Basic Region-leucine Zipper Research Articles

Hybrids of the bHLH and bZIP protein motifs display different DNA-binding activities in vivo vs. in vitro.

Minimalist hybrids comprising the DNA-binding domain of bHLH/PAS (basic-helix-loop-helix/Per-Arnt-Sim) protein Arnt fused to the leucine zipper (LZ) dimerization domain from bZIP (basic region-leucine zipper) protein C/EBP were designed to bind the E-box DNA site, CACGTG, targeted by bHLHZ (basic-helix-loop-helix-zipper) proteins Myc and Max, as well as the Arnt homodimer. The bHLHZ-like structure of ArntbHLH-C/EBP comprises the Arnt bHLH domain fused to the C/EBP LZ: i.e. swap of the 330 aa PAS domain for the 29 aa LZ. In the yeast one-hybrid assay (Y1H), transcriptional activation from the E-box was strong by ArntbHLH-C/EBP, and undetectable for the truncated ArntbHLH (PAS removed), as detected via readout from the HIS3 and lacZ reporters. In contrast, fluorescence anisotropy titrations showed affinities for the E-box with ArntbHLH-C/EBP and ArntbHLH comparable to other transcription factors (K d 148.9 nM and 40.2 nM, respectively), but only under select conditions that maintained folded protein. Although in vivo yeast results and in vitro spectroscopic studies for ArntbHLH-C/EBP targeting the E-box correlate well, the same does not hold for ArntbHLH. As circular dichroism confirms that ArntbHLH-C/EBP is a much more strongly α-helical structure than ArntbHLH, we conclude that the nonfunctional ArntbHLH in the Y1H must be due to misfolding, leading to the false negative that this protein is incapable of targeting the E-box. Many experiments, including protein design and selections from large libraries, depend on protein domains remaining well-behaved in the nonnative experimental environment, especially small motifs like the bHLH (60–70 aa). Interestingly, a short helical LZ can serve as a folding- and/or solubility-enhancing tag, an important device given the focus of current research on exploration of vast networks of biomolecular interactions.

Characterization of bZip-Type Transcription Factor AtfA with Reference to Stress Responses of Conidia ofAspergillus nidulans

Microorganisms growing in natural habitats are permanently confronted with a wide variety of external stresses. In fungi, including Aspergillus nidulans, one of the general and ultimate strategies to survive in harsh habitats is the development of stress-tolerant conidia. In this study, we found that one of the basic-region leucine zipper transcription factors, named AtfA, plays a crucial role in the acquisition of tolerance against oxidative and heat stress on conidia, but not on mycelia, although this factor is not involved in asexual development per se.

BACH1 Is a Specific Repressor of HMOX1 That Is Inactivated by Arsenite

Intracellular heme is a redox active molecule that can be detrimental to cells at high concentrations or under oxidizing conditions. To prevent accumulation, the inducible enzyme heme oxygenase-1 (HMOX1) catalyzes degradation of heme. In the absence of elevated intracellular heme or oxidative stress, the basic region leucine zipper transcriptional regulator BACH1 binds HMOX1 antioxidant response elements and represses transcription. Conversely, increased intracellular heme or sulfhydryl oxidation inactivate BACH1, permitting transcriptional induction of HMOX1. Here, we investigate the effect of BACH1 inactivation on the induction of HMOX1 and as a mechanism for broader gene induction. We show that BACH1 is inactivated at low micromolar arsenite concentrations and that BACH1 inactivation is necessary and sufficient for transcriptional induction of HMOX1. Because BACH1 is thought to interact with antioxidant response element motifs, we further examined the role of BACH1 as a regulator of inducible antioxidant gene expression by assessing the global profile of gene expression following BACH1 knockdown using small interfering RNA. The loss of BACH1 function in human keratinocytes results almost exclusively in HMOX1 induction, suggesting that BACH1 may function as a rheostat regulating levels of intracellular free heme.

Treatment of arthritis with a selective inhibitor of c-Fos/activator protein-1

To inhibit arthritis upstream of inflammatory cytokine release and matrix metalloproteinase (MMP) action, we designed de novo a small-molecule inhibitor of c-Fos/activator protein-1 (AP-1) using three-dimensional (3D) pharmacophore modeling. This model was based on the 3D structure of the basic region-leucine zipper domain of AP-1-DNA complex. Administration of this inhibitor prevented type II collagen-induced arthritis from day 21, before the onset of arthritis, or from day 27, resolved arthritis after its onset. Suppression of disease was accomplished by reducing the amounts of inflammatory cytokines and MMPs in vivo in sera and joints and in vitro in synovial cell and chondrocyte cultures. The primary action of this molecule was the inhibition of matrix-degrading MMPs and inflammatory cytokines including interleukin 1beta; this molecule also synergized with anti-tumor necrosis factor alpha to inhibit arthritis. Thus, selective inhibition of c-Fos/AP-1 resolves arthritis in a preclinical model of the disease.

A Novel Protein, Luman/CREB3 Reruitment Factor, Inhibits Luman Activation of the Unfolded Protein Response

Luman/CREB3 (also called LZIP) is an endoplasmic reticulum (ER)-bound cellular transcription factor. It has been implicated in the mammalian unfolded protein response (UPR), as well as herpes simplex virus reactivation from latency in sensory neurons. Here, we report the identification of a novel Luman recruitment factor (LRF). Like Luman, LRF is a UPR-responsive basic-region leucine zipper protein that is prone to proteasomal degradation. Being a highly unstable protein, LRF interacts with Luman through the leucine zipper region and promotes Luman degradation. LRF was found to recruit the nuclear form of Luman to discrete nuclear foci, which overlap with the nuclear receptor coactivator GRIP1 bodies, and repress the transactivation activity of Luman. Compared to LRF+/+ mouse embryonic fibroblast (MEF) cells, the levels of CHOP, EDEM, and Herp were elevated in LRF-/- MEF cells. We propose that LRF is a negative regulator of the UPR. For Luman, it may represent another level of regulation following Luman proteolytic cleavage on the ER and nuclear translocation. In addition to inducing rapid Luman turnover, LRF may repress the transactivation potential of Luman by sequestering it in the LRF nuclear bodies away from key cofactors (such as HCF-1) that are required for transcriptional activation.

Dual role of the arginine methyltransferase CARM1 in the regulation of c‐Fos target genes

Fos proteins, the prototypic members of basic region-leucine zipper (bZIP) transcription factors, bind to other bZIP proteins to form the activator protein-1 (AP-1) complex, which regulates the expression of a plethora of target genes. Notably, c-Fos target genes include members of the matrix metalloproteinase (MMP) gene family and c-fos is overexpressed in a number of metastatic cancers, suggesting its direct involvement in this process. Here, we reveal that c-Fos-mediated transcriptional activation is regulated by the protein arginine methyltransferase CARM1 and by all three members of the p160 protein family of coactivators. Carm1-deficient cells showed a dramatic reduction in the expression level of c-Fos target genes MMP-1b, -3, and -13, indicating a major role for CARM1 in regulating the expression of these genes. RNA interference combined with quantitative polymerase chain reaction demonstrated that CARM1 and p160 proteins synergize to activate expression of MMP-1b, -3, and -13 in vivo. Furthermore, we show that CARM1 also regulates MMP expression at the post-transcriptional level, either positively or negatively. Our data indicate that CARM1 can play a dual role in the expression of AP-1 target genes involved in cancer or other diseases by acting at the transcriptional as well as at the post-transcriptional levels.

DNA-binding specificity of the CYS3 transcription factor of Neurospora crassa defined by binding-site selection

The CYS3 transcription factor is a basic region-leucine zipper (bZIP) DNA-binding protein that is essential for the expression of a coordinately regulated group of genes involved in the acquisition and utilization of sulfur in Neurospora crassa. An approach of using binding-site selection from random-sequence oligonucleotides was used to define CYS3-binding specificity. The derived consensus-binding site of ATGGCGCCAT defines a symmetrical sequence (half-site A T G/t G/a C/t) that resembles that of other bZIP proteins such as CREB and C/EBP. By comparison, CYS3 shows a greater range of binding to a central core of varied Pur–Pyr–Pur–Pyr sequences than CREB as determined by gel shift assays. The derived CYS3 consensus binding sequence was further validated by demonstrating in vivo sulfur regulation using a heterologous promoter construct. The CYS3-binding site data will be useful for the genome-wide study of sulfur-regulated genes in N. crassa, which has served as a model fungal sulfur control system.

Multiple Nuclear Localization Signals Function in the Nuclear Import of the Transcription Factor Nrf2

Nuclear factor erythroid 2-related factor 2 (Nrf2) mediates the transcriptional response of cells to oxidative stress and is translocated into the nucleus following, or concomitant with, its activation by electrophiles or reactive oxygen species. The mechanism of its translocation into the nucleus is not entirely elucidated. Here we have identified two novel nuclear localization signal (NLS) motifs in murine Nrf2, one located near the N-terminal region (amino acid residues 42-53) and the other (residues 587-593) located near the C-terminal region. Imaging of green fluorescent protein (GFP)-tagged Nrf2 revealed that mutation(s) in any of these sequences resulted in decreased nuclear fluorescence intensity compared with the wild-type Nrf2 when Nrf2 activation was induced with the electrophile tert-butylhydroquinone. The mutations also impaired Nrf2-induced transactivation of antioxidant response element-driven reporter gene expression to the same extent as the Nrf2 construct bearing mutation in a previously identified bipartite NLS that maps at residues 494-511. When linked to GFP or to GFP-PEPCK-C each of the novel NLS motifs was sufficient to drive nuclear translocation of the fusion proteins. Co-immunoprecipitation assays demonstrated that importins alpha5 and beta1 associate with Nrf2, an interaction that was blocked by the nuclear import inhibitor SN50. SN50 also blocked tert-butylhydroquinone-induced nuclear fluorescence of GFP-Nrf2 in cells transfected with wild-type GFP-Nrf2. Overall these results reveal that multiple NLS motifs in Nrf2 function in its nuclear translocation in response to pro-oxidant stimuli and that the importin alpha-beta heterodimer nuclear import receptor system plays a critical role in the import process.

The role of Nrf2 in increased reactive oxygen species and DNA damage in prostate tumorigenesis

The impact of oxidative stress in human cancer has been extensively studied. It is accepted that elevated reactive oxygen species (ROS) promote mutagenic DNA damage. Even with an extensive armament of cellular antioxidants and detoxification enzymes, alterations to DNA occur that initiate cellular transformation. Erythroid 2p45 (NF-E2)-related factor 2 (Nrf2) is a basic-region leucine zipper transcription factor that mediates the expression of key protective enzymes through the antioxidant-response element (ARE). By analysing 10 human prostate cancer microarray data sets, we have determined that Nrf2 and members of the glutathione-S-transferase (GST) mu family are extensively decreased in human prostate cancer. Using the TRAMP transgene and Rb and Nrf2 knockout murine models, we demonstrated that the loss of Nrf2 initiates a detrimental cascade of reduced GST expression, elevated ROS levels and ultimately DNA damage associated with tumorigenesis. Based on overwhelming data from clinical samples and the current functional analysis, we propose that the disruption of the Nrf2-antioxidant axis leads to increased oxidative stress and DNA damage in the initiation of cellular transformation in the prostate gland.

CEBPA polymorphisms and mutations in patients with acute myeloid leukemia, myelodysplastic syndrome, multiple myeloma and non-Hodgkin's lymphoma

The transcription factor CCAAT/enhancer binding protein (C/EBP)α is a myeloid-specific transcription factor which is required for normal myeloid differentiation. C/EBPα is encoded by an intronless gene that is 2783 bp long and maps to human chromosome 19q13.1. C/EBPα is a member of the basic region leucine zipper (bZIP) class of DNA-binding proteins. The loss of function of C/EBPα has leukemogenic potential. Four types of polymorphisms and 25 mutations (3 already known mutations and 22 novel mutations) were detected in CEBPA (gene for the transcription factor CCAAT/enhancer binding protein (C/EBP) α) in analysed samples from 390 patients with myelodysplastic syndrome (MDS) and hematologic malignancies. CEBPA mutations were found in 14/152 (9.2%) of acute myeloid leukemia (AML) patients' samples, 6/143 (4.2%) of MDS patients' samples, 2/56 (3.6%) of non-Hodgkin's lymphoma (NHL) patients' samples and 2/39 (5.1%) of multiple myeloma (MM) patients' samples. No C/EBPα mutations were detected in healthy donors (41 individuals). We discuss how these mutations can affect the cellular function of C/EBPα and block the myeloid differentiation.

Trehalose Is an Important Mediator of Cap1p Oxidative Stress Response in Candida albicans

Trehalose, a nonreducing disaccharide which accumulates dramatically during stationary phase or under oxidative stress, is well known as a stress protectant in several organisms. Here we investigated the putative correlation of trehalose with Cap1p, which is a basic region-leucine zipper (bZip) transcription factor participating in oxidative stress tolerance in Candida albicans. HPLC-MS analysis showed that trehalose did not accumulate in the cap1/cap1 mutant during stationary phase. When the mutant was exposed to high concentration of H2O2, trehalose accumulation was still not induced. Under both of the conditions above, the cap1/cap1 mutant showed high sensitivity to H2O2, and the cell viability was rather low. Furthermore, when exogenous trehalose was added to the culture of the cap1/cap1 mutant, the tolerance of this strain to oxidative stress was increased. Real time reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that the transcript levels of TPS2 and TPS3 were increased in the wild type strain compared to that in cap1/cap1 mutant when exposed to H2O2. These results indicated that trehalose accumulation is important to the oxidative stress tolerance mediated by Cap1p in C. albicans.

T Helper Cell-specific Regulation of Inducible Costimulator Expression via Distinct Mechanisms Mediated by T-bet and GATA-3

Inducible costimulator (ICOS) expression is critical for T cell-mediated immunity. We showed previously that T cell receptor and CD28 co-engagement up-regulate ICOS expression in activated T cells via the induction of NFATc2. Here, we examined the regulation of ICOS expression by Th-specific transcription factors T-bet and GATA-3. Overexpression of T-bet or GATA-3 alone could enhance, and NFATc2 could further synergize with either of them to increase, icos transcription. Although T-bet acted on the icos promoter, GATA-3 operated via an icos 3'-un-translated region element. Interestingly, NFATc2 was found to bind promiscuously the icos promoter in developing Th0, Th1, and Th2 cells but became selectively associated with T-bet at the promoter and with GATA-3 at the 3'-untranslated region in fully differentiated Th1 and Th2 cells, respectively. Collectively, our results reveal a temporally evolving circuit in which the non-selectively expressed NFATc2 cooperates with Th-restricted T-bet or GATA-3 to direct transcription of a costimulatory gene via distinct regulatory elements in different Th cells undergoing differentiation.

The sucrose regulated transcription factor bZIP11 affects amino acid metabolism by regulating the expression of ASPARAGINE SYNTHETASE1 and PROLINE DEHYDROGENASE2

Translation of the transcription factor bZIP11 is repressed by sucrose in a process that involves a highly conserved peptide encoded by the 5' leaders of bZIP11 and other plant basic region leucine zipper (bZip) genes. It is likely that a specific signaling pathway operating at physiological sucrose concentrations controls metabolism via a feedback mechanism. In this paper bZIP11 target processes are identified using transiently increased nuclear bZIP11 levels and genome-wide expression analysis. bZIP11 affects the expression of hundreds of genes with proposed functions in biochemical pathways and signal transduction. The expression levels of approximately 80% of the genes tested are not affected by bZIP11 promoter-mediated overexpression of bZIP11. This suggests that <20% of the identified genes appear to be physiologically relevant targets of bZIP11. ASPARAGINE SYNTHETASE1 and PROLINE DEHYDROGENASE2 are among the rapidly activated bZIP11 targets, whose induction is independent of protein translation. Transient expression experiments in Arabidopsis protoplasts show that the bZIP11-dependent activation of the ASPARAGINE SYNTHETASE1 gene is dependent on a G-box element present in the promoter. Increased bZIP11 expression leads to decreased proline and increased phenylalanine levels. A model is proposed in which sugar signals control amino acid levels via the bZIP11 transcription factor.

C/EBPα:AP-1 leucine zipper heterodimers bind novel DNA elements, activate the PU.1 promoter and direct monocyte lineage commitment more potently than C/EBPα homodimers or AP-1

The basic-region leucine zipper (BR-LZ or bZIP) transcription factors dimerize via their LZ domains to position the adjacent BRs for DNA binding. Members of the C/EBP, AP-1 and CREB/ATF bZIP subfamilies form homodimeric or heterodimeric complexes with other members of the same subset and bind-specific DNA motifs. Here we demonstrate that C/EBPalpha also zippers with AP-1 proteins and that this interaction allows contact with novel DNA elements and induction of monocyte lineage commitment in myeloid progenitors. A leucine zipper swap:gel shift assay demonstrates that C/EBPalpha zippers with c-Jun, JunB or c-Fos, but not with c-Maf or MafB. To evaluate activities of specific homodimers or heterodimers we utilized LZs with acid (LZE) or basic (LZK) residues in their salt bridge positions. C/EBPalphaLZE:C/EBPalphaLZK preferentially binds a C/EBP site, c-JunLZE:c-FosLZK an AP-1 site and C/EBPalphaLZE:c-JunLZK a hybrid element identified as TTGCGTCAT by oligonucleotide selection. In murine myeloid progenitors, C/EBPalpha:c-Jun or C/EBPalpha:c-Fos LZE:LZK heterodimers induce monocyte lineage commitment with markedly increased potency compared with C/EBPalpha or c-Jun homodimers or c-Jun:c-Fos heterodimers, demonstrating a positive functional consequence of C/EBP:AP-1 bZIP subfamily interaction. C/EBPalpha:cJun binds and activates the endogenous PU.1 promoter, providing one mechanism for induction of monopoiesis by this complex.

Prevalence and Prognostic Implications of CEBPα Mutations in Pediatric AML.

CCAAT/enhancer binding protein-α (C/EBPα) is a transcription factor that regulates granulocytic differentiation. Molecular alterations of the CEBPα gene which alter its function have been identified in AML; their presence may have prognostic implications in AML. We evaluated the prevalence and prognostic significance of CEBPα mutations in a cohort of 428 pediatric AML patients treated on pediatric AML trials CCG-2941 and CCG-2961. Initial screening of 100 samples by direct sequencing of the entire coding region of the CEBPα gene identified mutations in 8/100 (8%) of the patients tested. All 8 mutations were insertional or deletional mutations. A multiplex micro-capillary electrophoresis was devised to facilitate further screening of an additional 328 samples. Of the 428 samples analyzed, 65 mutations were identified in 57 patients (13%). 16/65 mutations (25%) were insertions or deletions in the N-terminal region, 14 of which would cause a frameshift resulting in a premature stop codon. 32/65 (49%) mutations involved the second transactivation domain (TAD2); except for one case, all were 6 bp insertions, and except for one case, all occurred at the same location. 17/65 (26%) mutations were in-frame insertions (N=16) or deletions (N=1) in the basic region leucine zipper (bZip) domain. This resulted in the insertion of one to twelve amino acids, or the deletion of six amino acids, which would be expected to disrupt the leucine zipper. 6/57 (11%) patients with mutations harbored more than one distinct mutation; all such instances of combined mutations included at least one mutation in the N-terminal region coupled with at least one mutation in the bZip domain. Presence of mutations was correlated with laboratory and clinical characteristics and clinical outcome. There were no differences in median age, sex, race, median diagnostic blast % or median diagnostic WBC between those with and without CEBPα mutations, although such mutations were less common in the younger patients between ages of 0–2 years (p=0.02). CEBPα mutations were more common in those with normal karyotype (38% vs. 21%, p=0.03) and less common in those with 11q23 abnormalities (8% vs. 26%, p=0.01). Of the 57 patients with CEBPα mutations, 15% and 10% had t(8;21) and inv(16), respectively, no different than those without mutations. Four of the 57 patients with CEBPα mutations had FLT3/ITD (7%) compared to 41/363 (11%) of the CEBPα-WT patients (p=0.46). Response to induction therapy was similar for those with and without mutations (91% vs. 86%, p=0.43). Actuarial overall survival (OS) from study entry was 63% for those with and 52% for those without CEBPα mutations (P=0.12). Of those who achieved an initial remission, cumulative incidence of relapse from remission was 25% vs. 39% for those with and without CEBPα mutations (p=0.069) with a corresponding OS at 5 years from remission of 67% vs. 58%, (p=0.27). CEBPα mutations are frequent events in pediatric AML and are highly associated with normal karyotype. Analysis of a larger cohort of patients may determine whether those with CEBPα mutations have a lower relapse rate and improved survival.

Significant Role of Peptidyl-Prolyl cis/trans Isomerase, Pin1 in Acute Myeloid Leukemia with C/EBPα Mutations.

Transcription factor CCAAT enhancer binding protein α (C/EBPα) is crucial for the differentiation of granulocytes. Experimental data from animal models as well as patient samples suggest that loss of function or expression of C/EBPα provides a platform on which acute myeloid leukemia (AML) develops. C/EBPα is mutated in around 9% of acute myeloid leukemia. The mutations reported in C/EBPα are frame shift mutations at N-terminal domain and point mutations at basic region Leucine zipper. The mutant form of C/EBPα ie C/EBPα-p30 exhibits dominant negative function over the wild type protein. Peptidyl-prolyl cis/trans isomerase, Pin1 binds to and isomerizes the peptidyl-prolyl bond in specific phosphorylated Ser/Thr-Pro motifs. A growing number of studies show that Pin1 is overexpressed in many cancers and has significant role in tumorigenesis. In the present study we investigated the role of Pin1 in acute myeloid leukemia with C/EBPα mutation. Here we report C/EBPα-p30 could induce Pin1 transcription as assessed by quantitative Real-Time RT-PCR analysis. Affymetrix mRNA expression analysis show that Pin1 is upregulated in patients with acute myeloid leukemia. Silencing of Pin1 could overcome the dominant negative action of the C/EBPα-p30 over the C/EBPα-p42 transactivation capacity as analyzed by promoter assay. By silencing Pin1 with inhibitor against Pin1 (PiB), we could show myeloid differentiation in Kasumi-6 cells by FACS analysis with granulocytic specific markers. Western blot analysis shows that Pin1 inhibition by PiB could upregulate wild type C/EBPα protein level. Luciferase promoter assay for the Pin1 promoter shows that C/EBPα-p30 induces Pin1 promoter activity in association with E2F1. Mean while, wild type C/EBPα interferes with transactivation of the Pin1 promoter and downregulates Pin1 mRNA expression. We investigated the mechanism underlying the dominant negative action of C/EBPα-p30 over the wild type protein. We have previously shown that c-Jun expression is high in AML patients with C/EBPα mutation and c-Jun could block C/EBPα function by protein-protein interaction. Quantitative Real-Time RT-PCR analysis shows that overexpression of Pin1 induces c-Jun mRNA expression, while inhibition of Pin1 by the inhibitor PiB downregulates c-Jun mRNA expression. We show that c-Jun blocks the DNA binding and transactivation of wild type C/EBPα protein as assessed by gel shift assay and promoter assay respectively. In conclusion, inhibition of Pin1 leads to granulocytic differentiation of human myeloid cells. Our findings suggest inhibition of Pin1 as a novel strategy in treating AML patients with C/EBPα mutation.

C/EBPα:AP-1 Leucine Zipper Heterodimers Bind Novel DNA Elements, Activate the PU.1 Promoter, and Direct Monocyte Lineage Commitment More Potently Than C/EBPα Homodimers or AP-1.

The basic-region leucine zipper (BR-LZ or bZIP) transcription factors dimerize via their LZ domains to position the adjacent BRs for DNA-binding. Members of the C/EBP, AP-1, and CREB/ATF bZIP subfamilies form homodimeric or heterodimeric complexes with other members of the same subset and bind specific DNA motifs. Here we demonstrate that C/EBPα also zippers with AP-1 proteins and that this interaction allows contact with novel DNA elements and induction of monocyte lineage commitment in myeloid progenitors. A leucine zipper swap:gel shift assay demonstrates that C/EBPα zippers with c-Jun, JunB, or c-Fos, but not with c-Maf or MafB. In addition, C/EBPα co-ips c-Jun, JunB, or c-Fos, either expressed in 293T cells or as endogenous proteins in a myeloid cell line. Affinity of C/EBPα for AP-1 proteins appears weaker in this assay than affinity of C/EBPα for the related C/EBPβ, suggesting that interaction of C/EBP and AP-1 proteins is not primarily cross-inhibitory but instead generates heterodimers capable of binding novel DNA elements and inducing unique biological effects. To evaluate activities of specific homodimers or heterodimers we utilized LZs with acid (LZE) or basic (LZK) residues in their salt bridge positions. This approach is advantageous over efforts to fuse bZIP proteins together as zippering of tethered proteins may be sterically hindered and disruptive to the remainder of the protein. We find that C/EBPαLZE:C/EBPαLZK preferentially binds a C/EBP site, c-JunLZE:c-FosLZK an AP-1 site, and C/EBPαLZE:c-JunLZK a hybrid element identified as 5′-TTGCGTCAT by oligonucleotide selection. In contrast, c-Jun homodimers selected the consensus site 5′-TGACGTCAT, and C/EBPα homodimers selected 5′-NNGNNGCAAC. Note that 5′-TTGC in the selected hybrid site corresponds to 5′-GCAA in the C/EBPα site. In lineage-negative murine myeloid progenitors, either C/EBPα-ER or c-Jun-ER potently induce monocyte as compared to granulocyte development. Using LZE:LZK proteins linked to the ER ligand-binding domain, we find that C/EBPα:c-Jun or C/EBPα:c-Fos LZE:LZK heterodimers induce monocyte lineage commitment with markedly increased potency compared with C/EBPα or c-Jun homodimers or c-Jun:c-Fos heterodimers, demonstrating a positive functional consequence of C/EBP:AP-1 bZIP subfamily interaction. We propose that C/EBPα induction of monopoiesis requires zippering with endogenous c-Jun and that conversely induction of monopoiesis by c-Jun requires zippering with endogenous C/EBPα. Finally, using Ba/F3 lines stably expressing LZE:LZK ER fusion proteins, we find that C/EBPα:c-Jun binds the endogenous PU.1 promoter as assessed by chromatin immunoprecipitation and that exposure of cells expressing C/EBPαLZK-ER and c-JunLZE-ER to estradiol induces PU.1 mRNA expression as assessed by quantitative RT-PCR, whereas C/EBPαLZK-ER in combination with C/EBPαLZE-ER did not induce PU.1 mRNA in Ba/F3 cells. In addition, both endogenous C/EBPα and c-Jun were detected by the ChIP assay in the vicinity of the PU.1 promoter in 32Dcl3 myeloid cells. Thus, induction of PU.1 may contribute to stimulation of monopoiesis by C/EBPα:c-Jun heterodimers. In addition, as C/EBP and AP-1 proteins are widely expressed, zippering between these bZIP subfamilies may have broad biologic relevance. To gain insight into the spectrum of genes activated specifically by this complex, we have initiated microarray studies using our Ba/F3 cell lines.

A Rapid and Sensitive Method for Large-Scale Screening of CEBPA Mutations in AML Patients with Normal Karyotype.

In 45% of patients with de novo acute myeloid leukemia (AML) no cytogenetic abnormalities can be detected (normal karyotype AML, NK-AML). Recently, several molecular markers with prognostic significance have been described which define distinct subgroups of NK-AML patients. Mutations in the CEBPA gene have shown to occur at about 8% of NK-AML in Western countries and confer a favorable prognosis. The C/EBPα protein, a member of the family of basic region leucine zipper (bZIP) transcriptional regulators, is important for normal granulocytic differentiation and is frequently disrupted in AML. We retrospectively analyzed bone-marrow samples of 442 patients with de novo NK-AML for the presence of CEBPA mutations and established a fast and sensitive screening method. A multiplex-PCR-gene scanning assay for combined detection of CEBPA and NPM mutation has recently been described in which, however, the primers did not cover the whole CEBPA gene. CEBPA mutations have been found to be distributed over the entire CEBPA gene and their functional and clinical consequences are not yet clear. Therefore, we designed a rapid CEBPA specific multiplex PCR-gene scanning assay covering the entire coding region of the CEBPA gene. Four primer pairs were designed, fluorescently labeled and included in 2 multiplex PCR reactions. The PCR products were electrophoresed on a genetic analyzer and the amplicon sizes were compared to wildtype CEBPA of U937 cell line by fragment length analysis. In order to evaluate our method, we analyzed 120 patient samples in parallel by both multiplex PCR and sequencing analysis. Using sequencing analysis as a gold standard, all of the CEBPA mutations could be detected by multiplex PCR and fragment length analysis. Thus, our multiplex PCR assay reached a sensitivity of 100%. The specificity was 89% due to the false positive detection of a 6 basepair duplication polymorphism in 2.5% of patients (Wouters BJ et al, Blood, 2007). 322 patient samples were subsequently screened for CEBPA mutations by the multiplex PCR assay. In case of alterations in the fragment length analysis, the relevant CEBPA region was sequenced to identify the exact type of mutation. Among 442 patients with NK-AML, 32 patients (7%) showed CEBPA mutations. Taken together, we identified 47 mutations in 32 patients, of which 17 patients had a single CEBPA mutation and 15 patients had more than one CEBPA mutation. We identified 30 out of frame insertion/deletion nonsense mutations resulting in an N-terminal stopcodon and 14 in frame insertion/deletion mutations as well as 3 out of frame insertion/deletion mutations in the C-terminal bZIP region. The only limitation of this method might be that single basepair substitutions, which do not affect the length of the amplicon, cannot be detected. Substitutions in the CEBPA gene are, however, rare events and often silent. In 120 sequenced AML patients we did not find any non-silent substitution. We established a fast and sensitive screening method suitable for large-scale detection of CEBPA mutation and applicable for inclusion in routine AML diagnostics. This is so far the largest reported analysis of CEBPA mutations in patients with NK-AML and might provide further insights into the functional and clinical relevance of the different types of CEBPA mutations and their correlation to other molecular markers in NK-AML.

ATF5 promotes cell survival through transcriptional activation of Hsp27 in H9c2 cells

The activating transcription factor (ATF) family comprises a group of basic region-leucine zipper (bZIP) proteins, which have roles in development, differentiation, proliferation and apoptosis. Here we described that ATF5 could promote cell survival in heat shock stress and identified that heat shock protein 27 (Hsp27) as one of its target gene related to cell survival. The ectopic expression of ATF5 could significantly increase cell tolerance to elevated temperature and the CRE motif played an important role in upregulating Hsp27 gene promoter activity by ATF5. Also, inhibition of Hsp27 expression by RNAi could increase cell death in ATF5 ectopic expressing cells, indicating that Hsp27 involved in ATF5 mediated cell survival. Taken together, our results indicated that ATF5 promoted H9c2 cell survival in heat shock stress at least through activation of Hsp27.

Cancer chemoprevention by phytochemicals: potential molecular targets, biomarkers and animal models

Recent studies have strongly indicated that certain daily-consumed dietary phytochemicals could have cancer protective effects against transgenic mice cancer models and cancers mediated by carcinogens, irradiations and carcinogenic metabolites derived from exogenous or endogenous sources. The cancer-protective effects elicited by these dietary compounds are believed to be due at least in part to the induction of cellular defense systems including the detoxifying and antioxidant enzymes system, as well as the inhibition of anti-inflammatory and anti-cell growth signaling pathways culminating in cell cycle arrest and/or celldeath. In this review, we summarize the potential mechanisms including the modulation of nuclear factor kappaB (NF-kappaB), cyclooxygenases-2 (COX-2), activator protein-1 (AP-1), mitogen-activated protein kinases (MAPKs) and the induction of phase II cellular detoxifying and antioxidant enzymes mediated mainly by the antioxidant response elements (ARE) within the promoter regions of these genes through nuclear factor-erythroid 2-related factor 2 (Nrf2), a member of the Cap "n" collar (CNC) family of the basic region-leucine zipper transcription factor. In addition, we also review several animal models of carcinogenesis and cancer chemopreventive efficacy studies of these animal models using dietary chemopreventive compounds. Finally, we discuss the cellular signaling cascades mediated by Nrf2, NF-kappaB, AP-1, MAPKs and COX-2, which have been considered to play pivotal roles in tumor initiation, promotion and progression processes, and could be promising molecular targets for the design of drugs targeting cancer prevention and therapy.