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Coordinated functional balance of negative and positive transcription complexes maintain and accommodate gene expression in hearts during quiescent and hypertrophic conditions, respectively. Negative elongation factor (Nelf) complex has been implicated in RNA polymerase II (pol II) pausing, a widespread regulatory transcriptional phenomenon observed across the cardiac genome. Here, we examine the role of NelfA aka, Wolf-Hirschhorn syndrome candidate 2 (Whsc2), a critical component of the negative elongation complex in hearts undergoing pressure-overload induced hypertrophy. Alignment of high-resolution genome-wide occupancy data of NelfA, Pol II, TFIIB and H3k9ac from control and hypertrophied hearts reveal that NelfA associates with active gene promoters. High NelfA occupancy is seen at promoters of essential and cardiac-enriched genes, expressed under both quiescent and hypertrophic conditions. Conversely, de novo NelfA recruitment is observed at inducible gene promoters with pressure overload, accompanied by significant increase in expression of these genes with hypertrophy. Interestingly, change in promoter NelfA levels correlates with the transcript output in hypertrophied hearts compared to Sham, suggesting NelfA might be playing a critical role in the regulation of gene transcription during cardiac hypertrophy. In vivo knockdown of NelfA (siNelfA) in hearts subjected to pressure-overload results in early ventricular dilatation and dysfunction, associated with decrease in expression of inducible and cardiac-enriched genes in siNelfA hypertrophied compared to control hypertrophied hearts. In accordance, in vitro knockdown of NelfA in cardiomyocytes showed no change in promoter pol II, however significant decrease in in-gene and downstream pol II occupancy was observed. These data suggest an inhibited pol II progression in transcribing and inducible genes, which reflects as a decrease in transcript abundance of these genes. These results indicate that promoter NelfA occupancy is essential for pol II -dependent transcription. Therefore, we conclude that NelfA is required for active transcription and gene expression during cardiac hypertrophy.

// Sumit Mukherjee 1, 3, 5, * , Priya Ranjan Debata 5, 8, * , Rahman Hussaini 5 , Kaushiki Chatterjee 2, 4 , Juliet N.E. Baidoo 1, 3 , Samay Sampat 5 , Anita Szerszen 6 , Joseph P. Navarra 7 , Jimmie Fata 2 , Elena Severinova 6, 9 , Probal Banerjee 1, 5 and Mario R. Castellanos 6 1 Department of Chemistry, The College of Staten Island (CUNY), New York, NY, USA 2 CUNY Doctoral Program in Biology, CUNY Graduate Center, New York, NY, USA 3 CUNY Doctoral Program In Biochemistry, CUNY Graduate Center, New York, NY, USA 4 Department of Biology, The College of Staten Island (CUNY), New York, NY, USA 5 Center of Developmental Neuroscience, The College of Staten Island (CUNY), New York, NY, USA 6 Division of Research, Department of Medicine, Staten Island University Hospital (Northwell Health), New York, NY, USA 7 College of Pharmacy and Health Sciences, St. John’s University, New York, NY, USA 8 Current Address: Department of Zoology North Orissa University Baripada, Mayurbhanj, Odisha, India 9 Current Address: Cell Biology and Molecular Medicine, Rutgers University, Newark, NJ, USA * These authors contributed equally to this work Correspondence to: Probal Banerjee, email: probal.banerjee@csi.cuny.edu , probalbanerjee@yahoo.com Keywords: curcumin, cervical cancer, human papillomavirus, synergism, combination index Received: May 31, 2016 Accepted: September 02, 2016 Published: March 29, 2017 ABSTRACT Curcumin (from curry) (C) is highly potent against cervical cancer cells (CCC), but poor bioavailability has limited its clinical use. Similar natural polyphenols resveratrol (from grapes) (R), and epicatechin gallate (from green tea) (E) also display activity against CCC. By treating CCC (HeLa) with C, E, or R, or combinations of these compounds, we computed combination indices and observed a strong synergism among C, E, and R at the unique molar ratio 4:1:12.5. This combination, named as TriCurin, rapidly down regulated HPV18 E6 and NF-kB expression while concomitantly inducing the tumor suppressor protein p53 in HeLa cells. In the mouse c-Ha-ras and HPV16 E6, E7-expressing TC-1 CCC, both C and TriCurin elicited suppression of E6, induction of both p53 and acetyl-p53 (activated p53), and activation of caspase-3, but the TriCurin-evoked changes were several-fold greater than that produced by curcumin (4.7-fold for E6 inhibition, and 2-fold, 6-fold, and 1.7-fold for the induction of p53, acetyl-p53, and active caspase-3, respectively). Consequently, TriCurin was more potent in killing TC-1 and HeLa cells. Intralesional TriCurin treatment of tumors generated in mice by subcutaneously implanting the TC-1 CCC caused an 80–90% decrease in tumor growth. The ability of C to eliminate HeLa cells was significantly stabilized when delivered as TriCurin than when delivered alone. Topical application of TriCurin dispersed in a cream base afforded efficient transfer of C across the skin. Subcutaneous TriCurin injection yielded no adverse effect in tumor-naive healthy mice. Thus, TriCurin is a safe and promising therapeutic agent against HPV-associated disease.

Background An increase in serum cortisol has been identified as a risk factor for cardiac failure, which highlights the impact of glucocorticoid signaling in cardiomyocytes and its influence in the progression of failure. Dexamethasone, a synthetic glucocorticoid, is sufficient for induction of cardiomyocyte hypertrophy, but little is known of the glucocorticoid receptor (GR) genome‐binding and ‐dependent transcriptional changes that mediate this phenotype. Methods and Results In this study using high‐resolution sequencing, we identified genomic targets of GR and associated change in the transcriptome after 1 and 24 hours of dexamethasone treatment. We showed that GR associates with 6482 genes in the cardiac genome, with differential regulation of 738 genes. Interestingly, alignment of the chromatin immunoprecipitation and RNA sequencing data show that, after 1 hour, 69% of differentially regulated genes are associated with GR and identify as regulators of RNA pol II–dependent transcription. Conversely, after 24 hours only 45% of regulated genes are associated with GR and involved in dilated and hypertrophic cardiomyopathies as well as other growth‐related pathways. In addition, our data also reveal that a majority of genes (76.42%) associated with GR show incremental changes in transcript abundance and are genes involved in basic cellular processes that might be regulated by the dynamics of promoter‐paused RNA pol II, as seen in hearts undergoing hypertrophy. In vivo administration of dexamethasone resulted in similar changes in the cardiac transcriptome, as seen in isolated cardiomyocytes. Conclusions Our data reveal genome‐wide GR binding sites in cardiomyocytes, identify novel targets and GR‐dependent change in the transcriptome that induces and contributes to cardiomyocyte hypertrophy.

Adaptive change in gene expression is one of the earliest responses of heart to hypertrophy. Gene transcription is tightly regulated by systematic recruitment of factors including positive and negative elongation complexes of RNA Pol II. Recently we embarked on the task of deconstructing the transcriptional machinery in the heart, by examining Pol II and TFIIB dynamics across the genome in sham and hypertrophied hearts. In this study, we continue examining the active transcriptional code by measuring occupancy of two interdependent factors that belong to positive (PTEFb) and negative (NELF) elongation complexes, Cdk9 and NelfA, respectively. Studies in non-cardiac cells have implicated these two factors in the regulation of promoter (prom) clearance of pol II, where Cdk9 mediated phosphorylation of NelfA results in its release from paused pol II and initiation of productive elongation. As expected, we observed increase Cdk9 (>1.5) association with proms in 59.7% of expressed genes (8257 of 13816), while 6% show decrease in prom Cdk9 (

Bacteriophage T7 is a prototypic member of a large family of phages that use host RNA polymerase (RNAP) for expression of their early genes but rely on their own, single-subunit RNAP for middle and late viral gene expression. The early T7 genes include, in addition to gene 1 coding for T7 RNAP, several genes whose products participate in host transcription shutoff. The shutoff occurs concurrently with the beginning of transcription of viral genes by T7 RNAP. The products of two viral genes, Gp0.7 and Gp2, are implicated in host transcription shutoff (33-35, 39). Gp0.7 is a serine/threonine protein kinase (31, 36). Among the identified targets are components of translation (IF1, IF2, IF3, elongation factor G, and ribosomal proteins S1 and S6 [37, 38]) and transcription (RNAP β′ subunit [47]) machineries; RNase III (24), the enzyme responsible for mRNA processing; and RNase E (23), which controls mRNA decay. Several lines of evidence suggest that Gp0.7 plays an important role in bacteriophage development. First, even though the 0.7 gene is dispensable for T7 growth under standard laboratory conditions, it is essential at elevated temperatures or in nutrient-poor media (19), as well as in the presence of certain extrachromosomal genetic elements (16). Second, the protein kinase activity appears to be conserved: it is present in T7-like coliphages (25, 41), as well as in phages that infect Yersinia enterocolitica (32), Kluyvera cryocrescens (15), Klebsiella pneumoniae (D. Savalia, I. J. Molineux, and K. Severinov, unpublished observation), and Caulobacter crescentus (20). Gp2 is a 64-amino-acid-long protein that binds to and abolishes promoter recognition by host RNAP (28). Gene 0.7 becomes essential when Gp2 function is attenuated by a mutation in its binding site in a functionally dispensable domain of RNAP β′ (18), supporting the idea that phosphorylation of β′ by Gp0.7 is functionally relevant and suggesting that Gp2 and Gp0.7 may cooperate with each other during bacteriophage development. Phosphorylation is a common covalent modification of proteins. In eukaryotes, core components of all three nuclear RNAPs are phosphorylated (4, 7). Phosphorylation of the largest subunit, A190, which is homologous to β′, may regulate the function of yeast (Saccharomyces cerevisiae) RNAP I (14). However, with the exception of the largest RNAP II subunit C-terminal domain phosphorylation, RNAP phosphorylation sites have not been mapped and functional consequences of RNAP phosphorylation have not been established. Here, we used protein chemistry and mutational analysis to localize the Escherichia coli RNAP β′ phosphorylation site by T7 Gp0.7 and to study the functional consequences of phosphorylation using a phosphomimetic substitution in β′.

We used limited trypsin digestion to determine the domain organization of theEscherichia coliRNA polymerase σ70subunit. Trypsin-resistant fragments containing σ70conserved region 2 (σ702), and carboxy-terminal fragments containing conserved regions 3 and 4 (σ703 – 4) were identified by a combination of amino acid sequencing and mass spectrometry. The domains were studied for partial biochemical functions of σ70·σ702bound core RNA polymerase competitively with intact σ70. In contrast to σ702alone, the RNA polymerase holoenzyme formed with σ702specifically bound a single-stranded DNA oligomer with a sequence corresponding to the non-template strand of the −10 promoter element (the Pribnow box). σ702also forms crystals that are suitable for X-ray analysis. σ703 – 4bound the T4 AsiA protein with high affinity. The epitope for T4 AsiA on σ70was further localized to within σ70[551 – 608], comprising σ conserved region 4.2.

The 2.6 Å crystal structure of a fragment of the σ70 promoter specificity subunit of E. coli RNA polymerase is described. Residues involved in core RNA polymerase binding lie on one face of the structure. On the opposite face, aligned along one helix, are exposed residues that interact with the −10 consensus promoter element (the Pribnow box), including four aromatic residues involved in promoter melting. The structure suggests one way in which DNA interactions may be inhibited in the absence of RNA polymerase and provides a framework for the interpretation of a large number of genetic and biochemical analyses.

Ribonucleotide analogs bound in the initiating site of Escherichia coli RNA polymerase-promoter complex were cross-linked to the beta subunit. Using limited proteolysis and chemical degradation, the cross-link was mapped to a segment of beta between amino acids Val516 and Arg540. This region (Rif-cluster I) is known to harbor many rifampicin-resistant (RifR) mutations. The results demonstrate that Rif-culster I is part of the "5'-face" of the active center and provide structural basis for the long-known effects of RifR mutations on transcription initiation, elongation, and termination.

The structure of E. coli core RNA polymerase (RNAP) has been determined to approximately 23 A resolution by three-dimensional reconstruction from electron micrographs of flattened helical crystals. The structure reveals extensive conformational changes when compared with the previously determined E. coli RNAP holoenzyme structure, but resembles the yeast RNAPII structure. While each of these structures contains a thumb-like projection surrounding a channel 25 A in diameter, the E. coli RNAP holoenzyme thumb defines a deep but open groove on the molecule, whereas the thumb of E. coli core and yeast RNAPII form part of a ring that surrounds the channel. This may define promoter-binding and elongation conformations of RNAP, as E. coli holoenzyme recognizes promoter sites on double-stranded DNA, while both E. coli core and yeast RNAPII are elongating forms of the polymerase and are incapable of promoter recognition.

During the development of purification procedures for Escherichia coli RNA polymerase (RNAP), we noticed the consistent co-purification of a 110-kDa polypeptide. Here, we report the identification of the 110-kDa protein as the product of the hepA gene, a member of the SNF2 family of putative helicases. We have cloned the hepA gene and overexpressed and purified the HepA protein. We show in vitro that RNAP preparations have an ATPase activity only in the presence of HepA and that HepA binds core RNAP competitively with the promoter specificity sigma70 subunit with a 1:1 stoichiometry and a dissociation constant (Kd) of 75 nM. An E. coli strain with a disruption in the hepA gene shows sensitivity to ultraviolet light.

Mutations conferring streptolydigin resistance onto Escherichia coli RNA polymerase have been found exclusively in the beta subunit (Heisler, L. M., Suzuki, H., Landick, R., and Gross, C. A. (1993) J. Biol. Chem. 268, 25369-25375). We report here the isolation of a streptolydigin-resistant mutation in the E. coli rpoC gene, encoding the beta' subunit. The mutation is the Phe793--Ser substitution, which occurred in an evolutionarily conserved segment of the beta' subunit. The homologous segment in the eukaryotic RNA polymerase II largest subunit harbors mutations conferring alpha-amanitin resistance. Both streptolydigin and alpha-amanitin are inhibitors of transcription elongation. Thus, the two antibiotics may inhibit transcription in their respective systems by a similar mechanism, despite their very different chemical nature.

The Escherichia coli rpoB gene, which codes for the 1342-residue beta subunit of RNA polymerase (RNAP), contains two dispensable regions centered around codons 300 and 1000. To test whether these regions demarcate domains of the RNAP beta subunit, fragments encoded by segments of rpoB flanking the dispensable regions were individually overexpressed and purified. We show that these beta-subunit polypeptide fragments, when added with purified recombinant beta', sigma, and alpha subunits of RNAP, reconstitute a functional enzyme in vitro. These results demonstrate that the beta subunit is composed of at least three distinct domains and open another avenue for in vitro studies of RNAP assembly and structure.