Human p53 interacts with the elongating RNAPII complex and is required for the release of actinomycin D induced transcription blockage

The p53 tumour suppressor regulates the transcription initiation of selected genes by binding to specific DNA sequences at their promoters. Here we report a novel role of p53 in transcription elongation in human cells. Our data demonstrate that upon transcription elongation blockage, p53 is associated with genes that have not been reported as its direct targets. p53 could be co-immunoprecipitated with active forms of DNA-directed RNA polymerase II subunit 1 (RPB1), highlighting its association with the elongating RNA polymerase II. During a normal transcription cycle, p53 and RPB1 are localised at distinct regions of selected non-canonical p53 target genes and this pattern of localisation was changed upon blockage of transcription elongation. Additionally, transcription elongation blockage induced the proteasomal degradation of RPB1. Our results reveal a novel role of p53 in human cells during transcription elongation blockage that may facilitate the removal of RNA polymerase II from DNA

PloS one

In eukaryotes the TFIID complex is required for preinitiation complex assembly which positions RNA polymerase II around transcription start sites. On the other hand, histone acetyltransferase complexes including SAGA and ATAC, modulate transcription at several steps through modification of specific core histone residues. In this study we investigated the function of Drosophila melanogaster proteins TAF10 and TAF10b, which are subunits of dTFIID and dSAGA, respectively. We generated a mutation which eliminated the production of both Drosophila TAF10 orthologues. The simultaneous deletion of both dTaf10 genes impaired the recruitment of the dTFIID subunit dTAF5 to polytene chromosomes, while binding of other TFIID subunits, dTAF1 and RNAPII was not affected. The lack of both dTAF10 proteins resulted in failures in the larval-pupal transition during metamorphosis and in transcriptional reprogramming at this developmental stage. Surprisingly, unlike dSAGA mutations, dATAC subunit mutations resulted in very similar changes in the steady state mRNA levels of approximately 5000 genes as did ablation of both dTaf10 genes, indicating that dTAF10- and/or dTAF10b-containing complexes and dATAC affect similar pathways. Importantly, the phenotype resulting from dTaf10+dTaf10b mutation could be rescued by ectopically added ecdysone, suggesting that dTAF10- and/or dTAF10b-containing complexes are involved in the expression of ecdysone biosynthetic genes. Indeed, in dTaf10+dTaf10b mutants, cytochrome genes, which regulate ecdysone synthesis in the ring gland, were underrepresented. Therefore our data support the idea that the presence of dTAF10 proteins in dTFIID and/or dSAGA is required only at specific developmental steps. We propose that distinct forms of dTFIID and/or dSAGA exist during Drosophila metamorphosis, wherein different TAF compositions serve to target RNAPII at different developmental stages and tissues

Human embryo polarization requires PLC signaling to mediate trophectoderm specification

Apico-basal polarization of cells within the embryo is critical for the segregation of distinct lineages during mammalian development. Polarized cells become the trophectoderm (TE), which forms the placenta, and apolar cells become the inner cell mass (ICM), the founding population of the fetus. The cellular and molecular mechanisms leading to polarization of the human embryo and its timing during embryogenesis have remained unknown. Here, we show that human embryo polarization occurs in two steps: it begins with the apical enrichment of F-actin and is followed by the apical accumulation of the PAR complex. This two-step polarization process leads to the formation of an apical domain at the 8–16 cell stage. Using RNA interference, we show that apical domain formation requires Phospholipase C (PLC) signaling, specifically the enzymes PLCB1 and PLCE1, from the eight-cell stage onwards. Finally, we show that although expression of the critical TE differentiation marker GATA3 can be initiated independently of embryo polarization, downregulation of PLCB1 and PLCE1 decreases GATA3 expression through a reduction in the number of polarized cells. Therefore, apical domain formation reinforces a TE fate. The results we present here demonstrate how polarization is triggered to regulate the first lineage segregation in human embryos

Model Based Estimation of 2D Crystallization Kinetics From Concentration and CLD Measurements

Due to the fact that crystal size and shape influence relevant macroscopic properties of solid particles, the understanding and control of these quantities have increasing importance in particulate science. Crystallization, the primary crystal formation and purification process, is usually tracked real-time, in situ, by spectroscopic techniques and Focused Beam Reflectance Measurement (FBRM). This sensor can measure the chord length distribution (CLD) of a population of particles suspended in a solution. The CLD is related to both the size and shape of the particles and it is measured using a rotating infrared laser beam that emanates through the probe window inserted in the suspension. During its rotation, the beam hits the particles within the sample and is reflected back to the probe. The calculated length of laser-crystal intersection is the so-called chord length. FBRM can provide a large amount of useful information during crystallization processes, however, since the CLD is significantly different compared to the actual crystal size and shape distribution (CSD), it is normally not used for the quantification of the kinetics of crystal growth and nucleation. Usually off-line techniques (laser diffraction, microscopy, ultrasound) are exploited. In this study we develop and present a new, projection based forward 2D CSD➔CLD transformation technique. In addition, a 2D population balance model is employed to simulate the evolution of 2D CSD and solute concentration. The model equations are solved by a high resolution finite volume method, involving GPU acceleration for improved simulation time. Such model allows the use of FBRM data for the estimation of the kinetics of crystallization, without relying on off-line measurements of CSD. As model system succinic acid is used. This forms prism-like crystals in the presence of growth rate modifiers. Crystal breakage is minimized through reduced mixing rate and the kinetics of primary and secondary nucleation as well as the growth and dissolution of individual crystal facets were estimated by developing and solving a process optimization problem. The result of the parameter regression was a calibrated model, which simulates fairly the concentration and CLD variations too

Experimental and theoretical aspects of aluminum expanding laser plasma

The formation and dynamics of aluminum laser produced plasma are experimentally and theoretically investigated. The visible emitting regions of plasma form two structures of different expansion velocities. Such behavior is in agreement with the transient current recorded by a cylindrical Langmuir probe. Using the hydrodynamic model of scale relativity theory, the plasma dynamics at different time scales are numerically and analytically analyzed. © 2009 The Japan Society of Applied Physics

Mapping of chromosome territories by 3D-chromosome painting during early mouse development.

Following fertilization in mammals, the chromatin landscape inherited from the two parental genomes and the nuclear organization are extensively reprogrammed. A tight regulation of nuclear organization is important for developmental success. One main nuclear feature is the organization of the chromosomes in discrete and individual nuclear spaces known as chromosome territories (CTs). In culture cells, their arrangements can be constrained depending on their genomic content (e.g., gene density or repeats) or by specific nuclear constrains such as the periphery or the nucleolus. However, during the early steps of mouse embryonic development, much less is known, specifically regarding how and when the two parental genomes intermingle. Here, we describe a three-dimensional fluorescence in situ hybridization (3D-FISH) for chromosome painting (3D-ChromoPaint) optimized to gain understanding in nuclear organization of specific CTs following fertilization. Our approach preserves the nuclear structure, and the acquired images allow full spatial analysis of interphase chromosome positioning and morphology across the cell cycle and during early development. This method will be useful in understanding the dynamics of chromosome repositioning during development as well as the alteration of chromosome territories upon changes in transcriptional status during key developmental steps. This protocol can be adapted to any other species or organoids in culture