scholarly journals The Herpesvirus Saimiri Replication and Transcription Activator Acts Synergistically with CCAAT Enhancer Binding Protein Alpha To Activate the DNA Polymerase Promoter

2005 ◽  
Vol 79 (21) ◽  
pp. 13548-13560 ◽  
Author(s):  
Louise Wakenshaw ◽  
Matthew S. Walters ◽  
Adrian Whitehouse
Keyword(s):  
Dna Polymerase ◽  
Binding Protein ◽  
Lytic Replication ◽  
Early Gene ◽  
Herpesvirus Saimiri ◽  
Sequence Motif ◽  
Gene Promoters ◽  
Transcription Activator ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein

ABSTRACT The open reading frame (ORF) 50 gene product, also known as the replication and transcription activator (Rta), is an immediate-early gene which is well conserved among all gamma-2 herpesviruses and plays a pivotal role in regulating the latent-lytic switch. Herpesvirus saimiri (HVS) ORF 50a functions as a sequence-specific transactivator capable of activating delayed-early (DE) gene expression via binding directly to an ORF 50 response element (RE) within the respective promoter. Analysis of the ORF 50 REs have identified two distinct types within HVS gene promoters. The first comprises a consensus sequence motif, CCN9GG, the second an AT-rich sequence. Here we demonstrate that ORF 50a is capable of transactivating the DE ORF 9 promoter which encodes the DNA polymerase. Deletion analysis of the ORF 9 promoter mapped the ORF 50 RE to a 95-bp region situated 126 bp upstream of the initiation codon. Gel retardation analysis further mapped the RE to a 28-bp fragment, which was able to confer ORF 50 responsiveness on an enhancerless simian virus 40 minimal promoter. Furthermore, sequence analysis identified multiple CCAAT enhancer binding protein alpha (C/EBPα) binding sites within the ORF 9 promoter and specifically two within the close vicinity of the AT-rich ORF 50 RE. Analysis demonstrated that the HVS ORF 50a and C/EBPα proteins associate with the ORF 9 promoter in vivo, interact directly, and synergistically activate the ORF 9 promoter by binding to adjacent binding motifs. Overall, these data suggest a cooperative interaction between HVS ORF 50a and C/EBPα proteins to activate the DNA polymerase promoter during early stages of the lytic replication cycle.

scholarly journals Role of CCAAT/Enhancer-Binding Protein Alpha (C/EBPα) in Activation of the Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Lytic-Cycle Replication-Associated Protein (RAP) Promoter in Cooperation with the KSHV Replication and Transcription Activator (RTA) and RAP

2003 ◽  
Vol 77 (1) ◽  
pp. 600-623 ◽  
Author(s):  
Shizhen Emily Wang ◽  
Frederick Y. Wu ◽  
Masahiro Fujimuro ◽  
Jianchao Zong ◽  
S. Diane Hayward ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Binding Sites ◽  
Kaposi's Sarcoma ◽  
Binding Protein ◽  
Lytic Cycle ◽  
Transcription Activator ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein

ABSTRACT The Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded replication-associated protein (RAP, or K8) has been shown to induce both CCAAT/enhancer binding protein alpha (C/EBPα) and p21CIP-1 expression, resulting in G0/G1 cell cycle arrest during the lytic cycle. RAP and C/EBPα are also known to interact strongly both in vitro and in lytically infected cells. We recognized two potential consensus C/EBP binding sites in the RAP promoter and performed electrophoretic mobility shift assay (EMSA) analysis with in vitro-translated C/EBPα; this analysis showed that one of these sites has a very high affinity for C/EBPα. Luciferase (LUC) assays performed with a target RAP promoter-LUC reporter gene confirmed that C/EBPα can transcriptionally activate the RAP promoter up to 50-fold. Although RAP had no effect on its own promoter by itself, the addition of RAP and C/EBPα together resulted in a threefold increase in activity over that obtained with C/EBPα alone. Importantly, the introduction of exogenous Flag-tagged C/EBPα triggered RAP expression in BCBL-1 cells latently infected with KSHV, as detected by both reverse transcription-PCR and double-label immunofluorescence assay analyses, suggesting the presence of a self-reinforcing loop with C/EBPα and RAP activating each other. The RAP promoter can also be activated 50- to 120-fold by the KSHV lytic-cycle-triggering protein known as replication and transcription activator (RTA). C/EBPα and RTA together cooperated to elevate RAP promoter activity four- to sixfold more than either alone. Furthermore, the addition of RAP, C/EBPα, and RTA in LUC reporter cotransfection assays resulted in 7- to 15-fold more activation than that seen with either C/EBPα or RTA alone. Site-specific mutational analysis of the RAP promoter showed that the strong C/EBP binding site is crucial for C/EBPα-mediated transactivation of the RAP promoter. However, the C/EBP binding site also overlaps the previously reported 16-bp RTA-responsive element (RRE), and the same mutation also both reduced RTA-mediated transactivation and abolished the cooperativity between C/EBPα and RTA. Furthermore, in vitro-translated RTA, although capable of binding directly to the polyadenylated nuclear RNA (PAN) RRE motif, failed to bind to the RAP RRE and interfered with RRE-bound C/EBPα in EMSA experiments. Partial RTA responsiveness but no cooperativity could be transferred to a heterologous promoter containing added consensus C/EBP binding sites. A chromatin immunoprecipitation assay showed that all three proteins associated specifically with RAP promoter DNA in vivo and that, when C/EBPα was removed from a tetradecanoyl phorbol acetate-treated JSC-1 primary effusion lymphoma cell lysate, the levels of association of RTA and RAP with the RAP promoter were reduced 3- and 13-fold, respectively. Finally, RTA also proved to physically interact with both C/EBPα and RAP, as assayed both in vitro and by immunoprecipitation. Binding to C/EBPα occurred within the N-terminal DNA binding domain of RTA, and deletion of a 17-amino-acid basic motif of RTA abolished both the C/EBPα and DNA binding activities as well as all RTA transactivation and the cooperativity with C/EBPα. Therefore, we suggest that RTA transactivation of the RAP RRE is mediated by an interaction with DNA-bound C/EBPα but that full activity requires more than just the core C/EBP binding site.

scholarly journals CCAAT/Enhancer-Binding Protein-α Is Induced during the Early Stages of Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Lytic Cycle Reactivation and Together with the KSHV Replication and Transcription Activator (RTA) Cooperatively Stimulates the Viral RTA, MTA, and PAN Promoters

2003 ◽  
Vol 77 (17) ◽  
pp. 9590-9612 ◽  
Author(s):  
Shizhen Emily Wang ◽  
Frederick Y. Wu ◽  
Yanxing Yu ◽  
Gary S. Hayward
Keyword(s):  
Transcription Factor ◽  
Reporter Gene ◽  
Binding Sites ◽  
Binding Protein ◽  
Lytic Cycle ◽  
Transcription Activator ◽  
Cellular Transcription Factor ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein ◽  
Cellular Transcription

ABSTRACT During the immediate-early (IE) phase of reactivation from latency, the Kaposi's sarcoma-associated herpesvirus (KSHV) replication and transcription activator protein (RTA) (or ORF50) is thought to be the most critical trigger that upregulates expression of many downstream viral lytic cycle genes, including the delayed-early (DE) gene encoding the replication-associated protein (RAP) (or K8). RAP physically interacts with and stabilizes the cellular transcription factor CCAAT/enhancer-binding protein-α (C/EBPα), leading to upregulated expression of the cellular C/EBPα and p21CIP-1 proteins followed by G0/G1 cell cycle arrest. Furthermore, RTA also interacts with C/EBPα, and both RAP and RTA cooperate with C/EBPα to activate the RAP promoter through binding to a strong proximal C/EBP binding site that also serves as an RTA-responsive element (RRE). Here we show that C/EBPα also activates the IE RTA promoter in transient-cotransfection reporter gene assays and that addition of either RTA or RAP enhances the effect. Electrophoretic mobility shift assay and deletion analysis revealed three C/EBP binding sites that mediate cooperative transactivation of the RTA promoter by C/EBPα and RTA. Furthermore, chromatin immunoprecipitation assay results showed that the endogenous C/EBPα, RTA, and RAP proteins all associate with RTA promoter sequences in tetradecanoyl phorbol acetate-induced primary effusion lymphoma (PEL) cells. Induction of endogenous KSHV RTA mRNA in PEL cells by exogenously introduced C/EBPα was confirmed by reverse transcription-PCR analysis and by double-label indirect immunofluorescence assays. Reciprocally, expression of exogenous RTA also led to an increase of endogenous C/EBPα expression that could be detected by Western immunoblot assays even in KSHV-negative DG75 cells. Cotransfected RTA also increased positive C/EBPα autoregulation of the C/EBPα promoter in transient-cotransfection reporter gene assays. Finally, C/EBPα proved to strongly activate the promoters of two other KSHV DE genes encoding PAN (polyadenylated nuclear) RNA and MTA (ORF57), which was again mediated by C/EBP binding sites that also contribute to RTA activation. Overall, these results support a model in which the cellular transcription factor C/EBPα and RTA:C/EBPα interactions play important roles both upstream and downstream of the two major KSHV regulatory proteins RTA and RAP during the early stages of lytic cycle reactivation.

scholarly journals AU-rich element-binding protein negatively regulates CCAAT enhancer-binding protein mRNA stability during long-term synaptic plasticity in Aplysia

2012 ◽  
Vol 109 (38) ◽  
pp. 15520-15525 ◽  
Author(s):  
Yong-Seok Lee ◽  
Sun-Lim Choi ◽  
Heejung Jun ◽  
Se-Jeong Yim ◽  
Jin-A Lee ◽  
...  
Keyword(s):  
Synaptic Plasticity ◽  
Sensory Neurons ◽  
Binding Protein ◽  
Critical Role ◽  
Early Gene ◽  
Long Term Memory ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein ◽  
Element Binding Protein

The consolidation of long-term memory for sensitization and synaptic facilitation in Aplysia requires synthesis of new mRNA including the immediate early gene Aplysia CCAAT enhancer-binding protein (ApC/EBP). After the rapid induction of ApC/EBP expression in response to repeated treatments of 5-hydroxytryptamine (5-HT), ApC/EBP mRNA is temporarily expressed in sensory neurons of sensory-to-motor synapses. However, the molecular mechanism underlying the rapid degradation of ApC/EBP transcript is not known. Here, we cloned an AU-rich element (ARE)-binding protein, ApAUF1, which functions as a destabilizing factor for ApC/EBP mRNA. ApAUF1 was found to bind to the 3′ UTR of ApC/EBP mRNA that contains AREs and subsequently reduces the expression of ApC/EBP 3′ UTR-containing reporter genes. Moreover, overexpression of ApAUF1 inhibited the induction of ApC/EBP mRNA in sensory neurons and also impaired long-term facilitation of sensory-to-motor synapses by repetitive 5-HT treatments. These results provide evidence for a critical role of the posttranscriptional modification of ApC/EBP mRNA during the consolidation of synaptic plasticity.

scholarly journals Dysregulation of CCAAT/enhancer binding protein-alpha (CEBPA) expression in the bone marrow of acute myeloid leukemia patients

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Naglaa M. Hassan ◽  
Fadwa Said ◽  
Roxan E. Shafik ◽  
Mona S. Abdellateif
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Binding Protein ◽  
Response To Treatment ◽  
Pathological Features ◽  
Poor Prognostic Factor ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein ◽  
Acute Myeloid

Abstract Background Acute myeloid leukemia (AML) is a heterogeneous malignant disease characterized by accumulation of different types of mutations commonly the CCAAT/enhancer binding protein-alpha (CEBPA). However, the dysregulations of CEBPA expression in AML is still a debatable issue. The aim of the current study was to assess CEBPA gene expression in bone marrow (BM) aspiration specimens of 91 AML patients, compared to 20 control donors of bone marrow transplantation (BMT), using RT-PCR. Data were correlated with patients’ clinico-pathological features, response to treatment, progression-free survival (PFS), and overall survival (OS) rates. Results There was overexpression of CEBPA gene in AML patients compared to normal control [1.7 (0.04–25.6) versus 0.17 (0–4.78), respectively, P < 0.001]. Upregulation of CEBPA expression associated significantly with increased BM hypercellularity, total leucocyte counts, peripheral blood blast cell count, and poor PFS (P < 0.001, 0.002, 0.001, and 0.013, respectively). There was no significant association between CEBPA expression and any other relevant clinico-pathological features or OS rates (P = 0.610) of the patients. ROC analysis for biological relevance of CEBPA expression with AML showed that sensitivity and specificity of CEBPA expression at a cut-off value of 0.28 are 92.3% and 78.6%, respectively (P < 0.001). All patients who had CEBPA overexpression and mutant FLT3 showed BM hypercellularity, adverse cytogenetic risk, increased TLC, and PB blast cells count (P = 0.007, P < 0.001, 0.016, and 0.002, respectively). Conclusion CEBPA overexpression could be used as a genetic biological marker for AML diagnosis, as well as a poor prognostic factor for disease progression. It has no impact on OS rates of the patients.

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