scholarly journals p110 CUX1 Cooperates with E2F Transcription Factors in the Transcriptional Activation of Cell Cycle-Regulated Genes

2008 ◽  
Vol 28 (10) ◽  
pp. 3127-3138 ◽  
Author(s):  
Mary Truscott ◽  
Ryoko Harada ◽  
Charles Vadnais ◽  
François Robert ◽  
Alain Nepveu
Keyword(s):  
Cell Cycle ◽  
Dna Polymerase ◽  
Transcriptional Activation ◽  
Affinity Purification ◽  
Gene Promoter ◽  
Dominant Negative ◽  
In Vitro Assays ◽  
Dominant Negative Mutant ◽  
Dna Polymerase Α ◽  
Reporter Assays

ABSTRACT The transcription factor p110 CUX1 was shown to stimulate cell proliferation by accelerating entry into S phase. As p110 CUX1 can function as a transcriptional repressor or activator depending on promoter context, we investigated its mechanism of transcriptional activation using the DNA polymerase α gene promoter as a model system. Linker-scanning analysis revealed that a low-affinity E2F binding site is required for transcriptional activation. Moreover, coexpression with a dominant-negative mutant of DP-1 suggested that endogenous E2F factors are indeed needed for p110-mediated activation. Tandem affinity purification, coimmunoprecipitation, chromatin immunoprecipitation, and reporter assays indicated that p110 CUX1 can engage in weak protein-protein interactions with E2F1 and E2F2, stimulate their recruitment to the DNA polymerase α gene promoter, and cooperate with these factors in transcriptional activation. On the other hand, in vitro assays suggested that the interaction between CUX1 and E2F1 either is not direct or is regulated by posttranslational modifications. Genome-wide location analysis revealed that targets common to p110 CUX1 and E2F1 included many genes involved in cell cycle, DNA replication, and DNA repair. Comparison of the degree of enrichment for various E2F factors suggested that binding of p110 CUX1 to a promoter will favor the specific recruitment of E2F1, and to a lesser extent E2F2, over E2F3 and E2F4. Reporter assays on a subset of common targets confirmed that p110 CUX1 and E2F1 cooperate in their transcriptional activation. Overall, our results show that p110 CUX1 and E2F1 cooperate in the regulation of many cell cycle genes.

scholarly journals CDP/Cux Stimulates Transcription from the DNA Polymerase α Gene Promoter

2003 ◽  
Vol 23 (8) ◽  
pp. 3013-3028 ◽  
Author(s):  
Mary Truscott ◽  
Lélia Raynal ◽  
Peter Premdas ◽  
Brigitte Goulet ◽  
Lam Leduy ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Dna Polymerase ◽  
Transcriptional Activation ◽  
Gene Promoter ◽  
S Phase ◽  
Proteolytic Processing ◽  
Dna Polymerase Α ◽  
Stimulation Of

ABSTRACT CDP/Cux (CCAAT-displacement protein/cut homeobox) contains four DNA binding domains, namely, three Cut repeats (CR1, CR2, and CR3) and a Cut homeodomain. CCAAT-displacement activity involves rapid but transient interaction with DNA. More stable DNA binding activity is up-regulated at the G1/S transition and was previously shown to involve an N-terminally truncated isoform, CDP/Cux p110, that is generated by proteolytic processing. CDP/Cux has been previously characterized as a transcriptional repressor. However, here we show that expression of reporter plasmids containing promoter sequences from the human DNA polymerase α (pol α), CAD, and cyclin A genes is stimulated in cotransfections with N-terminally truncated CDP/Cux proteins but not with full-length CDP/Cux. Moreover, expression of the endogenous DNA pol α gene was stimulated following the infection of cells with a retrovirus expressing a truncated CDP/Cux protein. Chromatin immunoprecipitation (ChIP) assays revealed that CDP/Cux was associated with the DNA pol α gene promoter specifically in the S phase. Using linker scanning analyses, in vitro DNA binding, and ChIP assays, we established a correlation between binding of CDP/Cux to the DNA pol α promoter and the stimulation of gene expression. Although we cannot exclude the possibility that stimulation of gene expression by CDP/Cux involved the repression of a repressor, our data support the notion that CDP/Cux participates in transcriptional activation. Notwithstanding its mechanism of action, these results establish CDP/Cux as an important transcriptional regulator in the S phase.

scholarly journals Direct Repression of Cyclin D1 by SIP1 Attenuates Cell Cycle Progression in Cells Undergoing an Epithelial Mesenchymal Transition

2007 ◽  
Vol 18 (11) ◽  
pp. 4615-4624 ◽  
Author(s):  
Jakob Mejlvang ◽  
Marina Kriajevska ◽  
Cindy Vandewalle ◽  
Tatyana Chernova ◽  
A. Emre Sayan ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcription Factors ◽  
Cyclin D1 ◽  
Cell Cycle Progression ◽  
Epithelial Mesenchymal Transition ◽  
Gene Promoter ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
A431 Cells ◽  
Mesenchymal Transition

Zinc finger transcription factors of the Snail/Slug and ZEB-1/SIP1 families control epithelial-mesenchymal transitions in development in cancer. Here, we studied SIP1-regulated mesenchymal conversion of epidermoid A431 cells. We found that concomitant with inducing invasive phenotype, SIP1 inhibited expression of cyclin D1 and induced hypophosphorylation of the Rb tumor suppressor protein. Repression of cyclin D1 was caused by direct binding of SIP1 to three sequence elements in the cyclin D1 gene promoter. By expressing exogenous cyclin D1 in A431/SIP1 cells and using RNA interference, we demonstrated that the repression of cyclin D1 gene by SIP1 was necessary and sufficient for Rb hypophosphorylation and accumulation of cells in G1 phase. A431 cells expressing SIP1 along with exogenous cyclin D1 were highly invasive, indicating that SIP1-regulated invasion is independent of attenuation of G1/S progression. However, in another epithelial-mesenchymal transition model, gradual mesenchymal conversion of A431 cells induced by a dominant negative mutant of E-cadherin produced no effect on the cell cycle. We suggest that impaired G1/S phase progression is a general feature of cells that have undergone EMT induced by transcription factors of the Snail/Slug and ZEB-1/SIP1 families.

scholarly journals Protein kinase C-theta isoenzyme selective stimulation of the transcription factor complex AP-1 in T lymphocytes.

1996 ◽  
Vol 16 (4) ◽  
pp. 1842-1850 ◽  
Author(s):  
G Baier-Bitterlich ◽  
F Uberall ◽  
B Bauer ◽  
F Fresser ◽  
H Wachter ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Kinase C ◽  
Pkc Isoforms ◽  
Pkc Alpha ◽  
Transcriptional Complex ◽  
Induced Signal ◽  
Transcription Factor Complex

T-lymphocyte stimulation requires activation of several protein kinases, including the major phorbol ester receptor protein kinase C (PKC), ultimately leading to induction of lymphokines, such as interleukin-2 (IL-2). The revelant PKC isoforms which are involved in the activation cascades of nuclear transcription factors involved in IL-2 production have not yet been clearly defined. We have examined the potential role of two representative PKC isoforms in the induction of the IL-2 gene, i.e., PKC-alpha and PKC-theta, the latter being expressed predominantly in hematopoietic cell lines, particularly T cells. Similar to that of PKC-alpha, PKC-theta overexpression in murine EL4 thymoma cells caused a significant increase in phorbol 12-myristate 13-acetate (PMA)-induced transcriptional activation of full-length IL-2-chloramphenicol acetyltransferase (CAT) and NF-AT-CAT but not of NF-IL2A-CAT or NF-kappaB promoter-CAT reporter gene constructs. Importantly, the critical AP-1 enhancer element was differentially modulated by these two distinct PKC isoenzymes, since only PKC-theta but not PKC-alpha overexpression resulted in an approximately 2.8-fold increase in AP-1-collagenase promoter CAT expression in comparison with the vector control. Deletion of the AP-1 enhancer site in the collagenase promoter rendered it unresponsive to PKC-theta. Expression of a constitutively active mutant PKC-theta A148E (but not PKC-alpha A25E) was sufficient to induce activation of AP-1 transcription factor complex in the absence of PMA stimulation. Conversely, a catalytically inactive PKC-theta K409R (but not PKC-alpha K368R) mutant abrogated endogenous PMA-mediated activation of AP-1 transcriptional complex. Dominant negative mutant Ha-RasS17N completely inhibited the PKC-O A148E-induced signal, PKC-O. Expression of a constitutively active mutant PKC-O A148E (but not PKC-alpha A25E) was sufficient to induce activation of AP-1 transcription factor complex in the absence of PMA stimulation. Conversely, a catalytically inactive PKC-O K409R (but not PKC-alpha K368R) mutant abrogated endogenous PMA-mediated activation of AP-1 transcriptional complex. Dominant negative mutant Ha-enRasS17N completely inhibited in the PKC-O A148E-induced signal, identifying PKC-theta as a specific constituent upstream of or parallel to Ras in the signaling cascade leading to AP transcriptional activation.

scholarly journals Molecular cloning of the cDNA for the catalytic subunit of plant DNA polymerase α and its cell-cycle dependent expression

1997 ◽  
Vol 2 (11) ◽  
pp. 695-709 ◽  
Author(s):  
Masayuki Yokoi ◽  
Masaki Ito ◽  
Masako Izumi ◽  
Hiroshi Miyazawa ◽  
Hirokazu Nakai ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Molecular Cloning ◽  
Dna Polymerase ◽  
Catalytic Subunit ◽  
Dna Polymerase Α ◽  
Plant Dna ◽  
Cycle Dependent

Pivotal Role of Survivin in Leukemogenesis by E2A-HLF Chimeric Transcription Factor.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2988-2988
Author(s):  
Mayuko Okuya ◽  
Hidemitsu Kurosawa ◽  
Takayuki Matsunaga ◽  
Mitsuoki Eguchi ◽  
Yusuke Furukawa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Survivin Expression ◽  
Small Population ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Transcriptional Level ◽  
Survivin Promoter ◽  
M Phase ◽  
Negative Mutant

Abstract The E2A-HLF fusion transcription factor generated by the t(17;19)(q22;p13) translocation is found in a small population of pro-B cell ALL. Patients associated with this chimera share distinct clinical features such as hypercalcemia, coagulopathy and very poor prognosis due to resistance to intensive chemotherapy including aggressive conditioning for BMT, all of which are unusual for this type of ALL. We have previously demonstrated that inhibition of the trans-activation potential of the E2A-HLF chimera by the dominant negative mutant results in apoptosis in t(17;19)+ ALL cells but does not affect cell cycle. Moreover, E2A-HLF blocks apoptosis induced by cytokine deprivation in IL-3-dependent cells, suggesting that this fusion protein contributes to leukemogenesis by substituting for the anti-apoptotic function of cytokines. The present study shows that survivin is a downstream target molecule of E2A-HLF. Four t(17;19)+ ALL cell lines expressed survivin at high levels and down-regulation of E2A-HLF function by the dominant negative mutant suppressed survivin expression. In addition, forced expression of E2A-HLF in Nalm-6, a t(17;19)− ALL cell line, up-regulated survivin expression. Survivin is known to be expressed predominantly in the G2/M phase. Indeed, separation of the fractions enriched for in each phase of the cell cycle using a counterflow centrifugal elutriator revealed G2/M phase-dominant survivin expression in t(17;19) − ALL cells including Nalm-6. In t(17;19)+ ALL cells, however, survivin was expressed throughout the cell cycle. Moreover, Nalm-6 cells forced to express E2A-HLF showed cell cycle-independent survivin expression. Reporter assay revealed that E2A-HLF induced luciferase activity by transfecting with each reporter construct containing the survivin promoter at a different length from the initial ATG, suggesting that E2A-HLF induces survivin expression at the transcriptional level, but not by direct binding of E2A-HLF to the survivin promoter. To test whether survivin plays anti-apoptotic roles in t(17:19)+ cells, we used a survivin mutant lacking a phosphorylation site (T34A-survivin) and considered to inhibit survivin function in a dominant negative manner. T34A-survivin induced massive apoptosis throughout the cell cycle in t(17;19)+ cells. In contrast, T34A-survivin in t(17;19) − cells induced cell death in only a small population in G2/M phase. In addition to caspase-dependent pathways, T34A-survivin induced apoptosis in t(17;19)+ ALL cells through caspase-independent pathways, in which apoptosis-inducing factor (AIF) translocated from cytoplasm to the nucleus. These results indicate that cell cycle-independent up-regulation of survivin by the E2A-HLF chimera is indispensable for the survival of t(17;19)+ ALL cells, and that inhibition of survivin may offer an effective therapeutic strategy against this refractory ALL.

scholarly journals Expression of dominant-negative mutant DP-1 blocks cell cycle progression in G1.

1996 ◽  
Vol 16 (7) ◽  
pp. 3698-3706 ◽  
Author(s):  
C L Wu ◽  
M Classon ◽  
N Dyson ◽  
E Harlow
Keyword(s):  
Cell Cycle ◽  
Dna Binding ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Dominant Negative ◽  
Functional Domains ◽  
Dominant Negative Mutant ◽  
G1 Arrest ◽  
Wild Type ◽  
Cycle Progression

Unregulated expression of the transcription factor E2F promotes the G1-to-S phase transition in cultured mammalian cells. However, there has been no direct evidence for an E2F requirement in this process. To demonstrate that E2F is obligatory for cell cycle progression, we attempted to inactivate E2F by overexpressing dominant-negative forms of one of its heterodimeric partners, DP-1. We dissected the functional domains of DP-1 and separated the region that facilitate heterodimer DNA binding from the E2F dimerization domain. Various DP-1 mutants were introduced into cells via transfection, and the cell cycle profile of the transfected cells was analyzed by flow cytometry. Expression of wild-type DP-1 or DP-1 mutants that bind to both DNA and E2F drove cells into S phase. In contrast, DP-1 mutants that retained E2F binding but lost DNA binding arrested cells in the G1 phase of the cell cycle. The DP-1 mutants that were unable to bind DNA resulted in transcriptionally inactive E2F complexes, suggesting that the G1 arrest is caused by formation of defective E2F heterodimers. Furthermore, the G1 arrest instigated by these DP-1 mutants could be rescued by coexpression of wild-type E2F or DP protein. These experiments define functional domains of DP and demonstrate a requirement for active E2F complexes in cell cycle progression.

scholarly journals Involvement of JunD in transcriptional activation of the orphan receptor gene nur77 by nerve growth factor and membrane depolarization in PC12 cells

1994 ◽  
Vol 14 (12) ◽  
pp. 7731-7743
Author(s):  
J K Yoon ◽  
L F Lau
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Transcriptional Activation ◽  
Membrane Depolarization ◽  
Dominant Negative ◽  
Orphan Receptor ◽  
Early Gene ◽  
Dominant Negative Mutant ◽  
Transcription Activator ◽  
Nerve Growth

nur77, an immediate-early gene that encodes an orphan nuclear receptor, is rapidly and transiently induced by nerve growth factor (NGF) stimulation or membrane depolarization in the rat pheochromocytoma-derived cell line PC12. The Nur77 protein can act as a potent transcription activator and may function to regulate the expression of downstream genes in response to extracellular stimuli. We show here that activation of nur77 by NGF treatment and membrane depolarization is signalled through distinct pathways. These distinct signals appear to converge on the same transcription factors acting on the same promoter elements. We show that nur77 activation by both processes requires two cis-acting AP1-like elements, NAP1 and NAP2, which contain the core sequence TGCGTCA centered at 67 and 38 nucleotides upstream of the transcription start site. The NAP elements can confer inducibility by NGF and membrane depolarization on an otherwise unresponsive heterologous promoter. We identified JunD as a key mediator of nur77 activation by reason of the following observations. (i) JunD, but not CREB or other members of the Fos/Jun family, is a component of NAP binding activity in PC12 cell nuclear extracts. (ii) JunD, but not other Fos/Jun family members, specifically transactivates the nur77 promoter through the NAP elements (iii) A dominant-negative mutant of JunD effectively abolishes the activation of nur77 by either NGF treatment or membrane depolarization. These data draw a contrast between the regulation of nur77 with that of c-fos, in which the sequence requirements for activation by NGF treatment and membrane depolarization appear separable, and CREB appears to play a role in activation by both NGF and membrane depolarization.

scholarly journals HBP1 Repression of the p47phox Gene: Cell Cycle Regulation via the NADPH Oxidase

2004 ◽  
Vol 24 (7) ◽  
pp. 3011-3024 ◽  
Author(s):  
Stephen P. Berasi ◽  
Mei Xiu ◽  
Amy S. Yee ◽  
K. Eric Paulson
Keyword(s):  
Cell Cycle ◽  
Nadph Oxidase ◽  
Cell Cycle Regulation ◽  
Dominant Negative ◽  
Cellular Growth ◽  
Superoxide Production ◽  
Regulatory Subunit ◽  
Dominant Negative Mutant ◽  
Ros Generation ◽  
Cycle Regulation

ABSTRACT Several studies have linked the production of reactive oxygen species (ROS) by the NADPH oxidase to cellular growth control. In many cases, activation of the NADPH oxidase and subsequent ROS generation is required for growth factor signaling and mitogenesis in nonimmune cells. In this study, we demonstrate that the transcriptional repressor HBP1 (HMG box-containing protein 1) regulates the gene for the p47phox regulatory subunit of the NADPH oxidase. HBP1 represses growth regulatory genes (e.g., N-Myc, c-Myc, and cyclin D1) and is an inhibitor of G1 progression. The promoter of the p47phox gene contains six tandem high-affinity HBP1 DNA-binding elements at positions −1243 to −1318 bp from the transcriptional start site which were required for repression. Furthermore, HBP1 repressed the expression of the endogenous p47phox gene through sequence-specific binding. With HBP1 expression and the subsequent reduction in p47phox gene expression, intracellular superoxide production was correspondingly reduced. Using both the wild type and a dominant-negative mutant of HBP1, we demonstrated that the repression of superoxide production through the NADPH oxidase contributed to the observed cell cycle inhibition by HBP1. Together, these results indicate that HBP1 may contribute to the regulation of NADPH oxidase-dependent superoxide production through transcriptional repression of the p47phox gene. This study defines a transcriptional mechanism for regulating intracellular ROS levels and has implications in cell cycle regulation.

scholarly journals HIV-1 Vpr Induces the K48-Linked Polyubiquitination and Proteasomal Degradation of Target Cellular Proteins To Activate ATR and Promote G2 Arrest

2010 ◽  
Vol 84 (7) ◽  
pp. 3320-3330 ◽  
Author(s):  
Jean-Philippe Belzile ◽  
Jonathan Richard ◽  
Nicole Rougeau ◽  
Yong Xiao ◽  
Éric A. Cohen
Keyword(s):  
Ubiquitin Ligase ◽  
Affinity Purification ◽  
E3 Ubiquitin Ligase ◽  
E3 Ligase ◽  
Proteasomal Degradation ◽  
Dominant Negative ◽  
Cellular Proteins ◽  
Dominant Negative Mutant ◽  
M Phase ◽  
Hiv 1

ABSTRACT HIV-1 viral protein R (Vpr) induces cell cycle arrest at the G2/M phase by a mechanism involving the activation of the DNA damage sensor ATR. We and others recently showed that Vpr performs this function by subverting the activity of the DDB1-CUL4A (VPRBP) E3 ubiquitin ligase. Vpr could thus act as a connector between the E3 ligase and an unknown cellular factor whose ubiquitination would induce G2 arrest. While attractive, this model is based solely on the indirect observation that some mutants of Vpr retain their interaction with the E3 ligase but fail to induce G2 arrest. Using a tandem affinity purification approach, we observed that Vpr interacts with ubiquitinated cellular proteins and that this association requires the recruitment of an active E3 ligase given that the depletion of VPRBP by RNA interference or the overexpression of a dominant negative mutant of CUL4A decreased this association. Importantly, G2-arrest-defective mutants of Vpr in the C-terminal putative substrate-interacting domain displayed a decreased association with ubiquitinated proteins. We also found that the inhibition of proteasomal activity increased this association and that the ubiquitin chains were at least in part constituted of classical K48 linkages. Interestingly, the inhibition of K48 polyubiquitination specifically impaired the Vpr-induced phosphorylation of H2AX, an early target of ATR, but did not affect UV-induced H2AX phosphorylation. Overall, our results provide direct evidence that the association of Vpr with the DDB1-CUL4A (VPRBP) E3 ubiquitin ligase induces the K48-linked polyubiquitination of as-yet-unknown cellular proteins, resulting in their proteasomal degradation and ultimately leading to the activation of ATR and G2 arrest.

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