Mechanism Of Transcriptional Activation Research Articles

Analysis of Induction Mechanisms of an Insulin-Inducible Transcription Factor SHARP-2 Gene by (−)-Epigallocatechin-3-gallate

The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is an insulin-inducible transcription factor. In this study, we examined the mechanism(s) involved in the regulation of the rat SHARP-2 gene expression by (-)-epigallocatechin-3-gallate (EGCG). The induction of SHARP-2 mRNA by EGCG was repressed by pretreatments with inhibitors for either phosphoinositide 3-kinase (PI3K) or RNA polymerase II. Then, we examined a biological relationship between EGCG and transcription factor NF-κB interfering with the insulin action. The protein levels of the NF-κB were rapidly decreased by an EGCG treatment. Finally, the mechanism(s) of transcriptional activation of the rat SHARP-2 gene by both NF-κB and EGCG was analyzed. While overexpression of the NF-κB p65 protein decreased the promoter activity of the SHARP-2 gene, EGCG did not affect it. Thus, we conclude that EGCG induces the expression of the rat SHARP-2 gene via both the PI3K pathway and degradation of the NF-κB p65 protein.

E2F7, a novel target, is up-regulated by p53 and mediates DNA damage-dependent transcriptional repression

The p53 tumor suppressor protein is a transcription factor that exerts its effects on the cell cycle via regulation of gene expression. Although the mechanism of p53-dependent transcriptional activation has been well-studied, the molecular basis for p53-mediated repression has been elusive. The E2F family of transcription factors has been implicated in regulation of cell cycle-related genes, with E2F6, E2F7, and E2F8 playing key roles in repression. In response to cellular DNA damage, E2F7, but not E2F6 or E2F8, is up-regulated in a p53-dependent manner, with p53 being sufficient to increase expression of E2F7. Indeed, p53 occupies the promoter of the E2F7 gene after genotoxic stress, consistent with E2F7 being a novel p53 target. Ablation of E2F7 expression abrogates p53-dependent repression of a subset of its targets, including E2F1 and DHFR, in response to DNA damage. Furthermore, E2F7 occupancy of the E2F1 and DHFR promoters is detected, and expression of E2F7 is sufficient to inhibit cell proliferation. Taken together, these results show that p53-dependent transcriptional up-regulation of its target, E2F7, leads to repression of relevant gene expression. In turn, this E2F7-dependent mechanism contributes to p53-dependent cell cycle arrest in response to DNA damage.

Electric oscillation and coupling of chromatin regulate chromosome packaging and transcription in eukaryotic cells

Transcription in eukaryotic cells is efficiently spatially and temporally regulated, but how this genome-wide regulation is achieved at the physical level remains unclear, given the limited transcriptional resources within the nucleus and the sporadic linear arrangements of genes within chromosomes. In this article, we provide a physical model for chromatin cluster formation, based on oscillation synchronization and clustering of different chromatin regions, enabling efficient systemic genome-wide regulation of transcription. We also propose that the electromagnetic field generated by oscillation of chromatin is the driving force for chromosome packing during M phase. We further explore the physical mechanisms for chromatin oscillation cluster (COC) formation, and long-distance chromatin kissing. The COC model, which connects the dots between chromatin epigenetic modification and higher-order nuclear organization, answers many important questions, such as how the CCCTC-binding factor CTCF contributes to higher-order chromatin organization, and the mechanism of sequential transcriptional activation of HOX clusters. In the COC model, long non-coding RNAs function as oscillation clustering adaptors to recruit chromatin modification factors to specific sub-nuclear regions, fine-tuning transcriptional events in the chromatin oscillation clusters. Introns of eukaryotic genes have evolved to promote the clustering of transcriptionally co-regulated genes in these sub-nuclear regions.

Regulation of the transcriptional activation of CTRP3 in chondrocytes by c-Jun

Although C1qTNF-related protein 3 (CTRP3) is highly expressed in cartilage and is involved in the proliferation and migration of chondrocytes, the molecular mechanism governing the transcriptional regulation of CTRP3 in chondrocytes is largely unknown. Here, we demonstrated the mechanism of transcriptional activation of the CTRP3 gene in chondrogenic cells. We observed that c-Jun stimulated CTRP3 promoter activity in ATDC5 cells. Deletion analysis of the CTRP3 promoter showed that the c-Jun-responsive region is localized at the position between -359 and -176. Mutation of the AP-1 binding site in this region eliminated c-Jun-mediated CTRP3 promoter activity. A promoter enzyme immunoassay and a chromatin immunoprecipitation assay showed that c-Jun binds directly to the AP-1 binding site at the region -184/-177 of the mouse CTRP3 promoter. Thus, these results demonstrated that c-Jun is a cis-acting element for CTRP3 regulation in chondrogenic cells.

Role for gene looping in intron-mediated enhancement of transcription

Intron-containing genes are often transcribed more efficiently than nonintronic genes. The effect of introns on transcription of genes is an evolutionarily conserved feature, being exhibited by such diverse organisms as yeast, plants, flies, and mammals. The mechanism of intron-mediated transcriptional activation, however, is not entirely clear. To address this issue, we inserted an intron in INO1, which is a nonintronic gene, and deleted the intron from ASC1, which contains a natural intron. We then compared transcription of INO1 and ASC1 genes in the presence and absence of an intron. Transcription of both genes was significantly stimulated by the intron. The introns have a direct role in enhancing transcription of INO1 and ASC1 because there was a marked increase in nascent transcripts from these genes in the presence of an intron. Intron-mediated enhancement of transcription required a splicing competent intron. Interestingly, both INO1 and ASC1 were in a looped configuration when their genes contained an intron. Intron-dependent gene looping involved a physical interaction of the promoter and the terminator regions. In addition, the promoter region interacted with the 5' splice site and the terminator with the 3' splice site. Intron-mediated enhancement of transcription was completely abolished in the looping defective sua7-1 strain. No effect on splicing, however, was observed in sua7-1 strain. On the basis of these results, we propose a role for gene looping in intron-mediated transcriptional activation of genes in yeast.

Role of disorder in IκB–NFκB interaction

The paradigmatic transcription factors of the NFκB family provide an increasingly complex view of the mechanism of signal-mediated transcriptional activation. Although the primary event, phosphorylation and subsequent ubiquitin-dependent degradation of IκBα, the inhibitor of the canonical NFκB (p50/p65), is reasonably well understood, the means whereby the activation is turned off by postinduction repression are less well understood. Recent work highlighted in this review suggests that the inhibitor IκBα participates in the "stripping" of NFκB from the DNA, and that this process relies heavily on the disordered and weakly ordered segments of IκBα. Kinetic and equilibrium measurements in vitro as well as genetic screens in vivo convincingly demonstrate not only that IκBα greatly increases the dissociation rate of NFκB from DNA but also that further control of the process is mediated by the extremely short half-life of free IκBα, doubtless a result of the overall weakly folded nature of the free protein. These studies illustrate the versatility of protein systems that use not only well-structured proteins and protein complexes but also the full range of available weakly structured and disordered states to maximize functional efficiency and metabolic control.

The Mediator of RNA Polymerase II Transcription: Links to Transcription Elongation and Leukemogenesis

Promoter proximal pausing by initiated RNA polymerase II (Pol II) and regulated release of paused polymerase into productive elongation has emerged as a major mechanism of transcription activation. Reactivation of paused Pol II has been shown to correlate with recruitment of a Super‐Elongation Complex or SEC that contains the transcription elongation factors ELL/EAF and P‐TEFb, as well as a collection of additional proteins encoded by genes involved in translocations with the MLL gene in leukemia. Our lab has recently obtained evidence for a role of human Mediator in recruiting SEC and another ELL/EAF‐containing complex to genes. In particular, we have shown that a conserved N‐terminal domain in the Mediator subunit Med26 provides docking sites for ELL/EAF, P‐TEFb, and associated proteins as well as the general initiation factor TFIID. Our findings are consistent with the model that Med26 functions as a molecular switch that interacts first with the Pol II initiation complex through direct interactions with TFIID and then “exchanges” TFIID for complexes containing elongation factors ELL/EAF and P‐TEFb to facilitate the transition of Pol II into the elongation stage of transcription.

Molecular Mechanisms of Transcription Activation by Juvenile Hormone: A Critical Role for bHLH-PAS and Nuclear Receptor Proteins.

Juvenile hormone (JH) is responsible for controlling many biological processes. In several insect species JH has been implicated as a key regulator of developmental timing, preventing the premature onset of metamorphosis during larval growth periods. However, the molecular basis of JH action is not well-understood. In this review, we highlight recent advances which demonstrate the importance of transcription factors from the bHLH-PAS and nuclear receptor families in mediating the response to JH.

Promoter-associated RNAs and promoter-targeted RNAs.

The world of RNAs is much more complex than previously thought, and has rapidly emerged as one of the most actively researched topics in the life sciences. Recently, two findings in this field were reported and given special attention: promoter-associated RNAs (paRNAs), a novel class of RNAs with numerous potential functions; and promoter-targeted RNA-induced transcriptional gene regulation, a new regulatory mechanism to control transcription. In this review, we summarize the studies in these two areas, and outline the current understanding with respect to the potential biological functions of paRNAs, and the molecular mechanisms of promoter-targeted RNA-induced transcriptional gene silencing and activation. Additionally, we seek to integrate these two areas, as paRNAs may have potential biological links with promoter-targeted RNA-induced transcriptional gene regulation. Finally, we will discuss the significance of identifying paRNAs and the possible use of promoter-targeted RNAs in gene regulation and therapy.

Visualization of auxin-mediated transcriptional activation using a common auxin-responsive reporter system in the liverwort Marchantia polymorpha

The phytohormone auxin plays a pivotal role in various developmental aspects in land plants. However, little is known of the auxin response and distribution in non-vascular plants. In this study, we made transgenic plants of the liverwort Marchantia polymorpha which express the uidA (GUS) reporter gene under control of the soybean auxin-inducible promoter, ProGH3, and used it to indirectly monitor auxin-mediated transcriptional activation in planta. Transgenic plants carrying ProGH3:GUS showed GUS activity in an auxin-dependent manner. Histochemical GUS staining was observed at the bottom of gemma cups in the process of vegetative propagation. Significant GUS activity was also detected around the gametophyte-sporophyte junction as well as the developing sporophyte after fertilization. These results suggest that the activity of auxin is crucial in both gametophyte and sporophyte development in M. polymorpha, and that the mechanism for auxin-mediated transcriptional activation had already been established when plants emerged on the terrestrial environment.

A subset of human gliomas shows over-expression of KIT without its amplification

Receptor tyrosine kinase (RTK) encoded by proto-oncogene KIT is known to be involved in different types of cancers. Reportedly, KIT expression has been associated with higher grade of gliomas. Initial RT-PCR based KIT expression observed in low grade glioma cases evoked our interest to ascertain its status in glioma patients who underwent resection during 2008–2009. Contrary to earlier reports, over-expression of the RTK was observed in 32.5% glioma cases across low/high grades (n=40). Using quantitative PCR (qPCR), an up-regulation of the receptor (KIT) and its ligand (KITLG) was detected in most of the immunopositive cases at the transcript level. Sequence analysis of KIT showed two nucleotide substitutions in exons 10 and 17, in 4 and 2 cases, respectively though their pathological significance remained unclear. qPCR detected gene amplification in 2/13 glioma and allele loss in 1/13 glioma cases. This was in accordance with FISH results of these KIT positive neoplastic tissues. The data suggest that deranged expression of KIT is independent of gene amplification (p>0.05). Aberrant KIT expression is significantly associated with transcriptional up-regulation (p<0.001), though the precise mechanism(s) for transcriptional activation remain unclear.

Conserved Motifs in the Msn2-Activating Domain are Important for Msn2-Mediated Yeast Stress Response

The Msn2 and Msn4 transcription factors play crucial roles in the yeast general stress response. Previous studies identified several large functional domains of Msn2, mainly through crude truncations. Here, using bioinformatics and experimental approaches to examine Msn2 structure-function relationships, we have identified new functional motifs in the Msn2 transcriptional-activating domain (TAD). Msn2 is predicted to adopt an intrinsically disordered structure with two short structural motifs in its TAD. Mutations in these motifs dramatically decreased Msn2 transcriptional activity, yeast stress survival and Msn2 nuclear localization levels. Using the split-ubiquitin assay, we found that these motifs are important for the interaction of Msn2 with Gal11, a subunit of the mediator complex. Finally, we show that one of these motifs is functionally conserved in several yeast species, highlighting a common mechanism of Msn2 transcriptional activation throughout yeast evolution.

The Role of PPARγ in the Transcriptional Control by Agonists and Antagonists.

In recent years, peroxisome proliferator-activated receptor gamma (PPARγ) has been reported to be a target for the treatment of type II diabetes. Furthermore, it has received attention for its therapeutic potential in many other human diseases, including atherosclerosis, obesity, and cancers. Recent studies have provided evidence that the endogenously produced PPARγ antagonist, 2,3-cyclic phosphatidic acid (cPA), which is similar in structure to lysophosphatidic acid (LPA), inhibits cancer cell invasion and metastasis in vitro and in vivo. We recently observed that cPA negatively regulates PPARγ function by stabilizing the binding of the corepressor protein, silencing mediator of retinoic acid and thyroid hormone receptor. We also showed that cPA prevents neointima formation, adipocyte differentiation, lipid accumulation, and upregulation of PPARγ target gene transcription. We then analyzed the molecular mechanism of cPA's action on PPARγ. In this paper, we summarize the current knowledge on the mechanism of PPARγ-mediated transcriptional activity and transcriptional repression in response to novel lipid-derived ligands, such as cPA.

Two- and Three-Dimensional Single-Molecule Super-Resolution Imaging in Live Bacteria Cells

Single-molecule imaging has extended the resolution of fluorescence microscopy down to the nanometer scale. This super-resolution technique is non-invasive, tolerates simple sample preparation, and takes advantage of high-specificity labeling schemes. We will discuss recent studies focused on imaging protein structure and dynamics in live bacterial cells, with attention to the particular challenges that this system presents: the organisms are small, have short cell cycles, live in specific environments, and their organization is relatively poorly understood. We have developed methods to extend the capabilities of single-molecule fluorescence microscopy to study motion and localization in live Caulobacter crescentus, Vibrio cholerae, and Bacteroides thetaiotaomicron.FtsZ is an essential protein that polymerizes at the mid-cell, recruits the division machinery, and may generate constrictive forces necessary for cytokinesis. Based on astigmatism and on the natural dynamics of the protein, we resolve in two and three dimensions the midplane Z-ring formed by FtsZ, in C. crescentus. We further apply live-cell single-molecule imaging to two prokaryotes of biomedical interest, V. cholerae, and the gut symbiont B.thetaiotaomicron. The V. cholerae protein TcpP is a rare example of a membrane-bound transcription factor, and we have investigated the dynamics and localization of TcpP, its binding partner ToxR, and the toxT gene they regulate, in order to elucidate the mechanism of membrane-bound transcription activation. The starch utilization system (Sus) allows B. thetaiotaomicron to catabolize complex carbohydrates, and we have investigated the response of Sus proteins to stimuli with live anaerobic cell single-molecule imaging.

Natural Antisense Makes Sense for Gene-specific Activation in Brain

The upregulation of specific genes in vivo has been an elusive goal for gene therapy when compared with the wide repertoire of methods available to silence genes or modify mRNA splicing patterns. In the latest issue of Nature Biotechnology, Modarresi and colleagues1 accomplished in vivo upregulation of brain-derived neurotrophic factor (BDNF), a relevant therapeutic target for a number of neurodegenerative diseases. Rather than using small molecules or microRNA inhibitors, which could lead to activation of off-target genes, Modarresi et al.1 upregulated BDNF by inhibiting a natural antisense transcript (NAT) in response to the local delivery of oligonucleotides to the central nervous systems of mice.

M-CSF Induces Monocyte Survival by Activating NF-κB p65 Phosphorylation at Ser276 via Protein Kinase C

Macrophage colony-stimulating factor (M-CSF) promotes mononuclear phagocyte survival and proliferation. The transcription factor Nuclear Factor-kappaB (NF-κB) is a key regulator of genes involved in M-CSF-induced mononuclear phagocyte survival and this study focused at identifying the mechanism of NF-κB transcriptional activation. Here, we demonstrate that M-CSF stimulated NF-κB transcriptional activity in human monocyte-derived macrophages (MDMs) and the murine macrophage cell line RAW 264.7. The general protein kinase C (PKC) inhibitor Ro-31-8220, the conventional PKCα/β inhibitor Gö-6976, overexpression of dominant negative PKCα constructs and PKCα siRNA reduced NF-κB activity in response to M-CSF. Interestingly, Ro-31-8220 reduced Ser276 phosphorylation of NF-κBp65 leading to decreased M-CSF-induced monocyte survival. In this report, we identify conventional PKCs, including PKCα as important upstream kinases for M-CSF-induced NF-κB transcriptional activation, NF-κB-regulated gene expression, NF-κB p65 Ser276 phosphorylation, and macrophage survival. Lastly, we find that NF-κB p65 Ser276 plays an important role in basal and M-CSF-stimulated NF-κB activation in human mononuclear phagocytes.

Mechanisms of MEOX1 and MEOX2 Regulation of the Cyclin Dependent Kinase Inhibitors p21CIP1/WAF1 and p16INK4a in Vascular Endothelial Cells

Senescence, the state of permanent cell cycle arrest, has been associated with endothelial cell dysfunction and atherosclerosis. The cyclin dependent kinase inhibitors p21CIP1/WAF1 and p16INK4a govern the G1/S cell cycle checkpoint and are essential for determining whether a cell enters into an arrested state. The homeodomain transcription factor MEOX2 is an important regulator of vascular cell proliferation and is a direct transcriptional activator of both p21CIP1/WAF1 and p16INK4a. MEOX1 and MEOX2 have been shown to be partially functionally redundant during development, suggesting that they regulate similar target genes in vivo. We compared the ability of MEOX1 and MEOX2 to activate p21CIP1/WAF1 and p16INK4a expression and induce endothelial cell cycle arrest. Our results demonstrate for the first time that MEOX1 regulates the MEOX2 target genes p21CIP1/WAF1 and p16INK4a. In addition, increased expression of either of the MEOX homeodomain transcription factors leads to cell cycle arrest and endothelial cell senescence. Furthermore, we show that the mechanism of transcriptional activation of these cyclin dependent kinase inhibitor genes by MEOX1 and MEOX2 is distinct. MEOX1 and MEOX2 activate p16INK4a in a DNA binding dependent manner, whereas they induce p21CIP1/WAF1 in a DNA binding independent manner.

The 19S proteasome subcomplex promotes the targeting of NuA4 HAT to the promoters of ribosomal protein genes to facilitate the recruitment of TFIID for transcriptional initiation in vivo

Previous studies have implicated SAGA (Spt-Ada-Gcn5-acetyltransferase) and TFIID (Transcription factor-IID)-dependent mechanisms of transcriptional activation in yeast. SAGA-dependent transcriptional activation is further regulated by the 19S proteasome subcomplex. However, the role of the 19S proteasome subcomplex in transcriptional activation of the TFIID-dependent genes has not been elucidated. Therefore, we have performed a series of chromatin immunoprecipitation, mutational and transcriptional analyses at the TFIID-dependent ribosomal protein genes such as RPS5, RPL2B and RPS11B. We find that the 19S proteasome subcomplex is recruited to the promoters of these ribosomal protein genes, and promotes the association of NuA4 (Nucleosome acetyltransferase of histone H4) co-activator, but not activator Rap1p (repressor-activator protein 1). These observations support that the 19S proteasome subcomplex enhances the targeting of co-activator at the TFIID-dependent promoter. Such an enhanced targeting of NuA4 HAT (histone acetyltransferase) promotes the recruitment of the TFIID complex for transcriptional initiation. Collectively, our data demonstrate that the 19S proteasome subcomplex enhances the targeting of NuA4 HAT to activator Rap1p at the promoters of ribosomal protein genes to facilitate the recruitment of TFIID for transcriptional stimulation, hence providing a new role of the 19S proteasome subcomplex in establishing a specific regulatory network at the TFIID-dependent promoter for productive transcriptional initiation in vivo.

Recruitment of RPL11 at promoter sites of p53-regulated genes upon nucleolar stress through NEDD8 and in an Mdm2-dependent manner

Ribosomal proteins (RPs) activate the p53 tumour-suppressor protein upon disruption of the nucleolus. However, the exact mechanisms for p53 transcriptional activation through RPs are not well understood. We show that the RPL11 is rapidly but transiently recruited at promoter sites of p53-regulated genes upon nucleolar stress induced by actinomycin D (ActD). Characterisation of molecular events at p53 promoter sites shows that L11 is required for the recruitment of p53 transcriptional co-activators p300/CBP and p53 K382 acetylation. We found that direct binding to Mdm2 E3 ligase and NEDDylation of L11 are critical regulators for L11 promoter recruitment. Our data suggest that binding of L11 to Mdm2 at the promoter results in relief from Mdm2-mediated transcriptional repression of p53. Analysis of chromatin and RNA polymerase II markers suggests that L11 is involved in the initiation step of transcriptional activation. Furthermore, analysis of 36 ActD-induced genes shows that L11 and NEDD8 are global regulators of the p53 activation response. The studies provide insights on how nucleolar stress through L11 and NEDD8 can activate the transcriptional activity of p53.

Transcriptional activators and activation mechanisms

Transcriptional activators are required to turn on the expression of genes in a eukaryotic cell. Activators bound to the enhancer can facilitate either the recruitment of RNA polymerase II to the promoter or its elongation. This article examines a few selected issues in understanding activator functions and activation mechanisms.