GAL4 Fusion Proteins Research Articles

Synthetic regulatory RNAs selectively suppress the progression of bladder cancer

The traditional treatment for cancer is lack of specificity and efficacy. Modular synthetic regulatory RNAs, such as inhibitive RNA (iRNA) and active RNA (aRNA), may overcome these limitations. Here, we synthesize a new iRNA to bind the upstream activating sequence (UAS) of a minimal promoter that drives expression of artificial miRNAs (amiRNAs) targeting MYC, which represses the binding interaction between UAS and GAL4 fusion protein (GAL4-VP64) in GAL4/UAS system. The aRNA driven by a tumor-specific mutant human telomerase reverse transcriptase (hTERT) promoter is created to interact with iRNA to expose UAS again in bladder cancer. Without the aRNA, mRNA and protein levels of MYC, cell growth, cell apoptosis and cell migration were no significance in two bladder cancer cell lines, T24 and 5637, and human foreskin fibroblast (HFF) cells. The aRNA significantly inhibited the expression of MYC in mRNA and protein levels, as well as the proliferation and migration of the cancer cells, but not in HFF cells. These results indicated that regulatory RNAs selectively controlled the expression of amiRNAs and ultimately suppress the progression of bladder cancer cells without affecting normal cells. Synthetic regulatory RNAs might be a selective therapeutic approach for bladder cancer.

N- and C-terminal non-conserved residues contribute to transactivation by a sea anemone (Nematostella vectensis) NF-κB transcription factor

NF-κB is an evolutionarily conserved eukaryotic transcription factor that plays a role in many important developmental and immune-related processes by activating target gene expression. The goal of these experiments was to define the sequences required for a sea anemone NF-κB's intrinsic transactivation activity by using mutant proteins with serial deletions of the N- and C-terminal sequences. Deletion mutants were constructed that were missing the C-terminal 15, 32 or 47 amino acids (aa) or the N-terminal 17, 27 or 47 aa of the 440 aa NF-κB protein from the starlet sea anemone, Nematostella vectensis (Nv), a simple model organism in the phylum Cnidaria. These Nv-NF-κB mutants were expressed as GAL4 fusion proteins in yeast, and their transactivation activities were assessed by LacZ reporter gene assays. The deletion of 47 aa from either the N terminus or the C terminus of NF-κB completely inactivates the transactivation function of Nv-NF-κB. In addition, we identified proline-258 in the center of the protein as a key residue for the transactivation function of Nv-NF-κB. Taken together, these results demonstrate that non-conserved N- and C-terminal residues are both required for the transcriptional activating function of the sea anemone NF-κB protein, suggesting that it has a novel functional domain structure among known NF-κB proteins.

Zscan4 Is Regulated by PI3-Kinase and DNA-Damaging Agents and Directly Interacts with the Transcriptional Repressors LSD1 and CtBP2 in Mouse Embryonic Stem Cells

The Zscan4 family of genes, encoding SCAN-domain and zinc finger-containing proteins, has been implicated in the control of early mammalian embryogenesis as well as the regulation of pluripotency and maintenance of genome integrity in mouse embryonic stem cells. However, many features of this enigmatic family of genes are poorly understood. Here we show that undifferentiated mouse embryonic stem cell (ESC) lines simultaneously express multiple members of the Zscan4 gene family, with Zscan4c, Zscan4f and Zscan4-ps2 consistently being the most abundant. Despite this, between only 0.1 and 0.7% of undifferentiated mouse pluripotent stem cells express Zscan4 protein at a given time, consistent with a very restricted pattern of Zscan4 transcripts reported previously. Herein we demonstrate that Zscan4 expression is regulated by the p110α catalytic isoform of phosphoinositide 3-kinases and is induced following exposure to a sub-class of DNA-damage-inducing agents, including Zeocin and Cisplatin. Furthermore, we observe that Zscan4 protein expression peaks during the G2 phase of the cell cycle, suggesting that it may play a critical role at this checkpoint. Studies with GAL4-fusion proteins suggest a role for Zscan4 in transcriptional regulation, further supported by the fact that protein interaction analyses demonstrate that Zscan4 interacts with both LSD1 and CtBP2 in ESC nuclei. This study advances and extends our understanding of Zscan4 expression, regulation and mechanism of action. Based on our data we propose that Zscan4 may regulate gene transcription in mouse ES cells through interaction with LSD1 and CtBP2.

Function of the C. elegans T-box factor TBX-2 depends on SUMOylation

T-box transcription factors are critical developmental regulators in all multi-cellular organisms, and altered T-box factor activity is associated with a variety of human congenital diseases and cancers. Despite the biological significance of T-box factors, their mechanism of action is not well understood. Here we examine whether SUMOylation affects the function of the C. elegans Tbx2 sub-family T-box factor TBX-2. We have previously shown that TBX-2 interacts with the E2 SUMO-conjugating enzyme UBC-9, and that loss of TBX-2 or UBC-9 produces identical defects in ABa-derived pharyngeal muscle development. We now show that TBX-2 is SUMOylated in mammalian cell assays, and that both UBC-9 interaction and SUMOylation depends on two SUMO consensus sites located in the T-box DNA binding domain and near the TBX-2 C-terminus, respectively. In co-transfection assays, a TBX-2:GAL4 fusion protein represses expression of a 5xGal4:tk:luciferase construct. However, this activity does not require SUMOylation, indicating SUMO is not generally required for TBX-2 repressor activity. In C. elegans, reducing SUMOylation enhances the phenotype of a temperature-sensitive tbx-2 mutant and results in ectopic expression of a gene normally repressed by TBX-2, demonstrating that SUMOylation is important for TBX-2 function in vivo. Finally, we show mammalian orthologs of TBX-2, Tbx2, and Tbx3, can also be SUMOylated, suggesting SUMOylation may be a conserved mechanism controlling T-box factor activity.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-013-1336-y) contains supplementary material, which is available to authorized users.

ZKSCAN3 Is a Master Transcriptional Repressor of Autophagy

Autophagy constitutes a major cell-protective mechanism that eliminates damaged components and maintains energy homeostasis via recycling nutrients under normal/stressed conditions. Although the core components of autophagy have been well studied, regulation of autophagy at the transcriptional level is poorly understood. Herein, we establish ZKSCAN3, a zinc finger family DNA-binding protein, as a transcriptional repressor of autophagy. Silencing of ZKSCAN3 induced autophagy and increased lysosome biogenesis. Importantly, we show that ZKSCAN3 represses transcription of a large gene set (>60) integral to, or regulatory for, autophagy and lysosome biogenesis/function and that a subset of these genes, including Map1lC3b and Wipi2, represent direct targets. Interestingly, ZKSCAN3 and TFEB are oppositely regulated by starvation and in turn oppositely regulate lysosomal biogenesis and autophagy, suggesting that they act in conjunction. Altogether, our study uncovers an autophagy master switch regulating the expression of a transcriptional network of genes integral to autophagy and lysosome biogenesis/function.

The Metalloprotease Meprin β Generates Amino Terminal-truncated Amyloid β Peptide Species

The amyloid β (Aβ) peptide, which is abundantly found in the brains of patients suffering from Alzheimer disease, is central in the pathogenesis of this disease. Therefore, to understand the processing of the amyloid precursor protein (APP) is of critical importance. Recently, we demonstrated that the metalloprotease meprin β cleaves APP and liberates soluble N-terminal APP (N-APP) fragments. In this work, we present evidence that meprin β can also process APP in a manner reminiscent of β-secretase. We identified cleavage sites of meprin β in the amyloid β sequence of the wild type and Swedish mutant of APP at positions p1 and p2, thereby generating Aβ variants starting at the first or second amino acid residue. We observed even higher kinetic values for meprin β than BACE1 for both the wild type and the Swedish mutant APP form. This enzymatic activity of meprin β on APP and Aβ generation was also observed in the absence of BACE1/2 activity using a β-secretase inhibitor and BACE knock-out cells, indicating that meprin β acts independently of β-secretase.

The Unique Transcriptional Activation Domain of Nuclear Factor-I-X3 Is Critical to Specifically Induce Marker Gene Expression in Astrocytes

Transcription factors of the nuclear factor 1 (NFI) family regulate normal brain development in vertebrates. However, multiple splice variants of four NFI isoforms exist, and their biological functions have yet to be elucidated. Here, we cloned and analyzed human NFI-X3, a novel splice variant of the nfix gene, which contains a unique transcriptional activation (TA) domain completely conserved in primates. In contrast to previously cloned NFI-X1, overexpression of NFI-X3 potently activates NFI reporters, including glial fibrillary acidic protein (GFAP) reporter, in astrocytes and glioma cells. The GAL4 fusion protein containing the TA domain of NFI-X3 strongly activates the GAL4 reporter, whereas the TA domain of NFI-X1 is ineffective. The expression of NFI-X3 is dramatically up-regulated during the differentiation of neural progenitors to astrocytes and precedes the expression of astrocyte markers, such as GFAP and SPARCL1 (Secreted Protein, Acidic and Rich in Cysteines-like 1). Overexpression of NFI-X3 dramatically up-regulates GFAP and SPARCL1 expression in glioma cells, whereas the knockdown of NFI-X3 diminishes the expression of both GFAP and SPARCL1 in astrocytes. Although activation of astrocyte-specific genes involves DNA demethylation and subsequent increase of histone acetylation, NFI-X3 activates GFAP expression, in part, by inducing alterations in the nucleosome architecture that lead to the increased recruitment of RNA polymerase II.

Functional analysis of fish BCL-6 and Blimp-1 in vitro: Transcriptional repressors for B-cell terminal differentiation in fugu (Takifugu rubripes)

The transcriptional repressors BCL-6 and Blimp-1 are key regulators of B-cell terminal differentiation in mammals. We have previously identified the BCL-6 gene and Blimp-1 gene in fugu (Takifugu rubripes). In the present report, we conducted a functional analysis of fugu BCL-6 and Blimp-1 by using a one-hybrid reporter assay with Gal4 fusion proteins and Gal4DBD luciferase reporter gene. Results from the reporter assays in mammalian cell lines (HeLa, HEK-293, CV-1 and NIH3T3) and the fish cell line EPC show that Gal4-BCL6 and Gal4-Blimp1 strongly repress the transcription of the luciferase gene in all cell lines. Furthermore, deletion analyses show that the N-terminal region of BCL-6 has transcriptional repression activity; the BTB/POZ domain is an especially potent repression domain. In contrast to BCL-6, although the N-acidic domain and PR domain are insufficient for repression, most functional motifs of Blimp-1 are associated with transcriptional repression. These results suggest that BCL-6 and Blimp-1 are functional transcriptional repressors in fugu and that they regulate B-cell terminal differentiation in fugu.

Molecular Evidence for a Functional Ecdysone Signaling System in Brugia malayi

BackgroundFilarial nematodes, including Brugia malayi, the causative agent of lymphatic filariasis, undergo molting in both arthropod and mammalian hosts to complete their life cycles. An understanding of how these parasites cross developmental checkpoints may reveal potential targets for intervention. Pharmacological evidence suggests that ecdysteroids play a role in parasitic nematode molting and fertility although their specific function remains unknown. In insects, ecdysone triggers molting through the activation of the ecdysone receptor: a heterodimer of EcR (ecdysone receptor) and USP (Ultraspiracle).Methods and FindingsWe report the cloning and characterization of a B. malayi EcR homologue (Bma-EcR). Bma-EcR dimerizes with insect and nematode USP/RXRs and binds to DNA encoding a canonical ecdysone response element (EcRE). In support of the existence of an active ecdysone receptor in Brugia we also cloned a Brugia rxr (retinoid X receptor) homolog (Bma-RXR) and demonstrate that Bma-EcR and Bma-RXR interact to form an active heterodimer using a mammalian two-hybrid activation assay. The Bma-EcR ligand-binding domain (LBD) exhibits ligand-dependent transactivation via a GAL4 fusion protein combined with a chimeric RXR in mammalian cells treated with Ponasterone-A or a synthetic ecdysone agonist. Furthermore, we demonstrate specific up-regulation of reporter gene activity in transgenic B. malayi embryos transfected with a luciferase construct controlled by an EcRE engineered in a B. malayi promoter, in the presence of 20-hydroxy-ecdysone.ConclusionsOur study identifies and characterizes the two components (Bma-EcR and Bma-RXR) necessary for constituting a functional ecdysteroid receptor in B. malayi. Importantly, the ligand binding domain of BmaEcR is shown to be capable of responding to ecdysteroid ligands, and conversely, ecdysteroids can activate transcription of genes downstream of an EcRE in live B. malayi embryos. These results together confirm that an ecdysone signaling system operates in B. malayi and strongly suggest that Bma-EcR plays a central role in it. Furthermore, our study proposes that existing compounds targeting the insect ecdysone signaling pathway should be considered as potential pharmacological agents against filarial parasites.

CBP/p300 is a cell type-specific modulator of CLOCK/BMAL1-mediated transcription.

BackgroundPrevious studies have demonstrated tissue-specific regulation of the rhythm of circadian transcription, suggesting that transcription factor complex CLOCK/BMAL1, essential for maintaining circadian rhythm, regulates transcription in a tissue-specific manner. To further elucidate the mechanism of the cell type-specific regulation of transcription by CLOCK/BMAL1 at the molecular level, we investigated roles of CBP/p300 and tissue-specific cofactors in CLOCK/BMAL1-mediated transcription.ResultsAs shown previously, CBP/p300 stimulates CLOCK/BMAL1-mediated transcription in COS-1 cells. However, CBP/p300 repressed CLOCK/BMAL1-mediated transcription in NIH3T3 cells and knockdown of CBP or p300 expression by siRNA enhanced this transcription. Studies using GAL4-fusion proteins suggested that CBP represses CLOCK/BMAL1-mediated transcription by targeting CLOCK. We further investigated mechanisms of this cell type-specific modulation of CLOCK/BMAL1-mediated transcription by CBP by examining roles of co-repressor HDAC3 and co-activator pCAF, which are highly expressed in NIH3T3 and COS cells, respectively. CBP repressed CLOCK/BMAL1-mediated transcription in COS-1 cells when HDAC3 was overexpressed, but activated it in NIH3T3 cells when pCAF was overexpressed. CBP forms a complex with CLOCK by interacting with HDAC3 or pCAF; however, direct interaction of CBP with CLOCK was not observed.ConclusionOur findings indicate possible mechanisms by which CBP/p300 tissue-specifically acts cooperatively with pCAF and HDAC3 either as a co-activator or co-repressor, respectively, for CLOCK/BMAL1.

Eto2/MTG16 and MTGR1 are heteromeric corepressors of the TAL1/SCL transcription factor in murine erythroid progenitors

The TAL1 (or SCL) gene, originally discovered through its involvement by a chromosomal translocation in T-cell acute lymphoblastic leukemia, encodes a basic helix–loop–helix (bHLH) transcription factor essential for hematopoietic and vascular development. To identify its interaction partners, we expressed a tandem epitope-tagged protein in murine erythroleukemia (MEL) cells and characterized affinity-purified Tal1-containing complexes by liquid chromatography–tandem mass spectrometry analysis. In addition to known interacting proteins, two proteins related to the Eight-Twenty-One (ETO) corepressor, Eto2/Mtg16 and Mtgr1, were identified from the peptide fragments analyzed. Tal1 interaction with Eto2 and Mtgr1 was verified by coimmunoprecipitation analysis in Tal1, Eto2-, and Mtgr1-transfected COS-7 cells, MEL cells expressing V5 epitope-tagged Tal1 protein, and non-transfected MEL cells. Mapping analysis with Gal4 fusion proteins demonstrated a requirement for the bHLH domain of Tal1 and TAF110 domain of Eto2 for their interaction, and transient transfection and glutathione S-transferase pull-down analysis showed that Mtgr1 and Eto2 enhanced the other’s association with Tal1. Enforced expression of Eto2 in differentiating MEL cells inhibited the promoter of the Protein 4.2 (P4.2) gene, a direct target of TAL1 in erythroid progenitors, and transduction of Eto2 and Mtgr1 augmented Tal1-mediated gene repression. Finally, chromatin immunoprecipitation analysis revealed that Eto2 occupancy of the P4.2 promoter in MEL cells decreased with differentiation, in parallel with a decline in Eto2 protein abundance. These results identify Eto2 and Mtgr1 as authentic interaction partners of Tal1 and suggest they act as heteromeric corepressors of this bHLH transcription factor during erythroid differentiation.

PPARδ選択的アゴニストの創製と受容体-リガンド複合体構造情報を踏まえた選択性発現機構解明

A series of 3-(4-alkoxypheny)propanoic acid derivatives was prepared as candidate peroxisome proliferator-activated receptor (PPAR) delta-selective agonists, based on our previously discovered potent human PPARalpha/delta dual agonist TIPP-401 as a lead compound. Structure-activity relationship studies clearly indicated the importance of the chain length of the alkoxy group at the 4-position, and the n-butoxy compound exhibited the most potent PPARdelta transactivation activity and highest PPARdelta selectivity. The (S)-enantiomer of a representative compound (TIPP-204) exhibited extremely potent PPARdelta transactivation activity, comparable to that of the known PPARdelta-selective agonist GW-501516. To understand why TIPP-204 shows high selectivity for hPPARdelta among hPPAR subtypes, and why TIPP-401, a structurally related compound, is a hPPARalpha/delta dual agonist, computational docking of TIPP-401 to the ligand binding domains of hPPARalpha and hPPARdelta and X-ray structure analysis of TIPP-204-hPPARdelta ligand binding domain were carried out. The results allowed identification of certain amino acids as putative determinants of the hPPARdelta selectivity of TIPP-204. To confirm the significance of these amino acids, GAL4-fusion proteins of mutated hPPARdeltas and hPPARalphas were prepared, and the transactivation activity of TIPP-204 toward the mutants was evaluated. The amino acid(s) that predominantly influence the potency and selectivity of TIPP-204 are different from that of the well-known PPARdelta-selective agonist GW-501516, which belongs to a different chemical class. The significance of these amino acids was confirmed by the examination of the complex structure between TIPP-204 and hPPARdelta. The results revealed several interactions relevant to the hPPARdelta-selectivity of the two ligands and will be useful for logical hPPARdelta ligand design.

The Stability and Transactivation Potential of the Mammalian MafA Transcription Factor Are Regulated by Serine 65 Phosphorylation

The level of the MafA transcription factor is regulated by a variety of effectors of beta cell function, including glucose, fatty acids, and insulin. Here, we show that phosphorylation at Ser(65) of mammalian MafA influences both protein stability and transactivation potential. Replacement of Ser(65) with Glu to mimic phosphorylation produced a protein that was as unstable as the wild type, whereas Asp or Ala mutation blocked degradation. Analysis of MafA chimeric and deletion constructs suggests that protein phosphorylation at Ser(65) alone represents the initial degradation signal, with ubiquitinylation occurring within the C terminus (amino acids 234-359). Although only wild type MafA and S65E were polyubiquitinylated, both S65D and S65E potently stimulated transactivation compared with S65A. Phosphorylation at Ser(14) also enhanced activation, although it had no impact on protein turnover. The mobility of MafA S65A was profoundly affected upon SDS-PAGE, with the S65E and S65D mutants influenced less due to their ability to serve as substrates for glycogen synthase kinase 3, which acts at neighboring N-terminal residues after Ser(65) phosphorylation. Our observations not only illustrate the sensitivity of the cellular transcriptional and degradation machinery to phosphomimetic mutants at Ser(65), but also demonstrate the singular importance of phosphorylation at this amino acid in regulating MafA activity.

NAC1, a POZ/BTB protein that functions as a corepressor

We now demonstrate that NAC1 acts as a corepressor for other POZ/BTB proteins. NAC1 is a POZ/BTB motif containing transcriptional repressor protein. In a mammalian two hybrid assay in neuronal (N2A) cells and non-neuronal (HEK 293T) cells, VP16 activation domain tagged NAC1 resulted in significant reversal of transcriptional inhibition with the Gal4-ZID, Gal4-BCL6, Gal4-ZF5, and kelch proteins Gal4-MAYVEN and Gal4-NRP/B fusion proteins. We also observed similar results with another corepressor, BCoR Gal4 fusion protein. NAC1 potentiated ZF5 mediated repression in Gal4-DBD fusion transient assays. GST pulldown assays further confirmed protein–protein interactions between these proteins and NAC1. Both the NAC1 isoforms demonstrated selective interaction through the POZ/BTB domain but not with the non-POZ/BTB region. Endogenous NAC1 and BCL6 physically associated in CNS regions. Strikingly, NAC1 did not interact with the pro-myelocytic leukemia zinc finger protein (PLZF), another POZ/BTB protein that is not found in the adult brain. Therefore, we conclude that NAC1 functions as a corepressor for POZ/BTB proteins expressed in the mature CNS.

Function of Multiple Lis-Homology Domain/WD-40 Repeat-Containing Proteins in Feed-Forward Transcriptional Repression by Silencing Mediator for Retinoic and Thyroid Receptor/Nuclear Receptor Corepressor Complexes

Lis-homology (LisH) motifs are involved in protein dimerization, and the discovery of the conserved N-terminal LisH domain in transducin beta-like protein 1 and its receptor (TBL1 and TBLR1) led us to examine the role of this domain in transcriptional repression. Here we show that multiple beta-transducin (WD-40) repeat-containing proteins interact to form oligomers in solution and that oligomerization depends on the presence of the LisH domain in each protein. Repression of transcription, as assayed using Gal4 fusion proteins, also depended on the presence of the LisH domain, suggesting that oligomerization is a prerequisite for efficient transcriptional repression. Furthermore, we show that the LisH domain is responsible for the binding to the hypoacetylated histone H4 tail and for stable chromatin targeting by the nuclear receptor corepressor complex. Mutations in conserved residues in the LisH motif of TBL1 and TBLR1 block histone binding, oligomerization, and transcriptional repression, supporting the functional importance of the LisH motif in transcriptional repression. Our results indicate that another WD-40 protein, TBL3, also preferentially binds to the N-terminal domain of TBL1 and TBLR1, and forms oligomers with other WD-40 proteins. Finally, we observed that the WD-40 proteins RbAp46 and RbAp48 of the sin3A corepressor complex failed to dimerize. We also found the specific interaction UbcH/E2 with TBL1, but not RbAp46/48. Altogether, our results thus indicate that the presence of multiple LisH/WD-40 repeat containing proteins is exclusive to nuclear receptor corepressor/ silencing mediator for retinoic and thyroid receptor complexes compared with other class 1 histone deacetylase-containing corepessor complexes.

Modulation of Androgen Receptor Activation Function 2 by Testosterone and Dihydrotestosterone

The androgen receptor (AR) is transcriptionally activated by high affinity binding of testosterone (T) or its 5alpha-reduced metabolite, dihydrotestosterone (DHT), a more potent androgen required for male reproductive tract development. The molecular basis for the weaker activity of T was investigated by determining T-bound ligand binding domain crystal structures of wild-type AR and a prostate cancer somatic mutant complexed with the AR FXXLF or coactivator LXXLL peptide. Nearly identical interactions of T and DHT in the AR ligand binding pocket correlate with similar rates of dissociation from an AR fragment containing the ligand binding domain. However, T induces weaker AR FXXLF and coactivator LXXLL motif interactions at activation function 2 (AF2). Less effective FXXLF motif binding to AF2 accounts for faster T dissociation from full-length AR. T can nevertheless acquire DHT-like activity through an AR helix-10 H874Y prostate cancer mutation. The Tyr-874 mutant side chain mediates a new hydrogen bonding scheme from exterior helix-10 to backbone protein core helix-4 residue Tyr-739 to rescue T-induced AR activity by improving AF2 binding of FXXLF and LXXLL motifs. Greater AR AF2 activity by improved core helix interactions is supported by the effects of melanoma antigen gene protein-11, an AR coregulator that binds the AR FXXLF motif and targets AF2 for activation. We conclude that T is a weaker androgen than DHT because of less favorable T-dependent AR FXXLF and coactivator LXXLL motif interactions at AF2.

Inactivation and activation of replication initiation factors

The geminin-Cdt1 balance is critical for preventing re-replication in the same cell-cycle. Besides neutralization by geminin, human Cdt1 is degraded at the onset of S phase and after DNA damage. We have shown that the there are two redundant pathways for degrading Cdt1: a Skp2 dependent pathway that requires phosphorylation of Cdt1 by cyclin dependent kinases and Cul4 dependent pathway that requires the interaction of Cdt1 with PCNA through a QXXVXXF motif. Both pathways are used for degrading Cdt1 in S phase. The Cul4, PCNA dependent pathway alone is used for degrading Cdt1 after DNA damage. Thus PCNA, known as a matchmaker for many proteins involved in DNA and chromatin metabolism, is also involved in the degradation of some of these proteins. Origins of replication are expected to recruit initiation proteins like ORC and Cdc6 in eukaryotes and provide a platform for unwinding DNA. We have tested whether localization of initiation proteins onto DNA is sufficient for origin function. Different components of the ORC complex and Cdc6 stimulated pre-RC formation and replication initiation when fused to GAL4 DNA binding domain and recruited to plasmid DNA containing a tandem array of GAL4 binding sites. Replication occurred once per cell cycle and was inhibited by Geminin indicating that the plasmid was properly licensed during the cell cycle. The GAL4 fusion protein recruits other polypeptides of the ORC-Cdc6 complex and nascent strand abundance was highest near the GAL4 binding sites. Therefore the artificial origin recapitulates many of the regulatory features of physiological origins. Artificial recruitment of an eukaryotic cellular replication initiation factor to a DNA sequence can create a functional origin of replication providing a robust genetic assay for these factors and a novel approach to generating episomal vectors for gene-therapy.

Sumoylation and Acetylation Play Opposite Roles in the Transactivation of PLAG1 and PLAGL2*

PLAG1 (pleomorphic adenoma gene 1) and PLAGL2 (PLAG-like 2) are oncogenes involved in various malignancies. Thus the study of their regulatory mechanisms may lead to identification of novel therapeutic targets. In this study, we provide supporting evidence that sumoylation and acetylation regulate functions of PLAG1 and PLAGL2. A conserved transcriptional repression domain exists in both PLAG1 and PLAGL2, whose activity depends on the presence of three sumoylation motifs and an intact sumoylation pathway. In vivo sumoylation assays confirmed that lysines 244, 263, and 353 of PLAG1 and lysines 250, 269, and 356 of PLAGL2 are indeed sumoylation sites. Further study showed that sumoylation inhibits PLAG1-induced IGF-II expression in reporter assays. The repression mediated by sumoylation may be partially explained by its effect on the cellular localization of PLAG1 and PLAGL2, because sumoylation-deficient but not wild-type PLAG1 and PLAGL2 concentrate in the nucleolus. PLAG1 and PLAGL2 are also regulated by acetylation. They are acetylated and activated by p300 and deacetylated and repressed by HDAC7. Interestingly, the sumoylation-deficient mutant of PLAGL2 is acetylated at a lower level than its wild-type counterpart, suggesting that some of the lysine residues may be targets for both modifications. Finally, mutation of three lysine residues in sumoylation motifs significantly impairs the transformation ability of PLAG1 and PLAGL2, suggesting the essential roles of these sites in the oncogenic potential of PLAG proteins. Taken together, the activities of PLAG1 and PLAGL2 are tightly modulated by both sumoylation and acetylation, which have opposite effects on their transactivation. To our knowledge, this is the first demonstration that oncoproteins can be regulated by both sumoylation and acetylation.

Recruitment of ORC or CDC6 to DNA is sufficient to create an artificial origin of replication in mammalian cells

Origins of replication are expected to recruit initiation proteins like origin recognition complex (ORC) and Cdc6 in eukaryotes and provide a platform for unwinding DNA. Here we test whether localization of initiation proteins onto DNA is sufficient for origin function. Different components of the ORC complex and Cdc6 stimulated prereplicative complex (pre-RC) formation and replication initiation when fused to the GAL4 DNA-binding domain and recruited to plasmid DNA containing a tandem array of GAL4-binding sites. Replication occurred once per cell cycle and was inhibited by Geminin, indicating that the plasmid was properly licensed during the cell cycle. The GAL4 fusion protein recruits other polypeptides of the ORC-Cdc6 complex, and nascent strand abundance was highest near the GAL4-binding sites. Therefore, the artificial origin recapitulates many of the regulatory features of physiological origins and is valuable for studies on replication initiation in mammalian cells. We demonstrated the utility of this system by showing the functional importance of the ATPase domains of human Cdc6 and Orc1 and the dispensability of the N-terminal segments of Orc1 and Orc2 in this assay. Artificial recruitment of a eukaryotic cellular replication initiation factor to a DNA sequence can create a functional origin of replication, providing a robust genetic assay for these factors and a novel approach to generating episomal vectors for gene therapy.

Mutations of tumor necrosis factor α-responsive serine residues within the C-terminal transactivation domain of human transcription factor REL enhance its in vitro transforming ability

The human c-rel gene (REL), encoding an NF-kappaB transcription factor, is amplified or mutated in several human B-cell lymphomas and can transform chicken lymphoid cells in vitro. We have previously shown that certain deletions of C-terminal transactivation sequences enhance REL's transforming ability in chicken spleen cells. In this report, we have analysed the effect of single amino-acid changes at select serine residues in the C-terminal transactivation domain on REL's transforming ability. Mutation of either of two TNFalpha-inducible serine residues (Ser460 and Ser471) to nonphosphorylatable residues (alanine, asparagine, phenylalanine) made REL more efficient at transforming chicken spleen cells in vitro. In contrast, mutation of Ser471 to a phosphorylation mimetic aspartate residue impaired REL's transforming ability, even though it increased REL's inherent transactivation ability as a GAL4-fusion protein. Alanine mutations of several other serine residues within the transactivation domain did not substantially affect REL's transforming ability. Transactivation by GAL4-REL fusion proteins containing either transformation enhancing or nonenhancing mutations at serine residues was generally similar to wild-type GAL4-REL. However, more transforming mutants with mutations at either Ser460 or Ser471 differed from wild-type REL in their ability to transactivate certain kappaB-site reporter genes. In particular, the SOD2 promoter, encoding manganese superoxide dismutase, was activated less strongly by the more transforming REL mutant REL-S471N in transient assays, but REL-S471N-transformed chicken spleen cells had increased levels of MnSOD protein as compared to wild-type REL-transformed cells. Taken together, our results show that mutations of certain serine residues can enhance REL's transforming ability in vitro and suggest that these mutations increase REL-mediated transformation by altering REL's ability to modulate the expression of select target genes. Furthermore, phosphorylation of Ser471 may be involved in REL-mediated modulation of transformation-specific target gene expression. Lastly, these results suggest that similar mutations in the REL transactivation domain contribute to the development of certain human B-cell lymphomas.