DNA methylation-mediated silencing of PU.1 in leukemia cells resistant to cell differentiation
© Fernández-Nestosa et al.; licensee Springer. 2013
Received: 24 April 2013
Accepted: 13 August 2013
Published: 21 August 2013
In mice, the proviral integration of the Friend Spleen Focus Forming Virus (SFFV) within the PU.1 locus of erythroid precursors results in the development of erythroleukemia. SFFV integrates several kilobases upstream of the PU.1 transcription initiation start site leading to the constitutive activation of the gene which in turn results in a block of erythroid differentiation. In this study we have mapped and sequenced the exact location of the retroviral integration site. We have shown that SFFV integrates downstream of a previously described upstream regulatory element (URE), precisely 2,976 bp downstream of the URE-distal element. We have also found that SFFV persists integrated within the same location in resistant cell lines that have lost their differentiation capacity and in which case PU.1 remains silent. We have examined the methylation status of PU.1 and found that in resistant cells the nearby CpG islands remained methylated in contrast to a non-methylated status of the parental cell lines. Treatment with 5-aza-2′-deoxycytidine caused resistant cells to differentiate yet only when combined with HMBA. Altogether these results strongly suggest that methylation plays a crucial role with regard to PU.1 silencing. However, although demethylation is required, it is not sufficient to overcome the differentiation impasse. We have also showed that activation blockage of the Epo/Epo-R pathway remains despite of the absence of PU.1.
PU.1, the ETS transcription factor encoded by the Sfpi/PU.1 gene, is crucial for the regulation of hematopoietic development (Burda et al. 2010). The gene is already expressed at high levels in multipotential progenitors, including erythroblasts. Thereafter, PU.1 expression is tightly regulated and in mature hematopoietic cells is maintained in myeloid and B lymphoid cells but not in erythrocytes or T cells. Like other transcription factors and proteins, PU.1 can posses dual roles and play as an activator or repressor depending on its combination with variable binding-partners of each specific cell lineage. PU.1 holds a tumor suppresor activity during myeloid leukemia by promoting maturation of myeloid cells but acts as an oncogene when overexpressed in proerythroblasts by disrupting the erythroid differentiation program (Cook et al. 2004; Moreau-Gachelin et al. 1996; Rosenbauer et al. 2004).
Immortalized murine erythroleukemia (MEL) cells which are derived from transformed erythroblasts by the Friend complex virus constitute an appropriate and valuable model to study tumor cell reprogramming (Moreau-Gachelin 2006; Papetti and Skoultchi 2007). Insertion of the Friend spleen focus-forming virus (SFFV) upstream of the transcription start site of PU.1 leads to constitutive gene expression and to blockage of erythroid cell differentiation (Ruscetti 1999). MEL cells can be induced to reinitiate the differentiation program by the addition of chemical agents such as HMBA, in which case down regulation of PU.1 has been shown to be a critical event that takes place during early cell differentiation (Rao et al. 1997). We have previously reported on the establishment of HMBA-resistant cell lines (MEL-R) in which PU.1 remains silent even though the SFFV persists integrated within a similar location to the site found in MEL parental lines (Fernández-Nestosa et al. 2008). In this study, we have further characterized the concrete location of the SFFV integration site in both, parental and resistant cells, to confirm that no further changes have occurred in the resistant clones. The present analysis has revealed that the SFFV integration position in parental and resistant MEL cell lines is located downstream of the upstream regulatory element (URE) (Okuno et al. 2005), concretely at 2,976 bp from the distal element.
We have also studied the methylation status of the PU.1 promoter and determined that the four CpG islands close to the PU.1 promoter remain methylated in MEL-resistant cells in contrast to the non-methylated status of the parental cell lines.
Materials and methods
MEL-DS19 and MEL-resistant (MEL-R) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 units/ml of penicillin and streptomycin (Gibco). Cell differentiation was induced by exposing logarithmically growing cell cultures to 5mM of HMBA. MEL-R cells were routinely cultured in the presence of the differentiation inducer. Hemoglobinized cells were monitored by determining the proportion of benzidine-staining positive cells (B+) of cell cultures. In order to analyze the epigenetic changes of the PU.1 locus, MEL-DS19 and MEL-R cells were grown in the absence or presence of 0,4 μM or 0,8 μM 5-Aza-2′-deoxycytidine (5-azaC, Sigma).
PCR experiments were performed with MEL-DS19 and MEL-R genomic DNA samples to sequence the viral–host DNA junction. PCR amplifications were performed using 200 μM of each nucleotide, 0.4 μM of sense and antisense primers and 1.25 units of Taq DNA Polymerase (Invitrogen). The PCR program consisted of an initial denaturation at 95°C during 3 min, followed by 30 consecutive cycles at 94°C during 45 s, annealing at 62°C during 30 s, extension at 72°C during 30s and a final extension at 72°C during 10 min. Long-range PCR (LR-PCR) was performed using the LongRange PCR Kit (Qiagen) and amplifications were performed using 500 μM of each nucleotide, 0.4 μM of sense and antisense primers and 2 units of LongRange PCR Enzyme Mix. The LR-PCR conditions comprised: denaturation at 93°C during 3 min, followed by 35 repeated cycles at 93°C during 15 s, annealing at 62°C during 30 s and extension at 68°C during 8 min. The following Sfpi-1/PU.1 and SFFV-specific primers were used: PU.1 Fw: 5′-TCCGCTCAAGACCAGGTC-3′; PU.1 Rv: 5′-CCATGTAGCCTTCTGAGT-3′, SFFV 1: 5′-AAGAACAGATGGTCCCCAGA-3′; SFFV 2: 5′-AAGGCACAGGGTCATTTCAG-3′; SFFV 3: 5′-AAAGAGCTCACAA CCCCTCA-3′; SFFV 4: 5′-GCCCAACGTTAGCTGTTTTC-3′; SFFV-a 5′-CAGAACCAGACGCAGGCGCA-3′; SFFV-b 5′-TCCACCATCATGGGGCTTCTCA-3′; SFFV-c 5′-TCCGCTCAAGACCAGGTC-3′; SFFV-d 5′-AGAGAGGTGAGAGTCATGCAATG-3′. PCR products were resolved on agarose gels and visualized by ethidium bromide staining. The Sfpi-1/PU.1 and virus-specific primers were used for sequencing which was carried out by Secugen SL (CIB, Madrid).
Gene expression analysis
Total RNA was isolated from 1 × 107 cells using the TRIzol (GIBCO BRL) kit following the manufacturer’s instructions. For the semi-quantitative RT-PCR the reactions consisted of 5 μg of total RNA previously extracted from the MEL and MEL-R cells, which was reverse transcribed using the M-MLV reverse transcriptase (USB) as previously has been described (García-Sacristán et al. 2005). PCR amplifications were performed using 200 μM of each nucleotide, 0.5 μM of the sense and antisense primers plus 5 units of Recombi-Taq (LINUS). The amplification conditions encompassed: denaturation at 95°C during 2 min, followed by 30 cycles of 94°C during 1 min, annealing during 1 min was carried out at different temperatures depending on the primers used and extension was undertaken at 72°C during 1 min, with a final extension at 72°C during 5 min. The annealing temperatures were 55°C for the Shp1 primer pair and 65°C for the GAPDH. The following primers were used: Shp1 Fw 5′-CAGGATGGTGAGGTGGTTT-3′; Shp1 Rv 5 -CTCAAACTCCTCCC AGAAG-3′. For the quantitative real time PCR analysis RNA was isolated from 5 × 106 cells using the RNeasy Mini Kit (Qiagen) as described by the supplier. RNA samples were reverse-transcribed using SuperScript® II Reverse Transcriptase (RT) (Invitrogen). First-strand cDNA was synthesized from 2.0 μg of total RNA using the Superscript II (Invitrogen) in a final volume of 20 μl with 0.5 μg of Oligo dT (Invitrogen), 20 units of SUPERase In RNase Inhibitor (Ambion) and 200 units of Superscript II reverse transcriptase. The reaction mixture was incubated at 42°C during 50 min. Quantitative real time PCR was carried out in iQ5 system (Bio-Rad). The reaction mixture of 20 μl consisted of 1X iQ SYBR Green Supermix (Bio-Rad), 1 μl cDNA and 0.2 mM of each primer. The PCR protocol consisted of: 95°C during 5 min, followed by 50 cycles of 95°C during 30s and 60°C during 30s. The following primers were used: PU.1 Fwd 5′-GGGATCTGACC AACCTGGA-3′ and PU.1 Rev 5′-AACCAAGTCATCCGATGGAG-3′. The relative gene-expression quantification method was used to calculate the amount of mRNA expression change according to the comparative Ct method using β-actin as an endogenous control. Final results were determined as follows: 2-(ΔCt sample - ΔCt control), where the ΔCt values of the control and the sample were determined by subtracting the Ct value of the target gene from the value of the β-actin gene. All experiments were performed in triplicate; differences in cell input were compensated by normalization against the β-actin expression levels.
Sodium bisulfite conversion was undertaken with the EZ DNA Methylation-Direct Kit (Zymo Research). The DNA bisulfite conversion was performed directly from 8 × 104 cells according to the manufacturer’s instructions. Samples of 2 μl of bisulfite-treated DNAs were eluted in a 10 μl volume for each PCR reaction. Converted DNAs were amplified and sequenced by PCR using primers specific to the bisulfite-converted gDNA and the ZymoTaq™ DNA Polymerase (Zymo Research). The PCR protocol used was: 95°C during 10 min, followed by 35 cycles of 95°C during 30 s, 55°C during 30 s and finally 72°C during 40 s. The set of primers for bisulfite sequencing PCR (BSP) comprised: PU.1 bis DNA Fw 5′-GAAAGGAGATAAAATGTGGGAGAT-3′ and PU.1 bis DNA Rv 5′-CCAAATAATCCACTATTCTTTTAACCT-3′. The PCR products were separated and visualized in 1% agarose gels, followed by sequencing for methylation status evaluation. Sequencing was performed by Secugen SL (CIB, Madrid).
Measurement of DNA methylation by pyrosequencing
Sodium bisulfite modification of 0.5 μg of genomic DNA isolated from MEL-DS19 and MEL-R was carried out with the EZ DNA Methylation Kit (Zymo Reserch) following the manufacturer’s protocol. Samples of 2 μl bisulfite-treated DNA were eluted in a 15 μl volume for each PCR reaction. The region of interest was amplified by PCR using primers specific to the bisulfite-converted gDNA. As a control, the efficiency of the bisulfite conversion was assessed using primers for the non-converted DNA sequence. Pyrosequencing was performed using the following primers: Fw 5′-AGTTTGGTAGTTTTGGGATTAAAG-3′; Rev 5′ [Btn] ATTTCTTCTCAATCCCCTCTAA-3′; seq 5′-AAATTTATTTTTAAAATTAGGGA-3′. Pyrosequencing reactions and methylation quantification were performed in a PyroMark Q24 System version 2.0.6 (Qiagen). Graphic representation of methylation values showed bars identifying CpG sites that represented methylation percentages. Assay design reports, which include the target sequence region and primer sequences, are provided as Additional file 1.
Plasmid construction and DNA transfections
The PU.1-ER construct was generated by PCR using the Expand High Fidelity PCR System (Roche) to amplify the cDNA of PU.1 derived from MEL-DS19 cells. The primers contained BamHI recognition sites and consisted of the following sequences: PU.1-ER Fw 5′-GGGGCACCTGGTCCTGAG-3′ and PU.1-ER Rv 5′-CGCGGATCCGAGTGGGGCGGGAGGCG-3′. The expression vector producing the PU.1-ER fusion protein was constructed by subcloning the 869 bp BamHI fragment encoding the PU.1 cDNA into the pEBBpuro ER vector digested with the same enzyme. The inducible vector pEBBpuro GATA-1-ER (a generous gift from A. Skoultchi) was used to produce the conditionally active form of GATA-1. Stable transfectants of MEL-R were prepared as described previously (Vanegas et al. 2003). In short, recombinant DNA was introduced into exponentially growing MEL cells by lipofectine (Gibco) and after a 6 h incubation period cells were distributed into 96-well plates. The transfectants were selected and maintained in growth medium containing 5 μg/ml puromycin. Cell differentiation was tested by cell culture in the absence or presence of 5 mM HMBA with or without 10-7 M β-estradiol. Cell growth was measured on a daily basis by aliquot counting of the cultures with a Neubauer hemocytometer chamber.
Antibodies and immunoblot analysis
MEL and MEL-R cells (1 × 107) were pelleted, washed twice with cold PBS and lysed with Laemmli buffer (65 mM Tris–HCl pH 6.8, 10% glycerol, 5% 2-mercaptoethanol, 1% SDS) containing protease inhibitors (SIGMA). Proteins (20–50 μg) were resolved in a 10% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore). Primary antibodies included rabbit polyclonal anti-Sat1 antibody (1:1,000 Cell Signaling), rabbit polyclonal anti-Phospho-Sat1(Tyr 701) antibody (1:1,000 Cell Signaling), rabbit polyclonal anti-PU1 antibody (1:1,000; Santa Cruz) and mouse monoclonal anti-α-Tubulin antibody (1:10,000, Sigma).
Mapping the SFFV integration site within the PU.1 locus of MEL and MEL-R cell lines
Reactivation of the silenced PU.1 locus of MEL-R cells by 5-azaC treatment
Methylation status of CpG islands at the PU.1 promoter
5-azaC treatment induces MEL-R cell differentiation in the presence of HMBA
Ectopic expression of PU.1 limits the proliferative capacity yet is not required to induce MEL-R cell differentiation
MEL-R cells show similar expression levels of tyrosine phosphatase Shp-1 as those of the progenitor MEL cell lines
SFFV integration within the PU.1 locus of MEL and MEL-R cell lines
PU.1 proviral insertion is a major event that occurs during the second stage of the Friend disease and which leads to a block of the erythroid differentiation program. It was initially recognized that SFFV integrates upstream of the PU.1 transcriptional start site (Moreau-Gachelin et al. 1989; Paul et al. 1989) and later on the target for SFFV integration was precisely located at -14 kb of the URE in a non-conserved 500-bp spacer, lying exactly between two highly conserved homologous regions (Okuno et al. 2005). The mechanism by which SFFV induces permanent PU.1 expression in erythroleukemia cells was suggested to rely on the disruption of the URE at the non-homologous spacer. This interruption might prevent the 5′ distal region to down-regulate PU.1 expression, while the 3′ proximal element constitutively activates PU.1 transcription. In this study, we have identified, at the nucleotide level, the exact location of the SFFV integration site within the PU.1 locus of erythroleukemia cells. We provide proof that the integration site resides outside of the URE, concretely 2,976 bp apart, which therefore excludes the hypothesis of the negative/positive regulation of the URE 5′ proximal/3′ distal regions, respectively. Instead, it seems that the SFFV integration might block a negative regulation of the URE on the PU.1 promoter which together with the transcriptional enhancer elements supplied by the SFFV long terminal repeats (LTR) result in an unsolicited activation of the gene. On the other hand, as was predicted in our previous study (Fernández-Nestosa et al. 2008), the SFFV insertion of the resistant cell lines (MEL-R) occupied an identical position compared to the parental cell lines. This observation rules out the possibility that PU.1 silencing in MEL-R cells may well be a consequence of either the absence of SFFV or that proviral integration might have occurred at a different spot.
These findings are focused on the MEL-DS19 cell line derived from the MELC 745A strain, which in turn derives from an original Charlotte Friend’s lab strain (Ohta et al. 1976). Therefore, these results do not preclude the integration of SFFV at a different site of the PU.1 locus in other host genomes. Many models for the selection of integration-sites by a retrovirus favor their integration near to DNase I-hypersensitive sites characterized by an open chromatin conformation (Lewinski et al. 2006). Genome mapping has allowed the detection of putative SFFV integration sites extended over several kilobases upstream of the PU.1 coding region (Moreau-Gachelin et al. 1988; Paul et al. 1991). Proviral insertion at the 5′ end of the PU.1 locus has also been described in leukemia stem cells infected with the Friend virus (Hegde et al. 2012; Hasegawa et al. 2005). In this study we have identified and sequenced the genome-viral junction region of MEL-DS19 and MEL-R clones and plan to further extend a similar approach to other SFFV-infected cell lines. Finally, novel cis-regulatory elements outside of the URE, about 10kb upstream of the PU.1 transcriptional start site, have recently been described (Zarnegar et al. 2010). These elements can simultaneously act as enhancers in myeloid cells and repressors in T cells and sometimes have been described to neutralize the continuous URE enhancer function. These newly characterized elements overlap with the SFFV integration site described in this report and suggest other possible mechanisms that can mediate PU.1 regulation.
Methylation suppressed PU.1 expression of MEL-R cells
In this study we have demonstrated that 5′-CpG-3′ dinucleotides of the PU.1 promoter region are highly methylated in MEL-R cells, but not in the parental MEL-DS19 cell line. DNA methylation of CpG dinucleotides comprises one of the best characterized epigenetic marks that condition gene expression, usually producing a repressive transcription status with consequent gene silencing (Jones 2012). Analysis of the four CpG islands of the PU.1 promoter (Shearstone et al. 2011) showed a high level of methylation in MEL-R cells compared to the MEL parental cell lines. This hypermethylation could be responsible for the PU.1 inactivation of the resistant clones. Accordingly, reversal of this hypermethylated status caused by treatments with 5-azaC, a known antagonist of DNA methylases (Christman 2002), that have been reported to restore PU.1 expression (Amaravadi and Klemsz 1999; Tatetsu et al. 2007) induced the differentiation of MEL-R cell cultures. On the other hand, methylation of PU.1 in the MEL-DS19 cells was significantly lower than in MEL-R cell lines and the adding of 5-azaC to the cultures produced only minor alterations. The HMBA-induced differentiation did not modify the methylation status of PU.1 in the parental cell lines, as would have been expected based on the gradual silencing of PU.1 through erythroid differentiation. This truly represents a paradox as methylation prevents PU.1 expression in MEL-R cells leading to gene silencing and differentiation blockage. However, the gradual silencing of PU.1 during HMBA-induced erythroid differentiation has been reported not to consist of a methylation-related process (Shearstone et al. 2011). It has recently been reported that global DNA demethylation occurs continuously during mouse erythropoiesis in vivo, a process which is also associated to DNA replication and has been suggested to take place during differentiation of most somatic cells. The observed MEL-DS19 cell line PU.1 methylation status harmonizes with this model, even in the case of the HMBA-induced differentiation for which methylation of PU.1 is limited. On the contrary, PU.1 methylation of MEL-R clones follows the classical methylation concept as a robust mechanism for gene silencing. After methylation mark removal, PU.1 expression is restored and MEL-R cells become receptive to HMBA. This places emphasis on another controversy concerning the role of PU.1 in erythroleukemia cells. Differentiation blockage in murine erythroleukemia cells has been attributed to the inopportune expression of PU.1 (Rao et al. 1997). Erythroid differentiation is also partially blocked in PU.1 transgenic proerythroblasts (Moreau-Gachelin et al. 1996). However, previous findings have indicated a requirement of PU.1 expression for erythroid differentiation, Rosenbauer and coworkers demonstrated that the gradual activity reduction of PU.1, rather than the complete loss, can induce acute myeloid leukemia (AML) in mice (Rosenbauer et al. 2004). Two recent reports (Wontakal et al. 2011; Ridinger-Saison et al. 2012), on the other hand, have revealed the complexity of the pathways regulated by PU.1 in several hematopoietic lineages. PU.1 level differences are crucial for cell identity and even low concentrations of the protein can promote ubiquitous cellular functions (Wontakal et al. 2011). Altogether, these findings suggest that PU.1 is necessary for erythroid commitment, but contrasts with the downregulation requirement for terminal differentiation (Atar and Levi 2005; Rao et al. 1997; Yamada et al. 1997). PU.1 expression is abolished in the HMBA-resistant erythroleukemia cells and yet these cells are unable to differentiate (this study and (Fernández-Nestosa et al. 2008)). Low PU.1 levels might be necessary for erythroleukemia cells to differentiate, as probably is the case of MEL-DS19 cells. MEL-R cell lines, on the contrary, had a completely blocked (methylated) PU.1 expression and had lost their ability to respond to HMBA, a process that we have shown to be reversible by demethylating agents. We also observed that the expression of PU.1 driven by a conditional estrogen-activated element led to cell growth arrest and apoptosis, in agreement with earlier findings of MEL, K562 leukemia and myeloma cell lines (Aoyama et al. 2012; Tatetsu et al. 2007; Yamada et al. 1997). However, the ectopic expression of PU.1 could not have triggered the differentiation of MEL-R cells. It is most likely that additional factors, essential for erythroid differentiation, might have also been epigenetically silenced in resistant cell lines.
STAT-1 phosphorylation blockage of MEL-R cells was independent of PU.1 expression
In addition to the ability of PU.1 to block erythroid terminal differentiation by antagonism with GATA-1, the locus mechanism of action may also apply to the activation of SHP-1. Previous work had already shown that high levels of SHP-1 interfered with STAT1 phosphorylation, thus preventing the binding activity and blocking erythroid differentiation in SFFV-transformed erythroleukemia cell lines (Nishigaki et al. 2006). It has also been shown that SHP-1 expression is significantly reduced in PU.1-/- erythroid cells (Fisher et al. 2004). This data suggests that high PU.1 levels block differentiation signals although in an indirect way. We have observed that in MEL-DS19 cells, STAT1 α and β are neither tyrosine phosphorylated before nor after stimulation with erythropoietin (Epo). In addition, we also showed that SHP-1 was highly expressed in parental cell lines; nonetheless the expression was significantly reduced during the HMBA-induced differentiation. Unexpectedly, we have also noticed a failure of STAT1 to become phosphorylated in Epo-stimulated MEL-R cells in which PU.1 expression was completely abolished by DNA methylation. HMBA-resistant cell lines also showed a high SHP-1 expression. These results strongly suggest that in MEL-R cells the blocking of the STAT1 DNA binding activity is regulated by a different mechanism.
This work was supported by grants BFU2011-22489 from the Spanish Ministerio de Economia y Competitividad, AP/038170/11 from the AECI, Agencia Española de Cooperación Internacional and I-COOP0009 from the Agencia Estatal CSIC. MJFN was a recipient of a fellowship from the CONACYT, Paraguay. We thank Dr. Graciela Russomando for helpful discussions and continuous support. We also thank ML Martínez for technical assistance.
- Amaravadi L, Klemsz MJ: DNA methylation and chromatin structure regulate PU.1 expression. DNA Cell Biol 1999, 18(12):875-884. 10.1089/104454999314737View ArticleGoogle Scholar
- Aoyama S, Nakano H, Danbara M, Higashihara M, Harigae H, Takahashi S: The differentiating and apoptotic effects of 2-aza-5′-deoxycytidine are dependent on the PU.1 expression level in PU.1-transgenic K562 cells. Biochem Biophys Res Commun 2012, 420(4):775-781. 10.1016/j.bbrc.2012.03.071View ArticleGoogle Scholar
- Atar O, Levi BZ: PU.1 silencing leads to terminal differentiation of erythroleukemia cells. Biochem Biophys Res Commun 2005, 329(4):1288-1292. 10.1016/j.bbrc.2005.02.109View ArticleGoogle Scholar
- Burda P, Laslo P, Stopka T: The role of PU.1 and GATA-1 transcription factors during normal and leukemogenic hematopoiesis. Leukemia 2010, 24(7):1249-1257. 10.1038/leu.2010.104View ArticleGoogle Scholar
- Christman JK: 5-Azacytidine and 5-aza-2′-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy. Oncogene 2002, 21(35):5483-5495. 10.1038/sj.onc.1205699View ArticleGoogle Scholar
- Cmarik J, Ruscetti S: Friend Spleen Focus-Forming Virus Activates the Tyrosine Kinase sf-Stk and the Transcription Factor PU.1 to Cause a Multi-Stage Erythroleukemia in Mice. Viruses 2010, 2(10):2235-2257. 10.3390/v2102235View ArticleGoogle Scholar
- Cook WD, McCaw BJ, Herring C, John DL, Foote SJ, Nutt SL, Adams JM: PU.1 is a suppressor of myeloid leukemia, inactivated in mice by gene deletion and mutation of its DNA binding domain. Blood 2004, 104(12):3437-3444. 10.1182/blood-2004-06-2234View ArticleGoogle Scholar
- Fernández-Nestosa MJ, Hernández P, Schvartzman JB, Krimer DB: PU.1 is dispensable to block erythroid differentiation in Friend erythroleukemia cells. Leuk Res 2008, 32(1):121-130. 10.1016/j.leukres.2007.05.008View ArticleGoogle Scholar
- Fisher RC, Slayton WB, Chien C, Guthrie SM, Bray C, Scott EW: PU.1 supports proliferation of immature erythroid progenitors. Leuk Res 2004, 28(1):83-89. 10.1016/S0145-2126(03)00178-4View ArticleGoogle Scholar
- García-Sacristán A, Fernández-Nestosa MJ, Hernández P, Schvartzman JB, Krimer DB: Protein kinase clk/STY is differentially regulated during erythroleukemia cell differentiation: a bias toward the skipped splice variant characterizes postcommitment stages. Cell Res 2005, 15(7):495-503. 10.1038/sj.cr.7290319View ArticleGoogle Scholar
- Hasegawa M, Yamaguchi S, Aizawa S, Ikeda H, Tatsumi K, Noda Y, Hirokawa K, Kitagawa M: Resistance against Friend leukemia virus-induced leukemogenesis in DNA-dependent protein kinase (DNA-PK)-deficient scid mice associated with defective viral integration at the Spi-1 and Fli-1 site. Leuk Res 2005, 29(8):933-942. 10.1016/j.leukres.2005.01.016View ArticleGoogle Scholar
- Hegde S, Hankey P, Paulson RF: Self-renewal of leukemia stem cells in friend virus-induced erythroleukemia requires proviral insertional activation of Spi1 and Hedgehog signaling but not mutation of p53. Stem Cells 2012, 30(2):121-130. 10.1002/stem.781View ArticleGoogle Scholar
- Jones PA: Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat Rev Genet 2012, 13(7):484-492. 10.1038/nrg3230View ArticleGoogle Scholar
- Lewinski MK, Yamashita M, Emerman M, Ciuffi A, Marshall H, Crawford G, Collins F, Shinn P, Leipzig J, Hannenhalli S, Berry CC, Ecker JR, Bushman FD: Retroviral DNA integration: viral and cellular determinants of target-site selection. PLoS Pathog 2006, 2(6):e60. 10.1371/journal.ppat.0020060View ArticleGoogle Scholar
- Moreau-Gachelin F: Lessons from models of murine erythroleukemia to acute myeloid leukemia (AML): proof-of-principle of co-operativity in AML. Haematologica 2006, 91(12):1644-1652.Google Scholar
- Moreau-Gachelin F, Tavitian A, Tambourin P: Spi-1 is a putative oncogene in virally induced erythroleukemias. Nature 1988, 331: 277-280. 10.1038/331277a0View ArticleGoogle Scholar
- Moreau-Gachelin F, Ray D, Mattei MG, Tambourin P, Tavitian A: The putative oncogene Spi-1: murine chromosomal localization and transcriptional activation in murine acute erythroleukemias. Oncogene 1989, 4(12):1449-1456.Google Scholar
- Moreau-Gachelin F, Wendling F, Molina T, Denis N, Titeux M, Grimber G, Briand P, Vainchenker W, Tavitian A: Spi-1/PU.1 transgenic mice develop multistep erythroleukemias. Mol Cell Biol 1996, 16(5):2453-2463.View ArticleGoogle Scholar
- Nishigaki K, Hanson C, Ohashi T, Spadaccini A, Ruscetti S: Erythroblast transformation by the friend spleen focus-forming virus is associated with a block in erythropoietin-induced STAT1 phosphorylation and DNA binding and correlates with high expression of the hematopoietic phosphatase SHP-1. J Virol 2006, 80(12):5678-5685. 10.1128/JVI.02651-05View ArticleGoogle Scholar
- Ohta Y, Tanaka M, Terada M, Miller OJ, Bank A, Marks P, Rifkind RA: Erythroid cell differentiation: murine erythroleukemia cell variant with unique pattern of induction by polar compounds. Proc Natl Acad Sci U S A 1976, 73(4):1232-1236. 10.1073/pnas.73.4.1232View ArticleGoogle Scholar
- Okuno Y, Huang G, Rosenbauer F, Evans EK, Radomska HS, Iwasaki H, Akashi K, Moreau-Gachelin F, Li Y, Zhang P, Gottgens B, Tenen DG: Potential autoregulation of transcription factor PU.1 by an upstream regulatory element. Mol Cell Biol 2005, 25(7):2832-2845. 10.1128/MCB.25.7.2832-2845.2005View ArticleGoogle Scholar
- Papetti M, Skoultchi AI: Reprogramming leukemia cells to terminal differentiation and growth arrest by RNA interference of PU.1. Mol Cancer Res 2007, 5(10):1053-1062. 10.1158/1541-7786.MCR-07-0145View ArticleGoogle Scholar
- Paul R, Schuetze S, Kozak SL, Kabat D: A common site for immortalizing proviral integrations in Friend erythroleukemia: molecular cloning and characterization. J Virol 1989, 63(11):4958-4961.Google Scholar
- Paul R, Schuetze S, Kozak SL, Kozak CA, Kabat D: The Sfpi-1 proviral integration site of Friend erythroleukemia encodes the ets-related transcription factor Pu.1. J Virol 1991, 65(1):464-467.Google Scholar
- Rao G, Rekhtman N, Cheng G, Krasikov T, Skoultchi AI: Deregulated expression of the PU.1 transcription factor blocks murine erythroleukemia cell terminal differentiation. Oncogene 1997, 14(1):123-131. 10.1038/sj.onc.1200807View ArticleGoogle Scholar
- Ridinger-Saison M, Boeva V, Rimmele P, Kulakovskiy I, Gallais I, Levavasseur B, Paccard C, Legoix-Ne P, Morle F, Nicolas A, Hupe P, Barillot E, Moreau-Gachelin F, Guillouf C: Spi-1/PU.1 activates transcription through clustered DNA occupancy in erythroleukemia. Nucleic Acids Res 2012, 40(18):8927-8941. 10.1093/nar/gks659View ArticleGoogle Scholar
- Rosenbauer F, Wagner K, Kutok JL, Iwasaki H, Le Beau MM, Okuno Y, Akashi K, Fiering S, Tenen DG: Acute myeloid leukemia induced by graded reduction of a lineage-specific transcription factor, PU.1. Nat Genet 2004, 36(6):624-630. 10.1038/ng1361View ArticleGoogle Scholar
- Ruscetti SK: Deregulation of erythropoiesis by the Friend spleen focus-forming virus. Int J Biochem Cell Biol 1999, 31(10):1089-1109. 10.1016/S1357-2725(99)00074-6View ArticleGoogle Scholar
- Shearstone JR, Pop R, Bock C, Boyle P, Meissner A, Socolovsky M: Global DNA demethylation during mouse erythropoiesis in vivo. Science 2011, 334(6057):799-802. 10.1126/science.1207306View ArticleGoogle Scholar
- Tatetsu H, Ueno S, Hata H, Yamada Y, Takeya M, Mitsuya H, Tenen DG, Okuno Y: Down-regulation of PU.1 by methylation of distal regulatory elements and the promoter is required for myeloma cell growth. Cancer Res 2007, 67(11):5328-5336. 10.1158/0008-5472.CAN-06-4265View ArticleGoogle Scholar
- Vanegas N, García-Sacristán A, López-Fernández L, Párraga M, Del Mazo J, Hernández P, Schvartzman J, Krimer D: Differential expression of Ran GTPase during HMBA-induced differentiation in murine erythroleukemia cells. Leukemia Res 2003, 27: 607-615. 10.1016/S0145-2126(02)00231-XView ArticleGoogle Scholar
- Wontakal SN, Guo X, Will B, Shi M, Raha D, Mahajan MC, Weissman S, Snyder M, Steidl U, Zheng D, Skoultchi AI: A large gene network in immature erythroid cells is controlled by the myeloid and B cell transcriptional regulator PU.1. PLoS Genet 2011, 7(6):e1001392. 10.1371/journal.pgen.1001392View ArticleGoogle Scholar
- Yamada T, Kondoh N, Matsumoto M, Yoshida M, Maekawa A, Oikawa T: Overexpression of PU.1 induces growth and differentiation inhibition and apoptotic cell death in murine erythroleukemia cells. Blood 1997, 89(4):1383-1393.Google Scholar
- Zarnegar MA, Chen J, Rothenberg EV: Cell-type-specific activation and repression of PU.1 by a complex of discrete, functionally specialized cis-regulatory elements. Mol Cell Biol 2010, 30(20):4922-4939. 10.1128/MCB.00354-10View ArticleGoogle Scholar
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