YTHDF3 modulates hematopoietic stem cells by recognizing RNA m6A modification on Ccnd1

Abstract

Hematopoietic stem cells (HSC) give rise to the cells of the blood system over the whole lifespan. N6-methyladenosine (m⁶A), the most prevalent RNA modification, modulates gene expression via the processes of “writing” and “reading”. Recent studies showed that m⁶A “writer” genes (Mettl3 and Mettl14) play an essential role in HSC. However, which reader deciphers the m6A modification to modulate HSC remains unknown. In this study, we observed that dysfunction of Ythdf3 and Ccnd1 severely impaired the reconstitution capacity of HSC, which phenocopies Mettl3-deficient HSC. Dysfunction of Ythdf3 and Mettl3 results in a translational defect of Ccnd1. Ythdf3 and Mettl3 regulate HSC by transmitting m⁶A RNA methylation on the 5’ untranslated region of Ccnd1. Enforced Ccnd1 expression completely rescued the defect of Ythdf3-/- HSC and partially rescued Mettl3-compromised HSC. Taken together, this study identified, for the first time, that Ccnd1 is the target of METTL3 and YTHDF3 to transmit the m⁶A RNA methylation signal and thereby regulate the reconstitution capacity of HSC.

Introduction

Hematopoietic stem cells (HSC) generate all blood cells and themselves throughout life, a function that is achieved through differentiation and self-renewal.^1,2 Therefore, discovering the molecular mechanisms modulating HSC differentiation and self-renewal is of great importance to understand the nature of the blood system and hematopoietic malignancies. Although some studies have revealed several molecular mechanisms regulating HSC,³ the exact mechanisms are still not fully understood. In recent years, post-transcriptional chemical modifications, which are introduced at specific sites of RNA, have become an emerging field of interest.⁴ Among the various RNA alterations, N6-methyladenosine (m⁶A) is the most abundant nucleotide modification in messenger RNA (mRNA), which functions through the processes of “writing”, “erasing” and “reading”.⁵ A few genes are responsible for the process of "writing" the m⁶A code, including Mettl3, Mettl14, Wtap, Zc3h13, Kiaa1429, Rbm15 and Rbm15b^,6-8 with Mettl3 being the catalytic core;^6,9 m⁶A methylation can be reversed via active demethylation by m⁶A demethylases FTO or ALKBH5. YTH domain-carrying genes, including Ythdf1, Ythdf2 and Ythdf3,¹⁰ are responsible for the process of “reading” the m⁶A code by selectively binding to m⁶A-containing transcripts and have the function of modifying m⁶A.^10-13 Ythdf1 regulates the translation efficiency by binding m⁶A-modified mRNA,¹³ Ythdf2 decreases mRNA stability by recruiting the CCR4-NOT deadenylase complex,^12,14 and Ythdf3 facilitates translation or decay of m⁶A-modified mRNA through cooperation with Ythdf1 or Ythdf2.^11,15

Yao et al. reported that in the hematopoietic system, the METTL3 and METTL14 complex regulates the self-renewal capacity of HSC.¹⁶ Two more studies documented that Mettl3 modulates the differentiation and symmetric commitment of HSC by targeting Myc.^17,18 Given that m⁶A methylation exerts its effects through reader proteins, and that RNA m⁶A modification participates in diverse eukaryotic biological processes^19-20 and tumor initiation,^21,22 elucidating the function of m⁶A reader proteins will probably be crucial to uncovering the biological significance of the m⁶A modification. Two recent studies have shown that loss of Ythdf2 promotes HSC expansion and regeneration,^23,24 which is completely different from the phenotype of Mettl3 or Mettl14 dysfunctional HSC in which loss of these genes severely impairs HSC function.^16-18 It would, therefore, be intriguing to know on which gene(s) METTL3 writes the m⁶A signal and which reader recognizes this modification to regulate HSC function.

In this study, we observed that dysfunction of Ythdf3, but not of Ythdf1, impairs the reconstitution capacity of HSC, recapitulating the phenotype of Mettl3 dysfunctional HSC. The 5’-untranslated region (UTR) of Ccnd1 is the hub of METTL3 and YTHDF3 to transmit the m⁶A signal to regulate the translation of Ccnd1, and furthermore to modulate the reconstitution capacity of HSC. Enforced Ccnd1 expression completely rescued the functional defect of Ythdf3^-/- HSC, and partially rescued Mettl3-compromised HSC. Ectopic MYC only rescued the differentiation skewing of Mettl3-compromised HSC, but not the reconstitution capacity. Taken together, our study reveals, for the first time, that Ythdf3 deciphers the m⁶A modification on Ccnd1 to modulate HSC, and provides a reference for how the RNA m⁶A modification regulates stem cell function.

Methods

Mice

Mettl3^fl/fl, Ythdf1^-/- and Ythdf3^-/- mice were generated in Cyagen Biosciences Inc. (Guangzhou, China). All mice were kept in specific pathogen-free conditions and all procedures were approved by the Institutional Animal Care and Use Committee of Tsinghua University. Full details are supplied in the Online Supplementary Methods.

Results

Dysfunction of Ythdf3, but not Ythdf1, mildly disturbs the hematopoietic system

Given that m⁶A reader proteins are responsible for exerting the action of m⁶A, and that Ythdf2-deficient HSC exhibit increased self-renewal capacity,^23,24 which is not consistent with the phenotype of Mettl3^-/- HSC,^16-18 we generated Ythdf1 and Ythdf3 knockout mice to investigate the function of these two m⁶A readers in HSC (Figure 1A, B). Both strains of mice develop normally and can reproduce. Complete blood counts, including white blood cell, lymphocyte, neutrophil, red blood cell and platelet counts, revealed no difference between Ythdf1^-/-, Ythdf3^-/- and control mice (Figure 1C, D). In addition, the bone marrow cellularity of Ythdf1^-/- and Ythdf3^-/- was indistinguishable from that of control mice (Online Supplementary Figure S1A-D). We then sought to investigate the lineage composition, including T, B and myeloid cells, in peripheral blood and bone marrow of Ythdf1^-/- and Ythdf3^-/- mice. The results revealed no difference between Ythdf1^-/- and WT mice in either the peripheral blood (Figure 1E) or the bone marrow (Online Supplementary Figure S1G). As for Ythdf3^-/- mice, the percentages of B and myeloid cells remained stable in peripheral blood and bone marrow compared to those in WT mice (Figure 1F and Online Supplementary Figure S1H), while the percentages of CD4⁺ and CD8⁺ T cells decreased slightly in bone marrow (1.58±0.24 vs. 1.11±0.14, 1.62±0.26 vs. 1.15±0.14), but not in peripheral blood (10.94±1.50 vs. 10.54±1.65, 12.52±0.99 vs. 12.95±0.78) (Figure 1F and Online Supplementary Figure S1H). We then compared the frequencies of T cells in spleen and thymus between Ythdf3^-/- and WT mice, without finding a significant difference (Online Supplementary Figure S1I, J).

We next analyzed hematopoietic stem and progenitor cells of Ythdf1^-/- and Ythdf3^-/- mice, including common myeloid progenitors, granulocyte-macrophage progenitors, mega-karyocyte-erythroid progenitors, common lymphoid progenitors, multipotent progenitor cells and HSC. We observed that the frequencies and absolute numbers of these cells remained the same between Ythdf1^-/- and WT mice (Figure 1G, H and Online Supplementary Figure S1B, C). Likewise, the frequencies and absolute numbers of common myeloid, granulocyte-macrophage, megakaryocyte-erythroid, and common lymphoid progenitors, as well as multipotent progenitor cells in Ythdf3^-/- mice were comparable with those in WT mice (Figure 1I and Online Supplementary Figure S1E). However, the frequency and absolute number of Ythdf3^-/- HSC were iincreased significantly (64.31±10.34 vs. 106.01±6.99, 1.90±0.31 vs. 2.82 vs. 0.23) (Figure 1J and Online Supplementary Figure S1F), resembling those of Mettl3^-/- mice.^16,17 Cell cycle analysis revealed no difference between Ythdf3^-/- and WT HSC (Online Supplementary Figure S1K, L).

The reconstitution capacity of Ythdf3-deficient hematopoietic stem cells is impaired

To further investigate the reconstitution capacity of Ythdf1^-/- and Ythdf3^-/- HSC, 25 freshly isolated Ythdf1^-/-, Ythdf3^-/- and WT HSC were transplanted into lethally irradiated recipients together with 2.5×10⁵ competitor cells (Figure 2A). We observed no obvious differences of peripheral blood chimera and lineage distribution between Ythdf1^-/- and WT HSC (Figure 2B and Online Supplementary Figure S2A). By contrast, the reconstitution capacity of Ythdf3^-/- HSC was severely impaired (71.63±4.66 vs. 29.03±10.82 at the fourth month), including B, T and myeloid lineages (Figure 2C). The lineage distribution exhibited no obvious differentiation bias between Ythdf3^-/- and WT mice (Online Supplementary Figure S2B). We also counted the donor-derived HSC in the bone marrow of recipients at the end of the fourth month after transplantation and found no significant difference between Ythdf3^-/- and WT recipients (Online Supplementary Figure S2C, D).

Figure 1.Dysfunction of Ythdf3, but not Ythdf1, mildly disturbs the hematopoietic system. (A) Diagram of the production of Ythdf1 knockout mice. (B) Diagram of the production of Ythdf3 knockout mice. (C, D) The scatter plots show the differences in counts of white blood cells, lymphocytes, neutrophils, red blood cells and platelets between wildtype (WT) and Ythdf1^-/- mice (C), as well as WT and Ythdf3^-/- mice (D) (2 months old) as determined by an automatic peripheral blood analyzer. Six mice per group. Data are shown as mean ± standard error of mean (SEM). (E, F) The scatter plots depict the percentages of myeloid, B and T cells in peripheral blood for WT and Ythdf1^-/- mice (E), as well as for WT and Ythdf3^-/- mice (F) (2 months old). Six mice per group. Data are shown as mean ± SEM. (G-J) The scatter plots depict the numbers of common myeloid progenitors, granulocyte-macrophage progenitors, megakaryocyte-erythroid progenitors, common lymphoid progenitors, and multipotent progenitor cells per 10⁶ bone marrow cells in WT and Ythdf1^-/- mice (G), as well as in WT and Ythdf3^-/- mice (I) (2 months old), and the number of hematopoietic stem cells per 10⁶ bone marrow cells in WT and Ythdf1^-/- mice (H), and WT and Ythdf3^-/- mice (J) (2 months old). Six mice per group. Data are shown as mean ± SEM. WBC: white blood cells; LYM: lymphocytes; NEUT: neutrophils; RBC: red blood cells; PLT: platelets; CMP: common myeloid progenitors; GMP: granulocyte-macrophage progenitors; MEP: megakaryocyte-erythroid progenitors; CLP: common lymphoid progenitors; MPP: multipotent progenitor cells; HSC: hematopoietic stem cells.

Figure 2.The reconstitution capacity of Ythdf3-deficient hematopoietic stem cells is impaired. (A) Scheme of the competitive transplantation strategy for Ythdf1^-/- and Ythdf3^-/- hematopoietic stem cells (HSC). (B, C) Freshly isolated HSC (n=25) from 2-month-old knockout mice or wildtype (WT) mice were transplanted into lethally irradiated recipients together with 2.5×10⁵ competitor cells. Engraftment of donor cells was determined in overall (CD45.2⁺), myeloid (Mac-1⁺), B (B220⁺) and T (CD3⁺) cells every month after transplantation. The line plots depict the percentages of donor-derived cells (overall, B cells, myeloid cells, T cells) in recipient WT and Ythdf1^-/- mice (B), and WT and Ythdf3^-/- mice (C) at the indicated time points. Five mice per group. Data are shown as mean ± standard error of mean (SEM). (D) The scatter plots show the percentage of Ythdf3^-/- Lin^– Sca1⁺ cells that homed to the bone marrow relative to control. Five mice per group. Data are shown as mean ± SEM. (E) Freshly isolated LSK cells were infected by lentivirus carrying Ythdf3 shRNA or control shRNA; 4 days later, 10⁵ GFP⁺ cells were purified for western blot to evaluate the expression of YTHDF3. (F, G) Freshly isolated WT LSK cells were infected by lentivirus carrying Ythdf3 shRNA or control; 72 h later, 20,000 GFP⁺ cells were purified and transplanted into lethally irradiated recipients together with 2.5×10⁵ competitor cells. Chimerism in peripheral blood was evaluated every month until the fourth month. (F) Experimental design to evaluate the reconstitution capacity of Ythdf3 shRNA-carrying HSC. (G) Line plots depicting the percentages of GFP⁺ cells in donor-derived cells every month after transplantation. Six or seven mice per group. Data are shown as mean ± SEM. (H) Experimental design of the RNA-sequencing assay (see Methods). (I) These figures show the gene set enrichment analysis of HSC fingerprint genes in Ythdf3^-/- HSC versus WT HSC. The normalized enrichment score is |>0.3; P<0.05 represents a statistically significant difference. KO: knockout; PB: peripheral blood; HSCT: hematopoietic stem cell transplantation; NES: normalized expression score.

In order to rule out the possibility that the impaired reconstitution capacity of Ythdf3^-/- HSC was due to impaired homing capacity, we performed short-term homing assays for Ythdf3^-/- and WT HSC according to standard protocols.^25,26 The results showed no significant difference in homing efficiency between Ythdf3^-/- and WT Lin^- Sca1⁺ cells (Figure 2D).

To rule out the possibility that Ythdf3 is compensated for during the embryonic stage of Ythdf3^-/- mice, we generated two efficient short hairpin RNA (shRNA) against Ythdf3: shYthdf3-2 and shYthdf3-4 (Figure 2E). Freshly isolated WT LSK cells (Lin^- Sca1⁺ cKit⁺) were infected by Ythdf3 shRNA-carrying lentivirus. Seventy-two hours later, 20,000 green fluorescent protein (GFP)⁺ cells were purified and transplanted into lethally irradiated recipients together with 2.5×10⁵ competitor cells (Figure 2F). The results showed that the reconstitution capacity of Ythdf3 shRNA-carrying cells was severely impaired (shRNA-2: 6.13±2.82 vs. 60.51±8.33; shRNA-4: 6.74±4.55 vs. 60.51±8.33 at the fourth month), including the rconstitituion of B, T and myeloid lineages (Figure 2G and Online Supplementary Figure S2E). To further investigate the transcriptional difference between Ythdf3^-/- and WT HSC, we performed RNA sequencing for those cells (Figure 2H). With principal component analysis, we found that the expression patterns of the Ythdf3^-/- HSC were distinct from those of WT ones (Online Supplementary Figure S2F). Gene set enrichment analysis (GSEA) revealed that the HSC fingerprint genes were no longer enriched among Ythdf3^-/- HSC (Figure 2I), coinciding with the impaired reconstitution capacity of Ythdf3^-/- HSC. Furthermore, we analyzed apoptosis-related genes between Ythdf3^-/- and WT HSC by GSEA, and found no significant difference (Online Supplementary Figure S2G). We then wondered whether Ythdf3^-/- HSC undergo cell death during in vitro culture. Freshly isolated LSK cells from Ythdf3^-/- and WT mice were cultured in serum-free expansion medium with the cytokines, stem cell factor and thrombopoietin. The percentage of annexin V-positive cells was analyzed 24 h later (Online Supplementary Figure S2H) and found to be significantly increased in Ythdf3^-/- LSK cells (Online Supplementary Figure S2I,J). This result indicates that Ythdf3^-/- hematopoietic stem and progenitor cells are more sensitive to replication stress.

Given that ribosomes are the site of protein synthesis, we then evaluated protein synthesis-related signaling and observed that ribosome pathway and ribosome-related genes were downregulated in Ythdf3^-/- HSC (Online Supplementary Figure S3A, B). To further investigate this, we sought to measure protein synthesis in Ythdf3^-/- LSK cells. In this assay, LSK cells were incubated with a puromycin analog (OPP), which is incorporated into nascent polypeptide chains and then fluorescently labeled via a “click reaction”. The results showed that the protein synthesis of Ythdf3^-/-LSK cells was significantly decreased (Online Supplementary Figure S3C-E). Briefly, these data provide the confirmation that Ythdf3, but not Ythdf1, modulates the reconstitution capacity of HSC.

YTHDF3, but not YTHDF1, modulates the translation of CCND1

Our data indicate that the reconstitution capacity of Ythdf3^-/- HSC resembles that of Mettl3^-/- HSC,^16,17 and a previous study revealed that Ythdf3 plays a critical role in translating m⁶A-modified mRNA.¹¹ Moreover, two recent studies found that Ythdf2^-/- HSC exhibit enhanced recon-stitution capacity^23,24 and Ythdf3 shares targets with Ythdf2.¹¹ We speculated that Ythdf3 may modulate the targets of Ythdf2 oppositely to regulate the function of HSC. We therefore investigated the six genes (MYC, CCND1, AXIN2, MCL-1, CD133 and BCL2) which were significantly increased in Ythdf2^-/- HSC,²³ determining their expression in Ythdf3^-/- LSK cells by western blotting assays. The assays showed that only CCND1 was decreased, while MYC, MCL-1, CD133, AXIN2 and BCL2 remained stable compared to those in the controls (Figure 3A). Moreover, we found that knockout of Ythdf3 did not affect the mRNA expression level of Ccnd1 in LSK cells (Figure 3B). To further confirm this observation, we infected WT LSK cells by lentivirus carrying shRNA against Ythdf3 to evaluate the expression of Ccnd1 (Figure 3C). Knockdown of Ythdf3 significantly reduced the protein level of CCND1 (Figure 3D), but not the mRNA level (Figure 3E). A previous study showed that YTHDF3 promotes protein synthesis in synergy with YTHDF1,¹¹ so we wondered whether YTHDF1 is involved in CCND1 translation. We evaluated the protein expression of CCND1 in Ythdf1^-/- LSK cells and found no difference of CCND1 between Ythdf1^-/- and WT LSK cells (Online Supplementary Figure S4A), indicating that YTHDF3 modulates the translation of Ccnd1 via a YTHDF1-independent manner. In brief, the above data suggest that Ythdf3, but not Ythdf1, regulates Ccnd1 through post-transcriptional modification.

YTHDF3 promotes the translation of CCND1 by binding to the 5’ untranslated region

A previous study revealed that FTO demethylates m⁶A-modified Ccnd1 mRNA²⁷ and our results indicated that Ccnd1 is regulated post-transcriptionally by Ythdf3 (Figure 3A-E). We were tempted to speculate that Ccnd1 may be regulated by YTHDF3 through m⁶A modification. To test our hypothesis, we predicted the putative m⁶A motif on Ccnd1 by using SRAMP.²⁸ In silico analysis predicted four putative DRACH motifs in which m⁶A occurs preferably: two are located at the 5’UTR and two are located at the 3’UTR of Ccnd1 (Online Supplementary Figure S4B). It is notable that the first putative m⁶A motif (-180 to -184, GGATC) is completely conserved among mice, rats and humans, while the second putative one (-102 to -106, AGACT) is only conserved between mice and humans (Online Supplementary Figure S4C). However, the two putative ones (GGACT) at the 3’UTR of Ccnd1 are not conserved (Online Supplementary Figure S4C). This indicates that an m⁶A motif at the 5’UTR of Ccnd1 might play an important role in regulating Ccnd1 mRNA translation.

Figure 3.YTHDF3 promotes the translation of CCND1 through binding on the m⁶A site of the 5’ untranslated region. (A) Representative western blot showing the expression of YTHDF3, CCND1, CD133, BCL2, MYC, AXIN2 and MCL-1 in wildtype (WT) and Ythdf3^-/- LSK cells. Freshly isolated LSK cells from WT and Ythdf3^-/- mice were lysed in sodium dodecylsulfate loading buffer. Western blot analysis was performed with the indicated antibodies. (B) Histogram depicting the mRNA expression of Ccnd1 in WT and Ythdf3^-/- LSK cells. (C-E) Freshly isolated LSK cells were infected by lentivirus carrying Ythdf3 shRNA or control, and 4 days later, GFP⁺ cells were purified for western blot (5×10⁴ cells/well) and quantitative polymerase chain reaction (qPCR) (10⁵ cells) to evaluate the expression of Ccnd1. (C) Diagram showing the experimental design to evaluate the expression of Ccnd1. (D) Representative western blot showing the expression of YTHDF3 and CCND1. (E) Histogram depicting the mRNA expression of Ythdf3 and Ccnd1. Data are shown as mean ± standard error of mean (SEM). (F, G) Histograms showing the relative luciferase activity of Ccnd1-5’UTR-WT1 or Ccnd1-5’UTR-Mut1 (F) and Ccnd1-5’UTR-WT2 or Ccnd1-5’UTR-Mut2 (G) luciferase reporter in 293T cells transfected with control or YTHDF3 plasmid (see Online Supplementary Figure S3C and Methods). Firefly luciferase activity was measured and normalized to Renilla luciferase activity. Data are shown as mean ± SEM. (H, I) Freshly isolated LSK cells were infected by lentivirus carrying Pabpc1 shRNA, eIF4G2 shRNA or control, and 4 days later, GFP⁺ cells were purified for western blot to evaluate the expression of Ccnd1. Representative western blot showing the expression of PABPC1 and CCND1 (H), and eIF4G2 and CCND1 (I). (J, K) HEK293T cells were co-transfected with S-protein, Flag, and streptavidin-binding peptide (SFB)-tagged-YTHDF3 and MYC-PABPC1 or MYC-eIF4G2 plasmids for 24 h. Cell lysates were immunoprecipitated with S beads, and western blot analysis was performed with the indicated antibodies. The results show that YTHDF3 interacts with PABPC1 (J) and eIF4G2 (K). qPCR: quantitative polymerase chain reaction; IP: immunoprecipitation; WB: western blot.

We then cloned the 3’UTR of Ccnd1 into psiCHECK2 vector (Online Supplementary Figure S4D) and conducted a luciferase reporter assay. The results revealed that over-expression of Ythdf3 promotes the translation of luciferase reporters with both motifs (Online Supplementary Figure S4F-H). We then mutated the A at positions 1631 and 1532 to G, which is the key site of the m⁶A motif. We observed that the translation of luciferase reporter remained unchanged (Online Supplementary Figure S4G, H), suggesting that these two m⁶A motifs are not the direct region where m⁶A modification occurs.

We next cloned the 5’UTR of Ccnd1 containing the aforementioned DRACH motif into pGL3-Basic vector (Online Supplementary Figure S4E), and performed luciferase reporter assays. The results revealed that overexpression of Ythdf3 promoted the translation of luciferase activity with both m⁶A motifs (-180 to -184 and -102 to -106) (Figure 3F, G). We then mutated the A at positions -182 and -104 to G, and observed that luciferase activity was significantly decreased in both situations, wherein the A (-182) G mutation completely abolished the luciferase activity, while the A (-104) G mutation slightly reduced luciferase activity (Figure 3F, G).

A previous study indicated that two residues of YTHDF3, W438 and W492, contribute to the specific recognition of m⁶A modification.²⁹ We therefore mutated the W438 and W492 to A (alanine) to generate a YTHDF3 mutant (YTHDF3-Mut), and overexpressed YTHDF3-WT and YTHDF3-Mut plasmids in 3T3 cells. RNA immunoprecipitation (RIP) quantitative polymerase chain reaction (qPCR for Ccnd1 revealed that YTHDF3 could bind to Ccnd1 mRNA (Online Supplementary Figure S4I).

These results suggest that YTHDF3 regulates the translation of CCND1 by directly binding to the 5 ’UTR of Ccnd1, in which the -180 to -184 region is essential.

YTHDF3 promotes the translation of CCND1 by cooperating with PABPC1 and EIF4G2

Two previous studies showed that Ythdf3 plays a critical role in translating m⁶A-modified mRNA.^11,15 We observed that Ythdf3 deficiency resulted in a significant decrease of protein synthesis in hematopoietic stem and progenitor cells (Online Supplementary Figure S3E). A previous study revealed that YTHDF3 interacts directly with the translation factor PABPC1 and eIF4G2 to promote protein translation.²⁹ We then explored whether YTHDF3 promotes the translation of CCND1 by interacting with PABPC1 and eIF4G2. We generated two efficient shRNA against Pabpc1 and Eif4g2 separately (Online Supplementary Figure S4J, K). We infected WT LSK cells with lentivirus carrying shRNA against Pabpc1 and Eif4g2 to evaluate the expression of CCND1. The results showed that knockdown of PABPC1 and eIF4G2 significantly reduced the protein level of CCND1 (Figure 3H, I).

To explore whether YTHDF3 interacted directly with PABPC1 and eIF4G2, co-immunoprecipitation was performed using anti-Flag or Myc antibody in HEK293T cells, which showed that YTHDF3 interacts directly with PABPC1 and eIF4G2 (Figure 3J, K). In addition, using RIP-qPCR assays, we found that PABPC1 and eIF4G2 bind directly with the mRNA of Ccnd1 (Online Supplementary Figure S4L, M). Together, these results suggest that YTHDF3 promotes the translation of CCND1 by cooperating with PABPC1 and eIF4G2.

Ccnd1 is indispensable for maintaining hematopoietic stem cells

The above data indicate that Ccnd1 is the target of YTHDF3 to transmit the m⁶A signal. To investigate whether Ccnd1 plays a role in regulating HSC function, we generated two efficient guide RNA (gRNA) against Ccnd1 and cloned them (gRNA 2 and 3) (Figure 4A) into a self-made lentiviral vector (mCherry-labeled) (Online Supplementary Figure S5A). Cas9^flox/flox GFP mice³⁰ were crossed with Vav1-cre to generate mice expressing CAS9 in the blood system: Cas9^flox/flox; Vav1-Cre mice (hereafter named Cas9^+/+ mice). Freshly isolated LSK cells of Cas9^+/+ mice were infected by Ccnd1 gRNA-carrying lentivirus and 72 h later, 10,000 GFP⁺ mCherry⁺ cells were purified and transplanted into lethally irradiated recipients together with 2.5×10⁵ competitor cells (Online Supplementary Figure S5B). It was found that knockdown of Ccnd1 severely impaired the reconstitution capacity of HSC (Figure 4B and Online Supplementary Figure S5D), which recapitulates the phenotype of Mettl3^-/-16-17 and Ythdf3^-/- HSC (Figure 2C).

Forced Ccnd1 expression rescues the reconstitution capacity of Ythdf3-/- hematopoietic stem cells

Given that our data revealed that Ccnd1 is the hub of YTHDF3 to transmit the m⁶A signal in order to regulate its translation, and that knockdown of Ccnd1 impaired HSC, we wondered whether forced Ccnd1 expression could rescue the functional defect of Ythdf3^-/- HSC. We, therefore, cloned Ccnd1 complementary DNA into a lentiviral vector (GFP-labeled), producing efficient overexpression of CCND1 (Online Supplementary Figure S5C). Ythdf3^-/- LSK cells were infected with Ccnd1-overexpressing lentivirus, and 3,000 CD48^- Sca1⁺ GFP⁺ cells were FACS-purified 72 h after infection. These cells were then transplanted into lethally irradiated recipients together with 3×10⁵ competitor cells (Figure 4C). The results showed that enforced Ccnd1 expression rescued the reconstitution capacity of Ythdf3^-/- HSC, including B, T and myeloid cells (Figure 4D and Online Supplementary Figure S5E).

To further verify this finding, we simultaneously infected freshly isolated WT LSK cells with Ythdf3 shRNA-carrying lentivirus (GFP-labeled) and Ccnd1-overexpressing lentivirus (mCherry-labeled); 2,000 CD48^- Sca1⁺ GFP⁺ mCherry⁺ cells were FACS-purified 72 h after infection and subsequently transplanted into lethally irradiated recipients together with 3×10⁵ competitor cells (Figure 4E). It was found that enforced Ccnd1 expression completely rescued the reconstitution capacity of Ythdf3 shRNA-carrying HSC (Figure 4F and Online Supplementary Figure S5F), which is consistent with the aforementioned results (Figure 4D and Online Supplementary Figure S5E). Taken together, the above results show that Ccnd1 is the direct target of YTHDF3 to modulate HSC reconstitution capacity.

Figure 4.Overexpression of CCND1 rescues the reconstitution capacity of Ythdf3^-/- hematopoietic stem cells. (A) 3T3 cells were infected by lentivirus carrying Ccnd1 gRNA or control (GFP-labeled) and Cas9 virus (mCherry-labeled), and 6 days later, 10⁵ GFP⁺ mCherry⁺ cells were purified for western blot to evaluate the expression of CCND1. (B) Freshly isolated LSK cells of Cas9^+/+ mice were infected by Ccnd1 gRNA-carrying lentivirus, and 72 h later, 10,000 GFP⁺ mCherry⁺ cells were purified and transplanted into lethally irradiated recipients together with 2.5×10⁵ competitor cells. Engraftment of donor-derived cells was determined in overall (mCherry⁺) cells every month after transplantation. Three or four mice per group. Data are shown as mean ± standard error of mean (SEM). (C, D) Freshly isolated LSK cells from wildtype (WT) or Ythdf3^-/- mice were infected with Ccnd1-overexpressing or control virus (GFP-labeled), and 3,000 CD48^- Sca1⁺ GFP⁺ cells were FACS-purified 72 h after infection. These cells were then transplanted into lethally irradiated recipients together with 3×10⁵ competitor cells. (C) Experimental design to evaluate the role of CCND1 in regulating the reconstitution capacity of Ythdf3^-/- hematopoietic stem cells (HSC). (D) Line plots depicting the percentages of donor-derived cells in overall (GFP⁺) cells every month after transplantation. Five or six mice per group. Data are shown as mean ± SEM. (E, F) Freshly isolated LSK cells from WT mice were infected with Ccnd1-overexpressing virus (mCherry-labeled) and shYthdf3-carrying virus (GFP-labeled), and 2,000 CD48^- Sca1⁺ GFP⁺ mCherry⁺ cells were FACS-purified 72 h after infection. These cells were then transplanted into lethally irradiated recipients together with 3×10⁵ competitor cells. (E) Experimental design to evaluate the role of CCND1 in regulating the reconstitution capacity of shYthdf3 HSC. (F) Line plots depicting the percentages of donor-derived cells in overall (mCherry⁺) cells every month after transplantation. Six or seven mice per group. Data are shown as mean ± SEM.

Figure 5.Dysfunction of Mettl3 disturbs hematopoietic homeostasis and severely impairs hematopoietic stem cell reconstitution capacity. (A) Schematic illustration of the Mettl3 conditional knockout mice. (B) Representative western blot showing the protein expression of METTL3 and CCND1 in Mettl3^-/- and control LSK cells. (C) Histogram depicting the mRNA expression of Ccnd1 in Mettl3^-/- and control LSK cells. Data are shown as mean ± standard error of mean (SEM). (D, E) Histograms displaying the relative luciferase activity of Ccnd1-5’UTR-WT1 or Ccnd1-5’UTR-Mut1 (D) and Ccnd1-5’UTR-WT2 or Ccnd1-5’UTR-Mut2 (E) luciferase reporter in 293T cells transfected with control or METTL3 plasmid (see Online Supplementary Figure S4E and Methods). Firefly luciferase activity was measured and normalized to Renilla luciferase activity. Data are shown as mean ± SEM. (F) RNA immunoprecipitation quantitative polymerase chain reaction analysis detecting the binding of METTL3-WT or METTL3-Mut to the transcripts of Ccnd1 in 3T3 cells. (G, H) Thirty freshly isolated hematopoietic stem cells from Mettl3^-/- or control mice were transplanted into lethally irradiated recipients together with 3×10⁵ competitor cells. Engraftment of donor cells was determined in overall (CD45.2⁺), myeloid (Mac-1⁺), B (B220⁺) and T (CD3⁺) cells every month after transplantation. (G) Scheme of the competitive transplantation strategy. (H) Line plots depicting the percentage of donor-derived cells (overall, myeloid cells, B cells, T cells) in recipients at the indicated time points. Seven mice per group. Data are shown as mean ± SEM. cKO: conditional knockout; HSC: hematopoietic stem cells; PB: peripheral blood; HSCT: hematopoietic stem cell transplantation.

Mettl3 modulates Ccnd1 translation through the m6A motif at the 5’ untranslated region of Ccnd1

Given that Ythdf3 promotes the translation of Ccnd1 by recognizing the m⁶A modification at the 5’UTR, we next wondered whether METTL3 installs the m⁶A signal in the 5’UTR of Ccnd1 and furthermore modulates the translation of Ccnd1 through the same region. We performed a RIP-qPCR assay which showed that knockdown of Mettl3 reduced the binding of the m⁶A modification to Ccnd1 transcripts (Online Supplementary Figure S6A, B), which revealed that METTL3 installs the m⁶A signal on Ccnd1 mRNA. We then generated Mettl3^flox/flox mice (Figure 5A), and crossed Mettl3^flox/flox with Mx1-cre mice to generate Mx1-Cre; Mettl3^flox/flox mice. By administering polyinosine–polycytosine to these mice every other day for 10 days, we achieved total deletion of Mettl3 in LSK cells (Figure 5B) (hereafter named Mettl3^-/-). Meanwhile, we investigated the mRNA and protein expression of Ccnd1 in Mettl3^-/- LSK cells and found that deficiency of Mettl3 resulted in a significant reduction of protein level, but not of mRNA level of Ccnd1, in LSK cells (Figure 5B, C).

Figure 6.Ccnd1, but not Myc, is the target of METTL3 to regulate hematopoietic stem cell reconstitution capacity. (A, B) Freshly isolated LSK cells from wildtype (WT) or Mettl3^-/- mice were infected with Ccnd1-overexpressing or control virus (GFP-labeled), and 1,500 GFP⁺ CD48^- Sca1⁺ cells were FACS-purified 72 h after infection. These cells were then transplanted into lethally irradiated recipients together with 2.5×10⁵ competitor cells. (A) Experimental design to evaluate the role of CCND1 in regulating the reconstitution capacity of Mettl3^-/- hematopoietic stem cells (HSC). (B) Line plots depicting the percentage of GFP⁺ cells in donor-derived cells every month after transplantation. Four to six mice per group. Data are shown as mean ± standard error of mean (SEM). (C) 3T3 cells were infected by lentivirus carrying Mettl3 shRNA or control, and 4 days later, 10⁵ GFP⁺ cells were purified for western blot to evaluate the expression of METTL3: the quantitative plots are presented. (D, E) Freshly isolated WT LSK cells were infected by lentivirus carrying Mettl3 shRNA or control and 72 h later, 2,000 CD48^- Sca1⁺ GFP⁺ cells were purified and transplanted into lethally irradiated recipients together with 3×10⁵ competitor cells. (D) Experimental design to evaluate the role of Mettl3 in regulating the reconstitution capacity of HSC. (E) Line plots depicting the percentages of GFP⁺ cells in donor-derived cells every month after transplantation. Five mice per group. Data are shown as mean ± SEM. (F, G) Freshly isolated LSK cells from WT mice were infected with Ccnd1-overexpressing virus (mCherry-labeled) and shMettl3-carrying virus (GFP-labeled), and 2,000 CD48- Sca1⁺ GFP⁺ mcherry⁺ cells were FACS-purified 72 h after infection. These cells were then transplanted into lethally irradiated recipients together with 3×10⁵ competitor cells. (F) Experimental design to evaluate the role of CCND1 in regulating the reconstitution capacity of Mettl3-compromised HSC. (G) Line plots depicting the percentage of donor-derived cells in overall (mCherry⁺) cells every month after transplantation. (H-J) Freshly isolated LSK cells from WT mice were infected with Myc-overexpressing virus (mCherry-labeled) and shMettl3-carrying virus (GFP-labeled), and 2,000 CD48^- Sca1⁺ GFP⁺ mCherry⁺ cells were FACS-purified 72 h after infection. These cells were then transplanted into lethally irradiated recipients together with 3×10⁵ competitor cells. (H) Experimental design to evaluate the role of MYC in regulating the reconstitution capacity of Mettl3-compromised HSC. (I) Line plots depicting the percentages of mCherry⁺ cells in donor-derived cells every month after transplantation. (J) Scatter plot showing the lineage distribution of donor-derived mCherry^v cells in recipients at the first month. Five to seven mice per group. Data are shown as mean ± SEM. The gating strategy to generate these line plots is presented Online Supplementary Figure S5H. (K) This figure illustrates the proposed model of the reconstitution capacity of HSC reduced by a Mettl3→ RNA m⁶A→Ccnd1→Ythdf3 pathway.

To further confirm this result, we infected WT LSK cells with lentivirus carrying shRNA against Mettl3 to evaluate the expression of CCND1 (Online Supplementary Figure S6C) and found that knockdown of Mettl3 significantly reduced the level of CCND1 protein (Online Supplementary Figure S6D), but not mRNA (Online Supplementary Figure S6E).

To further test whether Mettl3 modulates Ccnd1 through m⁶A modification, we conducted a luciferase reporter assay as for Ythdf3. The results showed that Mettl3 regulated the translation of Ccnd1 through the 5’UTR region, especially the region from -180 to -184 (Figure 5D, E and Online Supplementary Figure S6F, H). Furthermore, based on published results, we constructed a plasmid to express the catalytic mutant METTL3 (METTL3-Mut, D395A).^9,31,32 RIP-qPCR for Ccnd1 revealed that METTL3 could bind to Ccnd1 mRNA (Figure 5F).

Taken together, these data suggest that METTL3 modulates the translation of Ccnd1 by binding directly to the m⁶A motif in the 5’ UTR region.

Dysfunction of Mettl3 disturbs hematopoietic homeostasis and severely impairs hematopoietic stem cell reconstitution capacity

To determine the influence of Mettl3 on the production of blood cells, we performed complete blood counts of Mettl3^-/- and littermate controls, which revealed that the white blood cell, lymphocyte, neutrophil, red blood cell and platelet counts were significantly decreased in Mettl3^-/- mice (Online Supplementary Figure S6I). Meanwhile, we observed that the frequency of Mettl3^-/- HSC was increased significantly compared to controls (Online Supplementary Figure S6K). Considering that bone marrow cellularity of Mettl3^-/- mice dropped significantly (Online Supplementary Figure S6J), we then counted the absolute number of HSC and found that the absolute number of HSC was still increased significantly in Mettl3^-/- mice (Online Supplementary Figure S6L), but not as dramatically as the frequency (Online Supplementary Figure S6K). It is notable that the expansion of HSC of Ythdf3^-/- mice was much less pronounced than that of Mettl3^-/- mice. The frequency of Ythdf3^-/- HSC increased by 1.65 times, while the frequency of Mettl3^-/- HSC increased by 82.01 times (Online Supplementary Figure S6M); the absolute number of Ythdf3^-/- HSC increased by 1.48 times, while Mettl3^-/- HSC increased by 14.51 times (Online Supplementary Figure S6N).

To further investigate the reconstitution capacity of Mettl3^-/- HSC, 30 freshly isolated Mettl3^-/- and control HSC were transplanted into lethally irradiated recipients together with 3×10⁵ competitor cells (Figure 5G). The results showed that Mettl3^-/- HSC failed to reconstitute the blood system (Figure 5H), which is consistent with previous reports.^16-18

To exclude the influence of homing, 30 freshly isolated HSC from either Mx1-Cre; Mettl3^flox/flox or control mice were transplanted into lethally irradiated recipients together with 3×10⁵ competitor cells. One month after transplantation, all recipients were administered polyinosine–polycytosine every other day for 10 days (Online Supplementary Figure S7A). It was found that deletion of Mettl3 severely decreased the reconstitution capacity of HSC (Online Supplementary Figure S7B1A, B). In brief, the above results indicate that Mettl3 is indispensable for maintaining hematopoietic homeostasis and the reconstitution capacity of HSC.

Ccnd1 is the target of METTL3 to regulate hematopoietic stem cell reconstitution capacity

Given that Ccnd1 is the hub of METTL3 and YTHDF3 to transmit the m⁶A signal to modulate HSC, and that forced Ccnd1 expression rescued Ythdf3^-/- HSC (Figure 4D), we then investigated whether forced Ccnd1 expression could rescue the reconstitution capacity of Mettl3^-/- HSC. We infected freshly isolated Mettl3^-/- LSK cells with Ccnd1-over-expressing lentivirus (GFP-labeled), and 1,500 CD48^- Sca1⁺ GFP⁺ cells were FACS-purified 72 h after infection and subsequently transplanted into lethally irradiated recipients together with 2.5×10⁵ competitor cells (Figure 6A). We could not detect Mettl3^-/--derived cells in the peripheral blood of recipients, while chimerism of the control group was 52.66% at the second month (Figure 6B). Thus, forced Ccnd1 expression could not restore the reconstitution capacity of Mettl3^-/- HSC.

Both previous studies^16-18 and our current results (Figure 5H and Online Supplementary Figure S7B) showed that Mettl3 deficiency resulted in severe impairment of HSC, indicating that METTL3-mediated m⁶A modification is pivotal in maintaining HSC. Chen et al. found that the cell function exhibited a Mettl3 dosage-dependent effect,³³ which is an interesting observation for exploring the functional target of Mettl3. We, therefore, generated one shRNA against Mettl3, which inhibited METTL3 by 71% (Figure 6C). We then infected freshly isolated WT LSK cells with Mettl3 shRNA-carrying lentivirus and 2,000 CD48^- Sca1⁺ GFP⁺ were FACS-purified 72 h after infection and transplanted into lethally irradiated recipients together with 3×10⁵ competitor cells (Figure 6D). The results revealed that knockdown of Mettl3 significantly impaired the reconstitution capacity of HSC, but still retained ~36% chimerism at the fourth month (Figure 6E). This result indicates that a certain amount of METTL3 can maintain HSC function to some extent. We infected freshly isolated WT LSK cells with Mettl3 shRNA-carrying lentivirus (GFP-labeled) and Ccnd1-overexpressing lentivirus (mCherry-labeled), and 2,000 CD48^- Sca1⁺ GFP⁺ mCherry+ cells were FACS-purified 72 h after infection. These cells were then transplanted into lethally irradiated recipients together with 3×10⁵ competitor cells (Figure 6F). It was found that enforced Ccnd1 expression partially rescued the reconstitution capacity of Mettl3 shRNA-carrying HSC (7.44±2.07 vs. 40.05±12.7 at the fourth month) including B, T and myeloid cells (Figure 6G and Online Supplementary Figure S7C).

Forced Myc expression cannot rescue the reconstitution capacity of Mettl3-/- hematopoietic stem cells in the long term

Previous studies showed that enforced Myc expression rescues the differentiation defects of Mettl3^-/- HSC.¹⁷ We therefore wondered whether forced Myc expression could rescue the reconstitution capacity of Mettl3^-/- HSC. We infected freshly isolated Mettl3^-/- LSK cells with Myc-over-expressing lentivirus (GFP-labeled), and 1,500 CD48^- Sca1⁺ GFP⁺ cells were FACS-purified 72 h after infection and subsequently transplanted into lethally irradiated recipients together with 2.5×10⁵ competitor cells (Online Supplementary Figure S7D, E). The results revealed that forced Myc expression could not rescue the reconstitution capacity of Mettl3^-/- HSC (Online Supplementary Figure S7F).

To confirm this result, we infected freshly isolated WT LSK cells using Mettl3 shRNA-carrying lentivirus (GFP-labeled) and Myc-overexpressing lentivirus (mCherry-labeled), and 2,000 CD48^- Sca1⁺ GFP⁺ mCherry⁺ cells were FACS-purified 72 h after infection. These cells were then transplanted into lethally irradiated recipients together with 3×10⁵ competitor cells (Figure 6H). The results showed that overexpression of MYC significantly improved the chimera of donor-derived cells at the end of the first month (Figure 6I), and lineage analysis showed that enforced MYC expression rescued the differentiation defect of Mettl3-compromised HSC (Figure 6J). However, the chimera of MYC-overexpressing cells dropped rapidly in the second and third months (Figure 6I). These results suggest that overexpression of MYC did indeed rescue the differentiation defect in the first month, but was not able to rescue the reconstitution capacity of Mettl3-compromised HSC in the long term.

Discussion

Our study provides the first experimental evidence that the reconstitution capacity of HSC is regulated by the Mettl3→ Ccnd1→ Ythdf3 pathway (Figure 6K). The 5’UTR of Ccnd1 is the hub for METTL3 and YTHDF3 to transmit the m⁶A modification. This study is of great significance in revealing how a RNA m⁶A writer and reader cooperate to modulate HSC. A more in-depth discussion is provided in the Online Supplementary Information.

Footnotes

• Received July 30, 2021
• Accepted November 25, 2021

Correspondence

*XZ and TC contributed equally as co-first authors

Ping Zhu zhuping301hospital@163.com

Hong Jiang jianghong961106@zju.edu.cn

Jianwei Wang jianweiwang@mail.tsinghua.edu.cn

Disclosures

No conflicts of interest to disclose.

Contribution

JW and HJ conceived the study and wrote the paper; JW, HJ, FZ and TC were responsible for the methodology; FZ, TC, LW, BZ, XW, SW, JS and ZP conducted the investigation; JW, HJ and MX performed the analyses and were responsible for the resources for this study; and JW acquired funding and supervised the study

Data-sharing statement

All raw sequencing data were deposited into the National Center for Biotechnology Information Gene Expression Omnibus with accession number GSE176458.

Funding

This work was supported by grant numbers Z181100001818005, 81870118, 91849106, 2018YFA0800200, 2017YFA0104000 and Z200022 to JW, 61773230, 61721003, and 2020YFA0906900 to XW., and 2020YFC2008900 to PZ from the National Key R&D Program of China or the Beijing Municipal Science & Technology Commission and the National Natural Science Foundation of China.

Acknowledgments

We thank the Beijing Advanced Innovation Center for Structural Biology, the Tsinghua-Peking Center for Life Sciences and the China Telecom Corporation Limited for facilities and financial support.