The dynamic regulation of hematopoietic stem cells: niche interactions, transcription factors, and pathological implications

Hematopoietic stem cells

Hematopoiesis is a lifelong, multistage process where hematopoietic stem cells (HSCs) generate all blood cell types from a multipotent state (Zhang et al. 2022).

The concept of HSCs originated from the work of Till and McCulloch, who demonstrated the ability of transplanted bone marrow cells to form cellular colonies and self-renew (Rodriguez y Baena et al. 2021). Today, human hematopoiesis is studied using xenotransplantation models where fetal human hematolymphoid organs are transplanted into immunodeficient mice to analyze long-term reconstitution capabilities (Pievani et al. 2018). The primary marker for isolating human HSCs is CD34, present in a small percentage of blood cells across different stages of development. CD34+ cells exhibit significant heterogeneity in terms of their differentiation potential. Advances in fluorescence-activated cell sorting and monoclonal antibodies have enabled the detailed characterization of HSCs by their surface markers, such as CD34, CD49f, CD90, and the absence of CD38, CD45RA, and lineage markers (Radu et al. 2023). HSCs are categorized into long-term HSCs (LT-HSCs), which are mostly quiescent and responsible for lifelong blood production, and short-term HSCs (ST-HSCs), which have limited self-renewal capacity and differentiate into multipotent progenitors (MPPs). MPPs further differentiate into lineage-committed progenitors, like common myeloid progenitors (CMPs) and common lymphoid progenitors (CLPs), leading to the formation of mature blood and immune cells (Pinho and Frenette 2019; Bozhilov et al. 2023) (Fig. 1).

Fig. 1
figure 1

Framework of the hematopoietic hierarchy. Hematopoietic stem cells are characterized by its ability to self-renew and to differentiate into all types of hematopoietic cells (multipotency). During the differentiation process, an HSC initially loses its self-renewal capacity. Subsequently, it gradually loses its potential to differentiate into various lineages, committing step-by-step to become a mature, functional cell of a specific lineage

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Hematopoietic niche under physiological and pathological conditions

The regulation of HSC self-renewal and differentiation involves both intrinsic stochastic processes and extrinsic factors such as cytokines (Wang and Ema 2015; Mann et al. 2022). The HSC niche is crucial in maintaining and controlling HSCs. Initially present in developmental regions such as the aorta-gonad-mesonephros (AGM) and yolk sac, HSC niches later localize to the placenta, fetal liver, spleen, and, postnatally, the bone marrow. Under conditions of hematopoietic stress, these niches can shift to extramedullary sites (Hayashi et al. 2019; Kandarakov et al. 2022; Li et al. 2022a; Barisas and Choi 2024).

The bone marrow niche comprises various cell types including endothelial cells, mesenchymal stromal cells (MSCs), osteoblasts, osteoclasts, adipocytes, and neuroglial cells (Ho and Méndez-Ferrer 2020) (Fig. 2). Endothelial cells are essential for HSC maintenance and regeneration by producing factors like stem cell factor (SCF), C-X-C motif chemokine 12 (CXCL12), and pleiotrophin (Smith et al. 2021). Deleting any of these factors from endothelial cells leads to a reduction in HSCs within the bone marrow. MSCs, although rare, play a critical role by supporting bone tissue regeneration and regulating HSPCs through direct interaction and the secretion of signaling molecules. They produce SCF and CXCL12, which are pivotal in maintaining HSC quiescence and facilitating their retention and homing, respectively (Hu et al. 2019; Chiarella et al. 2020). This essential function underscores their significance in steering the development and differentiation of the hematopoietic system. In a syngeneic MLL-AF9 AML transplantation model, MSCs underwent a shift toward an osteoblastic profile and concurrently decreased the expression of both factors (Hanoun et al. 2014).

Fig. 2
figure 2

The bone marrow niches housing hematopoietic stem cells (HSCs) constitute a complex and dynamic molecular network of interactions involving multiple cell types, including endothelial cells, mesenchymal stromal cells, osteoblasts, osteoclasts adipocytes, neuroglial cells, and mature hematopoietic cells. In bone marrow niches, hematopoietic stem cells (HSCs) maintain their self-renewal capacity, thanks to a pool of chemokines, including CXCL12 and SCF, which are produced by mesenchymal progenitors and endothelial cells

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The continuous remodeling of bone by osteoblasts, responsible for bone formation, and osteoclasts, responsible for bone resorption, dynamically affects the inner wall of bone and the endosteum region. These areas serve as osteoblastic niches crucial for the maintenance of hematopoietic stem cells (Li et al. 2022b).

In the bone marrow of mice, nestin-expressing stromal cells (NESCs) and Schwann cells play vital roles in providing a niche for normal hematopoietic stem cells. The non-myelinating Schwann glial cells are among the components of the bone marrow niche responsible for maintaining HSC quiescence by controlling the activation of latent transforming growth factor-beta (TGF-β) (Gautheron et al. 2023). Studies have shown that a decrease in NESCs and Schwann cells is observed in patients with myeloproliferative neoplasms and mouse models of the disease, whereas an increase in NESCs is noted in cases of acute myeloid leukemia (Cao-Sy et al. 2019). This underscores the complex relationship between niche components and hematopoietic malignancies.

With aging, the HSC niche undergoes functional changes, including microenvironmental aging, disrupted differentiation of bone marrow mesenchymal stromal cells, vascular remodeling, altered adrenergic signaling, and increased inflammation. These changes coordinately and dynamically affect the fate of HSCs and their progeny, ultimately resulting in a deficiency of lymphoid cells and an increase in myeloid cells (Ho and Méndez-Ferrer 2020).

Recent research indicates that acute AML disrupts the HSC niche, impairing healthy HSC function while providing a competitive advantage to leukemic stem cells (LSCs) (Villatoro et al. 2020). These interactions between leukemic blasts and niche components are being explored as potential targets for novel treatment strategies alongside induction chemotherapy.

Role of transcription factors in HSC regulation

Transcription factors play a pivotal role in regulating HSCs, with several key factors contributing to their maintenance, self-renewal, and differentiation. HoxB4 enhances HSC populations but can induce leukemia if misregulated, highlighting the need for precise control (Cusan et al. 2017). Conversely, factors like Gfi1 and Evi1 are essential for HSC self-renewal; their absence leads to a significant decline in HSC numbers (Goyama et al. 2008; Saito et al. 2013; Xie et al. 2023). c-Myc balances self-renewal and differentiation, with its loss leading to differentiation defects (Wilson et al. 2004; Sheng et al. 2021). The C/EBPalpha transcription factor influences myeloid differentiation, with its dysfunction linked to leukemia (Wesolowski et al. 2020). Other important factors include Meis1, which is crucial for HSC quiescence and is elevated in leukemia (Meriç and Kocabaş 2022), and HIF-1, which regulates adaptation to low oxygen levels (Takubo et al. 2010).

In addition, zinc finger proteins are a special class of transcription factors with the finger domain, which plays a significant role in hematopoiesis regulation. For example, GATA2 is a zinc finger transcription factor crucial for the development and maintenance of hematopoietic stem and progenitor cells. Complete knockout of Gata2 in mice leads to apoptosis of HSCs. In proliferating HSCs, Gata2 expression is activated by EVI1, and it has been demonstrated that haploinsufficiency of Gata2 impairs the cell cycle in mice. Mutations in GATA2, which result in abnormal expression, are frequently observed in familial diseases related to acute myeloid leukemia and myelodysplastic syndrome. This underscores the critical importance of GATA2 in maintaining proper blood system functions (Zhou et al. 2020b; Peters et al. 2023).

Also, PRDM16 is recognized for its significant roles in hematopoietic stem cell (HSC) function. Prdm16 zinc finger protein is expressed in HSCs and early progenitor cells, and its constitutive knockout is lethal at the perinatal stage. Research by Aguilo et al. indicated that Prdm16-deficient HSCs exhibit reduced bone marrow repopulating ability, a slight increase in cell cycling, and increased apoptosis (McGlynn et al. 2020).

B cell CLL/lymphoma 11A (BCL11A) is another C2H2 zinc-finger transcription factor expressed in the hematopoietic system and brain, essential for normal HSC function. Bcl11a-deficient HSCs exhibit cell cycle defects and a premature aging phenotype, which are associated with impaired HSC multilineage differentiation and self-renewal (Luc et al. 2016).

Recently, the Yin yang 1 (YY1) zinc finger protein has been recognized as critical in regulating HSC proliferation. Conditional knockout of Yy1 reduces the long-term repopulating ability of HSCs, while YY1 overexpression promotes their expansion. Additionally, through a distinct axis, YY1 binding to the Smc3 promoter suppresses its expression in HSPCs. Partial restoration of HSC numbers and quiescence occurs when one Smc3 allele is deleted in YY1 knockout mice. However, these HSCs cannot reconstitute blood following bone marrow transplantation if both YY1 and one Smc3 allele are missing (Lu et al. 2018, 2024).

Investigating the activity and dysregulation of transcription factors offers a promising avenue for enhancing HSC function in clinical settings, particularly for the treatment of leukemia. Here, I focus on Zinc Finger Protein 521, a transcription factor that has been progressively investigated over the last two decades due to its central role in maintaining the HSC pool within the bone marrow microenvironment. Recently, Znf521 overexpression has also been identified as a marker associated with poor prognosis in solid tumors, particularly in the progression of acute myeloid leukemia.

The transcription factor ZNF521/Zfp521

The ZNF521 gene is mapped on human chromosome 18 within the 18q11.2 region, spanning a length of 290 Kb. Its mRNA, which is 4869 nucleotides long, is comprised of eight exons (Fig. 3a, b). Notably, there is no canonical TATA motif adjacent to the transcription initiation site. The coding sequence and the arrangement of introns/exons show significant conservation in its murine counterpart, Evi3/Zfp521. Notably, within its genetic locus, there are several ultra-conserved non-coding elements believed to coordinate the expression of developmentally significant genes in vertebrates (Bond et al. 2008; Scicchitano et al. 2019). Initially, the gene encoding EVI3 was discovered as a frequent target of retroviral integration in murine AKXD B cell lymphomas. The integration site of Evi3 lies upstream of the first translated exon, leading to the upregulation of Evi3 through a promoter insertion mechanism. While EVI3 expression in normal B cells is subject to ontogenetic and developmental regulation, the modification of EVI3 expression caused by retroviral insertion could contribute to the development of murine B cell lymphoma (Justice et al. 1994). The EHZF/ZNF521 protein consists of 1311 amino acids and is characterized by 30 Kruppel-like zinc finger (ZF) motifs, which are arranged in clusters of 5–7 ZFs across the protein (Fig. 3c). It shares significant homology, exceeding 60%, with OAZ/ZNF423, another 30-ZF protein exhibiting molecular and functional resemblances (Bond et al. 2018). Notably, a sequence of 7 amino acids located just before ZF9 seems crucial for nuclear localization (Bond et al. 2004). The amino-terminal region of ZNF521 features a 12-amino acid motif, which is also found in various transcriptional co-repressors such as OAZ/ZNF423, friend of GATA (FOG)-1 and 2, BCL11A, and several members of the SALL family (Bond et al. 2004; Bernaudo et al. 2015). This domain is involved in binding to the nucleosome remodeling and histone deacetylase complex (NuRD), which is associated with transcriptional repression (Lin et al. 2004).

Fig. 3
figure 3

Schematic representation of the ZNF521/ZFP521 gene and its protein. a The ZNF521 gene, located on human chromosome 18 at the 18q11.2 region, spans 290 Kb. b Its mRNA is 4869 nucleotides long and consists of eight exons. c The Znf521/Zfp521 protein is characterized by the following features: 30 C2H2 zinc fingers distributed throughout the protein, a nuclear remodeling and histone deacetylation complex (NuRD) motif located at the N-terminal end, and a putative nuclear localization signal positioned between ZF8 and ZF9

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In humans, ZNF521 expression was detected in a variety of multipotent stem cells, as well as in differentiated tissues, including the brain, heart, kidney, spleen, lymph nodes, placenta, muscle, thymus, and fetal liver (Chiarella et al. 2021a).

Initially identified with restricted expression in human CD34+ progenitor cells (Bond et al. 2004), ZNF521 is now recognized for its widespread expression across various tissues, where it plays a role in regulating self-renewal and differentiation processes. The transcription factor can function as either an activator or a repressor, depending on the context. In the past decade, ZNF521 has been implicated in negatively impacting the osteogenic and adipogenic commitment of human adipose-derived mesenchymal stem cells, partly due to the inhibition of ZNF423 and EBF1 (Chiarella et al. 2018).

Similarly, Zfp521 functions by negatively modulating mesenchymal stem cell differentiation through repression of ZFP423, EBF1, and RUNX2 (Wu et al. 2009; Kang et al. 2012; Addison et al. 2014). Additionally, it negatively regulates apoptosis via BCL-2 in chondrocytes (Correa et al. 2010). Notably, forced expression of Zfp521 in embryonic stem cells results in skewed differentiation toward a self-renewing neural progenitor cell fate (Shen et al. 2011; Shahbazi et al. 2016). However, ZNF521 expression is also altered in cancer; for instance, high levels of ZNF521/Zfp521 are associated with a poor prognosis in medulloblastoma (Scicchitano et al. 2019). ZNF521 expression showed upregulation in gastric cancer samples. The Kaplan–Meier analysis revealed that elevated expression of ZNF521 correlated with poorer prognosis. Furthermore, the expression of ZNF521 was associated with the levels of infiltrating CD4+ T and CD8+ T cells, macrophages, neutrophils, and dendritic cells in gastric cancer. These findings also correlated with various immune marker sets (Li et al. 2022c). Prior research has demonstrated that ZNF521, downregulated by miR-802, inhibits the malignant progression of hepatocellular carcinoma by modulating Runx2 expression. Additionally, ZNF521 has been shown to inhibit apoptosis and promote the proliferation, migration, and invasion of gastric cancer cells by regulating microRNA-204-5p (Yang et al. 2020).

The impact of Zfp521/ZNF521 on physiological hematopoiesis

In the hematopoietic system, ZNF521/Zfp521 expression is detected in early hematopoietic stem cells (HSCs) but is not found in mature cells (Bond et al. 2004). Specifically, its expression is abundant in a limited subset of more undifferentiated CD34+ CD133+ cells, where its mRNA levels were found to be enriched 43-fold compared to the CD133− counterpart (Hemmoranta et al. 2006), as well as in CD34 + /CD33 − /CD38 − /Rholo/c kit+ cells (Eckfeldt et al. 2005) and slow-dividing CD34 + /CD38 − SCID-repopulating cells (Wagner et al. 2005). ZNF521 mRNA levels exhibit a rapid decline during the differentiation of CD34+ cells (Bond et al. 2004) and become scarce or undetectable in committed erythroid (Matsubara et al. 2009) and granulocytic precursors (Theilgaard-Mönch et al. 2005). Thus, ZNF521 may be regarded as part of the select group of genes whose expression delineates human hematopoietic stem and early progenitor cells.

Decoding the impact of Zfp521/Znf521 on hematopoietic stem and progenitor cells

Consistent findings have identified Zfp521 as a pivotal controller of both self-renewal and differentiation of hematopoietic stem cells during embryonic hematopoiesis (Fleenor et al. 2018).

Earlier investigations employing competitive bone marrow transplants (BMT) with whole fetal liver cells from Zfp521 − / − or Ctrl mice indicated a slight decrease in donor chimerism and reduced myeloid reconstitution from Zfp521 − / − donors in primary BMT recipients. Conversely, the introduction of ZFP521 into HSCs resulted in a significant preservation of progenitor activity in vitro (Garrison et al. 2017). Subsequently, upon analysis of Zfp521 − / − and littermate control mice at 3 weeks of age, it was observed by Fleenor et al. that Zfp521 − / − mice exhibit decreased frequencies and numbers of HSPCs. In particular, HSC populations lacking Zfp521 showed a reduction in cKit levels, while CD34 and CD135 cell surface antigens appeared dysregulated, which are markers used for phenotypic classification of LT-HSC, ST-HSC, and MPP populations. Zfp521-deficient mice exhibit heightened frequencies of GMP and decreased frequencies of MEP. Moreover, there is an elevation in LS lymphoid progenitors and a reduction in CLP. This phenomenon is attributed to changes in BM microenvironment cytokine levels and gene expression within resident HSPCs due to Zfp521 deficiency (Fleenor et al. 2018).

An interesting correlation between RNA-seq data and protein arrays strongly suggests elevated levels of cytokines that promote myeloid cell development, such as IL-1β, G-CSF, IFN-γ, and IL-17, accompanied by heightened signaling through their respective receptors in the HSC-enriched LSK compartment lacking Zfp521 (Lin neg cKit+ Sca1+). Moreover, IPA analysis identified transcriptome signatures indicative of key myeloid cell-associated transcription factors, including GATA1, CEBPα, and PU.1. However, it remains uncertain whether ZFP521 directly regulates these cytokines or if they contribute to the observed hematopoietic phenotypes. For example, it is noteworthy that two proinflammatory cytokines, IFN-γ and IL-17, were notably heightened in the absence of ZFP521. The upregulation of IFN-γ and IL-17 has previously been linked to compromised hematopoietic stem cell function and increased myelopoiesis. IFN-γ has also been associated with decreased erythropoiesis, which aligns with our observations of altered frequencies of GMP and MEP in Zfp521-deficient mice (De Bruin et al. 2014; Mojsilović et al. 2015; Fleenor et al. 2018).

In this context, Zfp521 revealed able to regulate HSCs through cell-extrinsic mechanisms in the BM microenvironment (Fleenor et al. 2018). Nevertheless, the absence of Zfp521 resulted in a reduction in the size of the HSC population and showed decreased long-term repopulating capacity when faced with regenerative stress, as emerged from serial transplantation assays using fetal liver cells (Garrison et al. 2017). Zfp521 plays a crucial role in maintaining the quiescent state and ability to self-renew of HSCs in adult hematopoiesis in vivo. In an elegant study, Li et al. (2021) effectively induced the conditional removal of Zfp521 in the hematopoietic compartments of adult mice through intraperitoneal injection of poly(I:C). They revealed that primary Zfp521-CKO mice exhibited a normal frequency of mature blood cells and HSPCs, suggesting that Zfp521 is not necessary in adult hematopoietic stem cells under normal, balanced conditions. Depleting Zfp521 does not impact the quiescence of HSCs and Lin−Sca-1+c-kit+ (LSK) cells during homeostasis. Nevertheless, it significantly diminishes the quiescence and viability of these cell populations under stress conditions. This reduction in quiescence and viability results in impaired self-renewal capacity of Zfp521-deficient HSCs under stress (Correa et al. 2010; Seriwatanachai et al. 2011; Al Dallal et al. 2016; Li et al. 2021). Furthermore, the absence of Zfp521 resulted in increased activation of Caspase-3 and decreased expression of the key apoptosis inhibitor BCL-2, likely explaining the heightened apoptosis observed in HPC and LSK cells in Zfp521-deficient mice (Yamasaki et al. 2010; Correa et al. 2010; Li et al. 2021). The data collected by Zhigang Li and colleagues from Zfp521-CKO chimeric mice clearly demonstrate that Zfp521 regulates HSC quiescence and self-renewal intrinsically within the HSC population. The evidence reported by Li and Fleenor describes Zfp521 as a protein involved in regulating the quiescence and self-renewal of HSCs through mechanisms that operate both internally and externally to the cells themselves. In addition, Zfp521 governs HSC quiescence by modulating Rela-mediated NF-κB signaling (Li et al. 2021). Specifically, the absence of Zfp521 resulted in the increased expression of Rela and Junb, which are essential components of the NF-κB pathway and its downstream target gene, respectively (Table 1). Additionally, there was a decrease in the expression of p57, which encodes cyclin-dependent kinase inhibitors. NF-κB signaling has a critical role in HSC quiescence. Changes in the negative regulatory pathways of NF-κB signaling disturb HSC quiescence and contribute to premature HSC depletion (Freire and Conneely 2018; Nakagawa et al. 2018). Deregulation of NF-κB activity leads to increased expression of proinflammatory cytokines, including TNF, IL1, IL6, and IFN-γ. Prolonged exposure of HSCs to these cytokines results in decreased self-renewal and quiescence. Similarly, constitutive NF-κB activation (Fleenor et al. 2018) reduces the HSC pool and impairs HSC functions (Liu et al. 2017; Li et al. 2021). Intriguingly, the depletion of Zfp521 led to enhanced expression of Rela by directly binding to its promoter. This interaction resulted in increased H3K9 acetylation and reduced H3K9me3 levels in the Rela promoter region, indicating heightened transcriptional activity of Rela. Additionally, knockdown of Rela via shRNA reversed the loss of quiescence observed in Zfp521-deficient HSPCs (Li et al. 2021).

Table 1 Key roles of ZFP521/ZNF521 in hematopoietic stem cells and cell lines
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Unveiling the molecular role of Zfp521/Znf521 in lymphocyte differentiation

ZNF521 is recognized for preserving the multipotency of primitive lympho-myeloid progenitors. It has the ability to inhibit the commitment of hematopoietic stem cells to the B cell lineage, which is driven by EBF1 (Mega et al. 2011). Specifically, the physical interaction between ZNF521 and EBF1 occurs at the carboxyl-terminal region of ZNF521, leading to the suppression of EBF1-dependent promoters and endogenous EBF1 target genes in B cells. The NuRD-binding domain found at the N-terminal end of ZNF521 is unnecessary to modulate EBF1 functionality, as evidenced by reporter assays performed with a deletion mutant lacking this motif. Furthermore, Mega et al. provide evidence that the overexpression of ZNF521 in human B-lymphoblastoid cells (IM-9, Roha9-MC3, and DeFew) suppresses EBF1-induced transcription and decreases the expression of endogenous B-lymphoid genes, including B29, mb1, VpreB, and λ5. Crucially, the authors illustrated that the suppression of ZNF521 significantly promotes the differentiation of B lymphocytes from both human and murine multipotent progenitors (Table 1). In experiments involving human CD34+ cells, transduction with the ZNF521-specific shRNA vector FG12-H11 followed by co-culture with MS-5 cells to induce B cell differentiation resulted in increased levels of CD10 and CD19 expression, particularly notable after 21 days, compared to cells infected with the lentiviral control (Ctrl) vector FG12. Similar findings were observed in mouse Lin− cells isolated from the bone marrow of C57BL/6 mice, where Zfp521 expression was blocked using a Zfp521-specific pLKo-puro-p15 vector. In this scenario, the Lin- modified cells were co-cultured with OP-9 cells in the presence of human IL-7 (10 ng/mL) and human Flt-3 L (10 ng/mL) to induce B cell differentiation. The analysis revealed a significant increase in CD19 and B220 cell surface antigens over time, with a peak observed after 15 days of culturing on the feeder stroma layer.

Role of Zfp521/Znf521 in erythrocyte differentiation

The influence of ZNF521 on the fate determination of hematopoietic progenitors extends beyond B-lymphoid commitment. Research conducted by Matsubara et al. (2009) suggests that ZNF521 interacts with GATA1 through the region spanning ZFs 21–26, operating at the level of the NuRD complex and repressing a GATA1-responsive promoter. As a result, when ZNF521 is silenced in erythromyeloid cell lines such as K-562 and HEL, it triggers spontaneous erythroid differentiation in these cells (Table 1). The molecular interplay between ZNF521 and GATA-1 appears to be essential for orchestrating the differentiation of erythroid cells and preserving the “stemness” of hematopoietic progenitors (Matsubara et al. 2009). GATA-1 possesses the capability to interact with various proteins through its ZF domains. Specifically, GATA-1 NF interacts with FOG-1 (friend of GATA-1) and c-Myb, while CF interacts with EKLF, Fli-1, PU.1, and EVI1. These interactions are pivotal in hematopoiesis as they modulate the transcriptional activation or repression of GATA-1 target genes (Swiers et al. 2006; Fujiwara 2017; Zhou et al. 2020a; Raghav and Gangenahalli 2021). According to the findings presented by Matsubara and colleagues, the complex formed by GATA-1 and ZNF521 suppresses globin gene expression during the early stages of erythroid differentiation through interaction with NuRD (Matsubara et al. 2009). While GATA-1 regulates the terminal differentiation of erythroid cells, GATA-2 is expressed in hematopoietic progenitors, governing the early stages of hematopoiesis. ZNF521 has been shown to interact with Smad1/4 in response to BMP signals and activate BMP-responsive genes, thereby promoting the proliferation of hematopoietic progenitors. Considering that GATA-2 expression is suppressed by GATA-1 during erythroid maturation, the inhibition of GATA-1 activity by ZNF521 might be crucial for sustaining the continuous expression of GATA-2, thus preserving hematopoietic progenitor cells (Matsubara et al. 2009).

Taken together, ZNF521 could play a significant role in maintaining the “stemness” of hematopoietic progenitors as summarized in Table 1.

The role of Zfp521 SUMOylation in stress-induced hematopoietic reconstitution

The hematopoietic reconstitution under stress conditions is also mediated by the SUMOylation of Zfp521 (Zhang et al. 2020). SUMOylation of proteins is a crucial post-translational modification necessary for ensuring many biological processes, such as transcription regulation, maintenance of genome integrity, and subcellular localization. However, it has also been found to be involved in some pathological processes (Yang et al. 2017). Previous research has highlighted the involvement of SUMOylation in the regulation of hematopoiesis through the modification of proteins like HIF-1a, STAT5, and c-Myb (Sæther et al. 2011; Li et al. 2014; Wang et al. 2021). Recently, Zhang and colleagues (2020) demonstrated that SUMOylation in Zfp521 does not influence hematopoiesis during normal conditions but can modulate hematopoietic regeneration under stress. Initially, they identified Lys-1146 as the site for SUMOylation on Zfp521 through biochemical and molecular analyses. Subsequently, they developed Zfp521 K1146R point mutation mice and observed that the deficiency of Zfp521 SUMOylation did not impact hematopoiesis under normal conditions. In this case, indeed, the counts of mature myeloid and lymphoid cells, in peripheral blood, spleen, and thymus, did not exhibit differences between Zfp521 mutant mice and Zfp521 wild-type mice. Moreover, the number of stem/progenitors cells in BM was comparable between wild-type and mutant mice. Finally, a significant reduction in the R2 population of the erythroid lineage in the BM and spleen of recipient mice undergoing transplantation was observed, indicating that the lack of Zfp521 SUMOylation hampers erythroid reconstitution under stress. Based on the evidence, SUMOylation could be involved in the regulation of hematopoietic reconstitution under stress, while not affecting hematopoiesis in normal conditions. In this context, the authors discussed the intriguing data obtained by considering three important biological events. Firstly, the proper SUMOylation of Zfp521 may not be necessary for the normal functioning of HSCs during homeostasis, but it could be crucial for enhancing their ability to respond to stress (Ganuza and McKinney-Freeman 2017; Zhang et al. 2020). Secondly, radiation exposure prior to bone marrow transplantation (BMT) could lead to damage in the microenvironment (niche) of the bone marrow, comprising both cellular components and various cytokines (Yu and Scadden 2016; Zhang et al. 2020). Consequently, the structural and functional interactions between HSCs and their niche might be compromised, resulting in inadequate extrinsic signals to support hematopoietic recovery in the Zfp521 K1146R mutant state. Thirdly, radiation-induced changes in the neuro-endocrine system of recipient mice could also play a critical role in erythroid differentiation (Stone et al. 2003; Zhang et al. 2020). Collectively, the differential role of Zfp521 SUMOylation in regulating erythroid reconstitution post-BMT, as opposed to its negligible impact on hematopoiesis in steady-state conditions, may stem from a complex interplay of intrinsic and extrinsic factors. These findings have unveiled a novel role of Zfp521 SUMOylation in facilitating erythroid reconstitution under stress, suggesting its potential as a promising therapeutic target in the future.

Evaluating the potential role of Znf521/Zfp521 as potential marker in acute myeloid leukemia

MLL-rearranged AML

Among adults, AML is the predominant form of leukemia, constituting approximately 80% of all cases (Vakiti and Mewawalla 2023). AML is a challenging and heterogeneous clonal disorder affecting hematopoietic stem/progenitor cells (HSPCs). Its hallmark features include compromised differentiation pathways and the uncontrolled proliferation of abnormal myeloid progenitors caused by the acquisition of various genetic mutations and chromosomal rearrangements (Trino et al. 2022; Abdel-Aziz 2023). One of the most aggressive variants of AML is identified by the presence of translocations that affect the mixed lineage leukemia gene (MLL, also referred to as KMT2A) (Nisticò and Chiarella 2023).

The MLL gene encodes a DNA-binding protein responsible for methylating histone H3 at lysine 4 (H3K4), thereby facilitating the activation of gene expression. This includes several Hox genes crucial for regulating the transcriptional program essential in the development and maintenance of hematopoiesis (Chinchole et al. 2022). The mixed lineage leukemia (MLL) locus is associated with more than 80 distinct rearrangements, resulting in a wide array of fusion partners, and is detected in various forms of leukemia, including both AML and acute lymphoid leukemia (ALL), and biphenotypic or chemotherapy-induced leukemias (Germano et al. 2017; Britten et al. 2019). One of the most recurrent fusion partners in MLL rearrangements (MLL-r) is AF9, mapping at chromosome 11q23. This prevalent translocation involves the fusion of the N-terminal portion of the MLL protein with the C-terminal fragment of the AF9 partner, arising from the t(9;11)(p22;q23) chromosomal anomaly. This event produces the oncogene MLL-AF9 (also known as KMT2A-MLLT3), found in approximately 5% of the adult AML cases and 25% of the de novo AML cases in children. The MLL-AF9 fusion protein triggers transformation in myeloid progenitors that undergo rapid cycling (Chiarella et al. 2014; Sparavier and Di Croce 2022; Heuts et al. 2023). The activation of MLL-AF9 converges on high expression of MLL target genes such as HOXA9 and MEIS1 critical for stem cell self-renewal, maintenance, and the suppression of genes associated with differentiation in MLL-induced leukemia. However, many other genes profoundly affected by MLL fusion proteins remain poorly characterized (Gundry et al. 2020).

Interestingly, recent studies examining gene expression profiles have shown a notable association between elevated ZNF521 mRNA levels and MLL-rearranged AML, prompting inquiry into the specific role of ZNF521 in this particular subtype of leukemia.

Role of ZNF521/ZFP521 in MLL-rearranged AML

Pediatric AML patients with MLL translocations demonstrate a significant upregulation of ZNF521 expression, irrespective of the fusion partner involved in the MLL translocation. The overexpression of ZNF521 emerges as a consistent transcriptional feature of MLL-rearranged AML across independent microarray datasets involving both adults and pediatric patients (Kohlmann et al. 2005; Jo et al. 2009; Pigazzi et al. 2011; Chiarella et al. 2021b).

Leukemia is characterized by a significant hindrance in hematopoietic differentiation, a consequence of MLL fusion protein expression. The primary impact of ZNF521 depletion is the promotion of myeloid differentiation in leukemia cells. This is evidenced by alterations in cell morphology, immunophenotype, and an increase in the expression of myeloid-specific genes in both MLL-rearranged cell lines and primary cells.

Specifically, the depletion of ZNF521 resulted in decreased cell viability in a panel of human MLL-rearranged AML cell lines, including THP-1, NOMO-1 (expressing MLL-AF9+), ML-2 (MLL-AF6+), and OCI-AML4 (MLL-ENL+).

This effect is due to the fact that ZNF521 knockdown triggers cell cycle arrest in MLL-rearranged AML cell lines by blocking the S phase, without inducing apoptosis. In addition, ZNF521 depletion induces myeloid differentiation in both MLL-rearranged AML cell lines and primary cells, as evidenced by increased levels of CD11b and CD14, along with a more mature macrophage-like morphology compared to control cells. Furthermore, the maturation induced by ZNF521 depletion was reinforced by the upregulation of mRNA levels of C/EBPA and PU.1, both of which are myeloid differentiation markers. Intriguingly, the importance of ZNF521 in maintaining an undifferentiated state associated with MLL-rearranged AML is highlighted by the significant decrease in ZNF521 expression upon treatment with specific differentiation-inducing agents, such as ATRA. When THP-1 and NOMO-1 AML cells were treated with all-trans retinoic acid (ATRA) and Securinine, ZNF521 mRNA and protein expression were found to be diminished, consequently promoting differentiation of MLL-rearranged cells (Germano et al. 2017).

This evidence underscores the central role of ZNF521 in sustaining AML cells in an undifferentiated state.

Gene Set Enrichment Analyses (GSEAs) conducted in THP-1 cells lacking ZNF521 revealed an abundance of downregulated genes linked to HSCs and embryonic stem cells (ESCs), as well as sets of genes associated with differentiation programs. Notably, the gene set resulting from ZNF521 depletion exhibited significant enrichment with genes that show increased expression in hematopoietic precursors under conditions where HOXA9 and MEIS1 are conditionally expressed. This includes genes targeted by HOXA9, which are known to be upregulated in hematopoietic stem cells. These results indicate that ZNF521 not only plays a part in fostering the self-renewal and preservation of HSCs but also sustains expression patterns associated with MLL-induced transformation. In this context, ZNF521 plays a critical role in MLL-mediated leukemia as it is a direct target of both MLL-AF9 and MLL-ENL fusion proteins. A specific genomic region spanning 555 base pairs in the 5′ ZNF521 promoter was discovered to be essential for its activation by MLL fusion proteins. Alterations in MLL-AF9 levels correspond to changes in ZNF521 expression across various human in vitro models (Germano et al. 2017).

However, ZNF521/Zfp521 is recognized as a universally conserved transcription factor enriched in HSCs in both human and murine hematopoiesis; its precise role in AML biology yet remains not completely clear.

In vitro experiments conducted by Chiarella et al. revealed that the simultaneous expression of ZNF521 and the MLL-AF9 fusion oncogene leads to a heightened proliferation of CB-CD34+ cells, likely attributed to an expanded progenitor cell population. Transcriptome analysis of CD34+ cells transduced with either MLL-AF9, ZNF521, or a combination of both revealed distinct sets of genes showing either up- or downregulation, which are associated with the leukemic phenotype, suggesting a synergistic effect between these two transcription factors. Specifically, transcription factors, epigenetic enzymes involved in chromatin remodeling, genes associated with cell cycle regulation, and transport proteins responsible for supplying transformed cells with the necessary metabolites were found to be modulated, supporting their high proliferation rate characteristic of leukemic blasts (Chiarella et al. 2021b). The suppression of ZNF521 in the MLL-AF9+ THP-1 cell line consistently replicated the effects observed in primary cells, resulting in impaired growth, an accumulation of cells in the G0/G1 phase of the cell cycle, and reduced clonogenicity (Garrison et al. 2017; Chiarella et al. 2021b). Based on these findings, it is evident that Znf521/Zfp521 plays a crucial role in promoting the progression of AML expressing the MLL-AF9 oncoprotein (Fig. 4a). Therefore, modulating its activity may have potential implications for mitigating + the aggressiveness of the disease.

Fig. 4
figure 4

ZNF521/ZFP521 promotes MLL-AF9 leukemogenesis in human and mouse hematopoietic stem cells. a Enforced expression of ZNF521 sustains cell expansion, increases the number of colony-forming cells, and activates MLL target genes in primary MLL-AF9-modified hematopoietic stem cells as well as in hematopoietic cell lines (THP-1 and NOMO-1). b The deficiency of Zfp521 caused a significant delay in mortality among both primary and secondary transplant recipients

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In vivo analysis of ZNF521/ZFP521 in animal models

The significance of ZNF521/ZFP521 in the context of MLL-AF9–mediated leukemia was investigated also by Garrison and colleagues through in vivo analysis. They transduced c-Kit + fetal liver cells from wild-type and Zfp521-deficient mice with MigR1-MLL-AF9-GFP retrovirus and then transplanted them into congenic recipients. Zfp521 deficiency resulted in a notable delay in mortality among primary transplant recipients, although ultimately all mice developed leukemia regardless of their genotype. A considerable postponement in mortality was also observed upon secondary transplantation, suggesting that ZFP521 loss prolongs the onset of leukemia, though it is not strictly essential for leukemogenesis or leukemic self-renewal (Fig. 4b). Furthermore, primary bone marrow cells expressing MLL-AF9 on a Zfp521-deficient background exhibited diminished colony-forming ability upon serial plating in methylcellulose. Correspondingly, MLL-AF9–expressing cells obtained from the peripheral blood of primary transplant recipients showed a slight decrease in blast-like cells, an increase in neutrophil terminal differentiation, and heightened apoptosis in the absence of Zfp521 (Garrison et al. 2017). Considering the significant role of Znf521/Zfp521 in MLL-AF9 leukemia progression, future challenges will involve investigating the potential for in vivo targeting of leukemia stem cells and developing small molecule drugs that inhibit the function of the ZNF521 protein as an alternative treatment approach.

Concluding remarks

Znf521 has been recognized as a new controller of hematopoietic regeneration. A thorough comprehension of both the factors influencing Znf521/zfp521 activity and the outcomes of its actions in hematopoietic regeneration is crucial for a deeper understanding of hematopoietic stem cell biology.

Such insights hold significant relevance for various clinical applications in hematopoietic regenerative medicine, including bone marrow transplantation, gene therapy, and in vitro expansion of HSCs.

Furthermore, it has recently been demonstrated that Znf521 is a critical factor for sustaining acute myeloid leukemia, especially those generated from the t(9;18) translocation. Henceforth, Znf521 could emerge as a promising target for future therapeutic interventions in the treatment of AML.