Oncogene (2009) 28, 3235–3245 & 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00 www.nature.com/onc ORIGINAL ARTICLE C/EBPa expression is partially regulated by C/EBPb in response to DNA damage and C/EBPa-deficient fibroblasts display an impaired G1 checkpoint

R Ranjan1, EA Thompson1, K Yoon2 and RC Smart1

1Cell Signaling and Cancer Group, Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, NC, USA and 2National Cancer Center, Division of Common Cancers, Lung Cancer Branch, Goyang-si, Gyeonggi-do, South Korea

We observed that CCAAT/-binding involved in homo- or hetero-dimerization (Ramji and (C/EBP)a is highly inducible in primary fibroblasts by Foka, 2002). The N-terminal region contains transcrip- DNA-damaging agents that induce strand breaks, alky- tion activation and regulatory domains that interact late and crosslink DNA as well as those that produce with basal apparatus and transcription bulky DNA lesions. Fibroblasts deficient in C/EBPa co-activators. There are six members of the C/EBP family À/À (C/EBPa ) display an impaired G1 checkpoint as and C/EBPs have important functions in fundamental evidenced by an inappropriate entry into the S-phase in cellular processes, including proliferation, apoptosis, response to DNA damage, and these cells also display an differentiation, inflammation, senescence and energy enhanced G1/S transition in response to mitogens. The metabolism (Ramji and Foka, 2002; Johnson, 2005). induction of C/EBPa by DNA damage in fibroblasts does C/EBPa mediates cell-cycle arrest associated with not require p53. Electrophoretic mobility shift assay terminal differentiation in adipocytes (Umek et al., (EMSA) analysis of nuclear extracts prepared from 1991; Lin and Lane, 1994), hepatocytes (Diehl et al., ultraviolet B (UVB)- and N-methyl-N0-nitro-N-nitroso- 1996) and myeloid cells (Radomska et al., 1998; Wang guanidine (MNNG)-treated fibroblasts showed increased et al., 1999) and it regulates the expression of binding of C/EBPb to a C/EBP associated with the differentiated phenotype in these cell and chromatin immunoprecipitation (ChIP) analysis also types. Consistent with its role in the regulation of showed increased C/EBPb binding to the C/EBPa differentiation, C/EBPa has been shown to be a tumor . To determine whether C/EBPb has a function suppressor in acute myeloid leukemia, in which it is in the regulation of C/EBPa, we treated C/EBPbÀ/À inactivated in B9% of AML cases through specific fibroblasts with UVB or MNNG. We observed that somatic (Pabst et al., 2001; Gombart et al., C/EBPa induction was impaired in both UVB- and 2002). The inactivating mutations in C/EBPa result MNNG-treated C/EBPbÀ/À fibroblasts. Our study shows in a block in granulocytic differentiation and contribute a novel function for C/EBPb in the regulation of C/EBPa to the uncontrolled proliferation of undifferentiated in response to DNA damage and provides definitive immature granulocytic blasts. Ectopic or forced expres- genetic evidence that C/EBPa has a critical role in the sion of C/EBPa inhibits cell-cycle progression in nearly DNA damage G1 checkpoint. all cell types examined (Hendricks-Taylor and Darling- Oncogene (2009) 28, 3235–3245; doi:10.1038/onc.2009.176; ton, 1995; Watkins et al., 1996; Halmos et al., 2002; published online 6 July 2009 Johnson, 2005; Shim et al., 2005); however, the intrinsic cellular signals and pathways that regulate C/EBPa Keywords: C/EBP; DNA damage response; G1/S transi- expression and its growth arrest properties are not fully tion; p53 understood. In some cases, these pathways are linked to cellular differentiation. C/EBPa expression is ablated or greatly diminished in a number of epithelial cancers, including lung (Halmos Introduction et al., 2002), skin (Oh and Smart, 1998; Shim et al., 2005), liver (Xu et al., 2001), head and neck (Bennett The CCAAT/enhancer-binding (C/EBPs) are et al., 2007), endometrial (Takai et al., 2005) and breast members of the basic leucine zipper (bZIP) class of (Gery et al., 2005), suggesting a tumor suppressor transcription factors that contain a C-terminal basic function. In many cases, the C/EBPa gene is silenced DNA-binding domain and a leucine zipper domain through promoter hypermethylation (Gery et al., 2005; Tada et al., 2006; Bennett et al., 2007). Causal or genetic evidence for a suppressor role of C/EBPa in epithelial Correspondence: Dr RC Smart, Cell Signaling and Cancer Group, tumorigenesis is lacking because of the absence of Department of Environmental and Molecular Toxicology, North C/EBPa mutations in epithelial tumors. Moreover, Carolina State University, Raleigh, NC 27695-7633, USA. E-mail: [email protected] genetically engineered mouse models to document the Received 23 December 2008; revised 23 May 2009; accepted 24 May suppressor function of C/EBPa in tumorigenesis 2009; published online 6 July 2009 have been problematic as germ line or lung-specific C/EBPa is regulated by C/EBPb in response to DNA damage R Ranjan et al 3236 deletion of C/EBPa is perinatally lethal (Wang et al., 1995; types of DNA damage with the induction of C/EBPa, Basseres et al., 2006). Recently, a genetically engineered and we present genetic evidence using C/EBPaÀ/À cells to mouse model in which C/EBPa was ablated in the show a role for C/EBPa in the G1 checkpoint. epidermis was successfully developed (Loomis et al., Importantly, we have identified a novel stress pathway, 2007). These mice survived and were highly susceptible in which C/EBPb has a role in the induction of C/EBPa to carcinogen-induced skin tumorigenesis, thus provid- in response to DNA damage. ing the first genetic evidence for C/EBPa as a suppressor of epithelial tumorigenesis. Surprisingly, the epidermal- specific C/EBPa knockout mice did not show alterations Results in stratified squamous differentiation or proliferation of epidermal keratinocytes, suggesting that their enhanced UVB induces C/EBPa in fibroblasts and C/EBPaÀ/À susceptibility to tumorigenesis is not related to alterations fibroblasts display alterations in the G1/S transition in keratinocyte differentiation (Loomis et al., 2007). and G1 checkpoint To prevent or reduce stress-induced injury and Initially, we attempted cell-cycle studies in wild-type cellular damage, cells have evolved intricate pathways and C/EBPaÀ/À primary keratinocytes; however, these that permit them to respond to both intrinsic and studies were not informative because of the inherent extrinsic stressors. In terms of DNA damage, cells complexity of using primary keratinocytes for cell-cycle respond by engaging cell-cycle checkpoints and repair- regulation studies owing to the presence of mixed ing damaged DNA in order to maintain genome populations of differentiating and proliferating primary integrity and to prevent heritable mutations, which can keratinocytes. To provide direct genetic evidence for lead to genomic instability, aging and cancer (Kastan C/EBPa in the G1 checkpoint and to extend the findings and Bartek, 2004; Sancar et al., 2004; Ishikawa et al., on UVB-induced expression of C/EBPa in keratinocytes 2006). In ultraviolet B (UVB)-treated skin keratinocytes, to another cell type, mouse dermal wild-type and C/EBPa expression is highly induced through a p53- C/EBPaÀ/À primary fibroblasts were used. Wild-type dependent pathway and the partial small interfering and C/EBPaÀ/À primary fibroblasts were exposed to a RNA (siRNA) knockdown of C/EBPa in the BALB/ single dose of UVB radiation (5 mJ/cm2). UVB radiation MK2 keratinocyte cell line resulted in a diminished produced a significant increase in C/EBPa protein levels G1 checkpoint after UVB-induced DNA damage in wild-type fibroblasts (Figure 1a) and C/EBPa was (Yoon and Smart, 2004). Although C/EBPa was highly not expressed in C/EBPaÀ/À fibroblasts (Figure 1b). In induced by UVB in keratinocytes, UVB treatment wild-type fibroblasts, C/EBPa was maximally induced at of HepG2, NRK and NIH3T3 cells failed to induce 6 h post UVB treatment and returned to control levels C/EBPa. Thus, C/EBPa, at least in keratinocytes, is by 24 h post UVB treatment. Elevated levels of C/EBPa regulated by stress involving DNA damage through a were detected as early as 1 h post UVB treatment (data p53 pathway, and it appears to have a role in the not shown). UVB also induced a modest transient maintenance of genomic stability through its role in the increase in C/EBPb at 6 h post UVB treatment G1 checkpoint. However, genetic evidence supporting a (Figure 1a). role for C/EBPa in the G1 checkpoint is lacking, and as To examine the effect of the genetic ablation of mentioned above, no other cell types other than C/EBPa on cell-cycle regulation, wild-type and keratinocytes have been reported to respond to DNA C/EBPaÀ/À and primary fibroblasts were synchronized damage with the induction of C/EBPa. by serum deprivation and then released into the cell In light of C/EBPa’s role in tumorigenesis and DNA cycle by the addition of serum-containing media. damage response, it is important to understand the Fibroblasts were either left untreated or treated with a stress pathways or mechanisms through which C/EBPa single dose of UVB (5 mJ/cm2) 4 h after release. Cells is regulated in response to DNA damage and the were pulsed with 5-bromo-20-deoxyuridine (BrdU) 1 h functional importance of C/EBPa’s induction. In this before each collection time point (4, 15, 18, 21 and 24 h study, we show that fibroblasts respond to multiple post release) and fluorescence-activated cell sorting

Figure 1 Ultraviolet B (UVB) radiation induces C/EBPa in primary fibroblasts and C/EBPa is involved in the G1/S transition as well as in 2 UVB-induced G1 checkpoint. (a) Primary fibroblasts were exposed to UVB (5 mJ/cm ), and cell lysates were prepared at various time points and immunoblot analysis was conducted. Nonspecific (NS) band is shown to confirm equal loading. (b) Primary fibroblasts from newborn wild-type or C/EBPaÀ/À were treated with UVB (5 mJ/cm2), and cell lysates were prepared at various time points and immunoblot analysis was conducted. (c and d) Wild-type (open column) and C/EBPaÀ/À (black column) fibroblasts were synchronized by serum deprivation for 28 h in 0.5% serum and then released into the cell cycle by the addition of serum-containing medium. Fibroblasts were either not treated (c) or treated with UVB (5 mJ/cm2) (d) at 4 h after the addition of serum-containing medium. Cells were pulse labeled with 5-bromo-20-deoxyuridine (BrdU) 1 h before collection, and then the cells were fixed, incubated with anti-BrdU antibody, stained with propidium iodine (PI) and subjected to FACS analysis. The number above each column pair represents the percentage increase in S-phase cells in C/EBPaÀ/À fibroblasts compared with wild type. Data represent mean±s.d (N ¼ 3 per time point per genotype). Two-factor ANOVA showed significant interaction between genotype and time for both untreated and treated cells (Po0.05). Significantly different from wild-type fibroblasts (*Po0.05) at the indicated time point as determined by Student’s t-test. (e) Representative scatter plots for untreated fibroblasts (for panel c) after release, showing the mean percentage of cells (N ¼ 3 per time point per genotype) in G1 (lower left), S (top), and G2M (lower right) phase of the cell cycle. (f) Representative scatter plots for UVB-treated fibroblasts (for panel d) showing the mean percentage of cells (N ¼ 3 per time point per genotype) in G1, S and G2/M phase. The data presented represent one of the four independent experiments all showing similar results.

Oncogene C/EBPa is regulated by C/EBPb in response to DNA damage R Ranjan et al 3237 (FACS) analysis was conducted to monitor entry into S- C/EBPaÀ/À and wild-type fibroblasts treated with UVB phase. Serum-released C/EBPaÀ/À and wild-type fibro- showed a significant decrease in the number of S-phase blasts not treated with UVB displayed a synchronized cells at 15 and 18 h compared with untreated control entry into S-phase. However, C/EBPaÀ/À fibroblasts cells and this decrease was followed by a recovery at 21 consistently displayed 10–20% more cells in and 24 h, indicating that cells from both genotypes S-phase than wild-type fibroblasts, indicating that engaged a G1 checkpoint response. However, UVB- C/EBPaÀ/À cells have an enhanced mitogen-induced treated C/EBPaÀ/À fibroblasts showed a significantly entry into S-phase (Figures 1c and e). Serum-released attenuated G1 checkpoint, as there were B70% more

∗ ∗ ∗ 20.1% 12.2% 41.9% Time (h) 0 6 12 18 24 ∗ α 56.6% C/EBP 70 70 ∗ C/EBPβ 60 60 19.9% ∗ NS 67.4% 50 50 UVB (5 mJ/cm2) 10.0% 40 40 ∗ 74.7% Time (h) 066 0 30 30 ∗ ∗ C/EBPα 20 -23.1% 20 -23.3% % of cells into S-phase C/EBPβ % of cells into S-phase 10 10 α-tubulin 0 0 4 15182124 h 4 15 18 21 24 h -/- Wild type C/EBPα Time after release (NO UVB) Time after release (UVB) UVB (5 mJ/cm2)

4 4 4 4 10 10 10 104 10 3.5% 50.6% 54.1% 32.0% 21.9% 3 3 3 3 10 10 10 103 10

2 2 2 2 10 10 10 102 10 Wild type

1 1 1 10 101 10 101 10 8.5%43.6% 5.6% 35.0% 10.7%48.9% 19.0% 65.6% 12.5% 87.8% 0 0 100 100 10 100 10 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 Propidium iodide-A Propidium iodide-A Propidium iodide-A Propidium iodide-A Propidium iodide-A 4 104 104 104 104 10 2.7% 60.8% 60.7% 38.3% 24.3% (NO UVB) Anti-BrdU 103 103 103 103 103

2 C/EBPα-/- 102 102 102 10 102

1 1 1 101 10 10 10 101 92.6%4.5% 36.5% 2.5% 29.1% 10.0% 42.5% 19.1% 64.0% 11.5% 0 0 0 100 10 10 10 100 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 Propidium iodide-A Propidium iodide-A Propidium iodide-A Propidium iodide-A Propidium iodide-A Time (h) 415182124

PropidiumIodide

4 4 4 104 104 10 10 10 3.5% 8.3% 20.3% 32.4% 41.3% 103 103 103 103 103

102 2 2 2 2 10 10 10 10 Wild type

1 1 1 1 10 10 101 10 10

0 87.8% 8.5% 83.0% 8.6% 72.0% 7.6% 6.9% 52.1% 6.4% 10 0 0 60.5% 0 ) 10 100 10 10 2 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 Propidium iodide-A Propidium iodide-A Propidium iodide-A Propidium iodide-A Propidium iodide-A

4 4 4 4 10 10 104 10 10 50.8% 58.6% 2.7% 14.6% 34.0% (UVB 5 mJ/cm Anti-BrdU 3 3 3 103 10 10 103 10

2 102 102 2 102 10 10 C/EBPα-/-

1 101 101 101 101 10 92.6% 4.5%80.4% 4.8% 62.6% 3.2% 46.3% 2.8% 35.6% 9.3% 100 100 100 100 100 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 Propidium iodide-A Propidium iodide-A Propidium iodide-A Propidium iodide-A Propidium iodide-A Time (h) 415182124

Propidium Iodide

Oncogene C/EBPa is regulated by C/EBPb in response to DNA damage R Ranjan et al 3238 aeBleomycin Time (h) 0 6 12 18 24 10 20 (µg/ml) Time (h) 0 6 126 12 5 mJ/cm2

CEBPα 10 mJ/cm2

α 20 mJ/cm2 -Tubulin

b UVB MNNG f No treatment UVB (6 h) Time (h) 0 6 12 18 6 12 18 01 2 4 0 1 2 4 CHX(h)

C/EBPα C/EBPα

N S C/EBPβ

Cisplatin c α-tubulin Conc (µM) 0 80 160 C/EBPα g 2.5

α * α-Tubulin 2 * d Camptothecin 1.5 1 3 (µM) 1 Time (h) 0 6 12 6 12 mRNA level C/EBPα 0.5

α-Tubulin Fold induction in C/EBP 0 0 6 12 18 Time (h) after UVB Figure 2 DNA-damaging agents induce C/EBPa in primary fibroblasts. (a) Primary fibroblasts were exposed to different doses of Ultraviolet B (UVB), and cell lysates were prepared at indicated time points and immunoblot analysis conducted. (b–e) Primary fibroblasts were treated with UVB (10 mJ/cm2), N-methyl-N0-nitro-N-nitrosoguanidine (MNNG) (35 mM), cisplatin (80 and 160 mM), camptothecin (1 and 3 mM), bleomycin (10 and 20 mg/ml), dimethyl sulfoxide (DMSO) or water alone, and immunoblot analysis was conducted. Nonspecific (NS) and a-tubulin band is shown to confirm equal loading. (f) Fibroblasts were either not treated (left panel) or treated (right panel) with UVB and 6 h later they were incubated with cycloheximide (50 mg/ml). Cells were harvested at indicated time points after the start of cycloheximide treatment and immunoblot analysis was conducted. (g) Total RNA was isolated from fibroblasts at different time points after UVB (5 mJ/cm2) treatment. Quantitative RT–PCR was conducted for C/EBPa and 18S mRNA levels. Data were normalized to 18S and analyzed using the comparative CT method. Data are expressed as mean±standard error (N ¼ 3) and each experiment was run in triplicate. Significantly different from time zero (*Po0.05) as determined by Student’s t-test.

C/EBPaÀ/À cells in S-phase at 15 and 18 h than similarly To determine whether the increases in UVB-induced treated wild-type cells, indicating inappropriate entry C/EBPa protein levels involve altered stability of into S-phase (Figures 1d and f). Collectively, these C/EBPa protein, untreated and UVB-treated fibroblasts results show that C/EBPa is highly induced by UVB in were incubated with cycloheximide, an inhibitor of fibroblasts and the genetic ablation of C/EBPa results protein synthesis, and the stability of the C/EBPa in an enhanced mitogen-induced G1/S transition in protein was examined over time. The degradation of untreated cells and a significantly diminished G1 C/EBPa protein was similar in both untreated checkpoint in response to DNA damage. (Figure 2f, left panel) and UVB-treated fibroblasts (Figure 2f, right panel). Similar results were observed DNA damage and the regulation of C/EBPa for C/EBPb (Figure 2f). To determine whether C/EBPa As shown in Figure 2a, UVB treatment of fibroblasts mRNA levels are increased by UVB treatment, we used with a dose of 5, 10 and 20 mJ/cm2 resulted in the TaqMan quantitative reverse transcription–PCR (qRT– induction of C/EBPa, with the higher doses displaying a PCR). UVB treatment (5 mJ/cm2) of primary fibroblast more prolonged induction of C/EBPa. To determine resulted in significant increases in C/EBPa mRNA whether C/EBPa can be induced by DNA-damaging level at 6 and 12 h post UVB treatment (Figure 2g), agents other than UVB, we treated fibroblasts with and this increase was blocked by actinomycin D, an N-methyl-N0-nitro-N-nitrosoguanidine (MNNG), a di- inhibitor of transcription (data not shown). These res- rect acting mutagen that methylates DNA; cisplatin, a ults indicate that the increased levels of C/EBPa in cancer therapeutic that crosslinks DNA; camptothecin, UVB-treated fibroblasts are due to increased transcrip- an alkaloid with antitumor activity that induces single- tion of C/EBPa. stranded DNA breaks by inhibiting topoisomerase I In keratinocytes, the p53 is enzyme; and bleomycin, an anti-neoplastic drug that essential for the UVB induction of C/EBPa (Yoon and induces both single- and double-stranded DNA breaks. Smart, 2004). As shown in Figure 3a, UVB treatment of As shown in Figures 2b–e, MNNG, cisplatin, camp- wild-type primary keratinocytes resulted in significant tothecin and bleomycin were inducers of C/EBPa. increases in the protein levels of C/EBPa and p53, as

Oncogene C/EBPa is regulated by C/EBPb in response to DNA damage R Ranjan et al 3239 a WT keratinocyte p53-/- keratinocytes from cells treated with UVB and MNNG displayed increased C/EBP binding to the C/EBP consensus C/EBPα sequence, and this increase appeared somewhat greater

p53 than the increase in C/EBPa and C/EBPb protein levels (Figure 4a, lower panel). As shown in Figure 4b, no p21 binding was detected with these nuclear extracts when a mutant C/EBP consensus sequence (TGCAGAGAC NS TAGTCTCTGCA) was used, and in competition Time (h)0 6 121824 0 6 12 18 24 studies, only the cold wild-type C/EBP consensus sequence could compete for binding. Supershift assays b WT fibroblasts p53-/- fibroblasts with antibodies to C/EBPa and C/EBPb, but not IgG control antibodies, showed that the increase in C/EBP α C/EBP DNA binding post UVB was due to both C/EBPa and p53 C/EBPb binding with C/EBPb that was present in all complexes (Figure 4c). Owing to alternative p21 start sites, C/EBPb protein can be present in three

NS isoforms, termed LAP* (liver-activating protein*); LAP Time (h) 0 6 121824 0 6 12 18 24 aka C/EBPb and LIP (liver inhibitory protein), which functions as a dominant-negative inhibitor of LAP*; Figure 3 Ultraviolet B (UVB) induction of C/EBPa does not and LAP. To begin to understand which C/EBPb require p53 in fibroblasts. (a) Primary keratinocytes were isolated from wild-type (WT) and p53À/À newborn mice and were treated isoforms are responsible for the increased binding in with UVB (10 mJ/cm2). Keratinocytes were harvested at the the EMSA analysis, we first conducted immunoblot indicated time points and immunoblot analysis was conducted. analysis of protein extracts from fibroblasts to examine (b) Primary fibroblasts isolated from WT and p53À/À newborn mice the levels of the three C/EBPb isoforms and then were treated with UVB (10 mJ/cm2). Fibroblasts were harvested at indicated time points and immunoblot analysis was conducted. overexpressed LAP and LIP and examined their DNA binding location using EMSA. As shown in Figure 4d, LAP is the predominate C/EBPb isoform in fibroblasts and is also the predominate C/EBPb-binding isoform in well as in the p53 target gene p21, whereas UVB UVB-treated fibroblasts (Figure 4e). As shown in treatment of p53À/À primary keratinocytes failed to Figure 4f, we also conducted EMSA analysis using the 0 significantly induce the expression of both C/EBPa and C/EBP-binding sequence 5 -(À188) GCGTTGCGCC 0 p21 protein (Figure 3a). These results verify that in ACGATCTCTC (À169)-3 , which was previously iden- keratinocytes, p53 is required for C/EBPa induction by tified as a bona fide C/EBP site in the C/EBPa promoter UVB. In contrast to keratinocytes, p53 was dispensable (Tang et al., 1999, 2004). We observed increases in for the UVB induction of C/EBPa in fibroblasts. As C/EBP binding after UVB or MNNG treatment to the shown in Figure 3b, UVB treatment of p53À/À fibroblasts C/EBPa promoter consensus oligomer (Figure 4f), efficiently induced C/EBPa and as anticipated failed to which is similar to those observed using the canonical induce the p53 target, p21, thus verifying the ablation of C/EBP consensus sequence (Figure 4a). This binding p53 activity (Figure 3b). These results indicate that p53 could compete with cold consensus sequence (data not is dispensable for the UVB induction of C/EBPa in shown). To determine whether the C/EBPb binds to this fibroblasts, indicating a p53-independent pathway that C/EBP site in the C/EBPa promoter in vivo,we contributes to the induction of C/EBPa in fibroblasts. conducted chromatin immunoprecipitation (ChIP) ana- lysis using a C/EBPb antibody to immunoprecipitate C/EBPb–DNA complexes. PCR was performed on the UVB increases C/EBPb binding to C/EBP consensus input DNA and C/EBPb-immunoprecipitated DNA sequence and is bound to the C/EBPa promoter with primers that flank the C/EBP site in the C/EBPa During the process of L1 pre-adipocyte differentiation, promoter. We observed that C/EBPb was bound to the both C/EBPb protein levels and DNA-binding activity C/EBPa promoter in the basal untreated state and that are increased, and C/EBPb has been shown to regulate UVB treatment consistently produced a modest increase C/EBPa levels during process (Christy et al., 1991; in C/EBPb binding (N ¼ 3) at the early time point Darlington et al., 1998; Tang et al., 1999, 2004). Because (Figure 4g). To further confirm the specificity of we observed that UVB produces an increase in C/EBPb immunoprecipitation of C/EBPb, we carried out ChIP protein levels, we initiated studies to examine whether analysis on the C/EBPa promoter in C/EBPbÀ/À C/EBPb has a function in the regulation of C/EBPa fibroblasts. We observed an absence of a PCR product expression in response to DNA damage. First, we used in the C/EBPb-immunoprecipitated samples, further electrophoretic mobility shift assay (EMSA) analysis to supporting our results that C/EBPb directly binds to examine whether nuclear extracts isolated from UVB- the C/EBPa promoter in wild-type fibroblasts. We also and MNNG-treated fibroblasts display increases in the conducted ChIP analysis on the C/EBPa promoter with binding of C/EBPb to a canonical C/EBP consensus a C/EBPa antibody to determine whether C/EBPa sequence (TGCAGATTGCGCAATCTGCA) (Osada is bound to its own promoter in vivo. We observed that et al., 1996). As shown in Figure 4a, nuclear extracts C/EBPa is bound to its own promoter at the basal state

Oncogene C/EBPa is regulated by C/EBPb in response to DNA damage R Ranjan et al 3240 β

Wild Type Mutant Competition IgG UVB MNNG probe probe WT MT anti-C/EBPα anti-C/EBPβ anti-C/EBP Time (h) 0 1 6 1 6 0606 06 016 016016 06 0 66

β-/- SS SS Wild Type C/EBP SS LAP* LAP β C/EBP LIP

α-tubulin C/EBPs C/EBPs

C/EBPs Time (h) 0 6 12 18 24 0 6 12 18 24 UVB 5 mJ/cm2

C/EBPα

C/EBPβ α-tubulin LAP LIP

β β

UVB MNNG β C/EBP UVB (6h) –––+ C/EBP Time(h) 00 11 6611 66 InputC/EBPβ IgG C/EBP Input IgG Time (h) 016 016016 6 6 6

-/- C/EBPβ LAP Wild type C/EBPβ (UVB 5 mJ/cm2) C/EBPs C/EBPβ LIP Input IgG C/EBPα C/EBPs Time (h) 016 016016

(UVB 5 mJ/cm2)

Figure 4 Ultraviolet B (UVB) and N-methyl-N0-nitro-N-nitrosoguanidine (MNNG) increase binding of C/EBPa and C/EBPb to the C/EBP consensus sequence and to the C/EBPa promoter in vivo.(a) Wild-type (WT) fibroblasts were treated with UVB (5 mJ/cm2)or MNNG (35 mM) and nuclear extracts were prepared. Electrophoretic mobility shift assay (EMSA) was conducted with 2 mg of nuclear extract and a labeled WT C/EBP consensus oligonucleotide probe. (Lower panel) Nuclear extract from above experiment was used to conduct immunoblot analysis. (b) WT fibroblasts were treated with UVB (5 mJ/cm2) and nuclear extracts were prepared. EMSA was conducted with 2 mg of nuclear extract and a labeled WT or mutant C/EBP consensus oligonucleotide probe. Competition assays were performed with cold WT or cold mutant (MT) C/EBP probe (50-fold in excess). (c) WT fibroblasts were treated with UVB (5 mJ/cm2) and nuclear extracts were prepared. EMSA was conducted with 2 mg of nuclear extract and a labeled WT C/EBP consensus oligonucleotide probe. Supershift assays were performed with anti-C/EBPa, anti-C/EBPb antibody or IgG (SS-Supershift). (d) Primary fibroblasts from WT and C/EBPbÀ/À mice were treated with UVB (5 mJ/cm2). Cells were harvested at the indicated time points and immunoblot analysis was conducted. (e) Nuclear extracts were prepared from non-treated and UVB (5 mJ/cm2) treated fibroblasts (lanes 1 and 2) and from HEK 293 cells transfected with pcDNA3.1-C/EBPb-LAP or pcDNA3.1-C/EBPb-LIP (lanes 3 and 4). EMSA was conducted with 2 mg of nuclear extract and a labeled C/EBP consensus oligonucleotide probe. (f) WT fibroblasts were treated with UVB (5 mJ/cm2) or MNNG (35 mM) and nuclear extracts were prepared at indicated time points. EMSA was conducted with 2 mgof nuclear extract and a labeled C/EBP consensus sequence corresponding to the C/EBP responsive element in the C/EBPa promoter. (g) WT or C/EBPbÀ/À fibroblasts were treated with UVB (5 mJ/cm2), and ChIP assay using a C/EBPb antibody was conducted as described in the Materials and methods section. Input control represents 5% DNA as compared with IgG or C/EBPb samples. (h)WT fibroblasts were treated with UVB (5 mJ/cm2), and ChIP assay using a C/EBPa antibody was conducted as described in the Materials and methods section. Input control represents 5% DNA as compared with IgG or C/EBPa samples.

and UVB treatment resulted in increased C/EBPa protein in C/EBPbÀ/À fibroblasts was impaired, as both binding (Figure 4h). Taken together, these results the level of protein induction was reduced and the time suggest that C/EBPa expression is regulated by C/EBPb course for its induction was altered. Although the in response to DNA damage and that C/EBPa has an induction of C/EBPa was impaired, the increase in p53 autoregulatory role. protein was comparable in wild-type and C/EBPbÀ/À fibroblasts (Figure 5a). The induction of C/EBPa was also impaired in C/EBPbÀ/À fibroblasts treated with a C/EBPb has a role in the UVB and MNNG induction lower dose of UVB (5 mJ/cm2) (Figure 5b) or with the of C/EBPa alkylating mutagen, MNNG (Figure 5c). To determine To determine whether C/EBPb has a functional role in whether C/EBPb deficiency has an effect on UVB- the regulation of C/EBPa in response to DNA damage, induced C/EBPa mRNA levels, we isolated RNA from we isolated dermal fibroblasts from wild-type and wild-type and C/EBPbÀ/À fibroblasts before and after C/EBPbÀ/À mice and treated these cells with UVB UVB treatment and examined C/EBPa mRNA levels (5 or 10 mJ/cm2) or MNNG (35 mM). As shown in using TaqMan qRT–PCR (Figure 5d). The UVB Figure 5a, UVB (10 mJ/cm2) induction of C/EBPa induction of C/EBPa mRNA was significantly decreased

Oncogene C/EBPa is regulated by C/EBPb in response to DNA damage R Ranjan et al 3241 β-/- Wild Type C/EBP Wild Type C/EBPβ-/-

C/EBPα C/EBPα

p53 p53

C/EBPβ C/EBPβ α-tubulin α-tubulin Time (h) 0 6 12 18 24 0 6 12 18 24 Time (h) 0 6 12 18 24 0 6 12 18 24 MNNG 35 µM UVB 10 mJ/cm2

2.5 Wild Type C/EBPβ-/-

-/- α Wild Type C/EBPβ 2 * C/EBPα * 1.5

p53 1 *

C/EBPβ mRNA level

α-tubulin 0.5 Fold induction in C/EBP Time (h) 0 6 12 18 24 0 6 12 18 24 UVB 5 mJ/cm2 0 0 6 12 18 Time (h) after UVB (5 mJ/cm2) Figure 5 C/EBPa is regulated by C/EBPb in response to DNA damage. (a–c) Primary fibroblasts from newborn wild type (WT) or C/EBPbÀ/À mice were treated with UVB (10 mJ/cm2), UVB (5 mJ/cm2)orN-methyl-N0-nitro-N-nitrosoguanidine (MNNG) (35 mM). Cells were harvested at the indicated time points and immunoblot analysis was conducted. (d) Primary fibroblasts from newborn WT or C/EBPbÀ/À mice were treated with UVB (5 mJ/cm2) and RNA isolated at the indicated time points. Quantitative RT–PCR was conducted for C/EBPa and 18S mRNA levels. Data were normalized using 18S and was analyzed using the comparative CT method. Data are expressed as mean±standard error (N ¼ 4) and each experiment was run in triplicate. Two-factor ANOVA showed significant interaction between genotype and time (Po0.05). Significantly different from WT fibroblasts (*Po0.05) at the indicated time point as determined by Student’s t-test. in C/EBPbÀ/À fibroblasts compared with that in wild- induction was altered; however, there was not a type fibroblasts (Figure 5d). Collectively, these results complete ablation of C/EBPa induction, indicating that indicate that in fibroblasts, C/EBPb functions down- other pathways are also involved. Although C/EBPb has stream of DNA damage to partially regulate C/EBPa been shown to regulate C/EBPa expression during pre- mRNA and protein expression. adipocyte differentiation (Christy et al., 1991; Darling- ton et al., 1998; Tang et al., 1999, 2004), to the best of our knowledge, our study is the first to show that Discussion C/EBPb functions downstream of DNA damage to regulate the induction of C/EBPa. Therefore, C/EBPb is Earlier we have shown that C/EBPa is a UVB/DNA a protein that can be activated by numerous cues, damage-inducible gene in mouse and human primary including those involving differentiation (Yeh et al., keratinocytes; however, UVB treatment did not induce 1995; Sterneck et al., 1997; Oh and Smart, 1998; Zhu C/EBPa in three other cell lines examined (HepG2, et al., 1999) and DNA damage (Ewing et al., 2008), as NRK or NIH3T3 cells) (Yoon and Smart, 2004), well as oncogenes (Sundfeldt et al., 1999; Rask et al., suggesting the induction of C/EBPa by UVB may be a 2000; Zhu et al., 2002; Shuman et al., 2004) and keratinocytes-specific effect. In keratinocytes, UVB inflammatory cytokines (Akira et al., 1990; Mukaida induction of C/EBPa is solely dependent on p53, and et al., 1990; Drouet et al., 1991). Recently, Ewing et al. this is mediated through p53 binding to a p53 response (2008) reported that C/EBPb represses p53 levels and element in the distal promoter of C/EBPa (Yoon and function to promote cell survival downstream of DNA Smart, 2004). The results presented in this study indicate damage. Therefore, emerging evidence indicate that that C/EBPa is a DNA damage-responsive gene in both C/EBPa and C/EBPb participate in DNA damage mouse primary fibroblasts and that p53 is dispensable response pathways. for the UVB induction of C/EBPa in fibroblasts. UVB treatment produced a modest increase in Instead, we observed that C/EBPa is regulated by C/EBPb levels, and ChIP analyses also showed modest C/EBPb in response to DNA damage and C/EBPa increases in C/EBPb binding to the C/EBPa promoter, likely has an autoregulatory role in response to DNA and yet C/EBPbÀ/À fibroblasts displayed a significantly damage. C/EBPa induction by DNA damage was impaired induction of C/EBPa mRNA and protein in impaired in C/EBPbÀ/À fibroblasts, both at the protein response to UVB or MNNG treatment. Our ChIP and mRNA levels and the time course for their results also showed that C/EBPb is constitutively bound

Oncogene C/EBPa is regulated by C/EBPb in response to DNA damage R Ranjan et al 3242 to the C/EBPa promoter in untreated fibroblasts. Taken Materials and methods together, these results suggest that post-translational modifications of C/EBPb may contribute to the regula- Cell lines and cell culture tion of C/EBPa in response to UVB. It is generally Mouse primary keratinocytes were isolated from the epidermis accepted that C/EBPb exists in a repressed state and, of newborn mice by overnight flotation of skin in trypsin at 1 post-translational modifications de-repress C/EBPb and 4 C (Hennings et al., 1980; Dlugosz et al., 1995) and dermal fibroblasts were isolated from the dermis after the removal of increase its transcriptional activity. For example, the epidermis from skin. Primary keratinocytes were cultured phosphorylation or deletion of the domain as described by Yoon and Smart (2004). For fibroblasts unfolds and induces conformational change in C/EBPb, isolation, the dermis was subjected to digestion in collagenase which results in de-repression and increased transactiva- (0.35%) for 25 min, followed by DNase (250 U per skin tion activity of C/EBPb (Kowenz-Leutz et al., 1994; sample) treatment for 5 min at 37 1C while shaking. Cells were Williams et al., 1995). In addition to phosphorylation, filtered and collected after centrifugation, and isolated C/EBPb is modified by acetylation and a mutant fibroblasts were plated in Eagle’s minimal essential medium C/EBPb that can no longer be acetylated on Lys-39 (BioWhittaker, Walkersville, MD, USA) (2 mM CaCl2) has an impaired ability to transactivate a C/EBPa supplemented with 10% fetal bovine serum (Sigma, St Louis, promoter reporter construct (Cesena et al., 2007, 2008). MO, USA), 100 U/ml of penicillin, 100 mg/ml of streptomycin and 0.25 mg/ml of amphotericin B (Gibco-Invitrogen, Post-translational modifications involving Carlsbad, CA, USA) in a 100-mm tissue culture dish. (Pless et al., 2008) and sumoylation (Kim et al., 2002; Upon confluence, fibroblasts were passaged and plated in a Eaton and Sealy, 2003; Berberich-Siebelt et al., 2006) 60-mm tissue culture dish. When cells reached 70% confluence, also have regulatory roles, and changes in the they were treated with UVB or other DNA-damaging agents. complex result in C/EBPb activation (Mo et al., 2004). Recently, it was reported that C/EBPb is involved in the Animals opening of chromatin, allowing other transcription Germline C/EBPaÀ/À mice do not survive; pups die prenatally or factors to bind to the gene promoter and increase gene survive only few hours after birth (Wang et al., 1995). A keratin expression (Plachetka et al., 2008). Further studies are 5-Cre, K5Cre, transgenic mouse line is used for generalized Cre- required to understand how C/EBPb is activated in mediated recombination or tissue-specific gene ablation. When floxed females carrying transgene K5Cre are mated with floxed response to DNA damage and how these events male animals, recombination occurs in all the tissues in all mice contribute to the regulation of C/EBPa. produced from the above mating pair (Ramirez et al., 2004). Previous studies in keratinocytes using siRNA Germline C/EBPaÀ/À pups were produced by mating epidermal knockdown of C/EBPa indicated that C/EBPa has a conditional C/EBPaÀ/À female (K5Cre;C/EBPafl/fl) (C57BL/ role in the G1 checkpoint in response to DNA damage 6;DBA;129SV) (Loomis et al., 2007) with C/EBPa floxed (C/ (Yoon and Smart, 2004). One goal of this study EBPafl/fl) male mice (C57BL/6;DBA;129SV) (Lee et al., 1997). was to use a genetic approach to confirm and verify Primers and PCR conditions used to genotype mice were published previously (Lee et al., 1997; Ramirez et al., 2004). the role of C/EBPa in the G1 checkpoint as siRNA p53 þ /À male mice were mated with p53 þ /À female mice to knockdown studies can be complicated by the uninten- À/À tional interaction of the siRNA with other unidentified generate p53 as well as wild-type newborn pups (C57BL/6). Primers and PCR conditions were published previously (Hulla targets. We observed a diminished G1 checkpoint et al., 2001). The C/EBPbÀ/À mice used in this study have been À/À response in C/EBPa fibroblasts compared with described previously (Sterneck et al., 1997). The C/EBPbÀ/À and wild-type fibroblasts in response to UVB treatment, wild-type new born pups were generated by mating C/EBPb þ /À and our results provide important genetic evidence females with C/EBPb þ /À males (C57BL/6;129/SV). For all other that C/EBPa is involved in DNA damage-induced G1 studies not using genetically modified mice, fibroblasts were checkpoint. Moreover, we observed that serum-deprived isolated from the dermis of wild-type 129SV mice. C/EBPaÀ/À fibroblasts display an enhanced mitogen- induced entry into S-phase compared with wild-type Treatment of cells fibroblasts, suggesting that C/EBPa has a direct role in The UV lamp (model EB 280C; Spectronics, Westbury, NY, the regulation of the G /S transition in response to USA) used for treating cells emits wavelengths between 280 1 and 320 nm with a spectrum peak at 312 nm. The intensity of mitogens. To our knowledge, there are no previous studies light emitted was measured by NIST Traceable Radiometer that have used synchronous cultures of primary cells Photometer (Model IL1400A, International Light Techno- genetically deficient in C/EBPa to define a functional role logies, Peabody, MA, USA). Cells were treated with UVB as for C/EBPa in the regulation of G1/S transition in response described by Yoon and Smart (2004). MNNG, camptothecin to mitogens. Proposed mechanisms for C/EBPa-induced and cycloheximide were dissolved in dimethyl sulfoxide. cell-cycle arrest involve interactions with cell-cycle proteins, Cisplatin (cis-Diammineplatinum (II)) and bleomycin were including Rb family members (Chen et al., 1996; Timchen- dissolved in water. Cells were treated with MNNG, cyclohex- ko et al., 1999), CDK4 and CDK2 (Wang et al., 2001), imide, camptothecin, bleomycin, dimethyl sulfoxide or water. E2F (Slomiany et al.,2000;Porseet al., 2001), p21 Cells were treated with cisplatin for 2 h, and then media was (Timchenko et al., 1996) and the SWI/SNF chromatin replaced with fresh media without cisplatin. Actinomycin D was dissolved in ethanol and cells were treated with remodeling complex (Muller et al., 2004). Further studies actinomycin D or ethanol. are required to understand the molecular mechanisms of C/EBPa’s involvement in the G1/S transition and how Preparation of cell lysates this impinges on the DNA damage-induced G1 checkpoint Nuclear extracts were prepared as previously described by and neoplasia. Schreiber et al. (1989). For preparation of whole-cell lysates,

Oncogene C/EBPa is regulated by C/EBPb in response to DNA damage R Ranjan et al 3243 cells were washed with cold phosphate-buffered saline and corresponding site were 50-(À324) GGCTGGAAGTGGGT harvested by scraping. Cells were collected by centrifugation GACTTA (À305)-30 and 50-(À115) CGCCTTCTCCTGT and protein was isolated in radioimmunoprecipitation assay GACTTTC (À134)-30 to produce a 210-bp PCR product. buffer as previously described (Ewing et al., 2008). EMSA and supershift Western blot analysis Nuclear extracts, 2 mgin10ml buffer C, were incubated with Protein from cell lysates was loaded onto a 12% polyacryla- 10 ml of master binding mix buffer with 32P-labeled C/EBP mide Tris-glycine gel (Invitrogen, Carlsbad, CA, USA), then probe (Santa Cruz Biotechnology) or 32P-labeled probe separated by electrophoresis and transferred to an Immobilon- corresponding to the C/EBP-responsive element in the P membrane (Millipore, Billerica, MA, USA). After incuba- C/EBPa promoter for 30 min at room temperature. For the tion in a blocking buffer (Oh and Smart, 1998), the membranes supershift assays, samples were treated as above but incubated were probed with rabbit polyclonal immunoglobulin G (IgG) with C/EBPa (sc-61), C/EBPb (sc-150) or IgG (sc-2027) (Santa raised against C/EBPa (sc-61), p53 (sc-6243), C/EBPb (sc-150), Cruz Biotechnology) antibody. For competition assays, p21 (sc-757) or mouse monoclonal raised against a-tubulin samples were incubated for 20 min with cold wild-type and (sc-8035) (1:2000) (Santa Cruz Biotechnology, Santa Cruz, cold mutant C/EBP consensus oligonucleotide probe (50-fold CA, USA) and then probed with a horseradish peroxidase- in excess) in 10 ml master mix binding buffer and then with linked secondary antibody (Amersham, GE Healthcare, wild-type 32P-labeled C/EBP probe for 20 min at room Piscataway, NJ, USA). Detection was carried out with an temperature. Samples were loaded onto 6% polyacrylamide enhanced chemiluminescence reagent (Perkin-Elmer Life gel and subjected to electrophoresis in 0.025 Â TBE buffer at Science, Waltham, MA, USA) followed by exposure of the 200 V for 5–7 h at 4–8 1C. membrane to the film. BrdU labeling and FACS analysis Quantitative real-time PCR When fibroblasts reached 30–40% confluence, cells were Total RNA was isolated from either control or UVB-treated synchronized by serum deprivation in 0.5% fetal bovine serum fibroblasts using TRI reagent (Sigma) and then purified by the for 28 h. Fibroblasts were released by adding 10% fetal bovine RNeasy Mini Kit (Qiagen, Valencia, CA, USA). cDNA was serum containing fibroblast medium. After 4 h of release, prepared from 50 ng RNA by ImProm-II Reverse Transcrip- fibroblasts were either left untreated or treated with UVB tion System (Promega, Madison, WI, USA) following as per (5 mJ/cm2). One hour before, the cells were harvested at each the manufacturer’s protocol. cDNA was used to perform time point, and were incubated with 10 mM BrdU. Cells were quantitative PCR using mouse C/EBPa TaqMan Gene then fixed in 70% alcohol, treated with 2 N HCl–Triton X-100 Expression Assays, 18S TaqMan Assays to denature DNA, followed by neutralization with Na2B4O7. (Applied Biosystems, Foster City, CA, USA) and TaqMan Cells were pelleted, resuspended in 0.5% Tween 20–1% bovine Universat PCR mix (Applied Biosystems). Data were analyzed serum albumin–phosphate-buffered saline with anti-BrdU- using the comparative CT method. fluorescein isothiocyanate antibody (1:50; Becton Dickinson, Franklin Lakes, NJ, USA) and 0.5 mg of RNase per ml, and ChIP assay incubated at 4 1C overnight. Cells were pelleted and resus- Primary fibroblasts were plated in 100 mm culture plates and pended in phosphate-buffered saline containing 5 mg/ml of were left untreated or treated with UVB dose of 5 mJ/cm2. propidium iodide (PI) and subjected to FACS analysis. After 1 and 6 h of UVB treatment, cells were treated with 1% formaldehyde, and thereafter ChiP assay was performed as per the manufacturer’s instruction (Upstate Biotechnology, Conflict of interest Millipore, Billerica, MA, USA). The formaldehyde-treated cells were lysed with SDS lysis buffer and sonicated to produce 200– 500 bp long DNA fragments. Samples were pre-cleared with The authors declare no conflict of interest. salmon sperm DNA/protein A, and then immunoprecipitated with polyclonal antibody against C/EBPb, C/EBPa or rabbit Acknowledgements IgG at 4 1C overnight. Immunoprecipitated DNA was de- crosslinked with 5 M NaCl and extracted by ethanol/chloroform This research was supported by grants from the National precipitation and amplified by PCR. Primer set for PCR was Cancer Institute (CA46637), the National Institute of Envir- designed to flank the C/EBP in the C/ onmental Health Sciences (ES12473), and a training grant EBPa promoter 50-(À188) GCGTTGCGCCACGATCTCTC from the NIEHS (ES007046). We thank Brian Sayers for his (À169)-30 (Tang et al., 1999, 2004). Primer set flanking the technical support with DNA-damaging agents.

References

Akira S, Isshiki H, Sugita T, Tanabe O, Kinoshita S, Nishio Y et al. enhancer binding protein alpha is epigenetically down-regulated in (1990). A nuclear factor for IL-6 expression (NF-IL6) is a member head and neck squamous cell carcinoma. Cancer Res 67: 4657–4664. of a C/EBP family. EMBO J 9: 1897–1906. Berberich-Siebelt F, Berberich I, Andrulis M, Santner-Nanan B, Basseres DS, Levantini E, Ji H, Monti S, Elf S, Dayaram T et al. Jha MK, Klein-Hessling S et al. (2006). SUMOylation interferes with (2006). Respiratory failure due to differentiation arrest and CCAAT/enhancer-binding protein beta-mediated c-myc repression, expansion of alveolar cells following lung-specific loss of but not IL-4 activation in T cells. J Immunol 176: 4843–4851. the transcription factor C/EBPalpha in mice. Mol Cell Biol 26: Cesena TI, Cardinaux JR, Kwok R, Schwartz J. (2007). CCAAT/ 1109–1123. enhancer-binding protein (C/EBP) beta is acetylated at multiple Bennett KL, Hackanson B, Smith LT, Morrison CD, Lang JC, lysines: acetylation of C/EBPbeta at lysine 39 modulates its ability Schuller DE et al. (2007). Tumor suppressor activity of CCAAT/ to activate transcription. J Biol Chem 282: 956–967.

Oncogene C/EBPa is regulated by C/EBPb in response to DNA damage R Ranjan et al 3244 Cesena TI, Cui TX, Subramanian L, Fulton CT, Iniguez-Lluhi JA, Loomis KD, Zhu S, Yoon K, Johnson PF, Smart RC. (2007). Genetic Kwok RP et al. (2008). Acetylation and deacetylation regulate ablation of CCAAT/enhancer binding protein alpha in epidermis CCAAT/enhancer binding protein beta at K39 in mediating gene reveals its role in suppression of epithelial tumorigenesis. Cancer Res transcription. Mol Cell Endocrinol 289: 94–101. 67: 6768–6776. Chen PL, Riley DJ, Chen Y, Lee WH. (1996). Retinoblastoma protein Mo X, Kowenz-Leutz E, Xu H, Leutz A. (2004). Ras induces mediator positively regulates terminal adipocyte differentiation through direct complex exchange on C/EBP beta. Mol Cell 13: 241–250. interaction with C/EBPs. Genes Dev 10: 2794–2804. Mukaida N, Mahe Y, Matsushima K. (1990). Cooperative interaction Christy RJ, Kaestner KH, Geiman DE, Lane MD. (1991). CCAAT/ of nuclear factor-kappa B- and cis-regulatory enhancer binding enhancer binding protein gene promoter: binding of nuclear factors protein-like factor binding elements in activating the interleukin-8 during differentiation of 3T3-L1 preadipocytes. Proc Natl Acad Sci gene by pro-inflammatory cytokines. J Biol Chem 265: 21128–21133. USA 88: 2593–2597. Muller C, Calkhoven CF, Sha X, Leutz A. (2004). The CCAAT Darlington GJ, Ross SE, MacDougald OA. (1998). The role of C/EBP enhancer-binding protein alpha (C/EBPalpha) requires a SWI/SNF genes in adipocyte differentiation. J Biol Chem 273: 30057–30060. complex for proliferation arrest. J Biol Chem 279: 7353–7358. Diehl AM, Johns DC, Yang S, Lin H, Yin M, Matelis LA et al. (1996). Oh HS, Smart RC. (1998). Expression of CCAAT/enhancer binding Adenovirus-mediated transfer of CCAAT/enhancer-binding pro- proteins (C/EBP) is associated with squamous differentiation in tein-alpha identifies a dominant antiproliferative role for this epidermis and isolated primary keratinocytes and is altered in skin isoform in hepatocytes. J Biol Chem 271: 7343–7350. neoplasms. J Invest Dermatol 110: 939–945. Dlugosz AA, Glick AB, Tennenbaum T, Weinberg WC, Yuspa SH. Osada S, Yamamoto H, Nishihara T, Imagawa M. (1996). DNA (1995). Isolation and utilization of epidermal keratinocytes for binding specificity of the CCAAT/enhancer-binding protein tran- oncogene research. Methods Enzymol 254: 3–20. scription factor family. J Biol Chem 271: 3891–3896. Drouet C, Shakhov AN, Jongeneel CV. (1991). Enhancers and Pabst T, Mueller BU, Zhang P, Radomska HS, Narravula S, transcription factors controlling the inducibility of the tumor Schnittger S et al. (2001). Dominant-negative mutations of CEBPA, necrosis factor-alpha promoter in primary macrophages. J Immunol encoding CCAAT/enhancer binding protein-alpha (C/EBPalpha), 147: 1694–1700. in acute myeloid leukemia. Nat Genet 27: 263–270. Eaton EM, Sealy L. (2003). Modification of CCAAT/enhancer- Plachetka A, Chayka O, Wilczek C, Melnik S, Bonifer C, Klempnauer binding protein-beta by the small ubiquitin-like modifier (SUMO) KH. (2008). C/EBPbeta induces chromatin opening at a cell-type- family members, SUMO-2 and SUMO-3. J Biol Chem 278: specific enhancer. Mol Cell Biol 28: 2102–2112. 33416–33421. Pless O, Kowenz-Leutz E, Knoblich M, Lausen J, Beyermann M, Ewing SJ, Zhu S, Zhu F, House JS, Smart RC. (2008). C/EBPbeta Walsh MJ et al. (2008). G9a-mediated lysine methylation alters the represses p53 to promote cell survival downstream of DNA damage function of CCAAT/enhancer-binding protein-beta. J Biol Chem independent of oncogenic Ras and p19(Arf). Cell Death Differ 15: 283: 26357–26363. 1734–1744. Porse BT, Pedersen TA, Xu X, Lindberg B, Wewer UM, Friis-Hansen Gery S, Tanosaki S, Bose S, Bose N, Vadgama J, Koeffler HP. (2005). L et al. (2001). E2F repression by C/EBPalpha is required for Down-regulation and growth inhibitory role of C/EBPalpha in adipogenesis and granulopoiesis in vivo. Cell 107: 247–258. breast cancer. Clin Cancer Res 11: 3184–3190. Radomska HS, Huettner CS, Zhang P, Cheng T, Scadden DT, Tenen Gombart AF, Hofmann WK, Kawano S, Takeuchi S, Krug U, Kwok DG. (1998). CCAAT/enhancer binding protein alpha is a regulatory SH et al. (2002). Mutations in the gene encoding the transcription switch sufficient for induction of granulocytic development from factor CCAAT/enhancer binding protein alpha in myelodysplastic bipotential myeloid progenitors. Mol Cell Biol 18: 4301–4314. syndromes and acute myeloid leukemias. Blood 99: 1332–1340. Ramirez A, Page A, Gandarillas A, Zanet J, Pibre S, Vidal M Halmos B, Huettner CS, Kocher O, Ferenczi K, Karp DD, Tenen DG. et al. (2004). A keratin K5Cre transgenic line appropriate for (2002). Down-regulation and antiproliferative role of C/EBPalpha tissue-specific or generalized Cre-mediated recombination. Genesis in lung cancer. Cancer Res 62: 528–534. 39: 52–57. Hendricks-Taylor LR, Darlington GJ. (1995). Inhibition of cell Ramji DP, Foka P. (2002). CCAAT/enhancer-binding proteins: proliferation by C/EBP alpha occurs in many cell types, does structure, function and regulation. Biochem J 365: 561–575. not require the presence of p53 or Rb, and is not affected by large Rask K, Thorn M, Ponten F, Kraaz W, Sundfeldt K, Hedin L et al. T-antigen. Nucleic Acids Res 23: 4726–4733. (2000). Increased expression of the transcription factors CCAAT- Hennings H, Michael D, Cheng C, Steinert P, Holbrook K, Yuspa SH. enhancer binding protein-beta (C/EBBeta) and C/EBzeta (CHOP) (1980). Calcium regulation of growth and differentiation of mouse correlate with invasiveness of human colorectal cancer. Int J Cancer epidermal cells in culture. Cell 19: 245–254. 86: 337–343. Hulla JE, French JE, Dunnick JK. (2001). Chromosome 11 allelotypes Sancar A, Lindsey-Boltz LA, Unsal-Kacmaz K, Linn S. (2004). reflect a mechanism of chemical carcinogenesis in heterozygous Molecular mechanisms of mammalian DNA repair and the DNA p53-deficient mice. Carcinogenesis 22: 89–98. damage checkpoints. Annu Rev Biochem 73: 39–85. Ishikawa K, Ishii H, Saito T. (2006). DNA damage-dependent cell Schreiber E, Matthias P, Muller MM, Schaffner W. (1989). Rapid cycle checkpoints and genomic stability. DNA Cell Biol 25: 406–411. detection of octamer binding proteins with ‘mini-extracts’, prepared Johnson PF. (2005). Molecular stop signs: regulation of cell-cycle from a small number of cells. Nucleic Acids Res 17: 6419. arrest by C/EBP transcription factors. J Cell Sci 118: 2545–2555. Shim M, Powers KL, Ewing SJ, Zhu S, Smart RC. (2005). Diminished Kastan MB, Bartek J. (2004). Cell-cycle checkpoints and cancer. expression of C/EBPalpha in skin carcinomas is linked to oncogenic Nature 432: 316–323. Ras and reexpression of C/EBPalpha in carcinoma cells inhibits Kim J, Cantwell CA, Johnson PF, Pfarr CM, Williams SC. (2002). proliferation. Cancer Res 65: 861–867. Transcriptional activity of CCAAT/enhancer-binding proteins is Shuman JD, Sebastian T, Kaldis P, Copeland TD, Zhu S, Smart RC controlled by a conserved inhibitory domain that is a target for et al. (2004). Cell cycle-dependent phosphorylation of C/EBPbeta sumoylation. J Biol Chem 277: 38037–38044. mediates oncogenic cooperativity between C/EBPbeta and Kowenz-Leutz E, Twamley G, Ansieau S, Leutz A. (1994). Novel H-RasV12. Mol Cell Biol 24: 7380–7391. mechanism of C/EBP beta (NF-M) transcriptional control: activa- Slomiany BA, D’Arigo KL, Kelly MM, Kurtz DT. (2000). tion through derepression. Genes Dev 8: 2781–2791. C/EBPalpha inhibits cell growth via direct repression of E2F-DP- Lee YH, Sauer B, Johnson PF, Gonzalez FJ. (1997). Disruption of the mediated transcription. Mol Cell Biol 20: 5986–5997. c/ebp alpha gene in adult mouse liver. Mol Cell Biol 17: 6014–6022. Sterneck E, Tessarollo L, Johnson PF. (1997). An essential role for Lin FT, Lane MD. (1994). CCAAT/enhancer binding protein alpha is C/EBPbeta in female reproduction. Genes Dev 11: 2153–2162. sufficient to initiate the 3T3-L1 adipocyte differentiation program. Sundfeldt K, Ivarsson K, Carlsson M, Enerback S, Janson PO, Proc Natl Acad Sci USA 91: 8757–8761. Brannstrom M et al. (1999). The expression of CCAAT/enhancer

Oncogene C/EBPa is regulated by C/EBPb in response to DNA damage R Ranjan et al 3245 binding protein (C/EBP) in the human ovary in vivo: specific Wang ND, Finegold MJ, Bradley A, Ou CN, Abdelsayed SV, Wilde increase in C/EBPbeta during epithelial tumour progression. Br J MD et al. (1995). Impaired energy homeostasis in C/EBP alpha Cancer 79: 1240–1248. knockout mice. Science 269: 1108–1112. Tada Y, Brena RM, Hackanson B, Morrison C, Otterson GA, Plass C. Wang X, Scott E, Sawyers CL, Friedman AD. (1999). C/EBPalpha (2006). Epigenetic modulation of tumor suppressor CCAAT/ bypasses granulocyte colony-stimulating factor signals to rapidly enhancer binding protein alpha activity in lung cancer. J Natl induce PU.1 gene expression, stimulate granulocytic differentiation, Cancer Inst 98: 396–406. and limit proliferation in 32D cl3 myeloblasts. Blood 94: 560–571. Takai N, Kawamata N, Walsh CS, Gery S, Desmond JC, Whittaker S Watkins PJ, Condreay JP, Huber BE, Jacobs SJ, Adams DJ. (1996). et al. (2005). Discovery of epigenetically masked tumor Impaired proliferation and tumorigenicity induced by CCAAT/ suppressor genes in endometrial cancer. Mol Cancer Res 3: enhancer-binding protein. Cancer Res 56: 1063–1067. 261–269. Williams SC, Baer M, Dillner AJ, Johnson PF. (1995). CRP2 (C/EBP Tang QQ, Jiang MS, Lane MD. (1999). Repressive effect of Sp1 on the beta) contains a bipartite regulatory domain that controls tran- C/EBPalpha gene promoter: role in adipocyte differentiation. Mol scriptional activation, DNA binding and cell specificity. EMBO J Cell Biol 19: 4855–4865. 14: 3170–3183. Tang QQ, Zhang JW, Daniel Lane M. (2004). Sequential gene XuL,HuiL,WangS,GongJ,JinY,WangYet al. (2001). Expression promoter interactions of C/EBPbeta, C/EBPalpha, and PPARgam- profiling suggested a regulatory role of liver-enriched transcription ma during adipogenesis. Biochem Biophys Res Commun 319: factors in human hepatocellular carcinoma. Cancer Res 61: 3176–3181. 235–239. Yeh WC, Cao Z, Classon M, McKnight SL. (1995). Cascade regulation Timchenko NA, Wilde M, Iakova P, Albrecht JH, Darlington GJ. of terminal adipocyte differentiation by three members of the C/EBP (1999). E2F/p107 and E2F/p130 complexes are regulated by family of leucine zipper proteins. Genes Dev 9: 168–181. C/EBPalpha in 3T3-L1 adipocytes. Nucleic Acids Res 27: 3621–3630. Yoon K, Smart RC. (2004). C/EBPalpha is a DNA damage-inducible Timchenko NA, Wilde M, Nakanishi M, Smith JR, Darlington GJ. p53-regulated mediator of the G1 checkpoint in keratinocytes. Mol (1996). CCAAT/enhancer-binding protein alpha (C/EBP alpha) Cell Biol 24: 10650–10660. inhibits cell proliferation through the p21 (WAF-1/CIP-1/SDI-1) Zhu S, Oh HS, Shim M, Sterneck E, Johnson PF, Smart RC. (1999). protein. Genes Dev 10: 804–815. C/EBPbeta modulates the early events of keratinocyte differentia- Umek RM, Friedman AD, McKnight SL. (1991). CCAAT-enhancer tion involving growth arrest and keratin 1 and keratin 10 binding protein: a component of a differentiation switch. Science expression. Mol Cell Biol 19: 7181–7190. 251: 288–292. Zhu S, Yoon K, Sterneck E, Johnson PF, Smart RC. (2002). CCAAT/ Wang H, Iakova P, Wilde M, Welm A, Goode T, Roesler WJ et al. enhancer binding protein-beta is a mediator of keratinocyte survival (2001). C/EBPalpha arrests cell proliferation through direct inhibi- and skin tumorigenesis involving oncogenic Ras signaling. tion of Cdk2 and Cdk4. Mol Cell 8: 817–828. Proc Natl Acad Sci USA 99: 207–212.

Oncogene