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Originally published In Press as doi:10.1074/jbc.M310023200 on January 27, 2004

J. Biol. Chem., Vol. 279, Issue 15, 15678-15687, April 9, 2004
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AML1/RUNX1 Increases During G1 to S Cell Cycle Progression Independent of Cytokine-dependent Phosphorylation and Induces Cyclin D3 Gene Expression*

Florence Bernardin-Fried{ddagger}, Tanawan Kummalue{ddagger}, Suzanne Leijen{ddagger}, Michael I. Collector{ddagger}, Katya Ravid§, and Alan D. Friedman{ddagger}

From the {ddagger}Division of Pediatric Oncology, Johns Hopkins University, Baltimore, Maryland 21231 and the §Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02128

Received for publication, September 9, 2003 , and in revised form, December 31, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
AML1/RUNX1, a member of the core binding factor (CBF) family stimulates myelopoiesis and lymphopoiesis by activating lineage-specific genes. In addition, AML1 induces S phase entry in 32Dcl3 myeloid or Ba/F3 lymphoid cells via transactivation. We now found that AML1 levels are regulated during the cell cycle. 32Dcl3 and Ba/F3 cell cycle fractions were prepared using elutriation. Western blotting and a gel shift/supershift assay demonstrated that endogenous CBF DNA binding and AML1 levels were increased 2-4-fold in S and G2/M phase cells compared with G1 cells. In addition, G1 arrest induced by mimosine reduced AML1 protein levels. In contrast, AML1 RNA did not vary during cell cycle progression relative to actin RNA. Analysis of exogenous Myc-AML1 or AML1-ER demonstrated a significant reduction in G1 phase cells, whereas levels of exogenous DNA binding domain alone were constant, lending support to the conclusion that regulation of AML1 protein stability contributes to cell cycle variation in endogenous AML1. However, cytokine-dependent AML1 phosphorylation was independent of cell cycle phase, and an AML1 mutant lacking two ERK phosphorylation sites was still cell cycle-regulated. Inhibition of AML1 activity with the CBF{beta}-SMMHC or AML1-ETO oncoproteins reduced cyclin D3 RNA expression, and AML1 bound and activated the cyclin D3 promoter. Signals stimulating G1 to S cell cycle progression or entry into the cell cycle in immature hematopoietic cells might do so in part by inducing AML1 expression, and mutations altering pathways regulating variation in AML1 stability potentially contribute to leukemic transformation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Core binding factor (CBF)1 is a family of transcription factors containing one of three CBF{alpha} subunits, RUNX1/AML1, AML2, or AML3, and a CBF{beta} subunit (1). The CBF{alpha} subunits bind a common DNA consensus site via the N-terminal Runt domain, which also mediates heterodimerization with CBF{beta} (2, 3). CBF{beta} does not contact DNA, but increases the DNA affinity of the {alpha}-subunits (4, 5). CBF activities are reduced in 30% of acute myeloid leukemia cases, due either to AML1 point mutations, AML1 gene deletion, or, most often, chromosomal abnormalities involving genes encoding AML1 or CBF{beta} (1).

Mice lacking either AML1 or CBF{beta} do not develop definitive hematopoiesis, indicating a critical role for these factors in pluripotent hematopoietic stem cells (6-10). During early hematopoiesis AML1 regulates myeloid and lymphoid lineage-specific genes, such as those encoding T-cell receptor {delta}, myeloperoxidase, and the M-CSF receptor (11-14), and AML1 is down-regulated during terminal neutrophilic differentiation (15). In addition, CBF/AML1 regulates the G1 to S cell cycle transition. CBF{beta}-SMMHC is a CBF oncoprotein capable of sequestering CBF{alpha} subunits in multimers, which form via its myosin (SMMHC) domain (16, 17). Expression of CBF{beta}-SMMHC from the zinc-responsive metallothionein (MT) promoter in the Ba/F3 pro-B lymphoid or 32Dcl3 myeloid cell lines reduces CBF/AML1 DNA binding, leading to the accumulation of hypophosphorylated retinoblastoma (Rb) protein, and slows cell proliferation during G1 (18). N-terminal CBF{beta} residues required for interaction with CBF{alpha} subunits are required for inhibition of proliferation by CBF{beta}-SMMHC (19). An AML1 DNA binding domain:KRAB transrepression domain fusion protein expressed inducibly in Ba/F3 or 32Dcl3 cells also blocks G1 progression (20). Similarly, AML1-ETO, a CBF oncoprotein, which binds DNA and represses transcription, slows G1 progression in myeloid cell lines, dependent upon its ability to bind DNA (17, 21, 22). Inhibition of proliferation by CBF{beta}-SMMHC or by KRAB-AML1-ER is overcome by exogenous cdk4, cyclin D2, or c-Myc (20, 23). Also, AML1 accelerates G1 progression when expressed stably in 32Dcl3 cells or when expressed as an AML1-ER fusion protein in Ba/F3 cells (20, 24). When co-expressed with CBF{beta}-SMMHC, AML1-ER stimulated proliferation more potently, dependent upon integrity of its transactivation domain (25). Finally, genetic changes that accelerate G1, loss of p16INK4a and p19ARF or expression of papillomavirus E7 protein, cooperate with CBF oncoproteins to induce acute leukemia in mice (26, 27).

Based on the conclusion that AML1 stimulates the G1 to S transition, we sought to determine whether AML1 levels are themselves regulated during the cell cycle. 32Dcl3 and Ba/F3 cell cycle fractions were prepared using elutriation, a method that avoids the cytotoxicity of chemical synchronizing agents. Endogenous or exogenous AML1 levels increased during G1 to S cell cycle progression, as did AML1 DNA binding activity. An AML1 variant lacking two ERK phosphorylation sites retained cell cycle variation, and IL-3 induced phosphorylation of endogenous AML1 independent of cell cycle phase. In addition, inhibition of AML1 repressed cyclin D3 mRNA expression, and AML1 bound and activated the cyclin D3 gene.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Generation of Stable Clones—32Dcl3 cells (28) were cultured in Iscove's modified Dulbecco's medium (IMDM) with 10% heat-inactivated fetal bovine serum (HI-FBS), 1 ng/ml murine interleukin-3 (IL-3, Peprotech.). Ba/F3 cells (29) were cultured in RPMI 1640 with 10% HI-FBS, and 1 ng/ml IL-3. CRE retroviral packaging cells (30) were cultured in Dulbecco's modified Eagle's medium with 10% heat-inactivated calf serum. All cultures contained penicillin-streptomycin. Zinc chloride, estradiol, or mimosine (Sigma) were added when indicated. 32Dcl3 differentiation was induced by washing twice with phosphate-buffered saline followed by culture in IMDM with 10% HI-FBS, and 20 ng/ml G-CSF (Amgen). 32Dcl3 or Ba/F3 cells were stably transfected by electroporation, and 32Dcl3 cells were transduced using CRE-AML1(86-217)ER cells as described and selected using 1.2 mg/ml G418 (total) or 2 µg/ml puromycin (18, 31). Single cell clones were isolated by limiting dilution. 293T cells were cultured in Dulbecco's modified Eagle's medium with 10% HI-FBS and were transiently transfected using LipofectAMINE 2000 (Invitrogen), as described (17).

Plasmids—The human AML1B cDNA, the longest splice variant of AML1 (32), was ligated into the polylinker of pMTCB6 (18) to generate pMT-AML1. pMT-Myc-AML1 was generated by inserting an oligonucleotide encoding Kozak's rules for translation initiation followed by a methionine and a Myc tag upstream of the AML1 cDNA. The oligonucleotide sense strand, with a KpnI-compatible 3'-end was 5'-CATCGATGCCGCCACCATGGAGCAGAAGCTCATCTCCGAGGAAGATCTCAGTAC. The sequence encoding the initiating Met and 11-residue Myc tag is underlined. Ligation into the polylinker upstream of the AML1 cDNA also inserted five additional residues, STEFA, between the tag and the AML1 cDNA. pMT-Myc-AML1-(1-217) was generated by 3'-deletion to a SmaI site, with insertion of a stop codon. A HindIII/BssHII segment of the AML1(S276A/S293A) cDNA, kindly provided by H. Hirai, was used to replace a similar segment in pMT-Myc-AML1 to generate pMT-Myc-AML1(S276A/S293A). pBabePuro-AML1-ER and pBabePuro-AML1 (86-217)-ER have been described (25). The numbering of these mutants reflects the AML1B sequence (32). pD3(-447)-Luc was prepared by ligating an XhoI/BamHI fragment from pD3GH (33) into p19LUC. pD3(-447)mAML1-LUC was prepared by replacing the AML1 site at -383, 5'-GACCACA-3', with 5'-GCTAGCA-3', by ligating two set of annealed oligonucleotides between the XhoI site at -447 and the SfoI site at -333. The ligation product was verified by DNA sequencing.

Elutriation and Fluorescence-activated Cell Sorting (FACS) Analysis—100-200 x 106 cells were washed and resuspended in 10 ml of elutriation medium (150 mM NaCl, 167 mM glucose, 0.3 mM EDTA, 1% bovine serum albumin). The cells were then injected into a JE-6B rotor and spun at 2000 rpm at 25 °C. The flow rate of elutriation medium was increased gradually and cell cycle fractions collected. Fractionation was monitored in each experiment by incubation of 5 x 105 cells with 11 µg/ml Hoechst 33258 dye in 0.7% Nonidet P-40, 4.7% formaldehyde followed by FACS analysis.

Western Blotting, Gel Shift Assay, and Northern Blotting—Total cellular protein extracts were prepared using Laemmli sample buffer and subjected to SDS-polyacrylamide gel electrophoresis and Western blotting as described (18). Extract corresponding to 1.5 x 106 cells were loaded in each lane. AML1 antiserum was kindly provided by H. Drabkin, CBF{beta} antiserum was kindly provided by N. Speck, and ER{alpha} and D3 antisera (MC-ER and C-16, Santa Cruz Biotechnology), c-Myc monoclonal antibody (9E10, Roche Applied Science), and cyclin E antiserum (Upstate Biotechnology) were obtained commercially. Equivalent amounts of extract were electrophoresed and subjected to Coomassie Blue dye staining to assess total protein content of each sample.

Nuclear extracts were prepared and subjected to gel shift and supershift assay using a radiolabeled CBF binding site from the myeloperoxidase (MPO) promoter and an N-terminal AML1 antiserum, kindly provided by S. Hiebert, as described (13). The wild-type and mutant murine MPO oligonucleotides were (12) WTMPO: 5'-CTAGACTGACCATTAACCACAACCAGTTG-3', and MutMPO: 5'-CTAGACTGACCAGGTAGCACAACCAGTTG-3'.

In addition the following oligonucleotides derived from the murine cyclin D genes, including 5'-CTAG overhangs, were annealed to antisense strands and used as gel shift competitors (potential AML1 binding sites are again underlined, and accession numbers with base pairs are listed): D1(-753): 5-CTAGGGCCCTTTGCAACCACCCCAGTGCGCC-3' (AF212040 [GenBank] , 587-613); D2(-541): 5'-CTAGCCATGGGGTTTGTGGTTCCCCTATCCG-3' (AF015788 [GenBank] , 405-431); D3(-383): 5'CTAGACAAAGTTATGACCACATTCCCTAGAG-3' (U43844 [GenBank] , 1291-1317).

Total cellular RNAs were prepared using Trizol reagent (Invitrogen) or RNAeasy (Qiagen) per the manufacturer's instructions. The RNAs, 10 µg per lane, were subjected to Northern blotting as described (31). Murine cyclin D1, D2, and D3 cDNAs were kindly provided by C. Sherr and the murine AML1 cDNA by Y. Ito (2). Densitometric analysis was carried out with the NIH Image 1.62 program.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Endogenous CBF DNA Binding and AML1 Increase During G1 to S Progression—We employed counterflow elutriation to fractionate 32Dcl3 and Ba/F3 cells into cell cycle fractions based on density. In this procedure, the flow of the elutriation media opposes the centrifugal force on the cells, establishing an equilibrium. As the flow rate is increased, denser cells come into position to exit the elutriation chamber. Use of elutriation allowed us to study cells in active, physiologic cell cycle, which did not prove possible by synchronizing cells followed by release of the chemical block as 32Dcl3 cells treated in this manner underwent apoptosis rather than continued cell cycle progression (not shown). A typical cell cycle fractionation profile and the proportion of cells in each fraction are shown (Fig. 1). Fr 18-20 contains G1 phase cells, Fr 21-24 contains late G1/early S cells, Fr 25-30 contains mainly S phase cells, and Fr 31-34 mainly G2/M phase cells. Cell cycle fractionation was monitored in each experiment. In some experiments the cells began to elute at 20 ml/min rather than 18 ml/min, as indicated in each figure.



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  		FIG. 1.
Cell cycle fractionation of 32Dcl3 cells. A, 2 x 108 32Dcl3 cells were subjected to counterflow elutriation. The cell cycle profile of the total cell population and of isolated fractions are shown. Approximately 80% of the input cells were recovered. B, proportion of the isolated cells in each fraction and their cell cycle phases are shown.

 
AML1 is expressed entirely in the cell nucleus (34). We first employed a gel shift assay to assess CBF DNA binding activity during the cell cycle, utilizing an equivalent amount of nuclear protein per sample (Fig. 2, A and B, left panels). Based on densitometric analysis, CBF DNA binding activity increased almost 4-fold as 32Dcl3 or Ba/F3 cells progressed from G1 to S. Of note, micrograms of nuclear protein per cell were not significantly different between the G1 and S phase samples. This pattern of CBF gel shift activity was reproduced in four separate experiments. To confirm that the indicated bands contain members of the CBF family, the peak fractions for each cell line were subjected to competition with increasing amounts of unlabelled wild-type CBF oligonucleotide or with an oligonucleotide carrying clustered point mutations in the CBF binding site (Fig. 2C). Specific competition was observed with the CBF complexes. To resolve AML1-specific DNA from among the CBF binding activity, equal amounts of the 32Dcl3 nuclear extracts were subjected to a supershift assay with normal rabbit serum, AML1 antiserum, or AML1 antiserum in the presence of its specific peptide (Fig. 2A, right panel). A specific supershift band was obtained from each fraction (arrow) which increased 2.3-fold as cells progressed from G1 to S. Gel shift assay indicated that Ba/F3 cells have substantially less overall CBF DNA binding activity than 32Dcl3 cells (Fig. 2B), perhaps accounting for our inability to detect a supershifted AML1 species with the Ba/F3 extracts, although the antibody did reduce the intensity of the CBF bands (Fig. 2B, right panel).



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  		FIG. 2.
CBF and AML1 DNA binding activity increases during G1 to S progression. A, nuclear extracts, 12 µg of total protein per lane, from the indicated 32Dcl3 cell cycle fractions were subjected to gel shift assay with a radiolabeled AML1 binding site from the myeloperoxidase gene, WTMPO, either alone (left panel) or in the presence of 1 µl of normal rabbit serum (R), AML1 antiserum (Ab), or AML1 antiserum, and 4 µg of its specific peptide (P) (right panel). The relative intensity of the CBF shift or the AML1 supershift bands between fractions is shown. B, nuclear extracts from the indicated Ba/F3 fractions were subjected to gel shift assay similarly. The right panel was exposed for a shorter time. C, 32Dcl3 fraction 25-28 and Ba/F3 fraction 24-25 were subjected to gel shift assay in the absence of competitor (-) or in the presence of 10-, 50-, and 200-fold excess of unlabelled wild-type (WT) or mutant (Mut) competitor. The two panels were exposed for similar times.

 
We next prepared total cellular proteins from 32Dcl3 and Ba/F3 cell cycle fractions and analyzed these for AML1 expression by Western blotting (Fig. 3, A and B). A slowly migrating nonspecific band was evident in the 32Dcl3 samples, and Coomassie Blue dye-stained protein fractions serve as a further control for protein loading. Densitometric analysis indicated that total cellular proteins increased 2-fold during the cell cycle, consistent with doubling of cell size. AML1 levels increased 2.7- or 2.6-fold from G1 (Fr 18-19) to S (Fr 28-29) or G2/M (Fr 32-33) in the 32Dcl3 fractions, relative to total cellular proteins, and increased ~1.8- or 1.9-fold in the same Ba/F3 fractions. Additional repetitions of this analysis with 32Dcl3 cells demonstrated a 3.0- or 3.5-fold increase (for an average of 3.0-fold) in AML1 expression during the cell cycle (see Fig. 8B). This degree of cell cycle variation is consistent with the EMSA studies for 32Dcl3 cells, but the increase in CBF DNA binding was greater than that for AML1 in Ba/F3 cells, perhaps reflecting a greater contribution of AML2 and AML3 to CBF cell cycle variation in these cells.



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  		FIG. 3.
Endogenous AML1 protein levels increase during the cell cycle. A, cellular proteins isolated from unfractionated cells (T) or from the indicated cell cycle fractions were subjected to Western blotting for AML1. The positions of molecular weight markers are shown (top panel). Equal volumes of each fraction were electrophoresed on a second gel and stained with Coomassie Blue dye (bottom panel). B, cell cycle fractions from Ba/F3 cells were analyzed similarly. Relative expression of AML1 in each sample based on densitometry, normalized for loading, is shown for both blots.

 



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  		FIG. 8.
Cytokine-dependent phosphorylation does not regulate AML1 expression during the cell cycle. A, 32Dcl3 cells were serum- and IL-3-starved for 3 h at room temperature in elutriation medium, and then returned to IMDM with fetal bovine serum and IL-3 at 37 °C, 5% CO2 for the indicated number of minutes. Total cellular proteins were then subjected to Western blotting for AML1. An extract from cells collected before starvation (pre) was also analyzed. Upper and lower bands in the AML1 doublet are indicated by arrows. B, the indicated 32Dcl3 cell cycle fractions were returned to IMDM/FBS/IL-3 for 4 h (left panels) or 6 h(right panels). Total cellular protein samples prepared prior to addition of growth medium (0 h) and after addition for the indicated times were subjected to Western blotting for AML1 and to Coomassie Blue staining. Expression of AML1 in each sample, corrected for loading, is shown below, with the 0 h samples being analyzed separately from the 4 or 6 h samples (+IL3). As there was not sufficient sample available for two of the Coomassie lanes, AML1 was normalized to the paired 0 or 6 h Coomassie signal, in view of the equality of the slowly migrating nonspecific band in the same paired fractions. C, total cellular proteins from the indicated 32D-Myc-AML1(S276A/S293A) cell cycle fractions were subjected to Western blotting using Myc tag antibody. Coomassie Blue dye staining of these fractions is also shown. Myc-AML1(S276A/S293A) expression in each sample, corrected for loading, is indicated below.

 
To determine whether the variation in AML1 protein levels during the cell cycle is paralleled by variation in AML1 RNA levels, total cellular RNAs were prepared from 32Dcl3 and Ba/F3 cell cycle fractions and 10 µg of each sample was subjected to Northern blotting (Fig. 4). AML1 mRNA increased only 1.3-fold in both Ba/F3 cells and 32Dcl3 cells from early G1 to late S phase, relative to {beta}-actin mRNA. Of note, RNA per cell increased 2-fold, on average, during the cell cycle. Thus, relative to total RNA, AML1 RNA increased 2.6-fold in each line. Comparison to {beta}-actin may be more relevant, as total RNA largely represents ribosomal RNA.



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  		FIG. 4.
Endogenous AML1 RNA levels do not vary during the cell cycle. Total cellular RNA prepared from the indicated Ba/F3 and 32Dcl3 cell cycle fractions, 10 µg per sample, were subjected to Northern blotting for murine AML1, cyclin D3, and {beta}-actin. Actin is 1892 and D3 is 1905 bp. Expression of AML1 RNA relative to {beta}-actin in each sample is shown.

 
AML1 Protein Levels Are Reduced in Cells Arrested in G1—Decreased expression of AML1 in early G1 suggests that cells arrested in G1 would have reduced levels of AML1 compared with proliferating cells. 32Dcl3 or Ba/F3 cells were exposed to mimosine, an agent, which induces the p27 cyclin-dependent kinase in hematopoietic cells (35). Mimosine was employed at 0.2 or 0.4 mM for 6 h, as exposure for longer times led to substantial apoptosis. At 0.4 mM, the G1/S ratio increased from 1.4 to 2.2 in Ba/F3 cells and from 1.1 to 1.6 in 32Dcl3 cells, with minimal apoptosis (Fig. 5, A and B). Total cellular proteins from these cultures were analyzed for endogenous AML1 expression by Western blotting (Fig. 5B). Exposure to 0.4 mM mimosine for 6 h reduced endogenous AML1 levels 1.7-fold in Ba/F3 cells and 2.5-fold in 32Dcl3 cells, relative to total protein content.



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  		FIG. 5.
Endogenous AML1 levels are reduced by G1 cell cycle arrest. A, cell cycle profiles of 32Dcl3 or Ba/F3 cells exposed to 0.4 mM mimosine (mim) for 6 h or of 32Dcl3 cells transferred from IL-3 to G-CSF for 1 or 2 days (D1, D2). B, total cellular proteins prepared from Ba/F3 or 32Dcl3 cells exposed to the indicated doses of mimosine for the indicated times were subjected to Western blotting for AML1 and to Coomassie Blue dye staining (left panels). Similar analysis was carried out on 32Dcl3 cells cultured in IL-3 or after transfer to G-CSF for 1 or 2 days (right panels). The G1/S ratio and the relative expression of AML1, corrected for loading, is shown for each sample. C, total cellular RNAs prepared from 32Dcl3 cells cultured in IL-3 or in G-CSF for 1 or 2 days and from 32Dcl3 and Ba/F3 cells cultured in 0.4 mM mimosine for 6 h were subjected to Northern blotting for murine AML1 and {beta}-actin. Expression of AML1 RNA relative to {beta}-actin in each sample is shown.

 
For comparison, 32Dcl3 cells were transferred from IL-3 to G-CSF, which induces differentiation concomitant with a potent G1 arrest (31). The G1/S ratio increased from 1.1 to 5.0 after 1 day (D1) and to 8.9 after 2 days (D2), without evident apoptosis (Fig. 5, A and B). AML1 levels were reduced 3-fold after 1 day and more than 10-fold after 2 days in G-CSF.

To determine whether reduced AML1 levels associated with G1 arrest are due to reduced AML1 RNA levels, total cellular RNAs were prepared from 32Dcl3 cells cultured in IL-3 or in G-CSF for 1 or 2 days and from 32Dcl3 or Ba/F3 cells cultured in 0.4 mM mimosine for 6 h. These RNAs were subjected to Northern blotting for AML1 and {beta}-actin (Fig. 5C). Relative to {beta}-actin, AML1 RNA increased in 32Dcl3 cells in response to exposure to mimosine for 6 h or to G-CSF for 2 days and only decreased 10% in Ba/F3 cells in response to mimosine.

Exogenous AML1 Levels Also Increase During G1 to S Progression—Based on our finding that endogenous AML1 levels are regulated during the cell cycle, we predicted that exogenous AML1 protein levels might be controlled similarly. To clearly distinguish exogenous AML1, we positioned a 17-residue tag, MEQKLISEEDLASTEFA (the Myc epitope is underlined), at the N terminus of the 480-residue AML1 cDNA just downstream of a segment containing Kozak's rules for optimum translational initiation. The myc-AML1 cDNA, lacking endogenous AML1 5'- or 3'-untranslated regions (UTRs), was then positioned downstream of the zinc-responsive metallothionein promoter in pMTCB6 to generate pMT-myc-AML1. This plasmid also contains the G418-resistance gene linked to the SV40 promoter. pMT-myc-AML1 was linearized and electroporated into 32Dcl3 cells. Two G418-resistant subclones expressing myc-AML1 in the presence of zinc chloride were identified by Western blotting. When a total cellular extract from zinc-treated 32D-Myc-AML1 cells was analyzed by Western blotting using an AML1 antibody, two doublets were detected, with the lower doublet co-migrating with AML1 detected from parental 32Dcl3 cells (Fig. 6A, left panel). Presence of a doublet may reflect AML1 phosphorylation (36). The increased size of exogenous myc-AML1 is due to the 17-residue Myc tag and to the fact that exogenous human AML1 contains 27 residues at it N terminus lacking in the endogenous murine protein. Of note, the remainder of human and murine AML1 are 97% identical (32). We observed that myc-AML1 was expressed at ~3-fold higher level than endogenous AML1. Use of the zinc-responsive MT promoter not only avoids generation of cell lines perturbed during isolation by high basal activity of AML1 but also allows control of myc-AML1 expression via use of suboptimal zinc concentrations (Fig. 6A, right panel). Even in the absence of zinc, basal expression of myc-AML1, especially the lower band of the doublet, is evident. 75 µM zinc chloride led to expression 2-3-fold below that detected in cells exposed to 100 µM zinc, presumably similar to endogenous AML1 levels. 32D-Myc-AML1 cells were therefore exposed to 75 µM zinc and subjected to elutriation. Cell cycle fractions were analyzed by Western blotting for myc-AML1, cyclin D3, and cyclin E (Fig. 6B). Compared with early G1 cells (Fr 18-20), Myc-AML1 levels increased 2.3-fold by mid-S phase (Fr 27-28) and 3.0-fold by late-S/G2/M phase (Fr 29-30). Cyclin D3 and E levels increased similarly. Analysis of a second 32Dcl3 subclone demonstrated a 3.4-fold increase in exogenous Myc-AML1 during cell cycle progression (not shown). Northern blot analysis indicated that AML1 RNA increased 1.8-fold relative to {beta}-actin, consistent with both transcriptional and post-transcriptional regulation of AML1 during G1 to S progression (Fig. 6C).



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  		FIG. 6.
Exogenous AML1 protein levels increase during the cell cycle. A, total cellular proteins prepared from 1 x 106 32Dcl3 (32D) cells or from 32D-Myc-AML1 cells exposed to 100 µM zinc chloride for 16 h were subjected to Western blotting using an AML1 antiserum (left panel). Extracts from 32D-Myc-AML1 cells exposed to the indicated concentrations of zinc chloride (µM) for 16 h were subjected to Western blotting using a Myc tag antibody. B, total cellular proteins prepared from the indicated 32D-Myc-AML1 cell cycle fractions, obtained after culture in 75 µM zinc chloride for 16 h, were subjected to Western blotting for myc-AML1 (using the Myc tag antibody), cyclin D3, or cyclin E. Coomassie Blue dye staining of these fractions is also shown (bottom panel). C, total cellular RNAs prepared from the indicated 32D-Myc-AML1 cell cycle fractions were subjected to Northern blotting for Myc-AML1 (using a human AML1 probe) and {beta}-actin. D, total cellular proteins prepared from the indicated 32D-AML1ER cell cycle fractions, obtained after culture for 16 h in 200 nM 4HT, were subjected to Western blotting for AML1-ER (using ER antiserum), CBF{beta}, and cylcin D3. Myc-AML1 or AML1-ER expression in each sample, corrected for loading, is shown in parts B, C, and D.

 
To evaluate the effect of cell cycle phase on the expression of exogenous AML1 by an independent strategy we utilized 32Dcl3 cells expressing AML1-ER from the MMLV retroviral LTR in pBabePuro (20). Western blot analysis of cell cycle fractions demonstrated that AML1-ER increased 1.9-2.3-fold by S phase (Fr 25-28 and Fr 29-31) and 4.5-fold by G2/M phase (Fr 32-34) relative to CBF{beta} levels, which remained constant throughout the cell cycle (Fig. 6D). Cyclin D3 levels again increased during G1 to S progression. Thus, exogenous AML1 levels varied similar to endogenous AML1 during the cell cycle despite lack of the AML1 promoter, expected to control transcription, or the AML1 5'- and 3'-untranslated RNA segments, expected to control translation efficiency and RNA stability.

Exogenous AML1 DNA Binding Domain Does Not Vary during the Cell Cycle—To begin to map the domain of AML1 responsible for its cell cycle variation, 32Dcl3 lines stably expressing either Myc-AML1-(1-217) from the MT promoter, retaining only the DNA binding domain and 87 N-terminal residues, or AML1-(86-217)-ER from the pBabePuro retroviral vector, retaining only the DNA binding domain, were subjected to elutriation and Western blot analysis (Fig. 7, A and B). In contrast to Myc-AML1 or AML1-ER, which increased 3.0- or 4.5-fold, Myc-AML1-(1-217) increased 1.5-fold and AML1-(86-217)-ER increased 1.6-fold during the cell cycle. Analysis of a second subclone expressing AML1-(86-217)-ER gave similar results (not shown).



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  		FIG. 7.
Expression of exogenous AML1 DNA binding domain varies minimally during the cell cycle. A, total cellular proteins from the indicated 32D-Myc-AML1-(1-217) cell cycle fractions were subjected to Western blotting using Myc tag antibody (top panel). Coomassie Blue dye staining of these fractions is also shown (bottom panel). B, similar analysis was carried out with 32D-AML1-(86-217) ER cells and ER antiserum. Myc-AML1 or AML1-ER expression in each sample, corrected for loading, is shown.

 
Phosphorylation of AML1 by ERK Does Not Mediate Its Cell Cycle Variation—Serines 276 and 293 are targets of cytokine-mediated, ERK-dependent phosphorylation in hematopoietic cells (36). In particular, these investigators found that exogenous HA-AML1 was expressed as four closely spaced bands in Ba/F3 cells, that the upper two bands were absent after withdrawal of IL-3 and serum, and that mutation of serines 276 and 293 to alanine prevented the appearance of the upper two bands. To determine whether cytokine-dependent phosphorylation of endogenous AML1 varied during the cell cycle, we first withdrew 32Dcl3 cells from serum and IL-3 for 3 h by transferring them at room temperature to the same buffer used for elutriation. Complete media was then added, the cells were returned to the 37 °C, 5% C02 incubator, and protein samples were prepared at various time intervals. These samples were subjected to Western blotting using AML1 antiserum (Fig. 8A). Before starvation, the majority of AML1 was in the upper band of a doublet, whereas after starvation the majority of AML1 was in the lower band. By 60 or 120 min the two bands were of equal intensity, and after 240 min the upper band again had a greater intensity. After serum and IL-3 withdrawal the cells potentially enter a temporary state not fully responsive to IL-3. Having found that 4 h are required to recover cytokine-dependent AML1 phosphorylation, we then sought to determine whether this varied among cell cycle fractions. 32Dcl3 cell cycle fractions were returned to IL-3 for 4 or 6 h and assayed for AML1 by Western blotting (Fig. 8B). Each fraction developed a doublet band indicative of phosphorylation, but the levels of AML1 after exposure to serum and IL-3 continued to vary during the cell cycle. In particular, AML1 levels increasing 1.4- or 3.6-fold in the 0 h fractions, comparing the earliest with the latest cell cycle fractions, and 4.6- or 3.3-fold in the samples exposed to IL-3 (for an average of 3.0- and 3.5-fold increase during the cell cycle in the two experiments). Thus, cytokine-dependent phosphorylation occurs during each cell cycle stage and does not lead to a rapid stabilization of AML1.

To further examine the role of ERK-dependent AML1 phosphorylation, 32Dcl3 lines stably expressing exogenous Myc-AML1(S276A/S293A) from the MT promoter were developed. This AML1 mutant increased more than 3-fold during cell cycle progression (Fig. 8B), similar data were obtained when this experiment was repeated. Thus, cytokine-mediated phosphorylation of AML1 does not account for the increase in endogenous or exogenous AML1 observed during G1 to S progression.

The Cyclin D3 Promoter Is Activated by AML1—Cyclin D3 mRNA levels did not vary, relative to {beta}-actin, in 32Dcl3 or Ba/F3 cell cycle fractions (Fig. 4). However, E2F1:DP-1, a heterodimeric factor with peak activity in late G1/early S phase, potently activated the murine cyclin D3 promoter,2 as predicted from the presence of an E2F consensus site (33). Therefore, we considered the possibility that the peak in CBF DNA binding activity in late G1/early S might be designed similarly to enable maximal cyclin D3 expression when needed in hematopoietic cells. We first examined Ba/F3 cells expressing dominant inhibitory AML1 oncoproteins (17, 18), CBF{beta}-SMMHC (INV) or AML1-ETO (A-E), from the MT promoter to assess the consequences of CBF inhibition on cyclin D1, D2, and D3 mRNA expression (Fig. 9A). Ba/F3 lines were employed rather than 32Dcl3 lines for this purpose as the MT promoter is leakier in 32Dcl3 cells, making it difficult to maintain high-level expression of these cell cycle-inhibitory oncoproteins. Cyclin D1 expression was not detected, as has been described previously for hematopoietic cell lines (37). Zinc did not affect cyclin D2 or D3 mRNA expression in parental Ba/F3 cells, but their expression was inhibited by both the INV or A-E proteins by 7 h, with D3 expression being affected the greatest. We selected candidate AML1 binding sites from the murine D1, D2, and D3 promoters based on the AML1 consensus, 5'-PuACCPuCA-3', and assessed their affinity for AML1 (Fig. 9B). In this assay, a strong AML1 site from the MPO promoter, WTMPO, was radiolabeled and competed with 5-, 10-, and 25-fold excess of unlabelled WTMPO, a version of this oligonucleotide mutant in the AML1 site, MutMPO, and each of the oligonucleotides derived from the cyclin D promoter regions. The best available site from the D1 promoter region, 5'-AACCACC-3', did not compete, from the D2 promoter region, 5'-AACCACA-3', competed modestly, and from the D3 promoter, 5'-GACCACA-3', competed to an even greater extent, almost as well as the WTMPO oligonucleotide. Differences in the affinities of the D2-541 and D3-383 sites may reflect affects of flanking sequences. To determine whether AML1 can transactivate the murine cyclin D3 promoter, a 447 bp promoter fragment, including the transcription start site was linked to luciferase and co-transfected with pCMV-AML1 and pCMV-CBF{beta} in 293T cells (Fig. 9C). AML1 activated the cyclin D3 promoter 2.3-fold in this context in multiple repetitions, and mutation of the AML1 binding site at -383 reduced this activation to 1.6-fold, suggesting a role for this binding site, although the differences in activity did not reach statistical significance.



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  		FIG. 9.
AML1 binds and activates the murine cyclin D3 promoter. A, total cellular RNAs prepared from parental Ba/F3 cells, Ba/F3 cells expressing CBF{beta}-SMMHC (INV), and Ba/F3 cells expressing AML1-ETO (A-E) from the MT promoter, were exposed to zinc for 0, 7, 24, or 48 h. Total cellular RNAs, 10 µg per sample, were then subjected to Northern blotting for cyclin D1, cyclin D2, cyclin D3, and {beta}-actin. B, 1 ng of radiolabeled WTMPO oligonucleotide, containing a strong AML1 binding site, was subjected to gel shift analysis with 12 µg of 32Dcl3 nuclear extract in the presence of no competitor (-) or 5, 10, or 25 ng of WTMPO, MutMPO, or the indicated oligonucleotides derived from the cyclin D1, D2, or D3 promoters. C, 293T cells in 6-well dishes were transiently transfected with 700 ng of pD3(-447)-LUC (WT) or pD3(-447)mAML1-LUC (m-383) together with either 70 ng of empty pCMV vector or 50 ng of pCMV-AML1B and 20 ng of pCMV-CBF{beta}. 48 h later cellular extracts were assay for luciferase. Activity obtained in the presence of AML1 and CBF{beta} divided by that obtain with empty vector is shown for each reporter. n = 7 for WT and 4 for m-383.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
A key finding of this study is that endogenous CBF DNA binding and AML1 protein levels reproducibly increase 3-fold, when normalized to total protein content, during the G1 to S cell cycle transition. In addition physiologic levels of exogenous AML1, expressed inducibly to avoid biasing cell cycle distribution, increased similarly, supporting the additional conclusion that AML1 protein stability is regulated during the cell cycle. Reduced expression of AML1 in G1-arrested cells is consistent with this conclusion. Regulation of AML1 transcription may also occur during cell cycle progression. These findings were confirmed in two growth factor-dependent hematopoietic cell lines. We have focused our studies on hematopoietic cells as these are most physiologically relevant in view of the finding that AML1-null mice specifically lack hematopoiesis (6, 7). To put the degree of AML1 variation in perspective, mdm2 levels increase 4-fold and cyclin B1 varies 10-fold during cell cycle progression in HeLa cells (38). AML1 levels remained high in G2/M phase compared with S phase cells, whereas CBF and AML1 DNA binding was reduced, suggesting that AML1 DNA binding activity is itself regulated during S to G2/M progression.

Several lines of experimentation using the 32Dcl3 and Ba/F3 cell lines had demonstrated that gene activation by AML1 stimulates their G1 to S progression. In particular, transrepression of AML1 target genes slowed G1 progression, an effect overcome by co-expression of regulators known to stimulate S phase entry, and exogenous AML1 accelerated the G1 to S transition, dependent upon the AML1 transactivation domain (18-20, 23, 25). It is therefore not surprising that hematopoietic cells require increased AML1 during late G1 and have developed mechanisms to reduce AML1 during the G1 arrest associated with differentiation. As differentiation is a multistep process, the reduced AML1 protein levels evident after G-CSF treatment may reflect mechanisms other than those utilized to regulate AML1 during the cell cycle.

It is intriguing that hematopoietic cells predominantly express cyclin D2 and D3, rather than D1, and that we have identified cis elements within the D2 and D3 promoters which bind AML1. On its own, AML1 only activated the D3 promoter 2.3-fold, perhaps reflecting the need to cooperate with other proteins to contribute most effectively. And mutation of the AML1 binding site at -383 only partially abrogated this activation, perhaps indicating contribution from additional AML1 binding sites. AML2 or AML3 may also contribute to regulation of the cyclin D3 promoter, as supershifting with AML1 antiserum did not eliminate cell cycle variation of residual CBF DNA binding activity (Fig. 2A), perhaps accounting in part for our inability thus far to detect interaction of AML1 with the cyclin D3 promoter by chromosomal immunoprecipitation. Interestingly, the C-terminal regions of AML2 or AML3 when knocked into the AML1 locus allow nearly normal in vivo hematopoiesis (39). Future experiments will further characterize regulation of the cyclin D3 gene by CBF family members.

As AML1 is expressed in pluripotent and lineage-restricted hematopoietic stem cells (40, 41), we speculate that stimulation of AML1 protein stability or RNA expression also plays a role in the entry of these cells into cell cycle from G0, as required to maintain hematopoietic homeostasis or during cytokine-mediated stress responses. In this regard, both c-Myc and B-Myb RNA levels increase when cells transition from G0 to G1, and during active proliferation B-Myb protein levels are maximal during S phase due to cyclinA/cdk2-mediated phosphorylation (42-44). Similarly, cyclin D levels increase upon cytokine-mediated cell cycle entry (45). Although Ba/F3 and 32Dcl3 cells divide rapidly, the reduced levels of cyclin D3 we detected in early cell fractions may indicate that after mitosis these lines enter a G0-like state and are transiently resistant to cytokine signaling. We recently found that CBF{beta}-SMMHC inhibits G1 to S progression in bone marrow-derived myeloid progenitors, just as it does 32Dcl3 and Ba/F3 cells (46), and AML1-ETO inhibits the proliferation of human and murine progenitors (47, 48). In addition, reduced megakaryocyte ploidy in adult AML1(+/-) mice may reflect inhibition of cell cycle progression (49). Future experiments will further address the role of AML1 in regulation of the cell cycle and cell cycle entry in hematopoietic stem cells.

Several mechanisms might operate to control AML1 protein stability. One possibility is that phosphorylation of AML1 by cdks such as cdk2, cdk4, or cdk6 increases its stability during late G1. AML1 contains three potential cdk2 phosphorylation sites ((S/T) (P/X) (K/R)) that are conserved in the murine and human proteins and 12 of the less well defined cdk4/6 phosphorylation sites ((S/T)P). Direct phosphorylation by cdk2 regulates cell cycle specific activity of the MEF transcription factor in COS cells (50). Cyclin E variation during the cell cycle depends upon ubiquitination by a specific ubiquitin ligase, which results in proteosome targeting and degradation (51). AML1 levels in 32Dcl3 cells were found to be increased by the MIG132 proteosome inhibitor (52). We replicated this finding using 32D-Myc-AML1 cells (not shown), although significant cellular toxicity ensued precluding cell cycle fractionation. Protein-protein interaction studies might identify a specific ubiquitin ligase or another protein, which stabilizes AML1 in late G1. Our deletional analysis of myc-AML1 and AML1-ER suggests that residues C-terminal to the DNA binding domain are involved in the regulation of AML1 stability. Of note, cyclin D3 interacts with AML1 via this region (53), potentially recruiting cdk4/6. We found that cytokine-dependent phosphorylation of serines 276 and 293 by ERK did not vary during the cell cycle nor alter AML1 stability. Future experiments will investigate whether additional AML1 phosphorylations or protein interactions control its stability during cell cycle progression or in response to cell cycle entry or exit. In this regard it is likely insufficient to characterize the ubiquitination or phosphorylation of a particular AML1 residue during the cell cycle as the unstable modified or unmodified form may be rapidly degraded. Rather, AML1 mutants resistant to modification might be identified which remain at high, constant levels throughout the cell cycle.

In a subset of acute myeloid leukemias, AML1 activities are reduced due to mutation or expression of dominant oncoproteins, potentially favoring transformation due to inhibition of differentiation and/or reduced apoptosis (13, 54, 55). Perhaps additional leukemias harbor mutations, which reduce AML1 level via protein modification, using the same biochemical pathway employed to reduce AML1 expression in G1 phase cells. In these leukemias, additional mutations stimulating G1 progression may be required (26, 27). Other acute leukemias harbor increased numbers of AML1 genes due to gene amplification or to trisomy 21 (56-59). In these leukemias, AML1 may facilitate transformation by stimulating cell cycle progression. Perhaps additional leukemias harbor alterations, which increase AML1 levels indirectly. Investigating the mechanisms underlying AML1 cell cycle variation might therefore uncover novel pathways contributing to leukemogenesis.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant CA098805 [GenBank] and the Children's Cancer Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Leukemia Society Scholar. To whom correspondence should be addressed: Johns Hopkins University, CRB 253; 1650 Orleans St., Baltimore, MD 21231. Tel.: 410-955-2095; Fax: 410-955-8897; E-mail: afriedm2{at}jhmi.edu.

1 The abbreviations used are: CBF, core binding factor; G-CSF, granulocyte-colony stimulating factor; ERK, extracellular signal-related kinase; IL-3, interleukin-3; MT, metallothionein; FACS, fluorescence-activated cell sorting; HI-FBS, heat-inactivated fetal bovine serum; MPO, myeloperoxidase; cdk, cyclin-dependent kinase. Back

2 F. Bernardin-Fried and A. D. Friedman, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Saul Sharkis for helpful discussion, H. Hirai for the AML1(S276A/S293A) cDNA, C. Sherr for the cyclin D cDNAs, and H. Drabkin, N. Speck, and S. Hiebert for antisera.



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The Hematopoietic Transcription Factor AML1 (RUNX1) Is Negatively Regulated by the Cell Cycle Protein Cyclin D3
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F. B. Fried and A. D. Friedman
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Expression Levels of TEL, AML1, and the Fusion Products TEL-AML1 and AML1-TEL versus Drug Sensitivity and Clinical Outcome in t(12;21)-Positive Pediatric Acute Lymphoblastic Leukemia
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F. M. Theriault, H. N. Nuthall, Z. Dong, R. Lo, F. Barnabe-Heider, F. D. Miller, and S. Stifani
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