al., 2009) and negatively regulates apoptosis via BCL-2 in mesenchymal stem cells (Correa et al., 2010). A forced expression of Zfp521 in embryonic stem cells led to a skewed differentiation toward a self-renewing neural progenitor cell fate (Kamiya et al., 2011; Shahbazi et al., 2016). In the hematopoietic system, ZNF521/Zfp521 is expressed in early HSC and absent in mature cells (Bond et al., 2004). Zfp521 contributes to maintaining the multipotency of primitive lympho-myeloid progenitors by inhibiting EBF1-driven commitment toward the B-cell lineage (Mega et al., 2011). ZFP521 inhibits erythroid differentiation in leukemia cell lines through direct binding with GATA-1 and inhibiting its activity (Matsubara et al., 2009). Garrison BS first reported that Zfp521^−/− HSC derived from fetal liver exhibited marked decrease in HSC frequency after two rounds of competitive serial transplantation, suggesting that Zfp521 regulates HSC self-renewal ability in vivo (Garrison et al., 2017). Zfp521 regulates the earliest stages of hematopoiesis and lymphoid cell development via a cell-extrinsic mechanism using the whole-body knockout (KO) mouse model (Fleenor et al., 2018). Given that Zfp521^−/− mice die within a few weeks of birth, the molecular mechanisms underlying its regulation of adult HSC function are yet to be fully characterized.

Herein, we investigated the function of Zfp521 in hematopoietic stem and progenitor cells (HSPCs) using conditional KO mouse models with loss of Zfp521 specifically in hematopoietic systems. We found that Zfp521 deficiency decreased the frequency of quiescent HSC and increased the proliferation of HSPC under stress. Consequently, the self-renewal capacity of Zfp521-deficient HSC was significantly lower than that of Zfp521-sufficient HSC in chimeric mice. Mechanistically, loss of Zfp521 induced downregulation of cyclin-dependent kinase inhibitors, including p21 and p57, by directly upregulating the expression of Rela, a critical effector of NF-κB pathway and crucial regulator of HSC quiescence (Nakagawa et al., 2018; Stein and Baldwin, 2013). Thus, our data indicated that Zfp521 is a critical regulator of the quiescence and self-renewal capacity of HSC through Rela-mediated NF-κB pathways.

Results

Generation of Zfp521 conditional KO mice

To the best of our knowledge, there are no reports on the cell-intrinsic regulation of adult hematopoiesis by Zfp521. To assess the role of Zfp521 in adult HSPC we first determined the expression level of Zfp521 in subsets of primitive and mature BM cells. Real-time polymerase chain reaction (RT-PCR) revealed that Zfp521 was highly enriched in HSC, especially in quiescent HSC, compared to progenitors and differentiated cells (Figure 1A), suggesting an important role of Zfp521 in HSC.

Figure 1.

Zfp521 loss does not alter adult hematopoiesis under homeostatic conditions

(A) Quantitative RT-PCR analysis of Zfp521 in hematopoietic cells from wild-type BM; results were normalized to expression of the control gene Actb and are presented relative to those of HSC, set as 1. The distinct subsets of primitive or mature cells were sorted by indicated cell surface markers: qHSC indicates quiescent HSC, Lin⁻Sca-1⁺c-Kit⁺CD48⁻CD150⁺Pyronin Y⁻; HSC, Lin⁻Sca-1⁺c-Kit⁺CD48⁻CD150⁺; LSK, Lin⁻Sca-1⁺c-Kit⁺; HPC, Lin⁻Sca-1⁻c-Kit⁺; CMP, Lin⁻Sca-1⁻c-Kit⁺CD34⁺CD16/32^-; GMP, Lin⁻Sca-1⁻c-Kit⁺CD34⁺CD16/32⁺; MEP, Lin⁻Sca-1⁻c-Kit⁺CD34⁻CD16/32^-; B cells, B220⁺; T cells, CD4⁺ or CD8⁺; Erythroblasts, CD71⁺Ter119⁺; Myeloid, Mac-1⁺Gr-1⁺.

(B) Schematic representation of the targeting strategy of Zfp521. Zfp521 locus, the targeting vector, floxed allele, and the KO loci. Exon 4 was flanked by loxP sites and a neomycin resistance gene (Neo) was inserted after exon 4, which generated Zfp521^fl/fl mice. Zfp521^fl/fl alleles were combined with Mx1-Cre to generate Zfp521^fl/fl; Mx1-Cre in which Zfp521 is specifically deleted in the hematopoietic system induced by intraperitoneal injections of poly (I:C). The targeting vector contained a diphtheria toxin fragment A (DTA) and Neo genes. The arrows indicate the position and direction of primers used for genotyping.

(C) Semiquantitative PCR analysis of the deletion of Zfp521(Zfp521⁻) and the loxP-flanked Zfp521 allele (Zfp521^fl) among genomic DNA in BM LSK cells from Zfp521^fl/fl and Zfp521-CKO mice. Molecular sizes are indicated in base pairs (bp).

(D and E) Quantitative RT-PCR analysis of Zfp521 mRNA in BM cells (D) or LSK cells (E) from Zfp521^fl/fl and Zfp521-CKO mice; Actb was used as an internal control and results are presented relative to those of HSC in Zfp521^fl/fl.

(F) Absolute number of WBC, neutrophils (NEs), lymphocytes (LYs), monocytes (MOs), eosinophils (EOs), and basophils (BAs) (left), as well as platelets (PLTs) and RBC, and the concentration of hemoglobin (Hb), in peripheral blood from Zfp521-CKO and Zfp521^fl/fl mice. Data are from two independent experiments (A,F; mean ± SD of n = 3 (A) or n = 7 (F) mice per genotype in each) or are representative of three independent experiments (C), three independent experiments (D,E; mean ± SD of n = 3 mice per genotype in each). ∗∗∗p < 0.001 (Student's t test in (D–F).

Since Zfp521^−/− mice die within a few weeks of birth (Kiviranta et al., 2013), we generated a Zfp521 conditional KO allele (Zfp521^fl/+) by inserting two loxP sites flanking Zfp521 exon4, which encodes around 85% of the amino acids of Zfp521. Deletion of exon4 could also result in a frameshift and trigger nonsense-mediated decay of the mutant mRNA transcript (Figure 1B). To investigate the function of Zfp521 in adult hematopoiesis, we generated inducible conditional KO mice (Zfp521^fl/flMx1-Cre) by crossing mice carrying a loxP-flanked (floxed) copy of the gene (Zfp521^fl/fl) with mice carrying the Mx1-Cre driver. Conditional deletion of Zfp521 was induced in Zfp521^fl/flMx1-Cre mice by three intraperitoneal injections of polyinosinic-polycytidylic acid (pIpC) (called “Zfp521-CKO mice” here). Control mice throughout this study (unless specified otherwise) consisted of Zfp521^fl/fl littermates that were treated identically to experimental mice, including receiving injections of pIpC to control for interferon-mediated effects (Baldridge et al., 2010). Two months after pIpC induction, we confirmed a high efficiency of Zfp521 deletion in Lin⁻Sca-1⁺c-Kit⁺ (LSK) cells by semiquantitative PCR analysis of genomic DNA isolated from LSK cells from Zfp521-CKO mice (Figure 1C). Accordingly, real-time PCR analysis showed only trace amounts of Zfp521 mRNA in BM and LSK cells from Zfp521-CKO mice (Figures 1D and 1E).

Zfp521 loss has no effect on adult hematopoiesis under homeostatic conditions

At 2 months of age, Zfp521^fl/flMx1-Cre mice and the littermate controls were administered three intraperitoneal injections of pIpC at a dosage of 8–10 mg/kg every second day. Two months after pIpC induction, Zfp521-CKO mice developed normal white blood cells (WBCs), red blood cells (RBCs), platelet counts, and hemoglobin (Hb) levels in the peripheral blood (PB) compared with control littermates (Figure 1F and Table S1). Analysis of the lineage distributions in the BM, spleen, and thymus from Zfp521-CKO mice and control littermates by cytometric analysis revealed that Zfp521-CKO mice had a normal frequency of myeloid cells, B cells, T cells, and RBC in these tissues (Figures S1A–S1C). The absolute number of long-term HSC (LT-HSC), short-term HSC (ST-HSC), multipotent progenitors (MPPs), LSK cells, HPC, common myeloid progenitors (CMPs), granulocyte/macrophage progenitors (GMPs), and megakaryocyte/erythroid progenitors (MEPs) in the BM of Zfp521-CKO mice was also comparable to that in Zfp521^fl/fl mice (Figures 2A and 2B). Furthermore, in the steady state, no significant difference was observed in the cell cycle and quiescence status of Zfp521-CKO HSC compared to that of Zfp521^fl/fl HSC (Figures 2C and 2D). These results suggest that Zfp521 loss did not interfere with adult hematopoiesis under the steady state.

Figure 2.

Depletion of Zfp521 does not affect normal hematopoiesis

(A) Flow cytometry of BM cells from Zfp521^fl/fl and Zfp521-CKO mice. Numbers adjacent to outlined areas indicate percent of c-Kit⁺Sca-1⁻ (HPC) or c-Kit⁺Sca-1⁺ cells (LSK cells) among Lin⁻ cells (left panel), or CD34⁻Flk2⁻ cells (LT-HSC) or CD34⁺Flk2⁻ (ST-HSC) or CD34⁺Flk2⁻ (MPP) (middle panel) among LSK cells, or CD16/32⁻CD34⁻ cells (MEP) or CD16/32⁺CD34⁺ cells (GMP) or CD16/32⁻CD34⁺ cells (CMP) among HPC (right panel).

(B) Total LSK cells, LT-HSC, ST-HSC, MPP, HPC, CMP, GMP, and MEP in BM from 8- to 12-week-old Zfp521^fl/fl and Zfp521-CKO mice.

(C) Flow cytometry analyzing the cell cycle in Zfp521^fl/fl and Zfp521-CKO LSK cells, and LT-HSC, stained with the DNA-binding dye DAPI and labeled with bromodeoxyuridine (BrdU). Numbers adjacent to outlined areas indicate percent cells in the GO-G1 (bottom left), S (top), or G2-M (bottom right) phases of the cell cycle. Right, cell cycle profile of cells at left.

(D) Flow cytometry analyzing the cell cycle of LSK cells, and LT-HSC from Zfp521^fl/fl and Zfp521-CKO mice. Numbers in quadrants indicate percent cells in various phases of the cell cycle: G1 (top left), S-G2-M (top right), G0 (bottom left), or other (bottom right). Right, cell cycle status of cells on the left. Data are representative of three experiments (A, C, D), or are obtained from three experiments (B and right panel of C, D; mean ± SD of n = 7–8 mice per genotype).

Depletion of Zfp521 impairs the size of HSC pools after transplantation

Although the expression of Zfp521 was higher in HSC than in differentiated cells, Zfp521 loss did not affect HSC number and proliferation in the homeostatic state. This prompted us to test the role of Zfp521 in HSC under stress. BM transplantation exposes HSC to replicative, oxidative, and inflammatory stresses (Baldridge et al., 2010; Harrison et al., 1990), eventually leading to impairment of HSC pool size. We first transplanted Zfp521-CKO or Zfp521^fl/fl BM cells into lethally irradiated wild-type recipient mice (CD45.1). Two months later, we confirmed that more than 95% of the BM cells in the recipient mice were donor-derived (Figure S2A). PB analysis revealed that Zfp521-CKO chimeric mice had a similar PB count compared to Zfp521^fl/fl chimeric mice (Figure S2B and Table S2). The frequency of myeloid cells, B cells, T cells, and RBC in the BM of Zfp521-CKO chimeric mice was comparable to that of Zfp521^fl/fl chimeric mice (Figures S2C and S2D). To determine whether Zfp521 is involved in maintaining HSPC under stress in vivo, we characterized the compartments of HSPC in the chimeric mice by flow cytometry. We found that the absolute number and frequency of HSC and HPC were significantly lower in Zfp521-CKO chimeric mice compared to Zfp521^fl/fl chimeric mice (Figures 3A, 3B, and 3E). Consistently, the total number of CD34⁻LSK and LT-HSC was also significantly lower in Zfp521-CKO chimeric mice (Figures 3A–3C). However, the absolute number and frequency of ST-HSC and MPP were not significantly altered in Zfp521-CKO chimeric mice (Figures 3A and 3C).

Figure 3.

Zfp521 loss impairs the size of HSC and HPC pools under stress in the chimeric mice

(A) Flow cytometry of BM cells from Zfp521^fl/fl and Zfp521-CKO chimeric mice. Numbers adjacent to outlined areas indicate percent c-Kit⁺Sca-1⁻ (HPC) or c-Kit⁺Sca-1⁺ cells (LSK cells) among Lin⁻ cells (far left), or CD48⁻CD150⁺ cells (HSC) (left), CD34⁻ cells (right), CD34⁻Flk2⁻ cells (LT-HSC), CD34⁺Flk2⁻ (ST-HSC), or CD34⁺Flk2⁻ (MPP) (far right) among LSK cells. SS Log (right), side scatter log value.

(B and C) Total LSK cells, CD34⁻LSK cells, HSC, LT-HSC, ST-HSC, and MPP in BM from 8- to 12-week-old Zfp521^fl/fl and Zfp521-CKO chimeric mice.

(D) Flow cytometry of HPC and myeloid progenitor cells from Zfp521^fl/fl and Zfp521-CKO chimeric mice. Numbers adjacent to outlined areas indicate percent CD16/32⁻CD34⁻ cells (MEP) (bottom left), CD16/32⁺CD34⁺ cells (GMP) (top right) or CD16/32⁻CD34⁺ cells (CMP) (bottom right) among HPC cells (left), or Flk2⁺ cells (LMPP) (right) among LSK cells (right).

(E and F) Total HPC, CMP, GMP, and MEP (E) and LMPP (F) in BM from 8- to 12-week-old Zfp521^fl/fl and Zfp521-CKO chimeric mice.

(G) In vitro assay of the colony-forming units of granulocyte, erythroid, macrophage, megakaryocyte colonies, granulocyte-macrophage colonies, granulocyte colonies (G), macrophage colonies (M), burst-forming unit-erythroid colonies (BFU-E), and total colony-forming cells (CFC) 10–12 d after plating of Zfp521^fl/fl and Zfp521-CKO chimeric BM.

(H and I) Loss of Zfp521 increases apoptosis in HPC and LSK cells. Flow cytometry images showing apoptosis in HPC and LSK cells obtained from Zfp521^fl/fl and Zfp521-CKO chimeric mice (H). The apoptotic profile of cells are presented on (I) H.

(J and K) Zfp521 deficiency elevates the activities of caspase 3 and caspase 7. Representative histograms for caspase 3/7 (J) and statistical results (K) are presented.

Data are representative of three experiments (A, D, H, and J) or, or are obtained from three experiments (B, C, E, F, I, and K; mean ± SD of n = 7–8 mice per genotype) or two experiments (G; mean ± SD of n = 3 mice per genotype). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (Student's t test in B, C, E–G, I, and K).

We next characterized myeloid progenitor cells in Zfp521^fl/fl and Zfp521-CKO chimeric mice. The frequency and total number of CMP, GMP, and MEP were significantly reduced in Zfp521-CKO chimeric mice compared to Zfp521^fl/fl chimeric mice (Figures 3D and 3E). Lymphoid-primed MPPs (Lin⁻Sca-1⁺c-Kit⁺Flk2⁺) have the ability to differentiate into T cells, B cells, and monocytes (Adolfsson et al., 2005). Zfp521-CKO chimeric mice also had a lower total number and frequency of lymphoid-primed MPPs compared to the Zfp521^fl/fl chimeric mice (Figures 3D and 3F).

In addition, a colony-forming unit assay was performed to identify the frequency of myeloid progenitors in the BM cells obtained from Zfp521-CKO and Zfp521^fl/fl chimeric mice. BM cells obtained from both genotypic chimeric mice were plated in methylcellulose medium supplemented with interleukin 3 (IL-3), interleukin 6 (IL-6), erythropoietin (EPO), and stem cell factor, and were cultured for 10–12 days. We observed that BM cells from Zfp521-CKO chimeric mice generated notably lower numbers of total colony-forming units, as well as colony-forming units of various myeloid precursors, including granulocyte-erythroid-macrophage-megakaryocyte colonies, granulocyte colonies, macrophage colonies, granulocyte-macrophage colonies, and burst-forming unit-erythroid colonies, than did Zfp521^fl/fl chimeric BM cells (Figure 3G). As the maintenance of HSC and HPC are closely associated with cell survival, the apoptosis of LSK cells and HPC from both genotypic chimeric mice was assessed by staining cells with annexin V. We found that the frequency of apoptosis was significantly higher in LSK cells and HPC from Zfp521-CKO chimeric mice than that in those from Zfp521^fl/fl chimeric mice at 1 month post-transplantation (Figures 3H and 3I). Meanwhile, the frequency of apoptosis remained higher in LSK and HPC cells from Zfp521-CKO chimeric mice at 4 month post-transplantation (Figures S3A and S3B). Similarly, the activities of caspase 3 and caspase 7 were significantly elevated in LSK cells and HSC from Zfp521-CKO chimeric mice (Figures 3J and 3K). Our results suggested that Zfp521 deficiency impaired the survival of HSC and HPC under stress.

Loss of Zfp521 impairs the long-term self-renewal capacity of HSC

To evaluate the effects of Zfp521 deficiency on the regenerative capacity of HSC under stress, Zfp521-CKO and Zfp521^fl/fl chimeric mice were intraperitoneally injected with 5-fluorouracil (5-FU) weekly; this induces proliferative stress on HSC and also excludes the potential effects of 5-FU on the non-hematopoietic tissues. We observed a significantly advanced 5-FU induced hematopoietic failure in Zfp521-CKO chimeric mice. At day 20, >80% of Zfp521-CKO chimeric mice died after the third injection of 5-FU, while more than 60% Zfp521^fl/fl chimeric were still alive (Figure 4A). Analysis of BM cells and HSPC from another cohort of 5-FU-treated Zfp521-CKO and Zfp521^fl/fl chimeric mice indicated that Zfp521-deficient HSCs failed to efficiently replenish BM cells and HSPC (Figures 4B and 4C). These data indicated that Zfp521 deficiency decreases the recovery of HSC after 5-FU treatment.

Figure 4.

Zfp521-deficient HSC have a significantly diminished repopulation capacity in the chimeric mice

(A) Survival of Zfp521^fl/fl and Zfp521-CKO chimeric mice after multiple injections (upward arrows) of 5-FU (50 mg per kg body weight), presented as Kaplan-Meier curves. ∗∗p < 0.01 (Mantel-Cox test).

(B and C) Total BM cells (B) and Lin⁻Sca-1⁺ cells (C) in Zfp521^fl/fl and Zfp521-CKO chimeric mice before (day 0) and on days 4, 7, and 10 after injection of 5-FU.

(D) Schematic representation of serial competitive transplantation studies.

(E) Flow cytometry of peripheral blood cells from chimeric CD45.1⁺ mice reconstituted with BM cells from Zfp521^fl/fl or Zfp521-CKO mice, assessed at indicated time.

(F) Ratio of donor-derived CD45.2⁺CD45.1⁻ peripheral blood (PB) cells to competitor cell-derived CD45.1⁺CD45.2⁺ PB cells from mice as in E, assessed before or at 1, and 4 month after transplantation.

(G–J) The ratio of donor-derived CD45.2⁺CD45.1⁻ from Zfp521^fl/fl or Zfp521-CKO vs. competitor-cell-derived CD45.1⁺CD45.2⁺ in myeloid cells (G), B cells (H), T cells (I), and granulocytes (J) in PB from first and secondary recipient mice were analyzed.

(K) Lineage differentiation in the mice as in E. Histogram shows percentage of donor-derived (CD45.2⁺CD45.1⁻) myeloid cells (Gr-1⁺Mac⁺), B cells (B220⁺), and T cells (CD3⁺) in PB analyzed 4 months after the first and secondary transplantation.

(L) Flow cytometric analysis of the frequency of LSK cells and HSC in the competitive repopulated recipient mice 4 months after secondary transplantation.

(M) Ratio of donor-derived cells to competitor cell-derived cells among total BM cells, Lin⁻ cells, HPC, LSK cells, and HSC from recipient mice 4 months after secondary transplantation as in F.

(N) Homing ability of Lin⁻Sca-1⁺ hematopoietic progenitor cells from Zfp521^fl/fl and Zfp521-CKO mice. Histograms showing the percentage of the recovered CFSE⁺ Lin⁻Sca-1⁺ in the BM 6 h after transplantation.

Data are obtained from one experiment (A-C, E-L; n = 8 per genotype (A-C) or mean ± SD of n = 4–5 recipient mice per donor genotype (E-L)) or two experiments (M,N; mean ± SD of n = 5 per donor genotype). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 (Student's t test in B, C, F-K, M, N).

To further assess the long-term self-renewal capacity of Zfp521-deficient HSC in a competitive environment, we performed competitive serial transplantation assays. An equal number of BM cells from Zfp521-CKO mice or their Zfp521^fl/fl littermate controls (all CD45.2⁺) were transplanted into lethally irradiated recipient mice (CD45.1⁺) together with competitor BM cells (CD45.1⁺CD45.2⁺). Four months after transplantation, BM cells were obtained from the primary transplanted mice and equal numbers of those cells were transplanted into a second set of lethally irradiated mice (Figure 4D). We assessed donor-derived (CD45.2⁺) and competitor-derived PB cells by flow cytometry every month after transplantation. Deletion of Zfp521 led to a progressive decrease in total donor chimerism (Figures 4E and 4F). Flow cytometry analysis of the myeloid, B cell, T cell, and granulocyte lineages in PB suggested that the repopulation of B-cell lineage was not altered but the differentiation of myeloid, T cell, and granulocyte lineages was impaired significantly after the second transplantation (Figures 4G–4J), further suggestive of a deficit in HSC function following Zfp521 loss. It is likely that depletion of Zfp521 decreased the myeloid linage differentiation under stress (Figure 4K). In addition, we determined the relative ratio of donor-derived versus competitor-derived cells in Lin-cells, HPC, LSK cells, and HSC in BM at 4 months after the second transplantation in both Zfp521-CKO and Zfp521^fl/fl chimeric mice. Frequencies of all subsets of primitive hematopoietic cells derived from Zfp521-CKO donor cells were significantly lower than that from Zfp521^fl/fl donor cells (Figures 4L and 4M). These data indicate that Zfp521-CKO HSC have a markedly reduced reconstitution ability after serial transplantation. To rule out the possibility that the impaired self-renewal capacity of HSC was due to HSC homing defects, we evaluated the homing capacity of Lin⁻c-Kit⁺ CSFE⁺ cells from Zfp521-CKO and Zfp521^fl/fl mice, as we described previously (Hou et al., 2015). The Zfp521-CKO and Zfp521^fl/fl BM cells had comparable homing abilities, indicating that Zfp521 loss does not affect the homing ability of BM cells (Figure 4N). Together, these results indicated that loss of Zfp521 impaired the long-term self-renewal capacity of HSC in a cell-intrinsic manner.

Zfp521 is required for the maintenance of HSC quiescence under stress

While it has been reported that Zfp521 does not modulate proliferation in osteoblasts (Hesse et al., 2010), other studies have reported that perturbations of Zfp521 expression disrupt the G1/S transition in the cell cycle (Al Dallal et al., 2016; Hiratsuka et al., 2016). To determine whether the depletion of Zfp521 would interfere with the cell cycle in HSC, we assessed in vivo incorporation of the thymidine analog bromodeoxyuridine (BrdU) to investigate the cell cycle kinetics of LSK cells and HSC from Zfp521-CKO and Zfp521^fl/fl chimeric mice.

While ~22% of LSK cells (and ~12% of HSC) from Zfp521^fl/fl were BrdU⁺, ~30% of LSK cells (and ~20% of HSC) from Zfp521-CKO mice incorporated BrdU (Figures 5A and 5B). These results indicated that the absence of Zfp521 promoted the proliferation of LSK cells and HSC. Additionally, depletion of Zfp521 also led to the accumulation of cells at the G2-M transition phase in HSC and LSK cells (Figures 5A and 5B). Taken together, these results suggested that Zfp521 negatively regulated the cell cycle in LSK cells and HSC in a cell context-dependent manner.

Figure 5.

Deletion of Zfp521 leads to the accumulation of cells in the S and G2-M phases of the cell cycle and loss of quiescence in HSC and LSK cells under stress in the chimeric mice

(A and B) Flow cytometry analyzing the cell cycle in LSKs (A), and LT-HSC (B) from Zfp521^fl/fl and Zfp521-CKO chimeric mice, stained with the DNA-binding dye DAPI and labeled with BrdU. Numbers adjacent to outlined areas indicate percent cells in the GO-G1 (bottom left), S (top), or G2-M (bottom right) phases of the cell cycle. Right, cell cycle profile of cells at left.

(C and D) Flow cytometry analyzing the cell cycle of LSK (C), and LT-HSC (D) from Zfp521^fl/fl and Zfp521-CKO chimeric mice. Numbers in quadrants indicate percent cells in various phases of the cell cycle: G1 (top left), S-G2-M (top right), G0 (bottom left), or other (bottom right). Right, cell cycle status of cells on the left. Data are representative of two experiments (mean ± SD of n = 5 mice per genotype). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 (Student's t test).

Several studies have reported that quiescence is essential for HSC to maintain their number and function (Cai et al., 2015; Hou et al., 2015; Liang et al., 2020; van Velthoven and Rando, 2019). We next investigated the fraction of LSK cells and HSC in G0 by assessing the ki67 expression and DNA content. As expected, the frequency of quiescent cells in LSK cells and HSC from Zfp521-CKO chimeric mice was ~1.5–2 fold lower than that in Zfp521^fl/fl chimeric LSK cells and HSC (Figures 5C and 5D). This result revealed that depletion of Zfp521 dysregulated the G0 phase in LSK cells and HSC. Thus, our data indicated that Zfp521 was crucial for the maintenance of LSK cell and HSC quiescence under stress, and that in its deficiency, quiescent cells preferred to enter the cell cycle.

Zfp521 deficiency affects the NF-κB pathway in HSC

To further characterize Zfp521-dependent genes and the molecular pathways involved in the regulation of HSC function, we conducted RNA-Seq analysis of HSC sorted from Zfp521^fl/fl and Zfp521-CKO chimeric mice 2 months after transplantation. Seventy-five differentially expressed genes were identified between Zfp521^fl/fl and Zfp521-CKO HSC with the criteria of |log2(foldchange)|>1 and an adjusted p value of <0.05 (Figure 6A and Table S3). The differentially expressed genes were classified into seven functional groups, including genes encoding products with nucleic acid binding transcription factor activity, binding activity, catalytic activity, and structural activity (Figure 6B). Notably, upregulated genes involved in several biological processes, such as chemokine signaling pathway, positive regulation of endothelial cell proliferation, NIK/NF-κB signaling, and angiogenesis (Figure 6C), whereas downregulated genes were enriched in cell differentiation, negative regulation of NIK/NF-κB signaling, angiogenesis, and embryo development, and so on (Figure 6D). Next, we used gene set enrichment analysis to determine whether specific predefined gene sets showed significant enrichment in HSC from Zfp521-CKO chimeric mice by analyzing a data set that included 19,000 genes. This analysis showed enrichment for a gene set associated with NF-κB signaling in Zfp521-deficient HSC (Figure 6E). Notably, a gene set related with DNA replication was significantly enriched in Zpf521-deficient HSC, which was consistent with the observed increase in cycling HSC in Zfp521-CKO chimeric mice (Figure 6F). Thus, our results suggested that dysfunction of HSC in Zfp521-deficient chimeric mice were possibly associated with alteration of the NF-κB and DNA replication signaling pathway.

Figure 6.

Genes and molecular pathways dysregulated in Zfp521-deficient chimeric mice HSC

(A) Heatmap of the differentially expressed genes (DEG) in HSC from Zfp521-CKO and Zfp521^fl/fl chimeric mice.

(B) Gene ontology analysis of the DEG according to the function of their products.

(C and D) Gene ontology analysis for the upregulated (C) and downregulated (D) genes in HSC from Zfp521-CKO and Zfp521^fl/fl chimeric mice.

(E and F) Gene set enrichment analysis of selected genes sets encoding products related to NF-κB signaling (E) and DNA replication (F), presented as enrichment score. Normalized enrichment score, 1.38, p = 0.032 (E), or 1.22, p = 0.043 (F).

(G) Quantitative real-time PCR analysis of selected genes in LT-HSC. Actb is used as the internal control, and the results are presented relative to those of Zfp521^fl/fl control HSC. Data are from three experiments (A) or are representative of one experiment (B-F) or two experiments (G; mean ± SD). ∗∗p < 0.01 (Student's t test in G).

Rela mediates Zfp521 function in regulating HSC quiescence

Pioneering studies have shown that constitutively active NF-κB signaling in HSC led to a decreased HSC pool, compromised HSC functions, and a hyper-proliferative phenotype associated with loss of quiescence (Nakagawa et al., 2015, 2018); this is associated with the downregulation of Cdkn1c (p57), and upregulation of Ccnd1 and Junb (Nakagawa et al., 2015, 2018). In Zfp521-CKO chimeric mice, we observed a similar phenotype, and our RNA-Seq profiling data, as well as quantitative RT-PCR data, revealed that Zfp521 deficiency in HSC was also associated with the downregulation of p57 and upregulation of Ccnd1 and Junb (Figure 6G). In contrast, the expression of genes encoding p21 (Cdkn1a) and p27 (Cdkn1b) was similar in Zfp521^fl/fl and Zfp521-CKO chimeric mice. These data implied that Zfp521 may regulate HSC quiescence by activating the NF-κB pathway under stress.

To explore the molecular mechanisms through which Zfp521 activates NF-κB signaling in HSC under stress, we screened the top 75 upregulated and downregulated genes in HSC from Zfp521-CKO mice (Figure 6A). Among these genes, the NF-κB subunit Rela was selected due to it displaying the most significant upregulation, with the log 2 (fold change) at 5.78, and due to its critical role in the maintenance of HSC homeostasis (Stein and Baldwin, 2013). We hypothesized that Zfp521 deficiency activates the NF-κB pathway by upregulating Rela, and further regulate the quiescence of HSC.

Given the upregulation of Rela in Zfp521-deficient HSCs, we intended to investigate whether Zfp521 can negatively regulated the expression of Rela in vitro. Knockdown of ZNF521 (the homolog of Zfp521 in humans) in 293T cells led to a striking upregulation of RELA expression, while, overexpression of ZNF521 inhibited the expression of RELA (Figures S4A and S4B). These results suggested that Zfp521 may act as a negative regulator of Rela. To explore whether Zfp521 regulates Rela expression through direct transcriptional repression, we performed dual-luciferase reporter assays with cells expressing the putative promoter of mouse Rela. The luciferase activity of the putative mouse Rela promoter was inhibited by Zfp521 overexpression, while it was activated by knockdown of Zfp521 (Figures 7A–7C). These results suggested that Zfp521 inhibited the expression of Rela through direct transcriptional repression of Rela by binding to its promoter. The luciferase activity of Rela promoter −1229 to +252 was significantly lower than that of promoter −496 to +252, thus we inferred that there are Zfp521 binding sites between −1229 to −496. To validate our hypothesis, we performed a chromatin-immunoprecipitation (ChIP) assay of BM cells harvested from 5-FU-treated mice. Our data showed that Zfp521 directly binds to the promoter of Rela (Figure 7D), which is consistent with the dual-luciferase reporter assays. Moreover, to investigate whether Zfp521 associated with the Rela promoter is related to transcriptional silencing, we performed ChIP assays of BM cells from 5-FU-treated mice using histone H3K9 acetylation and H3K9 trimethylation antibodies, which are epigenetic markers of transcriptionally active and silent chromatin, respectively. We observed an increased H3K9 acetylation and decreased H3K9me3 in the promoter region of Rela in Zfp521-deficient BM cells (Figures 7E and 7F). Taken together, our data indicated that Zfp521 directly binds to regulatory regions of the Rela promoter and repressed its transcription by facilitating transcriptional silencing of the Rela locus.

Figure 7.

Knockdown of Rela rescues Zfp521 deficiency-induced loss of quiescence in the chimeric mice

(A) Schematic representation of Rela and its promoter luciferase assay. The arrows indicates primers flanking the putative ZFP521-binding sites. Luciferase assay shows the activities of a series of truncated constructs of Rela promoter in 293T cells.

(B and C) Luciferase activities in 293T cells transfected with the Rela promoter plus a vector for overexpression (B) or knockdown of Zfp521 (C); results are presented relative to those of cells transfected with luciferase construct and empty vector.

(D) Chromatin-immunoprecipitation (ChIP) assay of the binding of endogenous Zfp521 to the upstream region of the Rela promoter in 5-FU-treated BM cells from wild-type mice using primers indicated in A; IgG is used as a negative control antibody.

(E and F) ChIP assay of trimethylated histone H3 lysine 9 (H3K9me3) (E) or acetylated histone H3 lysine 9 (H3K9ac) (F) in 5-FU-treated BM cells from Zfp521^fl/fl or Zfp521-CKO chimeric mice. IgG serves as a negative control.

(G) Summary of cell cycle status in LSK cells from lethally irradiated recipient mice reconstituted with Zfp521^fl/fl or Zfp521-CKO BM cells transduced with control vector or shRNA against Rela.

(H) Summary of frequency of BrdU⁺ LSK cells as described in G.

(I) Flow cytometry of BM cells from mice as described in G (numbers indicated frequency of HSC).

(J) Total number of HSC from mice as in G.

(K and L) The activities of caspase 3 and caspase 7 in HSC from mice as described in G. Representative histograms for caspase 3/7 (K) and statistical results (L) are presented.

(M and N) Total numbers of BM cells (M) and Lin⁻Sca-1⁺ cells (N) in another cohort of mice as described in G before (day 0) and on days 4, 7 and 10 after injection of 5-FU.

(O and P) Representative fluorescence-activated cell sorting plot (O) and quantification of the expression of NF-κB p65 and phosphorylated NF-κB p65 measured by indicated antibodies in LSK cells from mice as described in G (P).

(Q) Western blot analysis of the expression of NF-κB p65 and phosphorylated NF-κB p65 in Lin⁻ BM cells from mice as described in G. GAPDH was used as an internal control.

Data are from three experiments (mean ± SD of n = 3 (A-F), n = 5 mice per genotype (G, H, J, P)), or one experiments (mean ± SD of n = 5 (L), n = 3 mice per genotype (M, N)), or are representative of one experiments (K) or three experiments (I, O, Q). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 (One way analysis of variance [ANOVA] with Bonferroni post-tests in A, J, L, two-way ANOVA in C, G, H, M, N, P, Student's t test in B, D-F).

To determine whether the loss of quiescence observed in Zfp521-deficient LSK cells was mainly caused by upregulation of Rela, we investigated the effect of knockdown of Rela on the quiescence of Zfp521-deficient LSK cells in vivo. BM cells obtained from Zfp521^fl/fl and Zfp521-CKO mice were infected with control lentivirus or lentivirus carrying shRNAs against Rela, and these cells were transplanted into lethally irradiated recipient mice to generate chimeric mice. BM cells were collected from chimeric mice at 6 weeks after transplantation and analyzed by flow cytometry. Notably, the frequencies of quiescent cells and BrdU⁺ cells were significantly higher in Rela-specific shRNA-transduced Zfp521-CKO LSK cells than that in control vector-transduced Zfp521-CKO LSK cells in chimeric mice (Figures 7G and 7H). In addition, the frequency of LT-HSC was higher in BM cells from Rela-specific shRNA-transduced Zfp521-CKO chimeric mice than that in control vector-transduced Zfp521-CKO chimeric mice (Figures 7I and 7J). The activities of Caspase 3 and Caspase 7 was decreased in HSCs from Rela-specific shRNA-transduced Zfp521-CKO chimeric mice comparing with that in control vector-transduced Zfp521-CKO chimeric mice (Figures 7K and 7L). Furthermore, the capability of hematopoietic reconstitution of BM cells and HSPCs was better in 5-FU-treated Rela-specific shRNA-transduced Zfp521-CKO chimeric mice than that in control vector-transduced Zfp521-CKO chimeric mice (Figures 7M and 7N). Meanwhile, the expression level of NF-κB and phosphorylated NF-κB was higher in control vector-transduced Zfp521-CKO LSK cells than that in control vector-transduced Zfp521^fl/fl or Rela-specific shRNA-transduced Zfp521-CKO LSK cells (Figures 7O–7Q). These results indicated that knockdown of Rela rescued the hyper-activated NF-κB pathway and prevented the depletion of LSK cells and LT-HSC induced by the Zfp521 deficiency. In conclusion, these data suggested that Rela mediated or at least partly mediated Zfp521 deficiency-induced depletion of quiescence in LSK cells by regulating the NF-κB pathway.

Discussion

The role of endogenous Zfp521 in adult hematopoiesis has not been reported. In this study, we have identified a previously unrecognized role for Zfp521 in the maintenance of HSC and HPC in adult hematopoiesis. Using conditional deletion mouse models of Zfp521, we have determined that Zfp521 was essential for the maintenance of the quiescence and self-renewal capacity of HSC in vivo, and that it intrinsically regulated the proliferation of HSC and HPC. We have further identified Zfp521 as an upstream regulator of NF-κB signaling by transcriptional regulation of Rela, which was able to mediate the function of Zfp521 in regulating HSC quiescence.

Zfp521 has been consistently reported as a key regulator of HSC self-renewal and differentiation in embryonic hematopoiesis (Fleenor et al., 2018; Garrison et al., 2017). However, to the best of our knowledge, the in vivo role of Zfp521 in adult HSC has not yet been characterized. In our study, conditional deletion of Zfp521 in hematopoietic compartments was efficiently induced in adult mice by intraperitoneal injection of poly(I:C). We found that the primary Zfp521-CKO mice had a normal frequency of mature blood cells and HSPCs, indicating that Zfp521 is dispensable in adult HSC under homeostatic conditions. However, Zfp521 deficiency decreased the size of the HSC pool and exhibited a diminished long-term repopulating activity in response to regenerative stress, which is consistent with previously reports in a competitive serial transplantation assay with fetal liver cells (Garrison et al., 2017). Quiescence are critical for the maintenance of HSC (van Velthoven and Rando, 2019). We first revealed that Zfp521 depletion does not affect quiescence of HSC and LSK cells in the homeostatic state, but it significantly reduces quiescence and survival of those cell populations under stress. The decrease in quiescence and survival leads to the impaired self-renewal capacity of Zfp521-deficient HSC under stress conditions. All of the data we collected from the Zfp521-CKO chimeric mice demonstrated that Zfp521 regulated the HSC quiescence and self-renewal in an HSC-intrinsic manner. After analyze the Zfp521^−/− and littermate control mice at 3 weeks of age, Fleenor CJ, et al. found that Zfp521^−/− mice possess decreased frequencies and numbers of HSPC, which is due to the fact that Zfp521 deficiency changes BM microenvironment cytokine levels and gene expression within resident HSPC (Fleenor et al., 2018). Their data revealed that Zfp521 regulates HSC through cell-extrinsic mechanisms in the BM microenvironment. Together, these results indicate that Zfp521 regulates the quiescence and self-renewal of HSC in both intrinsic and extrinsic manners. Furthermore, it might make sense that the phenotypes of Zfp521^−/− are more obvious than that in Zfp521-CKO mice, it is likely due to the critical role of Zfp521 in regulation of adipocytes, osteoblasts, and osteoclasts in the BM microenvironment (Addison et al., 2014; Hesse et al., 2010; Kang et al., 2012a; Wu et al., 2009). Zfp521 depletion elevates T cell reconstitution at 8 and 12 weeks after transplantation in vivo (Garrison et al., 2017) and increases B-cell differentiation in vitro (Al Dallal et al., 2016). However, we only observed a slight increase in T cell frequency at 4 weeks after primary transplantation, and then a moderate decrease at 16 weeks after secondary transplantation. We also found that transplantation of Zfp521-deficient donor cells did not increase B-cell reconstitution, which is similar to the results of a previous study (Garrison et al., 2017).

Notably, long-term exposure of HSC to proinflammatory cytokines cause diminished self-renewal and quiescence (Baldridge et al., 2010; Essers et al., 2009; Sato et al., 2009). Indeed, deregulated NF-κB activity leads to increased expression of proinflammatory cytokines, including TNF, IL1, IL6, and IFN-γ (Boone et al., 2004; Coornaert et al., 2009; Dong et al., 2010; Harhaj and Dixit, 2012). Defects in the negative regulatory circuits of NF-κB signaling cause a loss of quiescence and pre-mature depletion of HSC (Nakagawa et al., 2015). Similarly, constitutive activation of NF-κB leads to a reduced HSC pool and compromised HSC functions (Nakagawa et al., 2018). These studies highlight the critical role of NF-κB signaling in HSC quiescence. In our study, Zfp521 deficiency led to the upregulation of Rela and Junb, the key effector of the NF-κB pathway and its downstream target gene, respectively, and downregulation of p57, whose genes encode cyclin-dependent kinase inhibitors. Notably, constitutive activation of the NF-κB pathway obviously impairs the quiescence of HSC and their capacity for self-renewal, and is associated with upregulation of Junb and downregulation of the p57 genes in HSC (Nakagawa et al., 2018). Zfp521 has revealed a vital role in epigenetic regulation of the transcription of its target genes by binding to chromatin and promoting chromatin remolding via its N-terminal NuRD motif, which binds to nucleosome remodeling and deacetylase corepressor complex (Addison et al., 2014; Al Dallal et al., 2016; Behrouznezhad et al., 2019; Yamasaki et al., 2010). Finally, our studies indicated that Zfp521 depletion promoted Rela expression by directly binding its promoter, which increased H3K9 acetylation and decreased H3K9me3 in the Rela promoter region, indicating an increase in the transcriptional activity of Rela. Meanwhile, shRNA-mediated knockdown of Rela reversed the loss of quiescence in Zfp521-deficient HSPC. Taken together, these results suggest that Zfp521 regulates HSC quiescence through control of Rela-mediated NF-κB signaling. Nevertheless, analysis of global gene expression in Zfp521-deficient and Zfp521-sufficient LT-HSC suggested that Zfp521 regulates the function of HSC through multiple molecular pathways. Further detailed research is needed to investigate whether other pathways are critical for mediating Zfp521 function in HSC.

We found that depletion of Zfp521 increased apoptosis in HPC and LSK cells under stress, which reflects the previously observed pro-survival activity of Zfp521 (Al Dallal et al., 2016; Correa et al., 2010; Seriwatanachai et al., 2011). Loss of Zfp521 increases Caspase-3 activation and decreases the expression of the key apoptosis inhibitor BCL-2 (Correa et al., 2010; Yamasaki et al., 2010), which is likely to be the reason for increased apoptosis in HPC and LSK cells in Zfp521-deficient mice. In summary, we have provided functional and molecular evidence that Zfp521 acts as a critical regulator of the quiescence and self-renewal of adult HSC in a cell-intrinsic manner, and Zfp521 may regulate HSC quiescence by activating the NF-κB pathway under stress.

Limitations of the study

This study demonstrated that Zfp521 was essential for the maintenance of the quiescence and self-renewal capacity of adult HSC under stress. Although we proposed that Zfp521 regulates the expression of Rela by binding to its promoter region, we could not specify the detailed binding site.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Yu Hou (houyuxn@vip.126.com).

Materials availability

Materials generated in this study will be made available upon reasonable request and may require a material transfer agreement.

Data and code availability

The RNA-seq data generated during this study are available at GEO (GSE163684).

Methods

All methods can be found in the accompanying Transparent Methods supplemental file.

Published: February 19, 2021