Self-assembly of promoter DNA and RNA Pol II machinery into transcriptionally active biomolecular condensates.

Transcription in the nucleus occurs in a concentrated, dense environment, and no reasonable biochemical facsimile of this milieu exists. Such a biochemical environment would be important for further understanding transcriptional regulation. We describe here the formation of dense, transcriptionally active bodies in vitro with only nuclear extracts and promoter DNA. These biomolecular condensates (BMCs) are 0.5 to 1 µm in diameter, have a macromolecular density of approximately 100 mg/ml, and are a consequence of a phase transition between promoter DNA and nuclear extract proteins. BMCs are physically associated with transcription as any disruption of one compromised the other. The BMCs contain RNA polymerase II and elongation factors, as well as factors necessary for BMC formation in vivo. We suggest that BMCs are representative of the in vivo nuclear environment and a more physiologically relevant manifestation of the preinitiation complex/elongation machinery.

Novel Pyrido[2',1':2,3]imidazo[4,5-c]quinoline Derivative Selectively Poisons Leishmania donovani Bisubunit Topoisomerase 1 to Inhibit the Antimony-Resistant Leishmania Infection in Vivo.

The unique bisubunit structure of Leishmania donovani topoisomerase 1B (LdTop1) is a potential drug target in the parasites unlike the monomeric Top1 from its human host counterpart. Here, we report the design, synthesis, and validation of a chimeric pyrido[2',1':2,3]imidazo[4,5-c]quinoline derivative (C17) as a novel antileishmanial agent that poisons topoisomerase 1-DNA covalent complexes (LdTop1cc) inside the parasites and inhibits Top1 religation activity both in the drug sensitive and antimony-resistant L. donovani clinical isolates. Importantly, the human Top1 is not sensitive to C17. Further, C17 overcomes the chemical instability of camptothecin (CPT) by generating persistent LdTop1cc-induced DNA breaks inside the parasites even after 12 h of drug removal. Intraperitoneal administration of C17 results in marked reduction of the Leishmania amastigotes from the infected spleen and liver of BALB/c mice. C17 confers a host protective immune-response up-regulating the Th1 cytokines facilitating parasite clearance which can be exploited for treating drug-resistant leishmaniasis.

MYC: a complex problem.

The MYC protooncogene functions as a universal amplifier of transcription through interaction with numerous factors and complexes that regulate almost every cellular process. However, a comprehensive model that explains MYC's actions and the interplay governing the complicated dynamics of components of the transcription and replication machinery is still lacking. Here, we review the potency of MYC as an oncogenic driver and how it regulates the broad spectrum of complexes (effectors and regulators). We propose a 'hand-over model' for differential partitioning and trafficking of unstructured MYC via a loose interaction network between various gene-regulatory complexes and factors. Additionally, the article discusses how unstructured-MYC energetically favors efficient modulation of the energy landscape of the transcription cycle.

TDP1 knockout Leishmania donovani accumulate topoisomerase 1-linked DNA damage and are hypersensitive to clinically used antileishmanial drugs.

Leishmania donovani, a unicellular protozoan parasite, causes a wide range of human diseases including fatal visceral leishmaniasis. Tyrosyl DNA-phosphodiesterase 1 (TDP1) hydrolyzes the phosphodiester bond between DNA 3'-end and a tyrosyl moiety of trapped topoisomerase I-DNA covalent complexes (Top1cc). We have previously shown Leishmania harbors a TDP1 gene (LdTDP1), however, the biological role of TDP1 remains largely unknown. In the present study, we have generated TDP1 knockout L. donovani (LdTDP1-/- ) promastigotes and have shown that LdTDP1-/- parasites are deficient in 3'-phosphodiesterase activities and were hypersensitive to Top1-poison like camptothecin (CPT), DNA alkylation agent like methyl methanesulfonate, and oxidative DNA lesions generated by hydrogen peroxide but were not sensitive to etoposide. We also detected elevated levels of CPT-induced reactive oxygen species triggering cell cycle arrest and cell death in LdTDP1-/- promastigotes. LdTDP1-/- promastigotes accumulate a significant change in the membrane morphology with the accumulation of membrane pores, which is associated with oxidative stress and lipid peroxidation. To our surprise, we detected that LdTDP1-/- parasites were hypersensitive to antileishmanial drugs like amphotericin B and miltefosine, which could be rescued by complementation of wild-type TDP1 gene in the LdTDP1-/- parasites. Notably, multidrug-resistant L. donovani clinical isolates showed a marked reduction in TDP1 expression and were sensitive to Top1 poisons. Taken together, our study provides a new role of LdTDP1 in protecting L. donovani parasites from oxidative stress-induced DNA damage and resistance to amphotericin B and miltefosine.

MYC assembles and stimulates topoisomerases 1 and 2 in a "topoisome".

High-intensity transcription and replication supercoil DNA to levels that can impede or halt these processes. As a potent transcription amplifier and replication accelerator, the proto-oncogene MYC must manage this interfering torsional stress. By comparing gene expression with the recruitment of topoisomerases and MYC to promoters, we surmised a direct association of MYC with topoisomerase 1 (TOP1) and TOP2 that was confirmed in vitro and in cells. Beyond recruiting topoisomerases, MYC directly stimulates their activities. We identify a MYC-nucleated "topoisome" complex that unites TOP1 and TOP2 and increases their levels and activities at promoters, gene bodies, and enhancers. Whether TOP2A or TOP2B is included in the topoisome is dictated by the presence of MYC versus MYCN, respectively. Thus, in vitro and in cells, MYC assembles tools that simplify DNA topology and promote genome function under high output conditions.

Dissecting transcriptional amplification by MYC.

Supraphysiological MYC levels are oncogenic. Originally considered a typical transcription factor recruited to E-boxes (CACGTG), another theory posits MYC a global amplifier increasing output at all active promoters. Both models rest on large-scale genome-wide "-omics'. Because the assumptions, statistical parameter and model choice dictates the '-omic' results, whether MYC is a general or specific transcription factor remains controversial. Therefore, an orthogonal series of experiments interrogated MYC's effect on the expression of synthetic reporters. Dose-dependently, MYC increased output at minimal promoters with or without an E-box. Driving minimal promoters with exogenous (glucocorticoid receptor) or synthetic transcription factors made expression more MYC-responsive, effectively increasing MYC-amplifier gain. Mutations of conserved MYC-Box regions I and II impaired amplification, whereas MYC-box III mutations delivered higher reporter output indicating that MBIII limits over-amplification. Kinetic theory and experiments indicate that MYC activates at least two steps in the transcription-cycle to explain the non-linear amplification of transcription that is essential for global, supraphysiological transcription in cancer.

Development of a metabolically stable topoisomerase I poison as anticancer agent.

We have recently reported a new chemotype of a potent topoisomerase I poison with compound 1 as a potential anticancer chemotherapeutic agent. During further optimization, it has been observed that compound 1 suffers from high intrinsic clearance in human liver microsomes. To overcome the metabolic instability of compound 1, we report design and synthesis of metabolically stable Top1 poison 3. Newly identified Top1 poison 3 exhibits t1/2 of 69.1 min in human liver microsomes in comparison to compound 1 with t1/2 of 9.9 min. Molecular dynamic study of the newly optimized Top1 poison 3 was performed to get the insight into the stability of the binding pose in the active site. Compound 3 was able to trap DNA-Top1 cleavage complex and found to be less cytotoxic in non-cancerous cell line as compared to compound 1.

Discovery and Mechanistic Study of Tailor-Made Quinoline Derivatives as Topoisomerase 1 Poison with Potent Anticancer Activity.

To overcome chemical limitations of camptothecin (CPT), we report design, synthesis, and validation of a quinoline-based novel class of topoisomerase 1 (Top1) inhibitors and establish that compound 28 ( N-(3-(1 H-imidazol-1-yl)propyl)-6-(4-methoxyphenyl)-3-(1,3,4-oxadiazol-2-yl)quinolin-4-amine) exhibits the highest potency in inhibiting human Top1 activity with an IC50 value of 29 ± 0.04 nM. Compound 28 traps Top1-DNA cleavage complexes (Top1ccs) both in the in vitro cleavage assays and in live cells. Point mutation of Top1-N722S fails to trap compound 28-induced Top1cc because of its inability to form a hydrogen bond with compound 28. Unlike CPT, compound 28 shows excellent plasma serum stability and is not a substrate of P-glycoprotein 1 (permeability glycoprotein) advancing its potential anticancer activity. Finally, we provide evidence that compound 28 overcomes the chemical instability of CPT in human breast adenocarcinoma cells through generation of persistent and less reversible Top1cc-induced DNA double-strand breaks as detected by γH2AX foci immunostaining after 5 h of drug removal.

Studies toward the synthesis of strevertenes A and G: stereoselective construction of C1-C19 segments of the molecules.

Stereoselective synthesis of common C1-C19 skeletons of pentane macrolides strevertenes A and G possessing 10 stereogenic centers has been achieved using a flexible and convergent strategy. The salient features of this synthetic study include the Evans aldol reaction for the constructions of C2, C3, C13, and C14 centers, CBS reduction for the generation of a C7 center, Hoveyda-Grubbs cross olefin metathesis for the synthesis of C8-C9 bond, and Wittig olefination for the installation of C16-C19 conjugated olefins.

PRMT5-mediated arginine methylation of TDP1 for the repair of topoisomerase I covalent complexes.

Human tyrosyl-DNA phosphodiesterases (TDP) hydrolyze the phosphodiester bond between DNA and the catalytic tyrosine of Top1 to excise topoisomerase I cleavage complexes (Top1cc) that are trapped by camptothecin (CPT) and by genotoxic DNA alterations. Here we show that the protein arginine methyltransferase PRMT5 enhances the repair of Top1cc by direct binding to TDP1 and arginine dimethylation of TDP1 at residues R361 and R586. Top1-induced replication-mediated DNA damage induces TDP1 arginine methylation, enhancing its 3'- phosphodiesterase activity. TDP1 arginine methylation also increases XRCC1 association with TDP1 in response to CPT, and the recruitment of XRCC1 to Top1cc DNA damage foci. PRMT5 knockdown cells exhibit defective TDP1 activity with marked elevation in replication-coupled CPT-induced DNA damage and lethality. Finally, methylation of R361 and R586 stimulate TDP1 repair function and promote cell survival in response to CPT. Together, our findings provide evidence for the importance of PRMT5 for the post-translational regulation of TDP1 and repair of Top1cc.

VEGF may contribute to macrophage recruitment and M2 polarization in the decidua.

It is increasingly evident that cytokines and growth factors produced in the decidua play a pivotal role in the regulation of the local immune microenvironment and the establishment of pregnancy. One of the major growth factors produced in the decidua is vascular endothelial growth factor (VEGF), which acts not only on endothelial cells, but also on multiple other cell types, including macrophages. We sought to determine whether decidua-derived VEGF affects macrophage recruitment and polarization using human endometrial/decidual tissue samples, primary human endometrial stromal cells (ESCs), and the human monocyte cell line THP1. In situ hybridization was used for assessment of local VEGF expression and immunohistochemistry was used for identification and localization of CD68-positive endometrial macrophages. Macrophage migration in culture was assessed using a transwell migration assay, and the various M1/M2 phenotypic markers and VEGF expression were assessed using quantitative real-time PCR (qRT-PCR). We found dramatic increases in both VEGF levels and macrophage numbers in the decidua during early pregnancy compared to the secretory phase endometrium (non-pregnant), with a significant increase in M2 macrophage markers, suggesting that M2 is the predominant macrophage phenotype in the decidua. However, decidual samples from preeclamptic pregnancies showed a significant shift in macrophage phenotype markers, with upregulation of M1 and downregulation of M2 markers. In THP1 cultures, VEGF treatment significantly enhanced macrophage migration and induced M1 macrophages to shift to an M2 phenotype. Moreover, treatment with conditioned media from decidualized ESCs induced changes in macrophage migration and polarization similar to that of VEGF treatment. These effects were abrogated by the addition of a potent VEGF inhibitor. Together these results suggest that decidual VEGF plays a significant role in macrophage recruitment and M2 polarization, and that inhibition of VEGF signaling may contribute to the shift in macrophage polarity observed in different pregnancy disorders, including preeclampsia.

Neutral Porphyrin Derivative Exerts Anticancer Activity by Targeting Cellular Topoisomerase I (Top1) and Promotes Apoptotic Cell Death without Stabilizing Top1-DNA Cleavage Complexes.

Camptothecin (CPT) selectively traps topoisomerase 1-DNA cleavable complexes (Top1cc) to promote anticancer activity. Here, we report the design and synthesis of a new class of neutral porphyrin derivative 5,10-bis(4-carboxyphenyl)-15, 20-bis(4-dimethylaminophenyl)porphyrin (compound 8) as a potent catalytic inhibitor of human Top1. In contrast to CPT, compound 8 reversibly binds with the free enzyme and inhibits the formation of Top1cc and promotes reversal of the preformed Top1cc with CPT. Compound 8 induced inhibition of Top1cc formation in live cells was substantiated by fluorescence recovery after photobleaching (FRAP) assays. We established that MCF7 cells treated with compound 8 trigger proteasome-mediated Top1 degradation, accumulate higher levels of reactive oxygen species (ROS), PARP1 cleavage, oxidative DNA fragmentation, and stimulate apoptotic cell death without stabilizing apoptotic Top1-DNA cleavage complexes. Finally, compound 8 shows anticancer activity by targeting cellular Top1 and preventing the enzyme from directly participating in the apoptotic process.

Total synthesis of marine natural products separacenes A and B.

A short and convergent route for the stereoselective total synthesis of separacenes A and B has been developed using (+)-methyl d-lactate and d-(-)-tartaric acid as the chiral pools. The characteristic features of this synthesis include the Trost-Rychnovsky alkyne rearrangement to construct the C7-C9 conjugated diene, the Horner-Wadsworth-Emmons olefination to form the C5-C6 and C11-C12 olefins and the Corey-Bakshi-Shibata reaction to install the C-13 hydroxy functionality.

Poly(ADP-ribose) polymers regulate DNA topoisomerase I (Top1) nuclear dynamics and camptothecin sensitivity in living cells.

Topoisomerase 1 (Top1) is essential for removing the DNA supercoiling generated during replication and transcription. Anticancer drugs like camptothecin (CPT) and its clinical derivatives exert their cytotoxicity by reversibly trapping Top1 in covalent complexes on the DNA (Top1cc). Poly(ADP-ribose) polymerase (PARP) catalyses the addition of ADP-ribose polymers (PAR) onto itself and Top1. PARP inhibitors enhance the cytotoxicity of CPT in the clinical trials. However, the molecular mechanism by which PARylation regulates Top1 nuclear dynamics is not fully understood. Using live-cell imaging of enhanced green fluorescence tagged-human Top1, we show that PARP inhibitors (Veliparib, ABT-888) delocalize Top1 from the nucleolus to the nucleoplasm, which is independent of Top1-PARP1 interaction. Using fluorescence recovery after photobleaching and subsequent fitting of the data employing kinetic modelling we demonstrate that ABT-888 markedly increase CPT-induced bound/immobile fraction of Top1 (Top1cc) across the nuclear genome, suggesting Top1-PARylation counteracts CPT-induced stabilization of Top1cc. We further show Trp205 and Asn722 of Top1 are critical for subnuclear dynamics. Top1 mutant (N722S) was restricted to the nucleolus in the presence of CPT due to its deficiency in the accumulation of CPT-induced Top1-PARylation and Top1cc formation. This work identifies ADP-ribose polymers as key determinant for regulating Top1 subnuclear dynamics.

Design, synthesis and evaluation of thiohydantoin derivatives as potent topoisomerase I (Top1) inhibitors with anticancer activity.

DNA topoisomerase I is a potential chemotherapeutic target. Here, we designed and synthesized a library comprising of hydantoin and thiohydantoin derivatives and tested them against human and Leishmania Top1. One of the thiohydantoin compounds with substituted thiophenyl as the central moiety (compound 15) exhibited potent inhibition of human Top1 (HTop1) through stabilization of Top1-DNA cleavage complexes and showed selective anticancer activity against human cervical carcinoma (HeLa) and breast carcinoma (MCF-7) cell lines. Molecular modeling studies with HTop1 rationalized the inhibitory mechanism of compound 15.

Asymmetric Total Synthesis of Bioactive Natural Lipid Mycalol.

A concise and convergent route for stereoselective total synthesis of promising anticancer natural lipid mycalol has been achieved using cheap and readily available l-arabinose as a chiral pool. The notable features of our synthesis comprised regioselective Wacker oxidation, Sharpless asymmetric dihydroxylation, Julia-Kocienski olefination, Wittig olefination, Zipper reaction, and Sonogashira reaction. Comparison of the spectroscopic data on a series of isomers supports the revised structure (Org. Lett. 2015, 17, 1652) instead of the one originally proposed.

Identification of human cytomegalovirus genes important for biogenesis of the cytoplasmic virion assembly complex.

UNLABELLED: Human cytomegalovirus (HCMV) has many effects on cells, including remodeling the cytoplasm to form the cytoplasmic virion assembly complex (cVAC), the site of final virion assembly. Viral tegument, envelope, and some nonstructural proteins localize to the cVAC, and cytoskeletal filaments radiate from a microtubule organizing center in the cVAC. The endoplasmic reticulum (ER)-to-Golgi intermediate compartment, Golgi apparatus, and trans-Golgi network form a ring that outlines the cVAC. The center of the cVAC ring is occupied by numerous vesicles that share properties with recycling endosomes. In prior studies, we described the three-dimensional structure and the extensive remodeling of the cytoplasm and shifts in organelle identity that occur during development of the cVAC. The objective of this work was to identify HCMV proteins that regulate cVAC biogenesis. Because the cVAC does not form in the absence of viral DNA synthesis, we employed HCMV-infected cells transfected with synthetic small interfering RNAs (siRNAs) that targeted 26 candidate early-late and late protein-coding genes required for efficient virus replication. We identified three HCMV genes (UL48, UL94, and UL103) whose silencing had major effects on cVAC development, including failure to form the Golgi ring and dispersal of markers of early and recycling endosomes. To confirm and extend the siRNA results, we constructed recombinant viruses in which pUL48 and pUL103 are fused with a regulatable protein destabilization domain (dd-FKBP). In the presence of a stabilizing ligand (Shield-1), the cVAC appeared to develop normally. In its absence, cVAC development was abrogated, verifying roles for pUL48 and pUL103 in cVAC biogenesis. IMPORTANCE: Human cytomegalovirus (HCMV) is an important human pathogen that causes disease and disability in immunocompromised individuals and in children infected before birth. Few drugs are available for treatment of HCMV infections. HCMV remodels the interior of infected cells to build a factory for assembling new infectious particles (virions), the cytoplasmic virion assembly complex (cVAC). Here, we identified three HCMV genes (UL48, UL94, and UL103) as important contributors to cVAC development. In addition, we found that mutant viruses that express an unstable form of the UL103 protein have defects in cVAC development and production of infectious virions and produce small plaques and intracellular virions with aberrant appearances. Of these, only the reduced production of infectious virions is not eliminated by chemically stabilizing the protein. In addition to identifying new functions for these HCMV genes, this work is a necessary prelude to developing novel antivirals that would block cVAC development.

PARP1-TDP1 coupling for the repair of topoisomerase I-induced DNA damage.

Poly(ADP-ribose) polymerases (PARP) attach poly(ADP-ribose) (PAR) chains to various proteins including themselves and chromatin. Topoisomerase I (Top1) regulates DNA supercoiling and is the target of camptothecin and indenoisoquinoline anticancer drugs, as it forms Top1 cleavage complexes (Top1cc) that are trapped by the drugs. Endogenous and carcinogenic DNA lesions can also trap Top1cc. Tyrosyl-DNA phosphodiesterase 1 (TDP1), a key repair enzyme for trapped Top1cc, hydrolyzes the phosphodiester bond between the DNA 3'-end and the Top1 tyrosyl moiety. Alternative repair pathways for Top1cc involve endonuclease cleavage. However, it is unknown what determines the choice between TDP1 and the endonuclease repair pathways. Here we show that PARP1 plays a critical role in this process. By generating TDP1 and PARP1 double-knockout lymphoma chicken DT40 cells, we demonstrate that TDP1 and PARP1 are epistatic for the repair of Top1cc. The N-terminal domain of TDP1 directly binds the C-terminal domain of PARP1, and TDP1 is PARylated by PARP1. PARylation stabilizes TDP1 together with SUMOylation of TDP1. TDP1 PARylation enhances its recruitment to DNA damage sites without interfering with TDP1 catalytic activity. TDP1-PARP1 complexes, in turn recruit X-ray repair cross-complementing protein 1 (XRCC1). This work identifies PARP1 as a key component driving the repair of trapped Top1cc by TDP1.

Deletion of the human cytomegalovirus US17 gene increases the ratio of genomes per infectious unit and alters regulation of immune and endoplasmic reticulum stress response genes at early and late times after infection.

Human cytomegalovirus (HCMV) employs numerous strategies to combat, subvert, or co-opt host immunity. One evolutionary strategy for this involves capture of a host gene and then its successive duplication and divergence, forming a family of genes, many of which have immunomodulatory activities. The HCMV US12 family consists of 10 tandemly arranged sequence-related genes in the unique short (US) region of the HCMV genome (US12 to US21). Each gene encodes a protein possessing seven predicted transmembrane domains, patches of sequence similarity with cellular G-protein-coupled receptors, and the Bax inhibitor 1 family of antiapoptotic proteins. We show that one member, US17, plays an important role during virion maturation. Microarray analysis of cells infected with a recombinant HCMV isolate with a US17 deletion (the ΔUS17 mutant virus) revealed blunted host innate and interferon responses at early times after infection (12 h postinfection [hpi]), a pattern opposite that previously seen in the absence of the immunomodulatory tegument protein pp65 (pUL83). Although the ΔUS17 mutant virus produced numbers of infectious particles in fibroblasts equal to the numbers produced by the parental virus, it produced >3-fold more genome-containing noninfectious viral particles and delivered increased amounts of pp65 to newly infected cells. These results suggest that US17 has evolved to control virion composition, to elicit an appropriately balanced host immune response. At later time points (96 hpi), ΔUS17 mutant-infected cells displayed aberrant expression of several host endoplasmic reticulum stress response genes and chaperones, some of which are important for the final stages of virion assembly and egress. Our results suggest that US17 modulates host pathways to enable production of virions that elicit an appropriately balanced host immune response.

Spatial relationships between markers for secretory and endosomal machinery in human cytomegalovirus-infected cells versus those in uninfected cells.

Human cytomegalovirus (HCMV) induces extensive remodeling of the secretory apparatus to form the cytoplasmic virion assembly compartment (cVAC), where virion tegumentation and envelopment take place. We studied the structure of the cVAC by confocal microscopy to assess the three-dimensional distribution of proteins specifically associated with individual secretory organelles. In infected cells, early endosome antigen 1 (EEA1)-positive vesicles are concentrated at the center of the cVAC and, as previously seen, are distinct from structures visualized by markers for the endoplasmic reticulum, Golgi apparatus, and trans-Golgi network (TGN). EEA1-positive vesicles can be strongly associated with markers for recycling endosomes, to a lesser extent with markers associated with components of the endosomal sorting complex required for transport III (ESCRT III) machinery, and then with markers of late endosomes. In comparisons of uninfected and infected cells, we found significant changes in the structural associations and colocalization of organelle markers, as well as in net organelle volumes. These results provide new evidence that the HCMV-induced remodeling of the membrane transport apparatus involves much more than simple relocation and expansion of preexisting structures and are consistent with the hypothesis that the shift in identity of secretory organelles in HCMV-infected cells results in new functional profiles.