YTHDF3 as a new player in hematopoietic stem cell regulation

Hematopoietic stem cells (HSC) are characterized by self-renewal and multipotent differentiation potential, and are required to maintain the hematopoietic system throughout life. Since HSC transplantation is a life-saving treatment for various hematopoietic disorders, dissecting the mechanisms underlying how intrinsic programs and extrinsic niche signals are orchestrated to regulate HSC in vivo would enable development of new methods for ex vivo HSC expansion, and would potentially benefit clinical treatment for blood disorders.

N6-methyladenosine (m⁶A) is the most prevalent messenger RNA (mRNA) modification in mammals.¹ The proteins involved in the deposition, removal, and execution of the m⁶A modification are referred to as m⁶A ‘writers’, ‘erasers’ and ‘readers’, respectively.² Over the last decade, the m⁶A modification has been demonstrated to play essential and broad roles in the regulation of RNA stability and translation in various types of cells, although the detailed mechanisms require further exploration and seem to be cell- and context-dependent.^2,3 In recent years, several studies have revealed that deficiency of the m⁶A writer Mettl3 or Mettl14 impairs self-renewal and differentiation capacity of HSC.^4-6 In contrast, two groups independently discovered that loss of Ythdf2 promotes HSC expansion and regeneration.^7,8 Given that the m⁶A readers are the primary component responsible for exerting the function of m⁶A-containing RNA deposited by METTL3, an important question is: which readers are responsible for the characteristics of Mettl3^-/- HSC?

As described in their paper in this issue of Haematologica, to test the hypothesis that other readers in the cytoplasm may be responsible for the hematopoietic phenotype in Mettl3^-/- mice, Zhang et al. first investigated the hematopoietic system in Ythdf1^-/- and Ythdf3^-/- mice.⁹ Both the frequency and the absolute number of HSC in Ythdf3^-/- mice, unlike in Ythdf1^-/- mice, were significantly increased when compared with those in wild-type mice. Of note, the magnitude of the increase in HSC in Ythdf3^-/- mice was much smaller than that in Mettl3^-/- mice. No other significant differences in hematopoietic cells were found between Ythdf3^-/- and the littermate mice. In contrast, bone marrow cellularity and hematopoietic cells declined markedly in Mettl3^-/- mice. Consistent with the severely impaired reconstitution ability of Mettl3^-/- HSC, the Ythdf3^-/- HSC exhibited poor reconstitution capacity with similar multilineage differentiation, comparable homing capacity, higher sensitivity to replication stress, and diminished protein synthesis.

Given that Ythdf3^-/- HSC resemble Mettl3^-/- HSC to some extent, and that targets of Ythdf3 and Ythdf2 overlap partially, Zhang et al. reasoned that Ythdf3 may mediate HSC via reversely regulating the targets of Ythdf2. Among six mRNA previously identified to be increased in Ythdf2^-/- HSC,⁸ only Ccnd1 was decreased in Ythdf3^-/- HSC. Based on these findings, the next question to be explored in the future is: how do YTHDF2 and YHTDF3 orchestrate the regulation of Ccnd1 expression and in what context do YTHDF2 and YTHDF3 each dominate such regulation? Knockdown assays confirmed that the protein level of Ccnd1, rather than the mRNA level, decreased significantly in both Ythdf3 shRNA-carrying and Mettl3 shRNA-carrying HSC. This result is congruent with the role of the m⁶A modification in regulating gene expression at a post-transcriptional level. The 5’ untranslated region of Ccnd1 in the -180 to -184 region was identified as the major m⁶A motif recognized by YTHDF3 and METTL3. YTHDF3 cooperated with PABPC1 and eIF4G2 to promote the translation of Ccnd1. Furthermore, knockdown of Ccnd1 recapitulated the compromised reconstitution capacity of Ythdf3^-/- HSC. On the other hand, forced expression of Ccnd1 completely rescued the reconstitution potential of Ythdf3 shRNA-carrying HSC, but not of Mettl3 shRNA-carrying HSC. Myc has previously been reported to be an important target gene of Mettl3, and enforced expression of Myc rescued the lineage differentiation bias of Mettl3^-/- HSC. To investigate the role of Myc in reconstitution potential, Zhang et al. over-expressed Myc in Mettl3^-/- HSC and evaluated the engraftment capacity of these cells. The result showed that forced expression of Myc failed to rescue the reconstitution ability of Mettl3^-/- HSC, although it consistently rescued the lineage differentiation bias of Mettl3^-/- HSC. These data indicate that, besides Ythdf3 and Myc, other unknown players are responsible for the characteristics of Mettl3^-/- HSC. Could the other m⁶A readers (e.g., YTHDC1 and YTHDC2) be the missing link?

The m⁶A modification has recently been discovered to play a critical role in the regulation of HSC, although the mechanisms are still not fully understood. Zhang et al. were able to establish that the Mettl3-Ccnd1-Ythdf3 axis regulates the characteristics of HSC. Their study uncovers the missing piece of the puzzle by revealing that the m⁶A reader YTHDF3 is partially responsible for Mettl3 deficiency in HSC, broadening the knowledge of how RNA m⁶A components elegantly combine to regulate HSC (Figure 1). Based on this discovery, there are several questions to be explored further in the future: (i) Besides mRNA, METTL3 has recently been discovered to influence chromatin state and transcription of mouse embryonic stem cells via YTHDC1-mediated regulation of chromosome-associated regulatory RNA. Is this regulatory mechanism also applicable to adult stem cells, for example HSC? (ii) Do the regulatory mechanisms that govern mouse HSC properties, such as self-renewal and differentiation, also regulate similar properties in human HSC? Although a study previously demonstrated the opposite role of Mettl3 in human hematopoietic stem and progenitor cells and in murine HSC, a series of experiments using purified human HSC, as opposed to human CD34⁺ hematopoietic stem and progenitor cells, which only contain a very small population of HSC, is warranted to answer this question. (iii) Given that Mettl3 deficiency leads to increased numbers and decreased differentiation of HSC, and that rapid differentiation of HSC in in vitro culture is a major challenge in the field, a pertinent question becomes: is it possible to inhibit METTL3 pharmacologically using small molecules to block differentiation of HSC in ex vivo cultures?

Figure 1.Proposed model of the Mettl3 → RNA m⁶A → Ccnd1 → Ythdf3 axis regulating the reconstitution capacity of mouse hematopoietic stem cells. METTL3, along with other components including METTL14 and WTAP, form a complex and deposit m⁶A to Ccnd1 mRNA in the nucleus. The m⁶A-containing Ccnd1 mRNA then travels from the nucleus to the cytoplasm for translation. YTHDF3 specifically recognizes the m⁶A motif, mainly at the 5’ UTR of Ccnd1 mRNA in the region from -180 to -184, and increases the expression of Ccnd1 by recruiting PABPC1 and eIF4G2 to enhance protein synthesis. The up- regulated expression of Ccnd1 is responsible for YTHDF3-mediated reconstitution capacity of mouse hematopoietic stem cells.

Footnotes

Correspondence

LIL@stowers.org

Disclosures

No conflicts of interest to disclose.

Contributions

XM wrote the editorial; LL supervised and edited it.