The Biological Effects of C/EBPα in K562 Cells Depend on the Potency of the N-terminal Regulatory Region, Not on Specificity of the DNA Binding Domain*

The transcription factor C/EBPα is more potent than C/EBPβ in inducing granulocitic differentiation and inhibiting BCR/ABL-expressing cells. We took a “domain swapping” approach to assess biological effects, modulation of gene expression, and binding to C/EBPα-regulated promoters by wild-type and chimeric C/EBPα/C/EBPβ proteins. Wild-type and N-C/EBPα+ C/EBPβ-DBD induced transcription of the granulocyte-colony stimulating factor receptor (G-CSFR) gene, promoted differentiation, and suppressed proliferation of K562 cells vigorously; instead, wild-type C/EBPβ and N-C/EBPβ+C/EBPα-DBD had modest effects, although they bound the G-CSFR promoter like wild-type C/EBPα and N-C/EBPα+C/EBPβ-DBD. Chimeric proteins consisting of the TAD of VP16 and the DBD of C/EBPα or C/EBPβ inhibited proliferation and induced differentiation of K562 cells as effectively as wild-type C/EBPα. Gene expression profiles induced by C/EBPα resembled those modulated by N-C/EBPα+C/EBPβ-DBD, whereas C/EBPβ induced a pattern similar to that of N-C/EBPβ+C/EBPα-DBD. C/EBPα activation induced changes in the expression of more cell cycle- and apoptosis-related genes than the other proteins and enhanced Imatinib-induced apoptosis of K562 cells. Expression of FOXO3a, a novel C/EBPα-regulated gene, was required for apoptosis but not for differentiation induction or proliferation inhibition of K562 cells.


The transcription factor C/EBP␣ is more potent than C/EBP␤ in inducing granulocitic differentiation and inhibiting BCR/ ABL-expressing cells. We took a "domain swapping" approach to assess biological effects, modulation of gene expression, and binding to C/EBP␣-regulated promoters by wild-type and chimeric C/EBP␣/C/EBP␤ proteins. Wild-type and N-C/EBP␣؉ C/EBP␤-DBD induced transcription of the granulocyte-colony stimulating factor receptor (G-CSFR) gene, promoted differentiation, and suppressed proliferation of K562 cells vigorously; instead, wild-type C/EBP␤ and N-C/EBP␤؉C/EBP␣-DBD had modest effects, although they bound the G-CSFR promoter like wild-type C/EBP␣ and N-C/EBP␣؉C/EBP␤-DBD. Chimeric proteins consisting of the TAD of VP16 and the DBD of C/EBP␣ or C/EBP␤ inhibited proliferation and induced differentiation of K562 cells as effectively as wild-type C/EBP␣. Gene expression profiles induced by C/EBP␣ resembled those modulated by N-C/EBP␣؉C/EBP␤-DBD, whereas C/EBP␤ induced a pattern similar to that of N-C/EBP␤؉C/EBP␣-DBD. C/EBP␣ activation induced changes in the expression of more cell cycle-and apoptosis-related genes than the other proteins and enhanced Imatinib-induced apoptosis of K562 cells. Expression of FOXO3a, a novel C/EBP␣-regulated gene, was required for apo-ptosis but not for differentiation induction or proliferation inhibition of K562 cells.
C/EBP␣ and C/EBP␤, two transcription factors of the CAAT/ enhancer-Binding protein family, regulate the proliferation and differentiation of various cell types (1). In hematopoietic cells, C/EBP␣ is expressed by myeloid progenitors and precursors but not by monocytes (2,3) and induces granulopoiesis and blocks monocytic differentiation upon expression in bipotential myeloid progenitors (4); consistent with this effect, loss of C/EBP␣ in vivo led to mice capable of producing monocytes but not mature granulocytes (5), and conditional knock-out of C/EBP␣ in hematopoietic stem cells/early progenitors increased the frequency of hematopoietic stem cells and blocked the transition of common myeloid progenitors into granulocyte-monocyte precursors (6).
The requirement of C/EBP␤ in hematopoietic cells is less well defined; C/EBP␤ knock-out mice exhibit a lymphoproliferative disorder and defective splenic macrophage activation but no apparent changes in granulopoiesis (7,8). Nevertheless, the knock-in of the C/EBP␤ gene at the C/EBP␣ locus restored granulocytic differentiation of C/EBP␣ knock-out mice but did not rescue their metabolic disorder, suggesting that C/EBP␤ has overlapping but not entirely redundant functions with C/EBP␣ (9).
Although C/EBP␣ expression is finely regulated in normal hematopoietic cells, its expression/activity is consistently altered in myeloid leukemias (10), suggesting that genetic or functional inactivation of C/EBP␣ is an important event in leukemogenesis (10,11). For example, transformation of myeloid precursor cells by p210 BCR/ABL and chronic phase-to-blast crisis transition in chronic myelogenous leukemia (CML) 8 is associated with decreased expression of C/EBP␣ by a mechanism that depends, in part, on inhibition of C/EBP␣ mRNA translation (12,13). C/EBP␣ is mutated in ϳ10% of acute myelogenous leukemia (AML) patients (14 -16); the identified mutants have reduced DNA binding and/or transactivation activity, suggesting that they suppress differentiation of blast cells through a dominant-negative effect or that reduced levels of functional protein are insufficient to promote blast cell differentiation. Genetic evidence indicates that expression of p30 C/EBP␣ (an isoform lacking the N-terminal transactivation domain) or of a DNA binding-deficient C terminus mutant in the absence of wild-type C/EBP␣ promotes the development of acute myeloid leukemia in mice (17,18). In AML patients with the t(8;21) translocation, the AML-1-ETO fusion protein appears to block differentiation by physically interacting with C/EBP␣ and suppressing its activity and by transcriptional repression of C/EBP␣ expression (19,20). C/EBP␣ expression is also downregulated via enhanced degradation in AML-overexpressing Trib2 (21) and via translation inhibition in AML with the inv16 fusion protein and the AML1-MDS1-EVI1 fusion gene (22,23). In AML with mutant FLT3, C/EBP␣ expression is in some cases down-modulated, but its function may also be inhibited by mitogen-activated protein kinase-dependent phosphorylation in the N terminus (24,25). Treatment of FLT3-ITD myeloid leukemia lines with a mitogen-activated protein kinase inhibitor induced partial differentiation and was associated with C/EBP␣ activation (25), and ectopic expression of C/EBP␣ in the t(8;21)-positive Kasumi cell line and in BCR/ABL-expressing cells induced granulocytic differentiation and suppressed proliferation in vitro and in leukemic mice (19,26,27), emphasizing the therapeutic potential of restoring expression of functional C/EBP␣ in leukemic cells.
Unlike C/EBP␣, much less is known about potential mechanisms of structural or functional inactivation of C/EBP␤ in AML and CML-BC cells. We found that expression of C/EBP␤ was induced in Imatinib (IM)-treated BCR/ABL-expressing cells and that levels of full-length C/EBP␤ were lower in CMLblast crisis than in CML-chronic phase or healthy individuals CD34ϩ progenitors (28). The effect of IM in myeloid precursor 32D-BCR/ABL-expressing cells is reminiscent of all-trans-retinoic acid in acute promyelocitic leukemia cells. In acute promyelocitic leukemia cells, C/EBP␤ expression was induced by all-trans-retinoic acid treatment and was required for all-transretinoic acid-dependent differentiation (29).
Consistent with these findings, ectopic expression of C/EBP␤ inhibited proliferation, induced differentiation, and suppressed leukemogenesis of myeloid precursor 32D-BCR/ ABL cells (28). However, C/EBP␤ was considerably less potent than C/EBP␣ (27), perhaps because C/EBP␣ exerts its biological functions not only via DNA binding and transcription activation of C/EBP-regulated genes but also via protein-protein interactions involving cell cycle regulators and chromatin remodeling proteins (30 -33). In addition, the less potent effects of C/EBP␤ may also reflect the subset of genes transcriptionally regulated by C/EBP␣, C/EBP␤, or both. C/EBP␣ and C/EBP␤ have a global homology at the protein level of 34%, but the homology in their TAD at the N terminus is only 24%, whereas it increases to 71% in the C terminus, where the DBD, the leucine-zipper domain, and the basic region reside (34).
To obtain additional insights on the mechanisms underlying the different potency of C/EBP␣ and C/EBP␤ in p210BCR/ABL expressing cells, we assessed the biological effects of 4-hydroxytamoxifen (4-HT)-regulated proteins consisting of C/EBP␣-C/EBP␤ hybrids or of VP16-C/EBP␣ or C/EBP␤ DBD chimera in K562 cells. We report here that the potent effect of C/EBP␣ in inhibiting proliferation and inducing differentiation of BCR/ABL-expressing cells depends on the strong activity of its TAD more than on different specificities of the DBDs; in addition to a subset of genes whose expression is modulated by C/EBP␣, C/EBP␤, and the chimeric proteins, expression of most C/EBP-regulated genes is influenced by their respective TAD or appears to be C/EBP␣or C/EBP␤-specific. In particular, C/EBP␣ modulates the expression of a subset of apoptosis and cell cycle-regulatory genes that includes FOXO3a; expression of FOXO3a is essential for the apoptosis-enhancing effect of C/EBP␣ but not for its roles in cell proliferation and differentiation.

EXPERIMENTAL PROCEDURES
Plasmids-C/EBP␣-ER and C/EBP␤-ER plasmids were previously described (26,27). N-C/EBP␣ϩC/EBP␤-DBD-ER and N-C/EBP␤ϩC/EBP␣-DBD-ER plasmids were generated as follows; a NotI site was inserted in C/EBP␤-ER plasmid with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) at nucleotide 623 upstream of the DBD; a NotI site was eliminated in C/EBP␣-ER plasmid at nucleotide 460, and a NotI site was inserted at nucleotide 810 upstream of the DBD. Each plasmid (sequenced to verify presence of mutations) was digested with NotI and BamHI to release the C/EBP␣ or C/EBP␤ DBDs, leaving the MigRI plasmids with the N terminus of C/EBP␣ or C/EBP␤ and the ER cassette. The C/EBP␣ DBD was directionally cloned into the NotI/BamHI-digested N terminus-C/EBP␤-ER-MigRI to obtain N-C/EBP␤ϩC/EBP␣-DBD-ER plasmid, whereas the C/EBP␤ DBD was directionally cloned into the NotI/BamHI-digested N terminus-C/EBP␣-ER-MigRI to obtain N-C/EBP␣ϩC/EBP␤-DBD-ER plasmid.
For retroviral infections, amphotropic Phoenix cells were transiently transfected with the indicated plasmids. The infectious supernatant was collected 48 h later and used to infect (a 48-h procedure) K562 cells. Twenty-four hours later, cells were sorted (EPICS Profile Analyzer; Coulter, Hialeah, FL) for green florescent protein (GFP) expression.
For shRNAs transfection, K562 cells (100 l at a density of 10 6 /ml) were resuspended in nucleofector V (Amaxa, Gaithersburg, MD) solution and mixed with 5 g of shRNA. The solution was added to Amaxa electrode cuvettes and electroporated in Amaxa Nucleofector II using program T-16. Then cells were diluted in 2 ml of Iscove's modified Dulbecco's medium and 24 h later seeded at 10 4 /ml and treated with 0.2 M IM. 12 h later, 250 nM 4-HT was added, and samples were collected after 24, 48, and 72 h for cell cycle and apoptosis analyses.
Cell Proliferation, Cell Cycle Analysis, Apoptosis, and Differentiation Assays-For proliferation and cell cycle analyses, K562 cells were washed with phosphate-buffered saline (PBS) and treated with 4-HT (250 nM; Sigma). Viable cells were counted by trypan blue exclusion.
For colony formation assays, cells were pretreated (1 h) with 250 nM 4-HT and plated in methylcellulose (5 ϫ 10 2 /plate) in the presence of 250 nM 4-HT. Colonies were counted 6 days later. Cell cycle distribution was determined by DNA content analysis of propidium iodide-stained nuclei as described (27).
Differentiation was monitored by May-Grunwald/Giemsa staining and by detection of CD11b and CD15 differentiation markers, as described (27). Images were visualized using an Olympus CK2 microscope with a 40ϫ/0.65 numeric aperture objective and were photographed using an Olympus SC35 type 12 camera (Olympus, Melville, NY). JPEG images were viewed using Adobe Photoshop (Adobe Systems, San Jose, CA), and contrast adjustments were made. For induction of apoptosis, K562 cells were seeded at 10 4 /ml and pretreated with 0.2 M IM for 12 h, then 250 nM 4-HT was added to IM-treated cells, and the percentage of apoptotic cells was evaluated 24, 48, and 72 h after 4-HT addition.
Reverse Transcription-PCR Analysis and Real-time Quantitative PCR-For RT-PCR analysis, RNA of untreated or 4-HTtreated cells was extracted using RNeasy Mini Kit (Qiagen, Valencia, CA) according to manufacturer's protocol. Extracted RNA was digested with DNase-RNase free (Roche Applied Science) for 1 h at 37°C and deactivated 15 min at 65°C. RT-PCR was performed with 200 ng of RNA using the ONE STEP RT-PCR kit (Qiagen) and oligodeoxynucleotides specific for granulocyte colony-stimulating factor (G-CSF) receptor and for FOXO3a cDNA (supplemental Table 1). cDNA samples were adjusted to yield relatively equal amplification of HPRT transcripts.
For real time quantitative-PCR, total RNA was isolated using the RNeasy Mini kit (Qiagen). Extracted RNA was digested with DNase-RNase free (Roche Applied Science) for 1 h at 37°C and deactivated for 15 min at 65°C. 2 g of total mRNA were reverse-transcribed, and the resulting first-strand cDNA was used as the PCR template. All reactions were done in triplicate. Primer pairs for all analyzed genes (supplemental Table 1) were designed using the ABI Primer Express software. Real time quantitative-PCR was performed using iQ SYBR Green supermix (Bio-Rad) on a MyIQ thermocycler (Bio-Rad) and quantified using MyIQ software (Bio-Rad). HPRT, a housekeeping gene with constant expression, was used as an internal control to normalize input cDNA. For relative comparison of each gene, we used the MyIQ software that analyzes the Ct value of real-time PCR data with ⌬⌬Ct method.
Luciferase Assay-293T cells were transiently transfected using ProFection Mammalian Transfection System-Calcium Phosphate (Promega, Madison, WI) with 3 g of reporter plasmid pTK-G-CSFR-luciferase (which contains four C/EBP␣ binding sites from the G-CSF receptor promoter), 3 g of the indicated C/EBP expression plasmid, and 1/50 Renilla luciferase plasmid to account for variation in transfection efficiencies. 24 h after co-transfection, 293T cells were treated with 250 nM 4-HT for 12 h. Firefly and renilla luciferase activity was recorded on a luminometer using the Dual-Luciferase Reporter Assay System (Promega). Results are expressed as -fold activation relative to empty vector after correction for Renilla luciferase activity and are representative of three independent experiments (performed in triplicate).
Microarray Analysis-RNA was isolated from 4-HT-or vehicle-treated C/EBP-ER K562 cells and purified with RNeasy Column (Qiagen). 5 g of total RNA were used to generate biotin-labeled cRNA using an oligo T7 primer in a reverse transcription reaction followed by in vitro transcription reaction with biotin labeled UTP and CTP. Ten micrograms of cRNA were fragmented and hybridized to HGU133 2.0 Plus arrays (Affymetrix, Santa Clara, CA) representing nearly 50,000 RNA transcripts and variants. Hybridized arrays were stained according to the manufacturer's protocols on a Fluidics Station 450 and scanned on an Affymetrix scanner 3000. All array images were visually inspected for defects and quality. Signal values were determined using the Gene Chip Operating System 1.0 (GCOS, Affymetrix). For each array, all probe sets were normalized to a mean signal intensity value of 100. A qualifier was considered detectable if the mean expression was greater than 50 signal units and the percentage of samples with a Present call, as determined by GCOS default settings, was greater than or equal to 25%. A qualifier was considered to be regulated if the difference between 4-HT-and vehicle-treated samples met the following criteria; 1) the qualifier was detected in at least 25% of the samples in either the vehicle or 4-HT-treated samples, 2) the -fold change was at least 2, and 3) the p value based on an t test was Յ0.05. For identification of the gene associated with specific biological processes, we used EPLtool, an automatic gene clustering tool developed by Wyeth Bioinformatics for Affymetrix qualifiers.
Chromatin Immunoprecipitation-ChIP assays were performed using the EZ-ChIP assay kit (Upstate Biotechnology, Inc.). Briefly, 3 ϫ 10 7 exponentially growing K562 cells (untreated and 4-HT-treated, 24 h) were cross-linked with 1% formaldehyde, incubated for 15 min, and treated with glycine at a final concentration of 125 mM for 5 min at room temperature. Cells were then washed with ice-cold PBS and resuspended in 1 ml of lysis buffer with a protease inhibitor containing mixture and sonicated at 24% power for 12 pulses of 10 s each in a Branson Sonifer 450 (Branson Ultrasonics, Danbury, CT). Chromatin was precleared with 50 l of protein A-agarose beads for 60 min at 4°C with rotation, and precleared lysates were immunoprecipitated with the anti-estrogen receptor ␣ antibody (12 g; SRA-1010 Stressgen) at 4°C overnight with rotation. Immunoprecipitations without antibody (no antibody control) and an anti-rabbit IgG were included with each experiment. Immune complexes were collected with 50 l of protein A-agarose beads for 60 min at 4°C with rotation (except for 10 l of supernatant of the no antibody control, saved as Input) and washed with the buffer recommended in the ChIP protocol. Immune complexes were next eluted using freshly prepared elution buffer (1% SDS and 0.1 M NaHCO 3 ). Cross-links were reversed by heating at 65°C overnight in the presence of 0.2 M NaCl. ChIP DNA (2 l) was next used as a template for real time quantitative-PCR using the following primers: human FOXO3a promoter primers (Ϫ655/Ϫ453), which encompass the putative C/EBP binding site TATTTCCACA at nucleotides Ϫ608 to Ϫ599 identified through both the AliBaba2 and PATCH programs; human FOXO3a promoter primers (Ϫ960/ Ϫ756), which do not contain putative C/EBP binding sites as the negative control (supplemental Table 1); human G-CSFR promoter primers, which include a canonical C/EBP binding site TGTTGCAATC at nucleotide Ϫ55 as the positive control (supplemental Table 1).
Recovered DNA was analyzed by real time quantitative-PCR with the primers in supplemental Table 1 to normalize ChIP-quantitative PCR data were analyzed with the Percent Input Method. Percent input was calculated by 100 ϫ 2( Ct Input Ϫ Ct Enriched ). Statistical Analyses-Data (presented as the means Ϯ S.D. of two or three experiments) were analyzed for statistical significance by the unpaired, 2-tailed Student's t test. p values of less than 0.05 were considered statistically significant.

Biological Effects of Wild-type and Chimeric C/EBP␣/␤ Proteins in K562
Cells-Activation of C/EBP␣-ER in K562 cells induces granulocytic differentiation rapidly and efficiently, whereas C/EBP␤-ER had no effects (33,36). To assess the mechanisms underlying such distinct effects, we generated retroviruses for two chimeric proteins by swapping the C terminus of C/EBP␣ and C/EBP␤ (Fig. 1A) and stably expressed wildtype and chimeric proteins into K562 cells (Fig. 1B). After treatment with 4-HT to activate ectopic C/EBP proteins, we first assessed the effects on K562 cell proliferation by cell counts and DNA content analyses. Compared with empty vector-transduced cells, activation of C/EBP␣-ER or N-C/EBP␣ϩC/EBP␤-DBD-ER led to statistically significant inhibition of proliferation (Fig. 1C). Likewise, DNA content analysis showed a statistically significant increase of G 1 phase cells and a decrease of S and G 2 /M phase cells only after a 72-h activation of C/EBP␣-ER or N-C/EBP␣ϩC/EBP␤-DBD-ER (Fig. 1D).
The effects on differentiation were assessed by morphology and expression of granulocytic markers. Activation of C/EBP␣-ER or N-C/EBP␣ϩC/EBP␤-DBD-ER promoted granulocytic differentiation of K562 cells, whereas activation of C/EBP␤-ER or N-C/EBP␤ϩC/EBP␣-DBD-ER induced the early appearance of metamyelocytes, as indicated by the presence of cells with "doughnut"-shaped nuclei, but the vast majority of these cells failed to undergo terminal differentiation ( Fig. 2A, Table 1).
Analysis of granulocytic differentiation markers confirmed these morphological changes; compared with empty vectortransduced cells, activation of C/EBP␣-ER or N-C/EBP␣ϩC/ EBP␤-DBD-ER led to a statistically significant induction of CD11b and CD15, whereas expression of these two markers did not increase in cells expressing wild-type C/EBP␤-ER or N-C/ EBP␤ϩC/EBP␣-DBD-ER (Fig. 2, B and C, respectively). Moreover, G-CSFR mRNA transcripts were readily detected by real-time or by semiquantitative RT-PCR upon activation of wild-type C/EBP␣-ER or the N-C/EBP␣ϩC/EBP␤-DBD-ER chimera (Fig. 2, D and E, respectively).
Effects of Wild-type and Chimeric C/EBP␣/␤ Proteins on the GCSF-R Promoter-To further assess the effects of wild-type and chimeric C/EBP␣ and C/EBP␤ proteins, we performed luciferase assays on the C/EBP␣-responsive G-CSFR promoter. After co-transfection of 293T cells with the G-CSFR promoterluciferase reporter (G-CSFR/Luc) plasmid, the Renilla luciferase control plasmid, to account for variation in transfection efficiencies and C/EBP plasmids or the MigRI empty vector, cells were treated with 4-HT for 12 h, and luciferase activity was measured thereafter. Results are expressed as -fold activation relative to vector alone after normalization for Renilla luciferase activity (Fig. 3A). C/EBP␣ and N-C/EBP␣ϩC/EBP␤-DBD transactivated the G-CSFR/Luc plasmid more effectively than C/EBP␤ or the N-C/EBP␤ϩC/EBP␣-DBD chimera; however, the results of these assays do not allow us to exclude differences in DNA binding that could account for the distinct effects of the chimeric proteins. Thus, quantitative ChIP assays were performed upon activation of C/EBP-ER proteins in K562 cells. As shown in Fig. 3B, quantitative PCR showed comparable binding of C/EBP␣, C/EBP␤, and the chimeric proteins to a segment of the G-CSFR promoter containing a canonical C/EBP␣ binding site, in sharp contrast with the inability of C/EBP␤ N terminus containing proteins to activate G-CSFR expression (Fig. 2, D  and E).
Effects of VP16-C/EBP␣/␤-DBD-ER Chimeric Proteins on the GCSF-R Promoter and the Proliferation and Differentiation of K562 Cells-To further address the possibility that the more potent biologic effects of C/EBP␣ depend on the stronger activity of its TAD more than on different specificities of the DBD, we generated K562 cell lines expressing chimeric proteins consisting of the VP16 TAD juxtaposed to the DBD of C/EBP␣ or C/EBP␤ fused to the tamoxifen-responsive estrogen receptor ligand binding domain (ER TAM ) (Fig. 4, A and B).
The herpes simplex virus type 1 (HSV-1) tegument protein VP16 is a structural protein of HSV-1 that activates transcription of the immediate early promoters of the virus (37,38). Although VP16 specifically activates promoters containing the so-called TAATGARAT element, specificity is conferred by the cellular DNA-binding protein(s) fused to the N-terminal domain of VP16 (39,40).
To assess the transactivation ability of the two VP16-C/ EBPs-DBD-ER chimeric proteins, we performed luciferase assays on the C/EBP␣-responsive G-CSFR promoter. After co-transfection of 293T cells with the G-CSFR/Luc plasmid, the Renilla luciferase control plasmid to account for variation in transfection efficiency, VP16-C/EBPs-DBD-ER expression vectors, or the MigRI empty vector, cells were treated with 4-HT for 12 h, and luciferase activity was measured thereafter. Results are expressed as -fold activation relative to vector only-transfected cells after normalization for Renilla luciferase activity. The assays show that the two chimeric proteins are essentially undistinguishable in the ability to transactivate the C/EBP␣-responsive G-CSFR promoter (Fig. 4C).

JOURNAL OF BIOLOGICAL CHEMISTRY 30841
We also assessed cell cycle distribution and methylcellulose colony formation of untreated and 4-HT-treated VP16-C/ EBP␣/␤-DBD-ER/K562 cells. After a 72-h treatment with 4-HT, either chimeric protein induced a marked increase in the number of G 1 cells and a decrease in the fraction of S and G 2 /M phase cells (Fig. 5D); likewise, both proteins markedly suppressed methylcellulose colony formation of K562 cells (Fig.  5E). Of note, the effects of the VP16-C/EBP␣/␤-DBD-ER proteins appear to be undistinguishable from those of wild-type C/EBP␣-ER and N-C/EBP␣ϩC/EBP␤-DBD-ER (compare Fig.  1 and Fig. 5).
As control, we also generated aVP16-C/EBP␣K298E-DBD-ERexpression plasmid in which the K298E mutation prevents DNA binding (27) to assess whether the effects of the chimeric VP16-C/EBP DBD-ER proteins were DNA binding-dependent. As shown in supplemental Fig. 1, activation of this chimeric protein failed to induce granulocytic differentiation or to suppress colony formation of K562 cells. Together, these studies further confirm that the markedly different biological effects of C/EBP␣ and C/EBP␤ in K562 cells depend on the potency of their transactivation domain.
Gene Expression Profiles Regulated by Wild-type and Chimeric C/EBP␣/␤ Proteins-The different transactivation activities of wildtype and chimeric C/EBP␣/␤ proteins should be reflected in the modulation of distinct patterns of gene expression; thus, we performed microarray hybridization assays using RNA of untreated and 4-HT-treated (12 h) C/EBP-ER K562 cells to assess overall patterns of gene expression and specific gene subsets regulated by wild-type and chimeric C/EBP␣/␤ proteins.
On analysis of the gene expression profiles modulated by the proteins that share the same DBD, C/EBP␣, and N-C/ EBP␤ϩC/EBP␣-DBD regulated 461 (2402;2211) common genes, whereas C/EBP␤ and N-C/EBP␣ϩC/EBP␤-DBD regulated 395 (2112;1841) common genes. 115 of these genes qualified as differentially expressed in all four cell lines (supplemental Fig. 2, panel C). Although a subset of genes is regulated by both C/EBP␣ and C/EBP␤, it is clear that C/EBP␣ is a more effective gene expression modulator than C/EBP␤ and that this higher activity is probably due to a more potent TAD, as the C/EBP␣and N-C/EBP␣ϩC/EBP␤-DBD-regulated gene expression profiles are very similar.
To validate the microarray hybridization data, expression of some genes differentially regulated by wild-type and chimeric C/EBP␣/␤ proteins in K562 cells was assessed by real time quantitative PCR; as shown in supplemental Fig. 3, levels of cyclin D1, cyclin D3, CDK6, and JAK2 mRNA were differentially modulated by the wild-type and chimeric C/EBP proteins, in agreement with the results of the microarray hybridization data.
We then asked to which functional groups the C/EBP-regulated genes belonged in order to correlate gene expression profiles and biological effects induced by wild-type and chimeric C/EBP␣/␤ proteins. Within the group of cell cycle-related genes, C/EBP␣ and N-C/EBP␣ϩC/EBP␤-DBD modulated the expression of 114 and 81 cell cycle-related genes, respectively; of these, 58 were unique for C/EBP␣ and 35 for N-C/EBP␣ϩC/ EBP␤-DBD chimeric protein, whereas 26 were modulated by both proteins. C/EBP␤ and N-C/EBP␤ϩC/EBP␣-DBD regulated 56 and 61 cell cycle-related genes, respectively; of these, 20 were unique for C/EBP␤ and 37 for the N-C/EBP␤ϩC/EBP␣-DBD chimeric protein, whereas 5 were regulated by both pro-teins, suggesting that the more potent inhibitory effects of C/EBP␣ and the N-C/EBP␣ϩC/EBP␤-DBD chimeric protein in proliferation and colony formation of K562 cells probably depend on a more complex pattern of cell cycle-related gene regulation.
The Effect of C/EBP␣ in Apoptosis Is, in Part, Dependent on FOXO3a Expression-Although a pro-apoptotic effect was not discerned upon C/EBP␣ activation in BCR/ABL-expressing cells in vitro and in leukemic mice (27), the more complex subset of apoptosis-related genes regulated by C/EBP␣ raised the possibility that, under certain circumstances, C/EBP␣ activation could also inhibit cell survival, possibly enhancing its antileukemia effects.
To address this possibility, K562 cells expressing C/EBP␣, C/EBP␤, or the chimeric proteins were pretreated for 12 h with a suboptimal concentration of IM (0.2 M) followed by incubation with 250 nM 4-HT, and apoptosis was assessed by propidium iodide staining to detect cells with hypodiploid DNA content. As shown in Fig. 6A, only activation of C/EBP␣ led to a statistically significant increase in the number of apoptotic cells. Among apoptosis-related genes modulated by C/EBP␣, it

Frequency of undifferentiated and differentiated myeloid cells in 4HT-treated C/EBP ER-transduced K562 cells
Numbers in parentheses are the number of cells.  OCTOBER 1, 2010 • VOLUME 285 • NUMBER 40 seemed especially interesting to further assess the role of FOXO3a, a transcription factor of the FoxO family that appears to play an important role in cell cycle arrest and apoptosis of normal and BCR/ABL-expressing hematopoietic cells (41)(42)(43). From the microarray data, FOXO 3a was differentially regulated by C/EBP␣, C/EBP␤, and N-C/EBP␣ϩC/EBP␤-DBD, but only C/EBP␣ was able to enhance the expression of five different FOXO3a qualifiers, one of which showed a 2.5-fold increase. Indeed, upon a 12-h 4-HT treatment of K562 cells expressing C/EBP␣, C/EBP␤, or the chimeric proteins, expression of FOXO3a mRNA was readily induced only by C/EBP␣ (Fig. 6B); the increased expression of FOXO3a was confirmed by anti-FOXO3a Western blotting (Fig. 6C). Of interest, both VP16-C/EBP␣/␤-DBD-ER chimeric proteins failed to activate FOXO3a mRNA expression (supplemental Fig. 4).

Biological Effect of C/EBP␣/␤ Chimera in K562 Cells
To assess whether FOXO3a is directly regulated by C/EBP␣, FOXO3a mRNA levels were measured in K562 cells treated with 250 nM 4-HT (to activate C/EBP␣) in the presence of the protein synthesis inhibitor cycloheximide. In control experiments using K562 cells transduced with the MigRI empty vector, co-treatment with 4-HT and cycloheximide had no effect on FOXO3a mRNA levels (not shown); by contrast, expression of FOXO3a mRNA was rapidly induced after C/EBP␣activation, and this early increase was not prevented by a 4-h treatment with cycloheximide (CHX, Fig. 6D), consistent with FOXO3a being a direct target of C/EBP␣.
Additional proof in support of FOXO3a being a transcriptional target of C/EBP␣ was obtained by ChIP assays; a search for C/EBP binding sites in the 5Ј-flanking region of the human FOXO3a gene identified a putative binding site at nucleotides Ϫ608 to Ϫ599 (underlined in Fig. 6E). PCR amplification of a segment including this site after anti-ER ChIP from 4-HT-treated K562 cells expressing wild-type or chimeric C/EBP␣-C/EBP␤ proteins demonstrated that each of these proteins was able to interact with the segment (nucleotides Ϫ655 to Ϫ453) of the FOXO3a promoter (Fig. 6E), although only C/EBP␣ was able to activate FOXO3a mRNA expression (Fig. 6B). C/EBP proteins did not interact with a more distal segment of the FOXO3a promoter lacking putative C/EBP binding sites (not shown).
To assess if the enhanced propensity of IM-treated C/EBP␣-expressing K562 cells to undergo apoptosis was associated with the ability of C/EBP␣ to induce FOXO3a expression, levels of FOXO3A were down-regulated by RNAi in empty vector-transduced or C/EBP␣-ER-expressing K562 cells (Fig. 7A), and the frequency of apoptotic cells was assessed after treatment with a suboptimal concentration of IM (0.2 M). Down-regulation of FOXO3a expression suppressed the increase in apoptosis induced by co-treatment of C/EBP␣-ER-expressing K562 cells with 4-HT and IM (Fig. 7B). Thus, under certain conditions (i.e. IM or chemotherapy activation of apoptotic pathways), C/EBP␣ may enhance apoptosis through FOXO3a-dependent mechanisms.
FOXO3a Expression Is Not Required for C/EBP␣-dependent Granulocytic Differentiation and Proliferation Inhibition in K562 Cells-Because expression of constitutively active FOXO3a induces erythroid differentiation of K562 cells (44), we also assessed the role of C/EBP␣-dependent FOXO3a expression in C/EBP␣-induced granulocytic differentiation of K562 cells. Thus, FOXO3a expression was down-regulated by RNAi in C/EBP␣-ER-expressing K562 cells (supplemental Fig. 5A), and the induction of granulocytic differentiation was assessed upon 4-HT treatment. K562 cells transfected with scramble or FOXO3a-specific shRNAs underwent granulocytic differentiation as indicated by morphology (supplemental Fig. 5B) and expression of the myeloid differentiation marker CD11b (supplemental Fig. 5C).
The role of FOXO3a expression for the proliferation inhibitory effects of C/EBP␣ were tested by assessing cell counts, cell cycle distribution, and methylcellulose colony formation of untreated and 4-HT-treated K562 cells transfected with scramble or FOXO3a specific shRNAs. Activation of C/EBP␣ suppressed proliferation (supplemental Fig. 5D) and induced a marked increase in the number of G 1 cells and a decrease in the fraction of S and G 2 /M phase cells (supplemental Fig. 5E) as effectively in scrambled and in FOXO3a shRNA-transfected cells; likewise, down-regulation of FOXO3a expression did not rescue the inhibition of methylcellulose colony formation induced by expression of functional C/EBP␣ in K562 cells (supplemental Fig. 5F).

DISCUSSION
Expression of C/EBP␣ and C/EBP␤ is reduced in BCR/ABLtransformed cell lines and in blast cells from patients with CML-blast crisis (27,28), suggesting that loss of C/EBP activity is necessary to suppress the differentiation potential of CML-blast crisis progenitors. Consistent with this hypothesis, ectopic expression of C/EBP␣ or C/EBP␤ induced granulocytic differentiation and inhibited proliferation of BCR/ABL-expressing cells in vitro and in mice, but the effects of C/EBP␤ were considerably less potent than those of C/EBP␣ (27,28). This contrasts with the apparent complete rescue of granulocytic differentiation in mice in which the C/EBP␣ gene was replaced by C/EBP␤ (9) and is probably due to the severe block of differentiation induced by BCR/ABL. Because C/EBP␣ and C/EBP␤ are highly homologous in their C terminus, where basic region, DNA binding, and leucine zipper domain reside, but are highly divergent in the N terminus, where the TAD is located, we took a domain-swapping approach to evaluate biological effects and gene expression profiles in K562 cells expressing 4-HT-regulated C/EBP␣-C/EBP␤ chimeric proteins.
Activation of C/EBP␣ or of the chimeric protein N-C/EBP␣ϩC/ EBP␤-DBD that shares the TAD of wild-type C/EBP␣, suppressed proliferation and promoted an increase in the number of G 1 phase cells, whereas C/EBP␤ or N-C/EBP␤ϩC/EBP␣-DBD-ER that shares the TAD of wildtype C/EBP␤ did not. Likewise, activation of C/EBP␣ and N-C/ EBP␣ϩC/EBP␤-DBD led to efficient granulocytic differentiation of K562 cells, whereas C/EBP␤ and N-C/EBP␤ϩC/EBP␣-DBD-ER did not. These effects correlated with those on the transactivation of the G-CSFR promoter. However, ChIP assays did not reveal any difference in the ability of C/EBP␣, C/EBP␤, or the chimeric proteins to interact with the G-CSFR promoter, suggesting that the TAD of C/EBP␣ is more potent of that of C/EBP␤, perhaps through the recruitment of interacting proteins capable of enhancing its transcription activation function.
In an attempt to distinguish between the role of C/EBP␣ N terminus-specific interactions versus TAD strength, we the assessed biological effects (induction of differentiation and inhibition of proliferation) and transcription activation of chimeric proteins consisting of the same N terminus (the VP16 TAD) fused to the C/EBP␣ or C/EBP␤ DBD. The effects of these two chimeric proteins were undistinguishable, confirming that the DBD does not confer specificity in modulating the transcription-dependent program required for proliferation inhibition and differentiation induction in K562 cells. Of equal importance, the effects of the VP16-C/ EBP␣/␤-DBD chimeric proteins were identical to those of wild-type C/EBP␣-or N-C/EBP␣ϩC/EBP␤-DBD, indicating that the gene expression program responsible for the ability of C/EBP␣ to induce granulocytic differentiation and to inhibit proliferation of K562 cells can be faithfully recapitulated by a chimeric protein in which the C/EBP DBD (which determines specificity of gene targets) is under the control of a strong TAD. Of interest, mutation of the DNA binding domain suppressed differentiation induction and colony formation inhibition by the VP16-C/EBP-DBD chimera, suggesting that C/EBP␣ can suppress proliferation through both transcriptional mechanisms and the interaction with cell cycle regulatory proteins (30 -33). The differences in the biological and biochemical effects of C/EBP␣, C/EBP␤, and chimeric proteins were reflected in the activation of distinct gene expression profiles, although a subset of genes was modulated by each protein.
The gene expression profile modulated by C/EBP␣ and N-C/ EBP␣ϩC/EBP␤-DBD was more complex than that regulated by C/EBP␤ and N-C/EBP␤ϩC/EBP␣-DBD, most likely reflecting the higher transactivation activity of C/EBP␣ and N-C/ EBP␣ϩC/EBP␤-DBD; however, the number of C/EBP␣-regulated genes was higher of that modulated by N-C/EBP␣ϩC/ EBP␤-DBD, raising the possibility that certain changes in gene expression may be due to a DBD-specific effect or to loss of C/EBP␣ N terminus protein-protein interactions in the N-C/ EBP␣ϩC/EBP␤-DBD chimeric protein.
Because gene expression profiles modulated by C/EBP proteins were analyzed 12 h post-4-HT treatment, the difference in the number of C/EBP␣and N-C/EBP␣ϩC/EBP␤-DBD-regulated genes might have been magnified by secondary changes possibly due to protein-protein interactions in the N terminus or DBD target specificity, although this would be inconsequential for the ability of N-C/EBP␣ϩC/EBP␤-DBD to inhibit proliferation and induce differentiation of K562 cells as effectively as wild-type C/EBP␣. Functional analysis of the gene subsets regulated by C/EBP␣, C/EBP␤, and the two chimeric proteins revealed that genes in the apoptosis pathway were especially modulated by C/EBP␣; this finding was of interest because activation of C/EBP␣, per se, does not promote apoptosis of BCR/ABL-expressing cells (27), but it enhanced IM-induced cell death of K562 cells. One of the genes regulated by C/EBP␣ and potentially required for its biological effects is FOXO3a, a transcription factor of the FoxO family with crucial roles in regulating the proliferation and survival of hematopoietic stem cells (41).
Of interest, expression of FOXO3a was not activated by N-C/ EBP␣-C/EBP␤-DBD or by the VP16-C/EBP-DBD chimeric proteins; this suggests that the transcription of some C/EBP␣ targets depends not only on the potency of the transactivation domain but also on specific protein interactions that may not be limited to the N-terminal region, although it cannot be excluded that changes in conformation of the chimeric proteins may have affected the interaction with cofactors necessary for FOXO3a transcription.
Although we did not demonstrate that C/EBP␣ transactivates the FOXO3a promoter, a direct role of C/EBP␣ in enhancing FOXO3a expression is suggested by ChIP assays demonstrating interaction with a C/EBP binding site in the human FOXO3a promoter and by the finding that C/EBP␣ induced FOXO3a mRNA expression in cells treated with cycloheximide to block de novo protein synthesis.
Recent studies have revealed that BCR-ABL inhibits FOXO3a activity via Akt-dependent phosphorylation, which promotes interaction with 14-3-3 protein and sequestration of FOXO3a into the cytoplasm (42,43), leading to lower expression of pro-apoptotic genes such TRAIL and BIM (44); furthermore, FOXO3A inhibition by BCR-ABL also affects the expression of cyclin D1 and D2 (45,46). Consistent with the pro-apoptotic effects of FOXO3a, downregulation of FOXO3A expression by RNAi blocked the increase in apoptosis induced by C/EBP␣ activation in IM-treated K562 cells. Based on the finding that expression of a constitutively active mutant of FOXO3a induced erythroid differentiation of K562 cells (47) and that proliferation regulatory  genes are among the FOXO3A targets (45,46), we suspected that FOXO3a was also involved in C/EBP␣-regulated granulocytic differentiation and proliferation of K562 cells; however, FOXO3a silencing did not rescue either effect, indicating that distinct C/EBP␣-regulated genes function as essential downstream effectors to enhance apoptosis susceptibility, promote differentiation, and inhibit proliferation. It remains to be established whether differentiation induction and proliferation inhibition are regulated by common C/EBP␣ target genes or are under the control of separate effectors.
In summary, we have used a C/EBP␣-C/EBP␤ domain swapping approach to demonstrate that (i) a strong TAD is responsible for the biological effects of C/EBP␣ in K562 cells, and (ii) the effects correlate with C/EBP␣-dependent activation of gene expression profiles, which include many cell cycle and apoptosis regulatory genes.
Although activation of C/EBP␣ is insufficient to induce apoptosis of BCR/ABL-expressing cells, it enhanced IM-induced cell death via expression of FOXO3a, further emphasizing the therapeutic potential of restoring functional C/EBP␣ alone or together with other anti-cancer agents in CML-blast crisis and perhaps other types of acute myeloid leukemia.