DNA Intercalators Inhibit Eukaryotic Ribosomal RNA Synthesis by Impairing the Initiation of Transcription.

In eukaryotes, ribosome biogenesis is driven by the synthesis of the ribosomal RNA (rRNA) by RNA polymerase I (Pol-I) and is tightly linked to cell growth and proliferation. The 3D-structure of the rDNA promoter plays an important, yet not fully understood role in regulating rRNA synthesis. We hypothesized that DNA intercalators/groove binders could affect this structure and disrupt rRNA transcription. To test this hypothesis, we investigated the effect of a number of compounds on Pol-I transcription in vitro and in cells. We find that intercalators/groove binders are potent inhibitors of Pol-I specific transcription both in vitro and in cells, regardless of their specificity and the strength of its interaction with DNA. Importantly, the synthetic ability of Pol-I is unaffected, suggesting that these compounds are not targeting post-initiating events. Notably, the tested compounds have limited effect on transcription by Pol-II and III, demonstrating the hypersensitivity of Pol-I transcription. We propose that stability of pre-initiation complex and initiation are affected as result of altered 3D architecture of the rDNA promoter, which is well in line with the recently reported importance of biophysical rDNA promoter properties on initiation complex formation in the yeast system.

Ureidothiophene inhibits interaction of bacterial RNA polymerase with -10 promotor element.

Bacterial RNA polymerase is a potent target for antibiotics, which utilize a plethora of different modes of action, some of which are still not fully understood. Ureidothiophene (Urd) was found in a screen of a library of chemical compounds for ability to inhibit bacterial transcription. The mechanism of Urd action is not known. Here, we show that Urd inhibits transcription at the early stage of closed complex formation by blocking interaction of RNA polymerase with the promoter -10 element, while not affecting interactions with -35 element or steps of transcription after promoter closed complex formation. We show that mutation in the region 1.2 of initiation factor σ decreases sensitivity to Urd. The results suggest that Urd may directly target σ region 1.2, which allosterically controls the recognition of -10 element by σ region 2. Alternatively, Urd may block conformational changes of the holoenzyme required for engagement with -10 promoter element, although by a mechanism distinct from that of antibiotic fidaxomycin (lipiarmycin). The results suggest a new mode of transcription inhibition involving the regulatory domain of σ subunit, and potentially pinpoint a novel target for development of new antibacterials.

CarD and RbpA modify the kinetics of initial transcription and slow promoter escape of the Mycobacterium tuberculosis RNA polymerase.

The pathogen Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, enacts unique transcriptional regulatory mechanisms when subjected to host-derived stresses. Initiation of transcription by the Mycobacterial RNA polymerase (RNAP) has previously been shown to exhibit different open complex kinetics and stabilities relative to Escherichia coli (Eco) RNAP. However, transcription initiation rates also depend on the kinetics following open complex formation such as initial nucleotide incorporation and subsequent promoter escape. Here, using a real-time fluorescence assay, we present the first in-depth kinetic analysis of initial transcription and promoter escape for the Mtb RNAP. We show that in relation to Eco RNAP, Mtb displays slower initial nucleotide incorporation but faster overall promoter escape kinetics on the Mtb rrnAP3 promoter. Furthermore, in the context of the essential transcription factors CarD and RbpA, Mtb promoter escape is slowed via differential effects on initially transcribing complexes. Finally, based on their ability to increase the rate of open complex formation and decrease the rate of promoter escape, we suggest that CarD and RbpA are capable of activation or repression depending on the rate-limiting step of a given promoter's basal initiation kinetics.

Structures of an RNA polymerase promoter melting intermediate elucidate DNA unwinding.

A key regulated step of transcription is promoter melting by RNA polymerase (RNAP) to form the open promoter complex1-3. To generate the open complex, the conserved catalytic core of the RNAP combines with initiation factors to locate promoter DNA, unwind 12-14 base pairs of the DNA duplex and load the template-strand DNA into the RNAP active site. Formation of the open complex is a multi-step process during which transient intermediates of unknown structure are formed4-6. Here we present cryo-electron microscopy structures of bacterial RNAP-promoter DNA complexes, including structures of partially melted intermediates. The structures show that late steps of promoter melting occur within the RNAP cleft, delineate key roles for fork-loop 2 and switch 2-universal structural features of RNAP-in restricting access of DNA to the RNAP active site, and explain why clamp opening is required to allow entry of single-stranded template DNA into the active site. The key roles of fork-loop 2 and switch 2 suggest a common mechanism for late steps in promoter DNA opening to enable gene expression across all domains of life.

Dynamic turnover of paused Pol II complexes at human promoters.

Paused RNA polymerase II (Pol II) that piles up near most human promoters is the target of mechanisms that control entry into productive elongation. Whether paused Pol II is a stable or dynamic target remains unresolved. We report that most 5' paused Pol II throughout the genome is turned over within 2 min. This process is revealed under hypertonic conditions that prevent Pol II recruitment to promoters. This turnover requires cell viability but is not prevented by inhibiting transcription elongation, suggesting that it is mediated at the level of termination. When initiation was prevented by triptolide during recovery from high salt, a novel preinitiated state of Pol II lacking the pausing factor Spt5 accumulated at transcription start sites. We propose that Pol II occupancy near 5' ends is governed by a cycle of ongoing assembly of preinitiated complexes that transition to pause sites followed by eviction from the DNA template. This model suggests that mechanisms regulating the transition to productive elongation at pause sites operate on a dynamic population of Pol II that is turning over at rates far higher than previously suspected. We suggest that a plausible alternative to elongation control via escape from a stable pause is by escape from premature termination.

Paused RNA polymerase II inhibits new transcriptional initiation.

RNA polymerase II (Pol II) pauses downstream of the transcription initiation site before beginning productive elongation. This pause is a key component of metazoan gene expression regulation. Some promoters have a strong disposition for Pol II pausing and often mediate faster, more synchronous changes in expression. This requires multiple rounds of transcription and thus cannot rely solely on pause release. However, it is unclear how pausing affects the initiation of new transcripts during consecutive rounds of transcription. Using our recently developed ChIP-nexus method, we find that Pol II pausing inhibits new initiation. We propose that paused Pol II helps prevent new initiation between transcription bursts, which may reduce noise.

Synergistic activation of Arg1 gene by retinoic acid and IL-4 involves chromatin remodeling for transcription initiation and elongation coupling.

All-trans Retinoic acid (RA) and its derivatives are potent therapeutics for immunological functions including wound repair. However, the molecular mechanism of RA modulation in innate immunity is poorly understood, especially in macrophages. We found that topical application of RA significantly improves wound healing and that RA and IL-4 synergistically activate Arg1, a critical gene for tissue repair, in M2 polarized macrophages. This involves feed forward regulation of Raldh2, a rate-limiting enzyme for RA biosynthesis, and requires Med25 to coordinate RAR, STAT6 and chromatin remodeler, Brg1 to remodel the +1 nucleosome of Arg1 for transcription initiation. By recruiting elongation factor TFIIS, Med25 also facilitates transcriptional initiation-elongation coupling. This study uncovers synergistic activation of Arg1 by RA and IL-4 in M2 macrophages that involves feed forward regulation of RA synthesis and dual functions of Med25 in nucleosome remodeling and transcription initiation-elongation coupling that underlies robust modulatory activity of RA in innate immunity.

The elongation factor Spt5 facilitates transcription initiation for rapid induction of inflammatory-response genes.

A subset of inflammatory-response NF-κB target genes is activated immediately following pro-inflammatory signal. Here we followed the kinetics of primary transcript accumulation after NF-κB activation when the elongation factor Spt5 is knocked down. While elongation rate is unchanged, the transcript synthesis at the 5'-end and at the earliest time points is delayed and reduced, suggesting an unexpected role in early transcription. Investigating the underlying mechanism reveals that the induced TFIID-promoter association is practically abolished by Spt5 depletion. This effect is associated with a decrease in promoter-proximal H3K4me3 and H4K5Ac histone modifications that are differentially required for rapid transcriptional induction. In contrast, the displacement of TFIIE and Mediator, which occurs during promoter escape, is attenuated in the absence of Spt5. Our findings are consistent with a central role of Spt5 in maintenance of TFIID-promoter association and promoter escape to support rapid transcriptional induction and re-initiation of inflammatory-response genes.

[Hypothetical SNP Markers That Significantly Affect the Affinity of the TATA-Binding Protein to VEGFA, ERBB2, IGF1R, FLT1, KDR, and MET Oncogene Promoters as Chemotherapy Targets].

The following hypothesis has been proposed: IF an SNP can significantly increase the expression of an oncogene by increasing the affinity of the TATA-binding protein (TBP) to its promoter, THEN this SNP can also reduce the apparent bioactivity of inhibitors of this oncogene during antitumor chemotherapy and vice versa. In the context of this hypothesis, the previously proposed method (http://beehive.bionet.nsc. ru/cgi-bin/mgs/tatascan/start.pl) was applied to analyze all SNPs found within the [-70; -20] regions (which harbor all proven TBP-binding sites) of the promoters of VEGFA, EGFR, ERBB2, IGF1R, FLT1, KDR, and MET oncogenes according to the human reference genome, hg19. For 83% of these SNPs, their effect on TBP affinity to the oncogene promoters required for assembly of preinitiation complexes was not significant. rs36208385, rs36208384, rs370995111, rs372731987, rs111811434, rs369547510, rs76407893, rs369728300, and rs72001900 can potentially serve as SNP markers to reduce the apparent bioactivity of oncogene inhibitors, while rs141092704, rs184083669, rs145139616, rs200697953, rs187746433, rs199730913, rs377370642, rs114484350, rs374921120, rs146790957, rs376727645, and rs72001900 can be the markers for enhancing this activity.

From indole to pyrrole, furan, thiophene and pyridine: Search for novel small molecule inhibitors of bacterial transcription initiation complex formation.

The search for small molecules capable of inhibiting transcription initiation in bacteria has resulted in the synthesis of N,N'-disubstituted hydrazines and imine-carbohydrazides comprised of indole, pyridine, pyrrole, furan and thiophene using the respective trichloroacetyl derivatives, carbohydrazides and aldehydes. Replacement of the indole moiety by smaller heterocycles linked by CONHNC linkers afforded a broad variety of compounds efficiently targeting the RNA polymerase-σ(70)/σ(A) interaction as determined by ELISA and exhibiting increased inhibition of the growth of Escherichia coli compared to Bacillus subtilis in culture. The structural features of the synthesized transcription initiation inhibitors needed for antibacterial activity were identified employing molecular modelling and structure-activity relationship (SAR) studies.

Where does transcription start? 5'-RACE adapted to next-generation sequencing.

The variability and complexity of the transcription initiation process was examined by adapting RNA ligase-mediated rapid amplification of 5' cDNA ends (5'-RACE) to Next-Generation Sequencing (NGS). We oligo-labelled 5'-m(7)G-capped mRNA from two genes, the simple mono-exonic Beta-2-Adrenoceptor (ADRB2R)and the complex multi-exonic Glucocorticoid Receptor (GR, NR3C1), and detected a variability in TSS location that has received little attention up to now. Transcription was not initiated at a fixed TSS, but from loci of 4 to 10 adjacent nucleotides. Individual TSSs had frequencies from <0.001% to 38.5% of the total gene-specific 5' m(7)G-capped transcripts. ADRB2R used a single locus consisting of 4 adjacent TSSs. Unstimulated, the GR used a total of 358 TSSs distributed throughout 38 loci, that were principally in the 5' UTRs and were spliced using established donor and acceptor sites. Complete demethylation of the epigenetically sensitive GR promoter with 5-azacytidine induced one new locus and 127 TSSs, 12 of which were unique. We induced GR transcription with dexamethasone and Interferon-γ, adding one new locus and 185 additional TSSs distributed throughout the promoter region. In-vitro the TSS microvariability regulated mRNA translation efficiency and the relative abundance of the different GRN-terminal protein isoform levels.

Removal of O-GlcNAcylation is important for pig preimplantation development.

Glucose has been recognized as an energy source for a long time, but it has recently been suggested that the hexosamine biosynthesis pathway (HBP) and downstream protein O-GlcNAcylation have important functions in mouse preimplantation development. Thus, whether or not O-GlcNAcylation was present and what functions O-GlcNAcylation has in pig preimplantation development were investigated in the present study. The expressions of mRNA of glutaminefructose-6-phosphate aminotransferase (Gfpt), O-GlcNAc transferase (Ogt) and O-GlcNAcase (Oga), which are involved in the HBP and O-GlcNAc cycling, were examined in pig parthenogenetic diploids at each preimplantation developmental stage. Gfpt and Ogt were detected in diploids at all stages. Though Oga was detected at all stages except the 4-cell stage, OGA proteins were detected in diploids from the 2-cell to blastocyst stage. Furthermore, O-GlcNAcylated proteins in MII oocytes and diploids were also detected by immunofluorescence at every stage. Inhibition of OGT by 4.0 mM BADGP did not affect development up to the blastocyst stage, while inhibition of OGA by 300 µM PUGNAc decreased the proportion of diploids beyond the 4-cell stage. Four-cell diploids cultured with PUGNAc until 48 h developed to the blastocyst stage after culture in a PUGNAc-free medium until 144 h after electrostimulation. RNA polymerase II (Pol II) phosphorylation, which indicates the onset of mRNA transcription, was detected in nuclei of diploids in the control group at 48 h but not in the PUGNAc-treated group. These results indicate that HBP and O-GlcNAcylation have important functions in pig preimplantation development and that inhibition of OGA is fatal for development. It is also suggested that OGA inhibition disrupts normal Pol II regulation and may cause a zygotic gene activation error.

Complex impacts of PI3K/AKT inhibitors to androgen receptor gene expression in prostate cancer cells.

BACKGROUND: Androgen deprivation therapy (ADT) is the first-line treatment to metastatic prostate cancer (PCa). However, sustained expression and function of the androgen receptor (AR) gene contribute to the progression of castration resistant prostate cancers (CRPC). Additionally, tumors can adapt the PI3K/AKT survival pathway to escape ADT. Co-targeting AR and PI3K/AKT signaling has been proposed to be a more effective therapeutic means for CRPC patients. Many clinical trials are ongoing to test whether PI3K/AKT inhibitors are beneficial to PCa patients. However whether these inhibitors have any impacts on the expressions of full length AR (AR-FL) and its splice variant (AR-V7) remains unclear. METHODS: Four human prostate cancer cell lines (LNCaP, LNCaP95, VCaP and 22Rv1) with different genetic backgrounds were treated with five PI3K/AKT inhibitors (LY294002, Wortmannin, BKM120, AKTi and AZD5363) and or AKT siRNA. AR and AR-V7 protein and mRNA levels were measured by immunoblotting and real-time PCR assays. AR gene transcription initiation, alternative RNA splicing and AR mRNA degradation rates were also determined. RESULTS: PI3K/AKT inhibitors had various impacts on AR protein expressions primarily through alterations of AR gene transcription initiation and RNA splicing. However, these effects remained unchanged in the presence RNA silencing of the AKT genes. CONCLUSION: PI3K/AKT inhibitors have off-target effects on AR gene expression in prostate cancer cells, which shall be considered when applying these inhibitors to PCa patients, particularly patients under ADT treatment.

Identification of TNF-α-responsive promoters and enhancers in the intestinal epithelial cell model Caco-2.

The Caco-2 cell line is one of the most important in vitro models for enterocytes, and is used to study drug absorption and disease, including inflammatory bowel disease and cancer. In order to use the model optimally, it is necessary to map its functional entities. In this study, we have generated genome-wide maps of active transcription start sites (TSSs), and active enhancers in Caco-2 cells with or without tumour necrosis factor (TNF)-α stimulation to mimic an inflammatory state. We found 520 promoters that significantly changed their usage level upon TNF-α stimulation; of these, 52% are not annotated. A subset of these has the potential to confer change in protein function due to protein domain exclusion. Moreover, we locate 890 transcribed enhancer candidates, where ∼50% are changing in usage after TNF-α stimulation. These enhancers share motif enrichments with similarly responding gene promoters. As a case example, we characterize an enhancer regulating the laminin-5 γ2-chain (LAMC2) gene by nuclear factor (NF)-κB binding. This report is the first to present comprehensive TSS and enhancer maps over Caco-2 cells, and highlights many novel inflammation-specific promoters and enhancers.

Synthesis and biological activity of novel bis-indole inhibitors of bacterial transcription initiation complex formation.

The increasing resistance of bacteria against clinically approved antibiotics is resulting in an alarming decrease in therapeutic options for today's clinicians. We have targeted the essential interaction between bacterial RNA polymerase and σ(70)/σ(A) for the development of lead molecules exhibiting a novel mechanism of antibacterial activity. Several classes of structurally related bis-indole inhibitors of bacterial transcription initiation complex formation were synthesized and their antimicrobial activities were evaluated. Condensation of indole-7- and indole-2-carbohydrazides with 7- and 2-trichloroacetylindoles or indole-7- and indole-2-glyoxyloyl chlorides resulted in the successful synthesis of 7,7'-, 2,2'-, 2,7'- and 3,2'-linked bis-indole derivatives with -CO-NH-NH-CO- and -CO-CO-NH-NH-CO- linkers. Indole-7-glyoxyloyl chlorides were reacted with hydrazine hydrate in different ratios to afford respective -CO-CO-NH-NH-CO-CO- bis-indole or hydrazide derivatives. The resulting compounds were found to be active against the ß'-CH-σ(70)/σ interaction in ELISA assays and inhibited the growth of both Gram-positive and Gram-negative bacteria. Structure-activity relationship (SAR) studies were performed in order to identify the structural features of the synthesized inhibitors required for biological activity.

Distinct functions of the RNA polymerase σ subunit region 3.2 in RNA priming and promoter escape.

The σ subunit of bacterial RNA polymerase (RNAP) has been implicated in all steps of transcription initiation, including promoter recognition and opening, priming of RNA synthesis, abortive initiation and promoter escape. The post-promoter-recognition σ functions were proposed to depend on its conserved region σ3.2 that directly contacts promoter DNA immediately upstream of the RNAP active centre and occupies the RNA exit path. Analysis of the transcription effects of substitutions and deletions in this region in Escherichia coli σ(70) subunit, performed in this work, suggests that (i) individual residues in the σ3.2 finger collectively contribute to RNA priming by RNAP, likely by the positioning of the template DNA strand in the active centre, but are not critical to promoter escape; (ii) the physical presence of σ3.2 in the RNA exit channel is important for promoter escape; (iii) σ3.2 promotes σ dissociation during initiation and suppresses σ-dependent promoter-proximal pausing; (iv) σ3.2 contributes to allosteric inhibition of the initiating NTP binding by rifamycins. Thus, region σ3.2 performs distinct functions in transcription initiation and its inhibition by antibiotics. The B-reader element of eukaryotic factor TFIIB likely plays similar roles in RNAPII transcription, revealing common principles in transcription initiation in various domains of life.

Transcriptional properties of mammalian elongin A and its role in stress response.

Elongin A was shown previously to be capable of potently activating the rate of RNA polymerase II (RNAPII) transcription elongation in vitro by suppressing transient pausing by the enzyme at many sites along DNA templates. The role of Elongin A in RNAPII transcription in mammalian cells, however, has not been clearly established. In this report, we investigate the function of Elongin A in RNAPII transcription. We present evidence that Elongin A associates with the IIO form of RNAPII at sites of newly transcribed RNA and is relocated to dotlike domains distinct from those containing RNAPII when cells are treated with the kinase inhibitor 5,6-dichloro-1-ß-d-ribofuranosylbenzimidazole. Significantly, Elongin A is required for maximal induction of transcription of the stress response genes ATF3 and p21 in response to several stimuli. Evidence from structure-function studies argues that Elongin A transcription elongation activity, but not its ubiquitination activity, is most important for its function in induction of transcription of ATF3 and p21. Taken together, our data provide new insights into the function of Elongin A in RNAPII transcription and bring to light a previously unrecognized role for Elongin A in the regulation of stress response genes.

Repression of class I transcription by cadmium is mediated by the protein phosphatase 2A.

Toxic metals are part of our environment, and undue exposure to them leads to a variety of pathologies. In response, most organisms adapt their metabolism and have evolved systems to limit this toxicity and to acquire tolerance. Ribosome biosynthesis being central for protein synthesis, we analyzed in yeast the effects of a moderate concentration of cadmium (Cd(2+)) on Pol I transcription that represents >60% of the transcriptional activity of the cells. We show that Cd(2+) rapidly and drastically shuts down the expression of the 35S rRNA. Repression does not result from a poisoning of any of the components of the class I transcriptional machinery by Cd(2+), but rather involves a protein phosphatase 2A (PP2A)-dependent cellular signaling pathway that targets the formation/dissociation of the Pol I-Rrn3 complex. We also show that Pol I transcription is repressed by other toxic metals, such as Ag(+) and Hg(2+), which likewise perturb the Pol I-Rrn3 complex, but through PP2A-independent mechanisms. Taken together, our results point to a central role for the Pol I-Rrn3 complex as molecular switch for regulating Pol I transcription in response to toxic metals.

Biochemical analysis of histone deacetylase-independent transcriptional repression by MeCP2.

MeCP2 is an abundant methyl-cytosine-guanine (CG)-binding protein and transcriptional repressor. We developed a biochemical system that exhibits CG methylation-specific transcriptional repression by purified human MeCP2. MeCP2 represses transcription by histone deacetylase (HDAC)-dependent and HDAC-independent mechanisms. Our system appears to recreate the HDAC-independent component of MeCP2-mediated repression and occurs via inhibition of the assembly of transcription preinitiation complexes. At a ratio of approximately one molecule of MeCP2 per two methyl-CG dinucleotides, as found in mammalian neurons, the magnitude of methylation-specific repression was greater than 10-fold. Notably, the HDAC inhibitor trichostatin A had no effect on MeCP2-mediated repression with either naked DNA or chromatin templates. We designed a CG-deficient core promoter that is resistant to MeCP2-mediated repression when placed in a plasmid lacking CG dinucleotides. By using this CG-deficient reporter as a reference, we found that eight CG dinucleotides in the core promoter region are sufficient for strong methylation-specific repression by MeCP2. In contrast, MeCP2 does not repress a construct with 13 CG dinucleotides located ∼1.7 kbp upstream of the promoter. Furthermore, by analysis of C-terminally truncated MeCP2 proteins, we found that binding of MeCP2 to methyl-CG dinucleotides is not sufficient for transcriptional repression. Hence, MeCP2-mediated repression is not due to the simple steric blockage of the transcriptional machinery. These experiments suggest that MeCP2 can function as a global methyl-CG-specific, HDAC-independent repressor. This HDAC-independent mechanism of MeCP2-mediated repression may be important in cells, such as mammalian neurons, that have high levels of CG methylation and MeCP2.

Inverse PPARß/δ agonists suppress oncogenic signaling to the ANGPTL4 gene and inhibit cancer cell invasion.

Besides its established functions in intermediary metabolism and developmental processes, the nuclear receptor peroxisome proliferator-activated receptor ß/δ (PPARß/δ) has a less defined role in tumorigenesis. In the present study, we have identified a function for PPARß/δ in cancer cell invasion. We show that two structurally divergent inhibitory ligands for PPARß/δ, the inverse agonists ST247 and DG172, strongly inhibit the serum- and transforming growth factor ß (TGFß)-induced invasion of MDA-MB-231 human breast cancer cells into a three-dimensional matrigel matrix. To elucidate the molecular basis of this finding, we performed chromatin immunoprecipitation sequencing (ChIP-Seq) and microarray analyses, which identified the gene encoding angiopoietin-like 4 (ANGPTL4) as the major transcriptional PPARß/δ target in MDA-MB-231 cells, previously implicated in TGFß-mediated tumor progression and metastatic dissemination. We show that the induction of ANGPTL4 by TGFß and other oncogenic signals is strongly repressed by ST247 and DG172 in a PPARß/δ-dependent fashion, resulting in the inhibition of ANGPTL4 secretion. This effect is attributable to these ligands' ability to induce a dominant transcriptional repressor complex at the site of transcription initiation that blocks preinitiation complex formation through an histone deacetylase-independent, non-canonical mechanism. Repression of ANGPTL4 transcription by inverse PPARß/δ agonists is functionally linked to the inhibition of cancer cell invasion into a three-dimensional matrix, as (i) invasion of MDA-MB-231 cells is critically dependent on ANGPTL4 expression, (ii) recombinant ANGPTL4 stimulates invasion, and (iii) reverses the inhibitory effect of ST247 and DG172. These findings indicate that a PPARß/δ-ANGPTL4 pathway is involved in the regulation of tumor cell invasion and that its pharmacological manipulation by inverse PPARß/δ agonists is feasible.