Induction of human fetal hemoglobin expression by adenosine-2’,3’-dialdehyde
- Yinghong He†1, 3,
- Gerhard Rank†2,
- Miaomiao Zhang1,
- Junyi Ju1,
- Ronghua Liu1,
- Zhen Xu1,
- Fiona Brown2,
- Loretta Cerruti2,
- Chi Ma1,
- Renxiang Tan1,
- Stephen M Jane2Email author and
- Quan Zhao1Email author
© He et al; licensee BioMed Central Ltd. 2013
Received: 9 August 2012
Accepted: 9 January 2013
Published: 14 January 2013
Pharmacologic reactivation of fetal hemoglobin expression is a promising strategy for treatment of sickle cell disease and β-thalassemia. The objective of this study was to investigate the effect of the methyl transferase inhibitor adenosine-2’,3’-dialdehyde (Adox) on induction of human fetal hemoglobin (HbF) in K562 cells and human hematopoietic progenitor cells.
Expression levels of human fetal hemoglobin were assessed by northern blot analysis and Real-time PCR. HbF and adult hemoglobin (HbA) content were analyzed using high-performance liquid chromatography (HPLC). DNA methylation levels on human gamma-globin gene promoters were determined using Bisulfite sequence analysis. Enrichment of histone marks on genes was assessed by chromosome immunoprecipitation (ChIP).
Adox induced γ-globin gene expression in both K562 cells and in human bone marrow erythroid progenitor cells through a mechanism potentially involving inhibition of protein arginine methyltransferase 5 (PRMT5).
The ability of methyl transferase inhibitors such as Adox to efficiently reactivate fetal hemoglobin expression suggests that these agents may provide a means of reactivating fetal globin expression as a therapeutic option for treating sickle cell disease and β-thalassemia.
Human globin genes undergo two switches during development, from embryonic (ε) to fetal (γ) and from fetal (γ) to adult (β) globin . Reactivation of fetal hemoglobin (HbF) in adults is one of the most effective strategies for treatment of sickle cell disease and β-thalassemia as increased fetal globin levels are associated with improved symptoms in hemoglobinopathy patients .
To date, compounds such as 5-aza-2-deoxycytidine (decitabine) , hydroxyurea (HU) , short-chain fatty acids (SCFAs) , and histone deacetylase (HDAC) inhibitors , have been used to increase HbF, although their effects are variable. However, the mechanism of action is not clear and remains controversial .
In previous studies, we determined that protein arginine methyltransferase 5 (PRMT5) coupled with Dnmt3a and related complexes played a critical role in human fetal globin gene repression [7, 8]. In this report, we have found that treatment with adenosine-2’,3’-dialdehyde (Adox), a methyltransferase inhibitor [9, 10], can induce fetal hemoglobin gene transcription in adult human bone marrow erythroid progenitor cells. This activity of Adox may be associated with inhibition of PRMT5.
Cell culture and reagents
K562 cells were cultured as described previously . To generate human bone marrow (BM) erythroid progenitor cells, isolated CD34+ cells were grown in StemSpan SFEM medium with 1X CC100 cytokine mix for 6 days, then reseeded into the same medium supplemented with SCF (20 ng/ml), EPO (1 U/ml), IL-3 (5 ng/ml), dexamethasone (2 μM), and β-estradiol (1 μM), and cultured for two more weeks . Cell surface marker analysis with CD71 and Glycophorin A indicated that the cultured cells were greater than 90% erythroid lineage. Human BM cells were collected under approval by the Melbourne Health Human Research Ethics Committee.
A 25 mM stock solution of adenosine-2’,3’-dialdehyde (Adox, Sigma) was prepared in 0.04 M HCl. Decitabine (Sigma) was prepared in phosphate-buffered saline (PBS). Working solutions of both reagents were prepared by dilution in PBS immediately prior to use.
Benzidine staining, Northern Blot, and DNA methylation analysis
K562 cells were treated with Adox (2.5 μM) for 2 days and washed twice with ice-cold phosphate-buffered saline. The cell pellets were then resuspended in ice-cold phosphate-buffered saline. Benzidine solution (0.1% 3,3’-diaminobenzidine containing hydrogen peroxide) was added at room temperature. Benzidine-positive cells were spun onto a glass plate, examined by light microscopy and photographed. Northern blot analysis of K562 cells was performed as described previously . Bisulfite sequence analysis was performed as described previously . PCR was performed with HiFi Taq polymerase (Roche) as follows: 30 cycles, 94°C for 20 s, 55°C for 20 s, and 68°C for 35 s. PCR products were cloned into pCRII (Invitrogen) followed by nucleotide sequencing using the Big-Dye Termination method (ABI). At lease 40 clones were sequenced for each CpG site.
ChIP assays were performed as described previously . H4R3me2s and histone H4 acetylation antibodies were purchased from Abcam. Each experiment was performed independently at least twice. The ChIP samples were analyzed by quantitative real-time PCR using FastStart Universal SYBR Green Master (Roche). A standard curve was prepared for the primers using serial titration of the input DNA. The percentage of ChIP DNA was calculated relative to the input DNA from primer-specific standard curves using Rotor-Gene 6000 Series Software 1.7.
Quantitative RT-PCR (Q-RT-PCR)
Total RNA was isolated from cells with Trizol reagent (Invitrogen). cDNA was generated using a reverse transcription system (Promega). The identities of the amplified bands were confirmed by sequencing. The PCR conditions and primers were described previously , and all samples were run in triplicate.
For bone marrow samples, plasmid DNA encoding γ-globin, β-globin or α-globin was used to generate the standard curve for determination of copy number. The number of molecules per nanogram total RNA from bone marrow cells was calculated from standard curves using Rotor-Gene 6000 Series Software 1.7.
HPLC for adult and fetal hemoglobin
Aliquots of one million cells were washed in phosphate-buffered saline. The pellets were lysed by repeated freeze-thaw. The supernatant was analyzed for HbF and adult hemoglobin (HbA) content by ion-exchange high-performance liquid chromatography (HPLC) using a Bio-Rad VARIANT β-thalassemia Short Program.
Results and discussion
DNA methylation has been shown to be important in regulation of globin gene expression. Because Adox can inhibit both DNA methylation and protein methylation including histone tail methylation, we performed bisulfite DNA sequencing experiments on globin genes. Using decitabine as a positive control, we found that Adox significantly reduced DNA methylation (Figure 2C). Together these results suggested that Adox was a potent inducer of γ-globin expression in K562 cells.
Adox is an odorless methyltransferase inhibitor that functions through a feedback loop . Adox can inhibit adenosylhomocysteine hydrolase activity thereby indirectly inhibiting methyltransferases that catalyze adenosylmethionine to adenosylhomocysteine . Mice can tolerate Adox at 100 μmol/kg without any ill-effect [16, 17]. Compared to DNA methylation inhibitors such as decitabine, Adox appears to function as an inhibitor of both DNA methylation and protein methylation. It is unclear how this compares to decitabine because we currently do not know the exact mechanism by which decitabine induces γ-globin expression .
DNA methylation plays a critical role in modulation of globin gene expression. Inhibitors of DNA methylation or histone deacetylation, such as decitabine and butyrate, have been shown to induce HbF [19, 20]. Compared to treatment of K562 cells, treatment of human BM cells with Adox triggered less reduction in methylation at the γ-globin gene, although there was more significant demethylation beyond the transcription initiation site (TIS) at CpG −53 and −50 (Figure 4D). This is similar to results obtained by 5-Azacytidine treatment . The fact that immortalized K562 cells resemble embryonic erythroid progenitors without expression of adult hemoglobin may contribute to this difference. Also, hypermethylation of the γ-globin promoter in BM cells may result in different requirements of Adox concentrations for inducing γ-globin expression in K562 and BM cells. Nevertheless, in this context, DNA hypomethylation produced by Adox treatment may not be a major event or direct trigger in the reactivation of γ-globin expression in human BM cells. Histone modification or repressor complex (e.g. NURD complex) reconstitution which might trigger histone modification changes may contribute to the γ-globin gene reactivation [21–23]. Alternatively, we cannot exclude the possibility that different erythroid specific transcription factors play roles upon γ-globin induction by Adox treatment [24, 25].
Histone methylation at H3K9, H3K27, H4K20, or symmetric methylation at H4R3 is normally associated with repression of transcription [26, 27]. We have previously demonstrated that histone H4R3me2s is an early histone mark induced by PRMT5 that can coordinately induce other histone methylation events such as H4K20me3, H3K9me3, H3K27me3, and deacetylation of histones . In the current results, in human bone marrow cells, Adox induced γ-globin expression independent of significant hypomethylation of the gene. This suggests that histone methylation, such as H4R3me2s, may play a more critical role in regulation of globin genes.
Our current studies indicat that Adox reactivates fetal hemoglobin expression efficiently. We speculate that reactivation of fetal globin by Adox may be through a mechanism involving inhibition of PRMT5 in both K562 and human bone marrow erythroid progenitor cells. These findings may contribute to the development of new reagents for reactivating fetal globin expression as a treatment for sickle cell disease and β-thalassemia.
We are grateful to members of the Jane and Zhao laboratories for helpful discussions. This work was supported by National Natural Science Foundation of China NSFC#31071118, NSFC#31170716, NSFC#81121062, BK2011052, NSFC#30270811 (QZ), NSFC#81060248 (YH), the Fundamental Research Funds for the Central Universities, and the Doctoral Program of Higher Education of China RFDP#20090091110033, The Cooley’s Anemia Foundation (QZ), and The National Health and Medical Research Council of Australia (SMJ).
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