Complex effects of flavopiridol on the expression of primary response genes

<p>Abstract</p> <p>Background</p> <p>The Positive Transcription Elongation Factor b (P-TEFb) is a complex of Cyclin Dependent Kinase 9 (CDK9) with either cyclins T1, T2 or K. The complex phosphorylates the C-Terminal Domain of RNA polymerase II (RNAPII) and negative elongation factors, stimulating productive elongation by RNAPII, which is paused after initiation. P-TEFb is recruited downstream of the promoters of many genes, including primary response genes, upon certain stimuli. Flavopiridol (FVP) is a potent pharmacological inhibitor of CDK9 and has been used extensively in cells as a means to inhibit CDK9 activity. Inhibition of P-TEFb complexes has potential therapeutic applications.</p> <p>Results</p> <p>It has been shown that Lipopolysaccharide (LPS) stimulates the recruitment of P-TEFb to Primary Response Genes (PRGs) and proposed that P-TEFb activity is required for their expression, as the CDK9 inhibitor DRB prevents localization of RNAPII in the body of these genes. We have previously determined the effects of FVP in global gene expression in a variety of cells and surprisingly observed that FVP results in potent upregulation of a number of PRGs in treatments lasting 4-24 h. Because inhibition of CDK9 activity is being evaluated in pre-clinical and clinical studies for the treatment of several pathologies, it is important to fully understand the short and long term effects of its inhibition. To this end, we determined the immediate and long-term effect of FVP in the expression of several PRGs. In exponentially growing normal human fibroblasts, the expression of several PRGs including FOS, JUNB, EGR1 and GADD45B, was rapidly and potently downregulated before they were upregulated following FVP treatment. In serum starved cells re-stimulated with serum, FVP also inhibited the expression of these genes, but subsequently, JUNB, GADD45B and EGR1 were upregulated in the presence of FVP. Chromatin Immunoprecipitation of RNAPII revealed that EGR1 and GADD45B are transcribed at the FVP-treatment time points where their corresponding mRNAs accumulate. These results suggest a possible stress response triggered by CDK9 inhibition than ensues transcription of certain PRGs.</p> <p>Conclusions</p> <p>We have shown that certain PRGs are transcribed in the presence of FVP in a manner that might be independent of CDK9, suggesting a possible alternative mechanism for their transcription when P-TEFb kinase activity is pharmacologically inhibited. These results also show that the sensitivity to FVP is quite variable, even among PRGs.</p

Analysis of ligation and DNA binding by Escherichia coli DNA ligase (LigA).

NAD+-dependent DNA ligases are essential enzymes in bacteria, with the most widely studied of this class of enzymes being LigA from Escherichia coli. NAD+-dependent DNA ligases comprise several discrete structural domains, including a BRCT domain at the C-terminus that is highly-conserved in this group of proteins. The over-expression and purification of various fragments of E. coli LigA allowed the investigation of the different domains in DNA-binding and ligation by this enzyme. Compared to the full-length protein, the deletion of the BRCT domain from LigA reduced in vitro ligation activity by 3-fold and also reduced DNA binding. Using an E. coli strain harbouring a temperature-sensitive mutation of ligA, the over-expression of protein with its BRCT domain deleted enabled growth at the non-permissive temperature. In gel-mobility shift experiments, the isolated BRCT domain bound DNA in a stable manner and to a wider range of DNA molecules compared to full LigA. Thus, the BRCT domain of E. coli LigA can bind DNA, but it is not essential for DNA nick-joining activity in vitro or in vivo

Evaluation of Annual Companion Crops for the Establishment of Perennial Forage Crops in Eastern Canada

The use of companion crops when establishing perennial forages is desirable as it often reduces weed growth and increases forage biomass in the seeding year. In eastern Canada, oat (Avena sativa L.) is the main species used as companion crop; although other species are used, they have not been systematically evaluated. A field study was established in 2019 at three sites in Québec, Canada, to contrast the use of six annual species as companion crops for the establishment of lucerne (Medicago sativa L.)-timothy (Phleum pratense L.) mixtures. Species evaluated include berseem clover (Trifolium alexandrinum L.), annual ryegrass (Lolium multiflorum Lamarck), forage pea (Pisum sativum L.), forage oat, Japanese millet [Echinochloa esculenta (A. Braun) H. Scholz], and sudangrass [Sorghum × drummondii (Nees ex. Steud.) Millsp. & Chase]. The control treatment consisted of the perennial species seeded without companion crop. Treatments were seeded at three dates (mid-May to early-June, mid-June to early-July, and early August) and evaluated during the seeding year based on biomass production and botanical composition. Overall, across sites, for the first two seeding dates, highest annual forage yields were observed with sudangrass, Japanese millet, and oat as companion crops. The use of these species increased yields by 1.8 to 2.5 Mg ha-1 on a dry matter basis (DM) compared to the control which yielded an average of 3.7 Mg DM ha-1. For the early August seeding, response varied significantly across sites. Annual yields were the highest with the use of oat at two sites (avg. of 2.4 Mg DM ha-1), whereas no differences between treatments were observed at the other site. Companion crop species which maximized total forage yields in the seeding year often reduced weed biomass, but also that of perennial species. The impact of treatments on the survival of perennial forages and their production during the first post-seeding year will be presented in a later publication

A Rigidifying Salt-Bridge Favors the Activity of Thermophilic Enzyme at High Temperatures at the Expense of Low-Temperature Activity

Although enzymes from thermophiles thriving in hot habitats are more stable than their mesophilic homologs, they are often less active at low temperatures. One theory suggests that extra stabilizing interactions found in thermophilic enzymes may increase their rigidity and decrease enzymatic activity at lower temperatures. We used acylphosphatase as a model to study how flexibility affects enzymatic activity. This enzyme has a unique structural feature in that an invariant arginine residue, which takes part in catalysis, is restrained by a salt-bridge in the thermophilic homologs but not in its mesophilic homologs. Here, we demonstrate the trade-offs between flexibility and enzymatic activity by disrupting the salt-bridge in a thermophilic acylphosphatase and introducing it in the mesophilic human homolog. Our results suggest that the salt-bridge is a structural adaptation for thermophilic acylphosphatases as it entropically favors enzymatic activity at high temperatures by restricting the flexibility of the active-site residue. However, at low temperatures the salt-bridge reduces the enzymatic activity because of a steeper temperature-dependency of activity

Toward halogen-free flame resistant polyethylene extrusion coated paper facings

Wire and cable coverings are potentially a major cause of fire in buildings and other installations. As they need to breach fire walls and are frequently located in vertical ducting, they have significant potential to increase the fire hazard. It is therefore important to understand the ignition and burning characteristics of cables by developing a model capable of predicting their burning behaviour for a range of scenarios. The fire performance of electrical cables is usually dominated by the fire performance of the sheathing materials. The complexity of the problem increases when cable sheathing incorporates fire retardants. One-dimensional pyrolysis models have been constructed for cable sheathing materials, based on milligram-scale and bench-scale test data by comparing the performance of three different software tools (ThermaKin, Comsol Multiphysics and FDS, version 6.0.1). Thermogravimetric analysis and differential scanning calorimetry were conducted on powdered cable coatings to determine the thermal degradation mechanism, the enthalpy of decomposition reactions, and the heat capacities of all apparent species. The emissivity and the in-depth absorption coefficient were determined using reflectance and transmittance measurements, with dispersive and non-dispersive spectrometers and integrating spheres. Bench-scale tests were conducted with a mass loss calorimeter flushed with nitrogen on samples in a horizontal orientation, for comparison with the pyrolysis model of non-flaming decomposition at an external heat flux of 50 kW m-2. The parameters determined through analysis of the milligram-scale data were used to construct a pyrolysis model that predicted the total mass loss from calorimeter tests in anaerobic conditions. A condensed phase pyrolysis model that accurately predicts in-depth temperature profiles of a solid fuel, and the mass flux of volatiles evolved during degradation of the fuel, is an essential component of a comprehensive fire model, which when coupled to a computational fluid dynamics code can be used to predict the burning processes in a fire scenario. Pyrolysis models vary considerably in complexity based on the assumptions incorporated into the development of the model

Integrated genome-wide chromatin occupancy and expression analyses identify key myeloid pro-differentiation transcription factors repressed by Myb

To gain insight into the mechanisms by which the Myb transcription factor controls normal hematopoiesis and particularly, how it contributes to leukemogenesis, we mapped the genome-wide occupancy of Myb by chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq) in ERMYB myeloid progenitor cells. By integrating the genome occupancy data with whole genome expression profiling data, we identified a Myb-regulated transcriptional program. Gene signatures for leukemia stem cells, normal hematopoietic stem/progenitor cells and myeloid development were overrepresented in 2368 Myb regulated genes. Of these, Myb bound directly near or within 793 genes. Myb directly activates some genes known critical in maintaining hematopoietic stem cells, such as Gfi1 and Cited2. Importantly, we also show that, despite being usually considered as a transactivator, Myb also functions to repress approximately half of its direct targets, including several key regulators of myeloid differentiation, such as Sfpi1 (also known as Pu.1), Runx1, Junb and Cebpb. Furthermore, our results demonstrate that interaction with p300, an established coactivator for Myb, is unexpectedly required for Myb-mediated transcriptional repression. We propose that the repression of the above mentioned key pro-differentiation factors may contribute essentially to Myb’s ability to suppress differentiation and promote self-renewal, thus maintaining progenitor cells in an undifferentiated state and promoting leukemic transformation

Reduction in Structural Disorder and Functional Complexity in the Thermal Adaptation of Prokaryotes

Genomic correlates of evolutionary adaptation to very low or very high optimal growth temperature (OGT) values have been the subject of many studies. Whereas these provided a protein-structural rationale of the activity and stability of globular proteins/enzymes, the point has been neglected that adaptation to extreme temperatures could also have resulted from an increased use of intrinsically disordered proteins (IDPs), which are resistant to these conditions in vitro. Contrary to these expectations, we found a conspicuously low level of structural disorder in bacteria of very high (and very low) OGT values. This paucity of disorder does not reflect phylogenetic relatedness, i.e. it is a result of genuine adaptation to extreme conditions. Because intrinsic disorder correlates with important regulatory functions, we asked how these bacteria could exist without IDPs by studying transcription factors, known to harbor a lot of function-related intrinsic disorder. Hyperthermophiles have much less transcription factors, which have reduced disorder compared to their mesophilic counterparts. On the other hand, we found by systematic categorization of proteins with long disordered regions that there are certain functions, such as translation and ribosome biogenesis that depend on structural disorder even in hyperthermophiles. In all, our observations suggest that adaptation to extreme conditions is achieved by a significant functional simplification, apparent at both the level of the genome and individual genes/proteins

Chromosome-Biased Binding and Gene Regulation by the Caenorhabditis elegans DRM Complex

DRM is a conserved transcription factor complex that includes E2F/DP and pRB family proteins and plays important roles in development and cancer. Here we describe new aspects of DRM binding and function revealed through genome-wide analyses of the Caenorhabditis elegans DRM subunit LIN-54. We show that LIN-54 DNA-binding activity recruits DRM to promoters enriched for adjacent putative E2F/DP and LIN-54 binding sites, suggesting that these two DNA–binding moieties together direct DRM to its target genes. Chromatin immunoprecipitation and gene expression profiling reveals conserved roles for DRM in regulating genes involved in cell division, development, and reproduction. We find that LIN-54 promotes expression of reproduction genes in the germline, but prevents ectopic activation of germline-specific genes in embryonic soma. Strikingly, C. elegans DRM does not act uniformly throughout the genome: the DRM recruitment motif, DRM binding, and DRM-regulated embryonic genes are all under-represented on the X chromosome. However, germline genes down-regulated in lin-54 mutants are over-represented on the X chromosome. We discuss models for how loss of autosome-bound DRM may enhance germline X chromosome silencing. We propose that autosome-enriched binding of DRM arose in C. elegans as a consequence of germline X chromosome silencing and the evolutionary redistribution of germline-expressed and essential target genes to autosomes. Sex chromosome gene regulation may thus have profound evolutionary effects on genome organization and transcriptional regulatory networks.National Institutes of Health (U.S.) (grant GM24663)National Institutes of Health (U.S.) (grant DK068429)National Institutes of Health (U.S.) (grant GM082971)National Institutes of Health (U.S.) (grant GM076378