FoxO Transcription Factor Is Delocalized and Inactivated in Acute Myeloid Leukaemia Patients.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1251-1251
Author(s):  
Daniela Cilloni ◽  
Cristina Panuzzo ◽  
Francesca Messa ◽  
Francesca Arruga ◽  
Paolo Nicoli ◽  
...  
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Mean Value ◽  
Binding Activity ◽  
Negative Regulator ◽  
Pi3k Inhibitor Ly294002 ◽  
Downstream Target ◽  
Dna Binding Activity ◽  
Blast Cells ◽  
Nuclear Signal

Abstract The FoxO family of transcription factors is regulated by PI3K/Akt induced phosphorylation resulting in nuclear exclusion and degradation. Nuclear FoxO transcribes proapoptotic molecules and cell cycle inhibitors. Although multiple mechanisms regulate FoxO activity, Akt seems to be crucial to its regulation and function. PI3K/Akt pathway has been reported to be abnormally activated in AML blast cells. The aim of this study was to investigate the function of FoxO in AML blast cells and the presence of alternative pathways responsible for FoxO3 inactivation other than PI3K-Akt. BM cells were collected from 35 AML patients at diagnosis and after chemotherapy and from 20 healthy donors. The expression levels of FoxO1, FoxO3, FoxO4 were tested by RQ-PCR, FoxO3 protein amount and localization by Western blot and immunofluorescence and the DNA binding activity by EMSA. Furthermore, downstream target genes transcribed by FoxO3 were quantified. Among these, Spred1 which codes for a negative regulator of RTK signal, including Ras mediated pathway triggered by FLT3. We have previously described the absence of Spred1 is AML patients and we have demonstrated that it promotes growth arrest and apoptosis in haematopoietic cells. Finally, BM cells were incubated with 5 mM of the PI3K inhibitor LY294002 and 20 mM PS1145, the inhibitor of IKK kinase also responsible for FoxO phosphorylation and with the combination LY294002 plus PS1145. We found that the amount of FoxO1, FoxO3 and FoxO4 mRNA are similar in AML patients and controls. Interestingly, while FoxO3 in control cells is localized in both, nucleous and cytoplasm, is completely cytoplasmatic in AML cells and it enters the nucleous after chemotherapy. The quantification of FoxO fluorescent signal in controls shows a mean value of intensity of 21.4±2 in the nucleous and 14,6±1.7 in the cytoplasm. By contrast, in AML cells is 8,2±4 in the nucleous and 18.1±4,6 in the cytoplasm. Additionally, FoxO3 DNA binding activity in AML patients is completely absent at diagnosis and reappears after therapy. Also the mRNA of the target gene Spred1 is rather undetectable at diagnosis (mean value 2−ΔΔCt= 0,009±0,3) and is upregulated during remission (mean value 2−ΔΔ= 2±1,5) or after LY29400 incubation (mean value =0,8±0,3). LY294002 and PS1145 results in FoxO partial nuclear relocalization with a nuclear signal of 15±3 and 12±3 respectively. Interestingly, the association of PS1145 and LY294002 induces a complete nuclear shuttle with a nuclear signal of 25±4, suggesting that both pathways are implicated in FoxO inactivation. Taken together these observations suggest that FoxO inactivation may be crucial for the apoptosis arrest observed in AML. These data demonstrate that also IKK pathway contributes to this effect, providing the rationale for a therapeutic strategy based on the combination of selective inhibitors such as FLT3 or Akt inhibitors or standard chemotherapy and the IKK inhibitor.

FoxO Transcription Factor Is Delocalized in CML Patients by Bcr-Abl Induced PI3K/AKT Activation and IKK Pathway and Can Be Reactivated by Imatinib Treatment.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1010-1010
Author(s):  
Daniela Cilloni ◽  
Cristina Panuzzo ◽  
Francesca Messa ◽  
Francesca Arruga ◽  
Paolo Nicoli ◽  
...  
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Mean Value ◽  
Binding Activity ◽  
Negative Regulator ◽  
Imatinib Treatment ◽  
Pi3k Inhibitor Ly294002 ◽  
Downstream Target ◽  
Dna Binding Activity ◽  
Nuclear Signal

Abstract The FoxO family of transcription factors is regulated by PI3K/Akt induced phosphorylation resulting in nuclear exclusion and degradation. Nuclear FoxO transcribes proapoptotic molecules and cell cycle inhibitors. Although multiple mechanisms regulate FoxO activity, Akt seems to be crucial to its regulation and function. In Chronic Myeloid Leukemia the TK activity of Bcr-Abl leads to the abnormal activation of downstream effectors including PI3K/Akt. The aim of this study was to investigate the role of FoxO in Bcr-Abl induced apoptotic arrest and cell growth, the consequence of imatinib (IM) treatment on FoxO activity and the alternative pathways responsible for FoxO3 inactivation other than PI3K-Akt. BM cells were collected from 20 CML patients at diagnosis and during IM treatment and from 20 healthy donors. The expression of FoxO1, FoxO3, FoxO4 were tested by RQ-PCR, FoxO3 protein amount and localization by Western blot and immunofluorescence and the DNA binding activity by EMSA. Downstream target genes transcribed by FoxO3 were quantified. Among these, Spred1 which codes for a negative regulator of RTK signal, including Ras mediated pathway triggered by Bcr-Abl. We have previously described the absence of Spred1 is CML cells and we have demonstrated that it promotes growth arrest and apoptosis in haematopoietic cells. Finally, BM cells and BV173 Ph+ cell line were incubated with 1 μM IM, 5 μM of the PI3K inhibitor LY294002 and 20 μM PS1145, the inhibitor of IKK kinase also responsible for FoxO phosphorylation, and with the combination of IM plus PS1145 and LY294002 plus PS1145. We found that the amount of FoxO1, FoxO3 and FoxO4 mRNA are similar in CML patients and controls. Interestingly, while FoxO3 in control cells is localized in both, nucleous and cytoplasm, is completely cytoplasmatic in Ph+ CML cells and it enters the nucleous during IM treatment. The quantification of FoxO fluorescent signal in controls shows a mean value of intensity of 21.4±2 in the nucleous and 14,6±1.7 in the cytoplasm. By contrast, in CML cells is 6.6±0.8 in the nucleous and 16.7±1.1 in the cytoplasm. Additionally, FoxO3 DNA binding activity in CML patients is completely absent at diagnosis and reappears during therapy or after IM incubation. Also the mRNA of the target gene Spred1 is rather undetectable at diagnosis (mean value 2−ΔΔCt= 0,001±0,09) and is upregulated during remission (mean value 2−ΔΔCt= 1,2±1,8) or after IM incubation (mean value = 0,7±0,9) or LY294002(1±0,7). Exposure to IM, LY294002 and PS1145 results in FoxO partial nuclear relocalization with a nuclear signal of 15±5, 17 ±3 and 12±2 respectively. Interestingly, the association of PS1145 and IM or PS1145 and LY294002 induces a complete nuclear shuttle with a nuclear signal of 23±4 and 24±4 respectively, suggesting that both pathways are implicated in FoxO inactivation. These observations suggest that FoxO inactivation may be crucial for Bcr-Abl induced proliferation and apoptosis arrest. The antiproliferative activity of IM may be mediated by FoxO3 re-localization. Nevertheless, we demonstrated that also IKK pathway contributes to this effect, providing the rationale for a therapeutic strategy based on the combination of IM plus an IKK inhibitor.

Analysis of the DNA-binding activity of p53 mutants using functional protein microarrays and its relationship to transcriptional activation

2010 ◽  
Vol 391 (2/3) ◽  
Author(s):  
Jitka Malcikova ◽  
Boris Tichy ◽  
Jiri Damborsky ◽  
Jitka Kabathova ◽  
Martin Trbusek ◽  
...  
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Target Genes ◽  
P53 Protein ◽  
Protein Microarrays ◽  
Binding Activity ◽  
Functional Protein ◽  
Downstream Target ◽  
Dna Binding Activity ◽  
Gene Assay

Abstract Sequence-specific DNA binding is the key function through which tumor suppressor p53 exerts transactivation of the downstream target genes, often being impaired in cancer cells by mutations in the TP53 gene. Functional protein microarray technology enables a high-throughput parallel analysis of protein properties within one experiment under the same conditions. Using an array approach, we analyzed the DNA binding activity of wild type p53 protein and of 49 variants. Our results show significant differences in the binding properties between the p53 mutants. The C-terminal mutant R337C displayed the highest DNA binding activity on the array. However, the same mutant showed only a partial activation in the reporter gene assay and almost no activation of downstream target genes after transfection of expression vector into cells lacking endogenous p53. These observations demonstrate that DNA binding itself is not sufficient for activating the p53 target genes in at least some of the p53 mutants and, therefore, in vitro studies might not always reflect in vivo conditions.

scholarly journals Direct activation of Sex-lethal transcription by the Drosophila runt protein

Development ◽  
1999 ◽  
Vol 126 (1) ◽  
pp. 191-200 ◽  
Author(s):  
S.G. Kramer ◽  
T.M. Jinks ◽  
P. Schedl ◽  
J.P. Gergen
Keyword(s):  
Transcriptional Activation ◽  
Target Genes ◽  
Specific Binding ◽  
Binding Activity ◽  
Transcriptional Activation Domain ◽  
Downstream Target ◽  
Early Promoter ◽  
Dna Binding Activity ◽  
Sex Lethal

Runt functions as a transcriptional regulator in multiple developmental pathways in Drosophila melanogaster. Recent evidence indicates that Runt represses the transcription of several downstream target genes in the segmentation pathway. Here we demonstrate that runt also functions to activate transcription. The initial expression of the female-specific sex-determining gene Sex-lethal in the blastoderm embryo requires runt activity. Consistent with a role as a direct activator, Runt shows sequence-specific binding to multiple sites in the Sex-lethal early promoter. Using an in vivo transient assay, we demonstrate that Runt's DNA-binding activity is essential for Sex-lethal activation in vivo. These experiments further reveal that increasing the dosage of runt alone is sufficient for triggering the transcriptional activation of Sex-lethal in males. In addition, a Runt fusion protein, containing a heterologous transcriptional activation domain activates Sex-lethal expression, indicating that this regulation is direct and not via repression of other repressors. Moreover, we demonstrate that a small segment of the Sex-lethal early promoter that contains Runt-binding sites mediates Runt-dependent transcriptional activation in vivo.

scholarly journals The retinoblastoma gene product RB stimulates Sp1-mediated transcription by liberating Sp1 from a negative regulator.

1994 ◽  
Vol 14 (7) ◽  
pp. 4380-4389 ◽  
Author(s):  
L I Chen ◽  
T Nishinaka ◽  
K Kwan ◽  
I Kitabayashi ◽  
K Yokoyama ◽  
...  
Keyword(s):  
Dna Binding ◽  
Gene Product ◽  
Binding Activity ◽  
Negative Regulator ◽  
Control Element ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Dna Binding Activity ◽  
Cell Extracts ◽  
Mobility Shift Assay

Studies have demonstrated that the retinoblastoma susceptibility gene product, RB, can either positively or negatively regulate expression of several genes through cis-acting elements in a cell-type-dependent manner. The nucleotide sequence of the retinoblastoma control element (RCE) motif, GCCACC or CCACCC, and the Sp1 consensus binding sequence, CCGCCC, can confer equal responsiveness to RB. Here, we report that RB activates transcription of the c-jun gene through the Sp1-binding site within the c-jun promoter. Preincubation of crude nuclear extracts with monoclonal antibodies to RB results in reduction of Sp1 complexes in a mobility shift assay, while addition of recombinant RB in mobility shift assay mixtures with CCL64 cell extracts leads to an enhancement of DNA-binding activity of SP1. These results suggest that RB is directly or indirectly involved in Sp1-DNA binding activity. A mechanism by which RB regulates transactivation is indicated by our detection of a heat-labile and protease-sensitive Sp1 negative regulator(s) (Sp1-I) that specifically inhibits Sp1 binding to a c-jun Sp1 site. This inhibition is reversed by addition of recombinant RB proteins, suggesting that RB stimulates Sp1-mediated transactivation by liberating Sp1 from Sp1-I. Additional evidence for Sp1-I involvement in Sp1-mediated transactivation was demonstrated by cotransfection of RB, GAL4-Sp1, and a GAL4-responsive template into CV-1 cells. Finally, we have identified Sp1-I, a approximately 20-kDa protein(s) that inhibits the Sp1 complexes from binding to DNA and that is also an RB-associated protein. These findings provide evidence for a functional link between two distinct classes of oncoproteins, RB and c-Jun, that are involved in the control of cell growth, and also define a novel mechanism for the regulation of c-jun expression.

The complex interactions of p53 with target DNA: we learn as we go

2003 ◽  
Vol 81 (3) ◽  
pp. 141-150 ◽  
Author(s):  
Ella Kim ◽  
Wolfgang Deppert
Keyword(s):  
Tumor Suppressor ◽  
Dna Binding ◽  
Target Genes ◽  
Dna Structure ◽  
Structural Features ◽  
Binding Activity ◽  
Tumor Suppressor P53 ◽  
Dna Binding Activity ◽  
Complex Interactions ◽  
Target Dna

The most import biological function of the tumor suppressor p53 is that of a sequence-specific transactivator. In response to a variety of cellular stress stimuli, p53 induces the transcription of an ever-increasing number of target genes, leading to growth arrest and repair, or to apoptosis. Long considered as a "latent" DNA binder that requires prior activation by C-terminal modification, recent data provide strong evidence that the DNA binding activity of p53 is strongly dependent on structural features within the target DNA and is latent only if the target DNA lacks a certain structural signal code. In this review we discuss evidence for complex interactions of p53 with DNA, which are strongly dependent on the dynamics of DNA structure, especially in the context of chromatin. We provide a model of how this complexity may serve to achieve selectivity of target gene regulation by p53 and how DNA structure in the context of chromatin may serve to modulate p53 functions.Key words: tumor suppressor p53, sequence-specific DNA binding, DNA conformation, chromatin, chromatin remodeling.

Identification of Cyclin D3 as a Direct Transcriptional Target of E2A Using Damid.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1618-1618
Author(s):  
John K. Choi ◽  
Siyuan Song ◽  
Jonathan Cooperman ◽  
Danielle L. Letting ◽  
Gerd A. Blobel
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
B Cell ◽  
Target Genes ◽  
Cell Development ◽  
Binding Activity ◽  
B Cell Development ◽  
Cyclin D3 ◽  
Dna Binding Activity ◽  
E Boxes

Abstract The transcription factor E2A is required for very early B cell development. The exact mechanism by which E2A promotes B cell development is unclear and cannot be explained by the known E2A targets, components of the pre-B cell receptor and cyclin dependent kinase inhibitors, indicating additional pathways and targets remain to be identified. We had previously reported that E2A can promote precursor B cell expansion, promote G1 cell cycle progression, and induce the expressions of multiple G1 phase cyclins including cyclin D3, suggesting that E2A induction of these genes may contribute to early B cell development. To better understand the mechanism by which E2A induces these cyclins, we characterized the relationship between E2A and the cyclin D3 gene promoter. E2A transactivated a luciferase reporter plasmid containing the 1kb promoter of cyclin D3 that contains two consensus E2A binding sites (E-boxes); however, deletion of the E-boxes did not disrupt the transactivation by E2A. We hypothesized three possible mechanisms: 1) indirect activation of cyclin D3 via another transcription factor, 2) binding of E2A to cryptic non-E-boxes, or 3) recruitment of E2A to the promoter via interaction with other DNA binding factor. To test the first possibility, promoter occupancy was examined using the DamID approach. In this approach, a fusion protein consisting of E. coli DNA adenosine methyltransferase (DAM) and a transcription factor of interest is expressed at low levels, resulting in specific methylation of adenosine residues within 2–5 kb of the transcription factor target sites. A fusion construct composed of E2A and DAM (E47Dam), was subcloned in lentiviral vectors, and used to transduce precursor B cell lines. The methylated adenosine residues were detected using a sensitive ligation-mediated PCR (LM-PCR) assay that required only 1 ug of genomic DNA and can detect methylation even if only 3% of the cells express E47Dam; no methylated adenosines were detected in control cells, indicating that all methylated residues resulted from E47Dam. Specific adenosine methylation was identified at the IgH intronic enhancer, a known E2A target site, but not at the non-target sites, CD19, HPRT, and GAPDH promoters. Specific methylation was detected at the cyclin D3 promoter but not 10 kb down-stream, despite similar concentrations of E-boxes at both sites. Chromatin immunoprecipitation analysis confirmed the DamID findings and further localized the binding to within 1 kb of the two E-boxes in the cyclin D3 promoter. To distinguish between the two remaining mechanisms (cryptic non-E-boxes versus recruitment via other DNA binding factors), two point mutations were introduced into E47Dam that disrupted its DNA binding activity. The mutated E47Dam continued to methylate at the cyclin D3 promoter. We conclude that E2A can be recruited to the cyclin D3 promoter, independent of E-boxes or E2A DNA binding activity. Our findings raise the possibility that some direct E2A target genes may lack functional E-boxes. Furthermore, mutated E2A, lacking an E2A DNA binding domain, that is seen in 6% of pediatric ALLs may still activate a subset of E2A target genes. Finally, our application of lentiviral vectors and LM-PCR to the DamID approach should permit analysis of primary human precursor B cells, despite the limitations in cell number and transduction efficiency.

scholarly journals Adenovirus E1B 55-Kilodalton Oncoprotein Inhibits p53 Acetylation by PCAF

2000 ◽  
Vol 20 (15) ◽  
pp. 5540-5553 ◽  
Author(s):  
Yue Liu ◽  
April L. Colosimo ◽  
Xiang-Jiao Yang ◽  
Daiqing Liao
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Target Genes ◽  
Binding Activity ◽  
Physical Interaction ◽  
Dna Binding Activity ◽  
C Terminus ◽  
Adenovirus E1b

ABSTRACT The adenovirus E1B 55-kDa protein binds to cellular tumor suppressor p53 and inactivates its transcriptional transactivation function. p53 transactivation activity is dependent upon its ability to bind to specific DNA sequences near the promoters of its target genes. It was shown recently that p53 is acetylated by transcriptional coactivators p300, CREB bidning protein (CBP), and PCAF and that acetylation of p53 by these proteins enhances p53 sequence-specific DNA binding. Here we show that the E1B 55-kDa protein specifically inhibits p53 acetylation by PCAF in vivo and in vitro, while acetylation of histones and PCAF autoacetylation is not affected. Furthermore, the DNA-binding activity of p53 is diminished in cells expressing the E1B 55-kDa protein. PCAF binds to the E1B 55-kDa protein and to a region near the C terminus of p53 encompassing Lys-320, the specific PCAF acetylation site. We further show that the E1B 55-kDa protein interferes with the physical interaction between PCAF and p53, suggesting that the E1B 55-kDa protein inhibits PCAF acetylase function on p53 by preventing enzyme-substrate interaction. These results underscore the importance of p53 acetylation for its function and suggest that inhibition of p53 acetylation by viral oncoproteins prevent its activation, thereby contributing to viral transformation.

scholarly journals Mth1 regulates the interaction between the Rgt1 repressor and the Ssn6-Tup1 corepressor complex by modulating PKA-dependent phosphorylation of Rgt1

2013 ◽  
Vol 24 (9) ◽  
pp. 1493-1503 ◽  
Author(s):  
Adhiraj Roy ◽  
Yong Jae Shin ◽  
Kyu Hong Cho ◽  
Jeong-Ho Kim
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Glucose Transporter ◽  
Binding Activity ◽  
Supporting Evidence ◽  
Dna Binding Activity ◽  
Rate Limiting Step ◽  
Corepressor Complex ◽  
Necessary And Sufficient ◽  
Rate Limiting

Glucose uptake, the first, rate-limiting step of its utilization, is facilitated by glucose transporters. Expression of several glucose transporter (HXT) genes in yeast is repressed by the Rgt1 repressor, which recruits the glucose-responsive transcription factor Mth1 and the general corepressor complex Ssn6-Tup1 in the absence of glucose; however, it is derepressed when Mth1 is inactivated by glucose. Here we show that Ssn6-Tup1 interferes with the DNA-binding ability of Rgt1 in the absence of Mth1 and that the Rgt1 function abrogated by Ssn6 overexpression is restored by co-overexpression of Mth1. Thus Mth1 likely regulates Rgt1 function not by modulating its DNA-binding activity directly but by functionally antagonizing Ssn6-Tup1. Mth1 does so by acting as a scaffold-like protein to recruit Ssn6-Tup1 to Rgt1. Supporting evidence shows that Mth1 blocks the protein kinase A–dependent phosphorylation of Rgt1 that impairs the ability of Rgt1 to interact with Ssn6-Tup1. Of note, Rgt1 can bind DNA in the absence of Ssn6-Tup1 but does not inhibit transcription, suggesting that dissociation of Rgt1 from Ssn6-Tup1, but not from DNA, is necessary and sufficient for the expression of its target genes. Taken together, these findings show that Mth1 is a transcriptional corepressor that facilitates the recruitment of Ssn6-Tup1 by Rgt1.

scholarly journals BCL11A promotes myeloid leukemogenesis by repressing PU.1 target genes

2021 ◽  
Author(s):  
Yoshitaka Sunami ◽  
Takashi Yokoyama ◽  
Seiko Yoshino ◽  
Tomoko Takahara ◽  
Yukari Yamazaki ◽  
...  
Keyword(s):  
Dna Binding ◽  
Myeloid Leukemia ◽  
Target Genes ◽  
Cell Interaction ◽  
Binding Activity ◽  
Hemoglobin Switching ◽  
Dna Binding Activity ◽  
Adult Hemoglobin

The transcriptional repressor, BCL11A, is involved in hematological malignancies, B-cell development, and fetal-to-adult hemoglobin switching. However, the molecular mechanism by which it promotes the development of myeloid leukemia remains largely unknown. We find that Bcl11a cooperates with the pseudokinase, Trib1, in the development of acute myeloid leukemia (AML). Bcl11a promotes the proliferation and engraftment of Trib1-expressing AML cells both in vitro and in vivo. ChIP-seq analysis showed that upon DNA-binding, Bcl11a is significantly associated with PU.1, an inducer of myeloid differentiation, and that Bcl11a represses several PU.1 target genes, such as Asb2, Clec5a, and Fcgr3. Asb2, as a Bcl11a target gene that modulates cytoskeleton and cell-cell interaction, plays a key role in Bcl11a-induced malignant progression. The repression of PU.1 target genes by Bcl11a is achieved by both sequence-specific DNA-binding activity and recruitment of corepressors by Bcl11a. Suppression of the corepressor components, HDAC and LSD1, reverses the repressive activity. Moreover, treatment of AML cells with the HDAC inhibitor, pracinostat, and LSD1 inhibitor, GSK2879552, resulted in growth inhibition both in vitro and in vivo. High BCL11A expression is associated with worse prognosis in human AML patients. Blocking of BCL11A expression upregulates the expression of PU.1 target genes, and inhibits the growth of HL-60 cells and their engraftment to the bone marrow, suggesting that BCL11A is involved in human myeloid malignancies via the suppression of PU.1 transcriptional activity.

scholarly journals Stress-induced binding of the transcriptional factor CHOP to a novel DNA control element.

1996 ◽  
Vol 16 (4) ◽  
pp. 1479-1489 ◽  
Author(s):  
M Ubeda ◽  
X Z Wang ◽  
H Zinszner ◽  
I Wu ◽  
J F Habener ◽  
...  
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Binding Activity ◽  
Control Element ◽  
Core Element ◽  
Transcriptional Activation Domain ◽  
Dna Binding Activity ◽  
Basic Region

CHOP (GADD153) is a mammalian nuclear protein that dimerizes with members of the C/EBP family of transcriptional factors. Absent under normal conditions, CHOP is induced by the stress encountered during nutrient deprivation, the acute-phase response, and treatment of cells with certain toxins. The basic region of CHOP deviates considerably in sequence from that of other C/EBP proteins, and CHOP-C/EBP heterodimers are incapable of binding to a common class of C/EBP sites. With respect to such sites, CHOP serves as an inhibitor of the activity of C/EBP proteins. However, recent studies indicate that certain functions of CHOP, such as the induction of growth arrest by overexpression of the wild-type protein and oncogenic transformation by the TLS-CHOP fusion protein, require an intact basic region, suggesting that DNA binding by CHOP may be implicated in these activities. In this study an in vitro PCR-based selection assay was used to identify sequences bound by CHOP-C/EBP dimers. These sequences were found to contain a unique core element PuPuPuTGCAAT(A/C)CCC. Competition in DNA-binding assays, DNase 1 footprint analysis, and methylation interference demonstrate that the binding is sequence specific. Deletions in the basic region of CHOP lead to a loss of DNA binding, suggesting that CHOP participates in this process. Stress induction in NIH 3T3 cells leads to the appearance of CHOP-containing DNA-binding activity. CHOP is found to contain a transcriptional activation domain which is inducible by cellular stress, lending further support to the notion that the protein can function as a positively acting transcription factor. We conclude that CHOP may serve a dual role both as an inhibitor of the ability of C/EBP proteins to activate some target genes and as a direct activator of others.

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