The gene CXXC5, encoding a retinoid-inducible nuclear factor (RINF), is located within a region at 5q31.2 commonly deleted in myelodysplastic syndrome and adult acute myeloid leukemia. RINF may act as an epigenetic regulator and has been proposed as a tumor suppressor in hematopoietic malignancies. However, functional studies in normal hematopoiesis are lacking, and its mechanism of action is unknown. Here, we evaluated the consequences of RINF silencing on cytokine-induced erythroid differentiation of human primary CD34+ progenitors. We found that RINF is expressed in immature erythroid cells and that RINF-knockdown accelerated erythropoietin-driven maturation, leading to a significant reduction (~45%) in the number of red blood cells, without affecting cell viability. The phenotype induced by RINF-silencing was dependent on tumor growth factor b (TGFb) and mediated by SMAD7, a TGFb-signaling inhibitor. RINF upregulates SMAD7 expression by direct binding to its promoter and we found a close correlation between RINF and SMAD7 mRNA levels both in CD34+ cells isolated from bone marrow of healthy donors and myelodysplastic syndrome patients with del(5q). Importantly, RINF knockdown attenuated SMAD7 expression in primary cells and ectopic SMAD7 expression was sufficient to prevent the RINF knockdown-dependent erythroid phenotype. Finally, RINF silencing affects 5’-hydroxymethylation of human erythroblasts, in agreement with its recently described role as a TET2-anchoring platform in mouse. Collectively, our data bring insight into how the epigenetic factor RINF, as a transcriptional regulator of SMAD7, may fine-tune cell sensitivity to TGFb superfamily cytokines and thus play an important role in both normal and pathological erythropoiesis.
In earlier work, we demonstrated that RINF loss-of-function affects granulopoiesis in normal and tumoral human hematopoietic cells.1 In keeping with its gene location at chromosome 5q31.2 (Online Supplementary Figure S1), a commonly deleted region associated with high risk in myeloid neoplasms,2 RINF loss of expression has been proposed to contribute to the development or progression of (pre)leukemia, such as myelodysplastic syndrome (MDS).1,3-5 We and others have demonstrated that RINF mRNA expression is an unfavorable6,7 and independent3 prognostic factor in acute myeloid leukemia (AML) as well as in solid tumors.8,9 However, its role in normal hematopoiesis has hitherto been poorly investigated and its contribution to the erythroid lineage and red blood cell (RBC) expansion is unknown.
RINF contains a nuclear localization signal that has been functionally validated10 and, in most studies, its subcellular localization is reported to be mainly or exclusively nuclear and it acts as a transcriptional cofactor.1,3,8,10-17 RINF associates strongly with chromatin1 through its conserved zincfinger domain (CXXC) which plays an essential role in providing the capacity to bind CpG islands.18,19 Interestingly, this domain is almost identical to the one harbored by TET1 and TET3, two epigenetic modulators involved in the erasure of DNA-methylation marks,20 pointing to the possibility that RINF might interfere with TET activities, hydroxy methylation, and gene transcription, as recently demonstrated in mice.12,15 RINF has also been reported to bind ATM, mediate DNA-damage-induced activation of TP533,10 and inhibit the WNT-b-catenin signaling pathway3,21-24 through a cytoplasmic interaction with disheveled proteins DVL and DVL2.21
Transforming growth factor b (TGFb) is a powerful and widespread cell growth inhibitor in numerous mammalian tissues.25,26 In the hematopoietic system, TGFb is known to regulate hematopoietic stem and progenitor cells (HSPC) and is also described as a potent inducer of erythroid differentiation and inhibitor of cell proliferation.27-30 TGFb signals through cell surface serine/threonine kinase receptors, mainly TGFbRI and TGFbRII. Activated TGFbRI phosphorylates SMAD2 and SMAD3 which translocate into the nucleus and form complexes that regulate transcription of target genes. TGFb can also elicit its biological effects by activation of SMAD-independent pathways.31,32 Inhibitory SMAD (SMAD6 and SMAD7) inhibit TGFb signaling. Importantly, a reduced SMAD7 expression sensitizes cells to the antiproliferative effects of TGFb and contributes to anemia in patients suffering from MDS, suggesting, firstly, that identifying transcriptional regulators of SMAD7 could enlighten our understanding of erythropoiesis and, secondly, that inhibiting TGFb signaling could be a therapeutic strategy that would mitigate ineffective hematopoiesis in disease states.33-35
In the present work, we used primary human CD34+ cells to demonstrate that RINF knockdown affects human erythropoiesis and mitigates RBC production through a mechanism that is mediated by SMAD7, the main inhibitor of TGFb signaling.
The methods for flow cytometric cell sorting of megakaryocyte- erythroid progenitor (MEP) cells, immunofluorescence studies, chromatin Immunoprecipitation (ChIP) experiments, and the primer sequences used for quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and ChIP-qPCR are described in the Online Supplementary Methods.
Primary culture of hematopoietic cells
Mononuclear cells were isolated from cord blood (CRB Saint- Louis Hospital, Paris, France) or adult bone marrow donors with written informed consent for research use, in accordance with the Declaration of Helsinki. The ethics evaluation committee of INSERM, the Institutional Review Board (IRB00003888), approved our research project (n. 16-319). Mononucleated cells were separated by Ficoll-Paque (Life Technologies) and purified with a human CD34 MicroBead Kit (cat. n. 130100453, Miltenyi Biotec). A two-step culture method for cell expansion and erythroid differentiation was used to obtain highly stage-enriched erythroblast populations.36,37 Briefly, CD34+ cells (purity 94-98%) were cultured for 7 days with interleukin (IL)6, IL3 (10 ng/mL), and stem cell factor (SCF, 100 ng/mL) to expand hematopoietic progenitors.37 CD36+ erythroid progenitors were purified using magnetic microbeads (CD36 FA6.152 from Beckman Coulter and anti-mouse IgG1 MicroBeads from Miltenyi Biotec). Then, erythropoietin (EPO) was added for 10-18 days to allow erythroid differentiation. For assays of colony-forming cells (CFC, counted at 11-14 days), CD34+ hematopoietic stem cells were seeded in methylcellulose (H4034, StemCell Technologies). TGFbRI inhibitor SB431542 was from Selleckchem (cat. n. S1067)38 and TGFb was from Peprotech (cat. n. 100-21).
Flow cytometry and differentiation analysis
To monitor end-stage erythroid differentiation of primary cells, cells were labeled with anti-human PE/Cy7-conjugated glycophorin A (GPA/CD235a) (cat n. A71564), PE-conjugated CD71 (IM2001U), and APC-conjugated CD49d (cat. n. B01682) from Beckman Coulter, PE-conjugated CD233/Band3 from IBGRL (cat. n. 9439-PE). Fluorescence-activated cell sorting (FACS) analysis was performed on an Accuri™ C6 Flow Cytometer (Becton-Dickinson). For morphological characterization cells were cytospun and stained with May-Grünwald- Giemsa. For the benzidine assay, 2x105 cells were incubated for 5 min with 0.01% benzidine and H2O2 at 0.3% (Sigma-Aldrich).
Culture of cell lines and treatments
K562 (ATCC#CCL243) and UT75.3 39 cells were cultured in RPMI 1640 medium and minimum essential medium-a supplemented with 2.5 ng/mL of granulocyte-macrophage colonystimulating factor (GM-CSF; Miltenyi Biotech), respectively. Both media were supplemented with 10% inactivated fetal bovine serum, 2 mM L-glutamine, 50 U/mL penicillin G and 50 mg/mL streptomycin (Life Technologies). To induce hemoglobin production, K562 cells were treated with hemin at 40 mM (Sigma-Aldrich). Erythroid maturation of UT75.3 was triggered by replacing GM-CSF with EPO 5 U/mL.
Lentiviral and retroviral transduction of hematopoietic cells
For shRNA-mediated RINF knockdown, we used the pTRIPDU3/GFP lentiviral vector40 which drives the same shRNA sequences downstream of the H1 promoter as our previously described pLKO.1/puroR vector.1 For RINF overexpression, the retroviral vector MigR/IRES-GFP was used with the previously described experimental conditions.1 Green fluorescent protein (GFP) sorting was performed on a FACSAriaIII (BD Biosciences). For SMAD7 overexpression, the retroviral vector pBABE-puro-SMAD7-HA (Addgene plasmid#37044) was used as previously described.1,41 For doxycycline-inducible lentiviral expression, SMAD7-HA cDNA was inserted into the pINDUCER21 vector by gateway technology (Addgene plasmid #46948) and shRNA sequences were inserted in the Tet-pLKOGFP vector (modified from Tet-pLKO-puro, Addgene plasmid# 21915, deposited by Dmitri Wiederschain).
Quantitative reverse transcriptase polymerase chain reaction
Cells were collected and stored directly at -80°C for RNA preparation with the TRIzol (Life Technologies) extraction protocol as previously described.1 First-strand cDNA synthesis (reverse trasncription) was carried out using a Transcriptor First Strand cDNA Synthesis Kit (cat. n. 489703000, Roche). RINF mRNA expression was detected using a Lightcycler® 480 ProbesMaster kit (cat. n. 4707494001, Roche). Relative mRNA expression was normalized to RLPL2, PPIA or ACTB gene expression in a two-color duplex reaction. For SMAD7, PU.1 and c-KIT mRNA detection, qRT-PCR was performed using SYBRGreen on a Light Cycler 480 machine (Roche) and gene expression was calculated by the 2-ΔΔCT method. Primer sequences are available in an Online Supplementary File.
Western blot analysis
Total cell extracts were prepared and western blotting was carried out as previously described.1 Blots were incubated with a primary polyclonal antibody and then with an appropriate peroxidase- conjugated secondary antibody (anti-rabbit and antimouse IgG horseradish peroxidase [HRP] antibodies from Cell Signaling, cat. n. 7074S, and 7076S, respectively) and anti-goat IgG HRP antibody from Southern Biotech (cat. n. 6160-05). Proteins were detected using a chemiluminescent system (Clarity Western ECL, cat. n. 170-5060, Biorad). Rabbit polyclonal antibodies were previously described for RINF,1 P85/PI3KR1,42 or commercially purchased for anti-RINF, anti-SMAD7 (cat. n. 16513-1-AP, cat. n. 25840-1-AP, ProteinTech), anti-b-actin (ACTB, cat. n. A1978, Sigma-Aldrich), anti-phospho-SMAD2/3 (cat. n. 8828, Cell Signaling), and anti-HSC70 mouse monoclonal antibody (SC-7298, Santa Cruz Biotechnology).
All analyses were performed using GraphPad Prism software. When the distribution of data was normal, the two-tailed Student t test was used for group comparisons. Contingency tables were established using the Fisher exact test, and the Pearson correlation coefficient was used to determine the correlation between the normally distributed RINF mRNA and SMAD7 mRNA expression values. Statistics were carried out on a minimum of three independent experiments. The statistical significance of P values is indicated in the Figures: not significant (ns): P>0.05; *P<0.05; **P<0.01; ***P<0.001. Error bars represent confidence intervals at 95% or standard deviations (SD) for the qRT-PCR analyses.
RINF is expressed during early human erythropoiesis
We employed cytokine-induced erythroid differentiation of CD34+ progenitor cells isolated from human cord blood or adult bone marrow to model human erythropoiesis in vitro. To obtain highly enriched populations of differentiating erythroblasts, cells were grown in a two-phase liquid culture (experimental design in Figure 1A) as previously described.36,37 We found that RINF protein was present at the earliest stages of erythroid differentiation and reached its highest level in progenitors and proerythroblasts (ProE) (Figure 1B). After that, the level of RINF protein decreased in the basophilic erythroblast stage and was barely detectable in late erythroblasts (i.e., polychromatic and orthochromatic erythroblast stages). Similarly, RINF mRNA levels were high in erythroid progenitors but decreased at the basophilic stage and onwards (Figure 1C). This temporal expression pattern mirrors RINF expression data extracted from three independent transcriptome datasets (microarray or RNA-sequencing) reported by Merryweather-Clarke et al.,43 An et al.,44 and Keller et al.45 with adult or cord-blood CD34+ cells (Online Supplementary Figure S2). Taken together, our data show that RINF expression peaks in erythroid progenitors and proerythroblasts during cytokine-induced erythropoiesis.
RINF silencing results in diminished red blood cell production without affecting cell viability
We next investigated the consequences of RINF knockdown on erythropoiesis and cell viability (see experimental design in Figure 2A). Knockdown experiments were performed with two previously validated short-hairpin RNA (shRINF#4 and shRINF#3) sequences1 driven by the lentiviral pTRIPDU3/GFP vector (Online Supplementary Figure S3A, B). We found that RINF was extinguished as early as 2 days after transduction (i.e., at day 0 of EPO) with shRINF#4 at both mRNA (Figure 2B) and protein (Figure 2C) levels, without affecting cell viability, assessed by trypan blue cell counting (Figure 2D) or cell growth up to day 10 of EPO (Figure 2E, left panel). However, in the last days of ex vivo culture (day 11-17), we noticed a reduction in total number of RBC produced (average decrease of ~45%) at 17 days with EPO (Figure 2E, right panel), which was particularly marked for four donors out of six studied.
To investigate how RINF knockdown affects the clonogenic capacity of HSPC, we next performed comparative colony-forming assays for the development and maturation of erythroid (burst-forming unit erythroid progenitors; BFU-E), myeloid (CFU-G, CFU-M, or CFU-GM) and mixed (CFU-GEMM) colonies. No statistically significant changes were noted in the total number of colonies or the number of CFU-M, CFU-GM, or CFU-GEMM (Online Supplementary Figure S3C). However, the proportions of granulocytic colonies (CFU-G) and BFU-E, were slightly reduced or increased, respectively (P<0.01, Fisher exact test). The number of MEP-sorted cells was also increased under RINF knockdown conditions (Online Supplementary Figure S3E), and this increase corresponded with an increase in small (more mature) BFU-E, findings that suggest an accelerated maturation. A more careful analysis of colony size revealed that the median size of BFU-E derived from CD34+ cells dropped by about 30% (i.e., from 0.101 to 0.071 mm2, unpaired t-test, P=0.007, n=3 donors) after RINF knockdown (Figure 2F). Concomitantly with the size reduction of large BFU-E (the most immature ones), we noted a slight but statistically significant increase in small BFU-E (the most mature ones), in agreement with accelerated maturation (Figure 2G).
Erythroid maturation is affected by RINF
We next investigated whether the reduced expansion observed from day 10 of EPO could be the consequence of accelerated maturation. Indeed, we observed that erythroid differentiation was accelerated under conditions of RINF knockdown using quantification by benzidine staining (Figure 3A), morphological analysis of cells stained with May-Grünwald-Giemsa (Figure 3B), and flow cytometry for the erythroid markers GPA and CD71 (Figure 3C, upper panel), and CD49d and Band3 (Figure 3C, lower panel). These differences were statistically significant (P<0.001) for CD34+ cells derived from both cord blood and adult bone marrow. The shRNA-RINF-induced acceleration of erythropoiesis was supported by the reduction in cell size (not shown) and the more rapid downregulation of c-KIT and PU.1 mRNA in RINF knockdown cells (Figure 3D). The accelerated maturation was also characterized by a pronounced reduction of ProE cells (∼2.8-fold less) enumerated at day 11 (Figure 3B). Despite a weaker efficiency of shRINF#3 to knockdown RINF expression compared to shRINF#4 (Online Supplementary Figure S3B, Figure 3E, left panel), we also confirmed that a similar phenotype, i.e., accelerated maturation (as shown by increased benzidine staining) (Figure 3E, right panel) and a reduction in the total number of RBC produced at day 17 (Figure 3F), was obtained with another shRNA sequence targeting RINF.
RINF controls erythroid maturation and red blood cell expansion in a transforming growth factor b-dependent manner
We then wondered whether TGFb, a well-known inducer of erythroid maturation and inhibitor of RBC expansion, could be involved in the RINF-dependent phenotype. In agreement with an important role for autocrine TGFb signaling in the early stages of erythropoiesis, both TGFB1 and TGFBRI were highly expressed in erythroid progenitors (Figure 4A). To functionally validate an involvement of the TGFb signaling pathway during RINF knockdown-dependent acceleration of erythropoiesis, we performed liquid cell culture experiments of primary CD34+ cells in the presence of SB431542, a potent and selective inhibitor of TGFbRI38 (Figure 4B). Strikingly, in the presence of SB431542, the maturation of RINF knockdown cells (evidenced by a faster acquisition of erythroid cell surface markers such as CD49d/Band3 at day 10 of EPO) was not accelerated and SMAD2 protein was not phosphorylated/activated after 3 days of EPO (Figure 4C, lanes 5 and 6), supporting the efficiency of the inhibitor. Conversely, in the absence of inhibitor, RINF-silencing gave rise to a faster rate of phosphorylation of SMAD2 protein, here noted at 8 h of EPO (Figure 4C compare lanes 1 and 2, or the kinetics of pSMAD2 waves in both conditions, right panel), indicating a higher sensitivity to autocrine TGFb signaling, which would be mediated through SMAD2, as previously reported in hematopoietic cells.35 A weaker RBC expansion was also noted for RINF knockdown cells, even when TGFb1 (5 ng/mL) was added as late as day 11 of EPO treatment (Figure 4D). Moreover, even for the two donors whose RINF silencing was more subtle (donors 1 and 2, Figure 2E), RINF knockdown cells were more sensitive than control cells to TGFb1 (Figure 4E, F). This was especially pronounced at low doses (0.1 ng/mL), at which we found faster acquisition of GPA (at day 6 of EPO) and reduced RBC production (at day 17 of EPO) (Figure 4F). Taken together, these data suggest that RINF knockdown cells were more sensitive to TGFb and that the level of RINF in erythroid progenitors influences the upcoming RBC production, at least in the presence of TGFb.
Identification of SMAD7 as a RINF target gene candidate
To identify molecular mechanisms by which RINF might modulate responsiveness to TGFb, we reanalyzed our previously published gene expression microarray datasets of K562 and UT75.3 cells transduced with shRINF#4 or shControl.7 A relatively discrete set of 193 gene candidates (Online Supplementary Table S1) were downregulated by RINF knockdown in both cell lines. Strikingly, ingenuity pathway analyses revealed that this gene list was enriched in genes belonging to the TGFb signaling pathway (Online Supplementary Figure S4C). Considering its well-known inhibitory function on TGFb-signaling and its low expression in MDS, SMAD7 appeared as a promising candidate for functional investigation.33,34 Encouragingly, SMAD7 was also downregulated by RINF knockdown in a third hematopoietic cell line, MV4-11 (Online Supplementary Figure S4E, right panel) and the downregulation of SMAD7 was confirmed by qRT-PCR analyses in K562 cells, even though the knockdown appeared moderate (~45%, P<0.001) in these independent experiments (Online Supplementary Figure S4D, right panel).
We next measured RINF levels in two cell line models of erythroid maturation, the K562 cell line treated with hemin and the UT75.3 cell line treated with EPO39 after GM-CSF withdrawal (Online Supplementary Figure S4A, B). In agreement with our findings in primary cells, RINF protein expression was downregulated upon treatment-induced erythroid differentiation, as early as 10 h after treatment with hemin for K562 cells, and after 2 days with EPO for UT75.3 cells (Online Supplementary Figure S4A). Moreover, RINF knockdown accelerated the erythroid maturation program (Online Supplementary Figure S4B) triggered by hemin in K562 cells (noted after 1 day of treatment), or EPO in UT75.3 cells (at 4 days of treatment). We also investigated whether RINF overexpression could delay hemoglobinization. To this end, we used the retroviral system MigR/IRESGFP vector1 (Online Supplementary Figure S5A, upper panel) to drive ectopic expression from full-length RINF cDNA. We found high constitutive expression of RINF protein for both cell lines (Online Supplementary Figure S5A), without cell toxicity or consequences on cell proliferation (not shown). When we evaluated erythroid differentiation using benzidine staining (Online Supplementary Figure S5B) we found that RINF overexpression reduced hemoglobinization in both cell lines (P<0.001), consistent with our knockdown experiments (Online Supplementary Figure S4A, B).
RINF controls SMAD7 transcription directly
Our qRT-PCR analyses demonstrated a robust increase (approximately 5-fold) of SMAD7 mRNA levels in cells overexpressing RINF (Figure 5A). To investigate whether RINF-mediated induction of SMAD7 mRNA was direct and occurred at the transcriptional level, we performed ChIP experiments with anti-RINF antibodies. To this end, five sets of primers were designed for regions encompassing the SMAD7 promoter (Figure 5B). As shown in Online Supplementary Figure S5C, we found that RINF binds directly to several regions (R1 to R4) of the SMAD7 promoter and that this binding was increased in cells overexpressing RINF. Moreover, H3K4me3 histone marks, quantified by ChIP-qPCR (Figure 5C), were increased by RINF overexpression in four proximal regions (R2-R4) of the SMAD7 transcription start site, indicating that chromatin was in a more active transcriptional state.
To investigate functional relationships between RINF and SMAD7, we next performed “rescue” experiments in K562 cells. We employed retroviral transduction to express SMAD7 ectopically (Figure 5D). Our data demonstrated that SMAD7 overexpression did not affect endogenous expression of RINF mRNA or protein (Figure 5E, F), but did reduce hemoglobinization (Figure 5G, lane 3), mimicking the effect of ectopic RINF (Online Supplementary Figure S5B). We did not detect any change in cell viability or proliferation in the absence of hemin. Importantly, under conditions in which SMAD7 was ectopically expressed, RINF knockdown did not result in acceleration of hemoglobinization (Figure 5G). Taken together, these data indicate that RINF binds the SMAD7 promoter directly to control its expression, and that SMAD7 is an effector downstream of RINF during erythroid maturation.
RINF and SMAD7 mRNA expression are correlated in bone marrow from adult donors and patients with myelodysplastic syndrome
We investigated whether RINF and SMAD7 mRNA expression correlated in primary human CD34+ cells from healthy donors and donors with MDS. To this end, we performed qRT-PCR on CD34+ cells isolated from adult bone marrow donors (n=11) and found a highly significant correlation between RINF mRNA and SMAD7 mRNA (Pearson, rho=0.684, P=0.02) (Figure 6A). We confirmed these data by analyzing an independent microarray dataset from Pellagatti et al.46 As shown in Figure 6B, RINF and SMAD7 mRNA levels correlated strongly in CD34+ cells isolated from healthy donors (Pearson rho=0.784, P<0.001, n=17). Interestingly, this correlation also reached statistical significance in CD34+ cells from MDS -5q patients (Pearson rho=0.603, P<0.001, n=47) but not in other MDS patients, i.e., without del(5q) or 5q-. We then compared the intensity of this correlation with the other 5q genes (n=48 genes) from the commonly deleted regions associated with high risk, and the CXXC5 probe sets were those correlating best with the SMAD7 probe set in both the healthy control group (rho=0.819, and rho=0.784) and the MDS patients’ cohort with del(5q) (Table 1) (Online Supplementary Figure S6A). Reinforcing the previously reported relevance of SMAD7 in MDS pathophysiology,33,34 SMAD7 expression was not only extinguished in CD34+ MDS samples compared to normal samples (in the microarray dataset from Gerstung et al.,47 (Online Supplementary Figure S6C), but patients with the lowest SMAD7 expression had a significantly shorter overall survival (Online Supplementary Figure S6D).
RINF-silencing alters genome-wide hydroxymethylation
Since murine RINF has recently been described as a necessary platform for TET2 activity,15,48 we wondered whether RINF-silencing could affect the genome-wide 5hmC of human immature erythroid cells. As shown in Figure 6C, we observed a statistically significant loss of 5hmC detected by immunofluorescence or flow cytometry (Figure 6D) in knockdown primary cells.
A RINF/SMAD7 axis controls erythroid maturation and red blood cell expansion in primary cells
We next investigated whether RINF knockdown altered SMAD7 expression in primary erythroid progenitors. As shown in Figure 7A, B, a 60-70% knockdown of RINF led to a statistically significant knockdown of SMAD7 (by 40-45%) in CD34+ cells isolated from cord blood (n=3 donors, paired Student t-test, P<0.001). Accordingly, a 30% knockdown of SMAD7 protein level was detected by immunofluorescence in primary erythroid progenitors (P<0.0001) (Figure 7C). For a direct demonstration of the relevance of this finely tuned regulation of SMAD7 mediated by RINF protein in primary HSPC, we next performed experiments in which we knocked down RINF and induced SMAD7 expression ectopically. To this end, we first transduced CD34+ cells with the doxycyclineinducible vector pInducer21/SMAD7-HA (Figure 7D), and then with the pTRIPUD3 vector that drives shRNA/RINF or shRNA/Control expression. Notably, in the presence of doxycycline, SMAD7 expression (Figure 7E, F) robustly prevented both the RINF knockdown-dependent accelerated maturation (Figure 7G) and reduction of RBC numbers (Figure 7H).
An increasing body of work suggests that RINF is involved in the maturation, function, or development of various cell types such as neural stem cells,21 endothelial cells,49 myoblasts,14 myofibroblasts,24 osteoblasts,23 as well as in kidney development,22 wound healing,24 and hair regrowth.50 Here, we report that the epigenetic factor RINF is a transcriptional regulator of SMAD7 which fine-tunes TGFb sensitivity of erythroid progenitors, findings that bring insight into molecular barriers that may prevent effective erythropoiesis.
Our in vitro erythropoiesis experiments demonstrated that RINF is expressed in human erythroid progenitors (i.e., BFU-E and CFU-E) and proerythroblasts (ProE) but not in the last stages. Loss of RINF expression does not affect cell viability or cell proliferation but accelerates erythroid maturation, and noticeably reduces RBC expansion. The average reduction in RBC number was estimated at ~45% in six experiments (Figure 2F) or one population doubling (i.e., 1.03, for the 4 donors #3-6), suggesting that RINF knockdown cells could skip one division (in the presence of TGFb). Our data indicate that RINF-dependent erythroid maturation is also dependent on TGFb signaling at physiological levels. This cytokine, a potent inducer of erythroid maturation and growth inhibition,27,29,51,52 is known to be enriched in the bone marrow microenvironment53 and we surmise that the RINF/SMAD7 regulation axis that we describe here is likely to exert a more pronounced effect in vivo than in our serum-free culture conditions (i.e., without exogenous TGFb). Moreover, since the mechanism of action is SMAD7-dependent, RINF downregulation may also sensitize hematopoietic cells to other cytokines of the TGFb superfamily which may act on late-stage erythropoiesis in vivo (such as activins and GDF11).54-57 In this manner, RINF could be one of the players regulating the dynamic balance between long-term expansion of the erythroid pool versus fast production of RBC for immediate physiological needs.
Two recent studies suggest that murine RINF could act as an anchoring platform necessary for TET2 (which lacks a CXXC domain) and its activity at CpG sites (5’- hydroxy methylation), in plasmacytoid dendritic cells15 and mouse embryonic stem cells.48 Conversely, an earlier study indicated that RINF inhibits TET2 and hydroxymethylation of genomic DNA in a caspase-dependent manner (in mouse embryonic stem cells), and another one indicated that RINF binds to unmethylated (and not methylated) CpG sites,18 probably reflecting a more complex mechanism of action.12 We here provide the first experimental evidence (to our knowledge) that RINF can regulate genome-wide hydroxy methylation in human cells. The role of RINF that we describe here could pave the way to future studies that will be necessary to better understand the complex molecular epigenetic events that occur during normal and ineffective erythropoiesis12,15,48,58 and that may involve TET enzymes.59
Several correlative studies have implicated RINF as a candidate in the pathogenesis of MDS/AML.1,3,5,60 MDS is a complex and heterogeneous malignant hematologic disorder in which the loss of several genes is suspected to lead to the pathophysiology.4 CXXC5 is located at chromosome 5q31.2, in a commonly deleted region associated with high risk in MDS and AML (Online Supplementary Figure S1).1,4 Even though it is uncertain that RINF loss of expression in itself can cause the development of preleukemia, our functional data in human CD34+ cells indicate that loss of RINF may exacerbate cytopenia of the erythroid lineage. Of importance, the functional contribution of Cxxc5/Rinf loss of function to MDS development was recently described in a mouse model, in which its invalidation (by random insertional mutation) was demonstrated to cooperate with Egr1 haploinsufficiency to promote MDS.4 This study indicated that the Cxxc5 gene could act as a tumor suppressor gene contributing to del(5q) myeloid neoplasms. Although RINF/CXXC5 is not frequently mutated in patients with MDS/AML,60 several recurrent chromosomal anomalies or gene mutations could lead to RINF loss of expression and contribute in the long-term to ineffective erythropoiesis and/or myeloid transformation (legend of Online Supplementary Figure S7).
Our findings point to a molecular mechanism in which RINF functions as a negative modulator of TGFb signaling by upregulating and maintaining SMAD7. Consequently, RINF loss of expression will sensitize human erythroid progenitors to autocrine/paracrine TGFb-induced growth inhibition as a mechanism to prevent expansion of the erythroid pool. Our data also suggest that this signaling would be at least partly mediated through SMAD2 activation and phosphorylation (Figure 4C), in agreement with a previous report on human bone marrow.35 The in vivo relevance of these findings is strengthened by the correlated RINF and SMAD7 mRNA expression in human bone marrow CD34+ cells. This correlation was also observed in MDS patients with del(5q) or 5q- but not in other forms of MDS, in which SMAD7 was silenced in most of the patients’ samples, whatever the RINF expression level. This extinction of SMAD7 in other MDS could be explained by various mechanisms such as miRNA21 expression.33 However, since RINF knockdown leads to global loss of 5hmC, it is tempting to speculate that the loss of RINF could lead to hypermethylation of the SMAD7 promoter in HSPC, a process that could have gone unnoticed in previous studies. Interestingly, the SMAD7 promoter is known to be silenced by hypermethylation in non-hematopoietic tissues, and hypomethylating agents such as azacytidine can revert this hypermethylation, and alleviates TGFb-induced diseases in several pathological models such as atherosclerosis,61 renal fibrosis62 and liver fibrosis.63 Thus, further studies should investigate whether the SMAD7 promoter is demethylated after treatment with hypomethylating agents (azacytidine or decitabine) in human HSPC isolated from MDS patients, and to what extend this mechanism could contribute to the efficacy of these epigenetic drugs to sustain erythropoiesis in the long-term. In support of this concept, TET2 mutation is a good indicator of response to azacytidine treatment.64
Previous studies have underscored the importance of SMAD7 regulation in MDS.33,34 Our present data complement these studies by demonstrating, first, a direct transcriptional mechanism by which SMAD7 is regulated in hematopoietic cells and, second, that the residual level of SMAD7 in MDS is associated with the severity of the disease and patients’ survival (Online Supplementary Figure S6D). These data are particularly relevant in the context of understanding how responsiveness to TGFb in the bone marrow microenvironment is fine-tuned,28 and in a context-specific manner.30 Even though this mechanism is unlikely to be solely responsible for TGFb-hypersensitivity, our findings pave the way for novel research avenues in blood disorders characterized by ineffective erythropoiesis such as myelofibrosis,65 Fanconi anemia,66 b-thalassemia, 57,67 and MDS,55 and for which novel therapies based on TGFb inhibitors are particularly promising.56,68 Finally, beyond its role during erythropoiesis, the RINF/SMAD7 regulation axis described here could, at least partly, contribute to the pleiotropic effects of RINF described during wound healing,24 fibrosis,61-63,69 immunity, 15 and tumor biology,1,3,6-9 and deserves to be investigated in these physiological processes.
- Received June 18, 2020
- Accepted November 16, 2020
FP is a holder of patents describing methods for RINF mRNA detection (WO2009/151337 and WO2012/010661).The other authors declare that they have no potential conflicts of interest.
AA and GM performed most of the experiments. DS and SZ performed the ChIP experiments. YZ, VF, IM, and E-FG performed some experiments and discussed the data. AA, GM, FV, EL, MF, AK, OD, and CL discussed the data. ES-B, ID-F, DB, OH, and PM supervised the study. FP conceived the study and supervised the experiments. AA, GM, and FP analyzed the data, created the figures and wrote the manuscript.
This work was supported by INSERM, Paris-Descartes University, the Ligue Nationale Contre le Cancer (LNCC), the Cochin Institute, and the Laboratory of Excellence GR-EX. AA was supported by LNCC, Société Française d'Hématologie, Fondation pour la Recherche Médicale, and Boehringer Society (travel grant). GM was supported by a fellowship grant from the Ministère de l’Enseignement Supérieur et de la Recherche and Société Française d’Hématologie. ES-B is supported by the National Center for Scientific Research (CNRS) and her team was supported by LNCC and Fondation de France. We are also grateful for support from the French-Norwegian exchange program (Aurora to FP).
The authors thank Pr. Jérôme Larghero and Thomas Domet from Centre de Ressources Biologiques / Banque de Sang de Cordon de l’AP-HP (Saint-Louis Hospital) for providing cord blood units, Anne Dubart-Kupperschmitt for the pTRIPDU3/GFP vector,
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