2. Factors Regulating the Homeostatic Function of Hematopoietic Stem Cells
An increasing number of regulators have been reported to change HSC function. In general, these regulators can be categorized into several groups according to their functions.
2.1. RNA-Binding Protein
RNA-binding proteins bind specific RNAs to manipulate RNA bioactivity, which in turn regulates cell function. An increasing number of studies have revealed the regulatory functions of RNA-binding proteins in HSCs (Figure 1).
Figure 1. Different mechanisms of RNA-binding protein interacting with its target RNA. U6 biogenesis 1 (USB1) excises U6 and U6atac small nuclear RNAs, and USB1 deadenylates miRNAs to avoid their degradation. Wrong splicing of SF3B1 mutants results in nonsense-mediated decay of RNA. TRAF6 ubiquitinates of hnRNPA1, causing abnormal alternative splicing of Arhgap1. Igf2bp2 regulates translation of mRNA through Lin28b/Hmga2 pathway.
As one type of RNA-binding protein, splicing factors participate intron removal from mRNA, which enables proteins to be accurately translated. Four genes (SF3B1
) encode splicing factors, and mutations in these genes are frequently reported in leukemia 
. For example, a common mutation in SF3B1
(K700E) contributes to the expansion of long-term HSCs (LT-HSCs) and the phenotype acquisition of myelodysplastic syndrome (MDS). More than 80% of patients with myelodysplastic syndrome with ring sideroblasts (MDS-RS) carry mutations in the SF3B1
gene; during bone marrow transplantation, SF3B1
-mutated MDS-RS HSCs differentiate into characteristic ring sideroblasts 
. One study demonstrated that the K700E mutation contributed to abnormal 3′ splice-site selection and increased the nonsense-mediated decay of RNA 
. Lieu et al. showed that this mutation promotes the mis-splicing of MAP3K7 and ultimately accelerates the death of erythrocytes and leads to the acquisition of the MDS phenotype 
. In addition, mutations in SF3B1
exert convergent effects by enhancing autophagy in hematopoietic stem and progenitor cells (HSPCs) and increasing the cell death rate through the hyperactivation of NF-κB signaling 
. A study reported that heterozygous mice carrying the SRSF2
P95H mutation exhibit a significant reduction in the number of HSPCs and an increased number of HSPC differentiation defects under both steady-state conditions and after transplantation 
. Fang et al. reported that hnRNPA1, an auxiliary splicing factor, is a substrate of TRAF6. Ubiquitination of hnRNPA1 by TRAF6 regulates the alternative splicing of Arhgap1, which is critical for the hematopoietic defects observed in TRAF6-expressing HSPCs 
In addition to these splicing factors, other RNA-binding proteins have also been reported to regulate the activity of HSCs. U6 biogenesis 1 (USB1) was discovered to be a 3′→5′ exoribonuclease that removes 3′-terminal uridine bases from U6 small nuclear RNA transcripts. In addition, USB1 has been recently reported to play a critical role in deadenylating microRNAs (miRNAs) and retarding their degradation, and mutations in USB1 can affect miRNA levels during blood development by inhibiting the removal of 3′-end adenylated tails, which impairs hematopoietic development 
. Igf2bp2, which is located downstream of the Lin28b/Hmga2 pathway, is an RNA-binding protein that regulates the stability and translation of mRNA. A recent study showed that Igf2bp2-dependent gene regulation in young HSCs and a decline in its function in aged HSCs both contributed to the acquisition of distinct phenotypes associated with HSC aging 
. Musashi 2 (Msi2), a translational inhibitor, maintains the stem cell compartment mainly by regulating the proliferation of primitive progenitors downstream in LT-HSCs. Through cell cycle and gene expression analyses, the decreased proliferation capacity of ST-HSCs and lymphoid myeloid-primed progenitors (LMPPs) was discovered in Msi2-defective mice. Moreover, HSCs with Msi2 knocked out exhibited significant defects in competitive repopulation experiments 
. By overexpressing MSI2, one study showed that pro-self-renewal phenotypes of HSCs can be induced, resulting in a 17-fold increase in the number of ST-HSCs and a 23-fold increase in the number of LT-HSCs. Furthermore, in a comprehensive analysis of MSI2-RNA interactions, MSI2 was shown to dampen aryl hydrocarbon receptor (AHR) signaling directly in cord blood HSPCs. This regulatory effect was mediated through the posttranscriptional downregulation of key components involved in the canonical AHR pathway 
. Zfp36l2 is a critical modulator of definitive hematopoiesis. Zfp36l2-KO mice showed a significant reduction in the levels of both definitive multilineage and hematopoietic progenitors, suggesting that Zfp36l2 functions as an essential target to control the stability of mRNA in HSPCs 
Taken together, these observations identify a class of RNA-binding proteins that maintain the self-renewal and differentiation of HSCs, and these RNA-binding proteins regulate the self-renewal and differentiation of HSC by regulating RNA transcription, splicing and translation. These intrinsic regulatory mechanisms provide a rich molecular basis for later nutritional interventions to extend HSC homeostasis.
2.2. Epigenetic Regulation of HSC
Several components of the m6A machinery, including METTL3, ALKBH5, a m6A eraser α-ketoglutarate-dependent dioxygenase (FTO) and the m6A reader YTH N6-methyladenosine RNA-binding protein 2 (YTHDF2), are commonly dysregulated in leukemia 
. It has been reported that the m6A writer enzyme METTL3 is highly expressed in acute myeloid leukemia (AML) cells, and its loss promotes cell differentiation and decreases cell proliferation in HSPCs 
. One study showed that either the downregulation of YTHDF2 or the activation of FTO inhibited CXCR4
decay in cord blood (CB) HSCs, promoting their homing and engraftment activity 
. YTHDF2 is an m6A reader that recognizes m6A-modified transcripts and participates in m6A-mRNA degradation. Recent studies have revealed that YTHDF2 functions as a repressor of inflammatory pathways in HSCs and plays an essential role in long-term HSC maintenance 
. Moreover, its inactivation leads to HSC expansion and ameliorates AML, while YTHDF2 deficiency exerts no effect on normal HSC function 
. These results suggest a potential strategy to reduce leukemic stem cell (LSC) survival.
In vivo experiments showed that m6A demethylation by ALKBH5 fine-tunes the activity of splicing factor encoded by SF3B1
and other epigenetic regulators, indicating that ALKBH5 may delay HSC leukemic transformation in patients with MDS 
. STED2, a histone H3 lysine 36 methyl-transferase, plays an important role in the pathogenesis of hematologic malignancies. A recent study showed that STED2 deficiency facilitated the self-renewal of NHD13+
HSPCs, which promoted the transformation of MDS into AML 
. The Ψ writer PUS7 plays a dual role by modifying and activating a network of tRNA-derived small fragments (tRFs) that target the translation initiation complex. When PUS7 is inactivated in embryonic stem cells, the translational regulation mediated by tRFs is impaired, resulting in increased protein biosynthesis. Thus, the disruption of the posttranscriptional regulatory mechanism leads to a reduced commitment of hematopoietic stem cells and an increased susceptibility to MDS development in humans 
Highly expressed in adult HSCs, MLLT3 (also known as AF9) is a component of super elongation complex 6 and interacts with DOT1L to exert its effects. MLLT3 sustains the abundance of H3K79me2 and is an HSC maintenance factor that connects histone readers with other modification factors to regulate the expression of HSC-specific genes. Moreover, in a mouse model, the sustained expression of MLLT3 led to balanced multilineage reconstitution in both the primary and secondary recipients 
These epigenetic regulators regulate the division pattern of HSCs, which subsequently controls the self-renewal and differentiation of HSCs (Figure 2), thus maintaining the normal development and homeostasis of the hematopoietic system.
Figure 2. Factors involved in the regulation of different states of HSCs in epigenetics. In normal HSCs, YTHDF2, the m6A reader, maintains long-term HSCs, while α-ketoglutarate-dependent dioxygenase (FTO) acts against YTHDF2, inhibiting CXCR4 decay to promote homing and engraftment activity of HSCs. In addition, MLLT3 sustains H3K79me2 level to maintain HSCs. In the pathogenesis of hematopoietic malignancies, METTL3 increases the HSPC growth in acute myeloid leukemia (AML). ALKBH5 delays HSC leukemic transformation in myelodysplastic syndromes (MDS), and STED2 deficiency facilitates the transformation of MDS into acute myeloid leukemia (AML). Moreover, inactivation of the Ψ “writer” PUS7 also contributes to the impairment of HSCs and the development of MDS.
2.3. Other Factors Regulating HSC Dormancy and Maintenance
Generally, cells in organisms must strike a balance between self-renewal and differentiation. In a stable environment, most HSCs are in a state of quiescence, as frequent differentiation of HSCs reduces the HSC pool. HSCs maintain dormancy through the action of multiple transcription factors. SIRT7 facilitates the transcription of DNA, and its expression is reduced in aged HSCs. Researchers found that its inactivation reduced the number of HSCs in a quiescent state and compromised HSC regenerative capacity. SIRT7 upregulation increased the regenerative capacity of aged HSCs 
. Hu et al. demonstrated that steroid receptor coactivator 3 (SRC-3) was highly expressed in HSCs. SRC-3−/−
mice were used to measure the function of SRC-3, and it was revealed that SRC-3 maintained the quiescence of HSCs and removed ROS from mitochondria 
. Adenosine-to-inosine RNA editing and the enzyme adenosine deaminase play crucial roles in hematopoietic cell development and differentiation. Specifically, antizyme inhibitor 1 (Azin1) has been observed to undergo extensive editing in hematopoietic stem and progenitor cells (HSPCs). This editing process leads to an amino acid change, resulting in the translocation of the Azin1 protein (AZI) to the nucleus. In the nucleus, AZI exhibits an enhanced binding affinity for DEAD box polypeptide 1 and modulates the expression of multiple hematopoietic regulators, ultimately promoting HSPC differentiation 
Zpf70 is a transcription factor that regulates the self-renewal and differentiation of hematopoietic stem cells. By using CRISPR/Cas9 technology to delete Zfp90, researchers found that Zpf70 promotes HSC self-renewal in a Hoxa9-dependent manner. Specifically, Zfp90 binds to the promoter of Hoxa9 to initiate its expression via the NURF complex 
. Autophagy suppresses the metabolism of hematopoietic stem cells by selectively eliminating functional and healthy mitochondria. This process is crucial for maintaining the quiescent state and stemness of cells. Additionally, autophagy becomes increasingly important with age, as it preserves the regenerative capacity of aging hematopoietic stem cells 
. HSCs with a high receptor tyrosine kinase Tie2 expression tend to be quiescent and resist undergoing apoptosis. The binding of Tie2 with its ligand angiopoietin-1 (Ang-1) maintains the long-term repopulation of HSCs in vivo 
. The activation of mitophagy, a quality control mechanism in mitochondria, is crucial for the self-renewal and expansion of Tie2+
hematopoietic stem cells (HSCs). The PPAR (peroxisome proliferator-activated receptor)-fatty acid oxidation pathway actively promotes the expansion of Tie2+
HSCs by facilitating the recruitment of Parkin to mitochondria 
. One study reported that the activity of chaperone-mediated autophagy (CMA) in HSCs decreased with age and showed that the genetic or pharmacological activation of CMA restored the functionality (lower intracellular ROS levels, increased PK and GAPDH activities, etc.) of HSCs in both old mice and elderly humans 
. These findings focused on the regulatory mechanisms of HSCs mediated by enhancer–promoter interactions and provided a theoretical basis for the future exploration of HSCs in this spatial field, leading to greater resolution of the multidimensional genetic regulatory mechanisms underlying HSC production.
2.4. Phase Separation
Phase separation has recently become a hot topic since a consensus has been reached suggesting that biomolecular condensates with membranes are reaction platforms for biological functions 
. Limited research on the influence of phase separation on HSCs has been reported, and the mechanisms established are shown in Figure 3
. One study showed that the transdifferentiation of spermatogonial stem cells (SSCs) into induced neural stem-cell-like cells (iNSCs) is mediated by the methylation reader protein YTHDF1 interacting with IKBα/β mRNAs, which promotes the formation of phase-separated condensates. The formation of condensates inhibits the translation of IKBα/β mRNAs and activates nuclear factor κB (NF-κB) p65, which ultimately facilitates the transdifferentiation of SSCs mediated through the expression of Eya1 
. Shao et al. revealed a mechanism in embryonic stem cells (ESCs), in which promoter-associated RNAs and their binding proteins cooperatively initiate the phase separation of polymerase condensates to promote transcription. Specifically, the paraspeckle protein PSPC1 combines with RNA, which functions as a multivalent molecule. This interaction results in the formation of transcript condensates and leads to the subsequent phosphorylation and release of RNA polymerase (Pol) II, increasing the activity of the polymerase 
Figure 3. Several proteins participating in the formation of phase separation in stem cells. YTHDF1 interacts with IKBα/β mRNAs to form phase separation, which ultimately promotes the transformation from spermatogonial stem cells (SSC) to induced neural stem-cell-like cells (iNSC). Phase separation of OCT4 reorganizes the structure of topological-associated domains (TADs), which manipulates induced pluripotent stem cell (iPSC) generation. PSPC1 binds RNA and forms transcription condensates, increasing transcription process in embryonic stem cells (ESC).
During the development of acute promyelocytic leukemia (APL), the aberrant phase separation of PML/RARα caused by neddylation of the RARα moiety results in the failure of PML nuclear body (NB) assembly, which is essential for tumor suppression. Moreover, when PML/RARα was deneddylated, phase separation was reinitiated and functional NBs were formed, and PML/RARα-driven leukemogenesis was also impeded 
. Phase separation has been found to influence epigenetic modifications in cancer stem cells (CSCs), specifically regulating their self-renewal activity. It has been hypothesized that phase separation promotes the tumorigenicity of CSCs through ubiquitination. For example, the mutation of speckle-type POZ protein (SPOP), which prevents the recruitment of ligase substrates, facilitates the accumulation of proto-oncoproteins and triggers the phase separation of SPOP, maintaining the ubiquitination of ubiquitin-dependent proteins 
. OCT4 is known as a master transcription factor for somatic cell reprogramming. Wang et al. recently demonstrated that the phase separation of OCT4 contributes to topological-associated domain (TAD) reorganization, which affects the efficiency of induced pluripotent stem cell (iPSC) generation 
The dysregulation of protein homeostasis plays a pivotal role in the aging process of hematopoietic stem cells (HSCs). In a recent study, researchers examined the proteome of HSCs and identified crucial protease inhibitors. Their findings revealed that the genetic elimination of prolyl isomerase resulted in the accelerated aging of HSCs. Interestingly, prolyl isomerase facilitates phase separation, thereby enhancing cellular stress resistance. These discoveries highlight the significance of maintaining the protein balance in HSCs and shed light on the role of prolyl isomerase in regulating HSC aging 
. This study linked a family of widely expressed chaperones to phase transitions and revealed that macromolecular condensation dynamics drive the aging of blood stem cells.