An RNA Polymerase III-Dependent Heterochromatin Barrier at Fission Yeast Centromere 1

Heterochromatin formation involves the nucleation and spreading of structural and epigenetic features along the chromatin fiber. Chromatin barriers and associated proteins counteract the spreading of heterochromatin, thereby restricting it to specific regions of the genome. We have performed gene expression studies and chromatin immunoprecipitation on strains in which native centromere sequences have been mutated to study the mechanism by which a tRNAAlanine gene barrier (cen1 tDNAAla) blocks the spread of pericentromeric heterochromatin at the centromere of chromosome 1 (cen1) in the fission yeast, Schizosaccharomyces pombe. Within the centromere, barrier activity is a general property of tDNAs and, unlike previously characterized barriers, requires the association of both transcription factor IIIC and RNA Polymerase III. Although the cen1 tDNAAla gene is actively transcribed, barrier activity is independent of transcriptional orientation. These findings provide experimental evidence for the involvement of a fully assembled RNA polymerase III transcription complex in defining independent structural and functional domains at a eukaryotic centromere

Nonlinear pharmacokinetics of therapeutic proteins resulting from receptor mediated endocytosis

Receptor mediated endocytosis (RME) plays a major role in the disposition of therapeutic protein drugs in the body. It is suspected to be a major source of nonlinear pharmacokinetic behavior observed in clinical pharmacokinetic data. So far, mostly empirical or semi-mechanistic approaches have been used to represent RME. A thorough understanding of the impact of the properties of the drug and of the receptor system on the resulting nonlinear disposition is still missing, as is how to best represent RME in pharmacokinetic models. In this article, we present a detailed mechanistic model of RME that explicitly takes into account receptor binding and trafficking inside the cell and that is used to derive reduced models of RME which retain a mechanistic interpretation. We find that RME can be described by an extended Michaelis–Menten model that accounts for both the distribution and the elimination aspect of RME. If the amount of drug in the receptor system is negligible a standard Michaelis–Menten model is capable of describing the elimination by RME. Notably, a receptor system can efficiently eliminate drug from the extracellular space even if the total number of receptors is small. We find that drug elimination by RME can result in substantial nonlinear pharmacokinetics. The extent of nonlinearity is higher for drug/receptor systems with higher receptor availability at the membrane, or faster internalization and degradation of extracellular drug. Our approach is exemplified for the epidermal growth factor receptor system

Predicting the F(ab)-mediated effect of monoclonal antibodies in vivo by combining cell-level kinetic and pharmacokinetic modelling

Cell-level kinetic models for therapeutically relevant processes increasingly benefit the early stages of drug development. Later stages of the drug development processes, however, rely on pharmacokinetic compartment models while cell-level dynamics are typically neglected. We here present a systematic approach to integrate cell-level kinetic models and pharmacokinetic compartment models. Incorporating target dynamics into pharmacokinetic models is especially useful for the development of therapeutic antibodies because their effect and pharmacokinetics are inherently interdependent. The approach is illustrated by analysing the F(ab)-mediated inhibitory effect of therapeutic antibodies targeting the epidermal growth factor receptor. We build a multi-level model for anti-EGFR antibodies by combining a systems biology model with in vitro determined parameters and a pharmacokinetic model based on in vivo pharmacokinetic data. Using this model, we investigated in silico the impact of biochemical properties of anti-EGFR antibodies on their F(ab)-mediated inhibitory effect. The multi-level model suggests that the F(ab)-mediated inhibitory effect saturates with increasing drug-receptor affinity, thereby limiting the impact of increasing antibody affinity on improving the effect. This indicates that observed differences in the therapeutic effects of high affinity antibodies in the market and in clinical development may result mainly from Fc-mediated indirect mechanisms such as antibody-dependent cell cytotoxicity

Patterns and Mechanisms of Ancestral Histone Protein Inheritance in Budding Yeast

Tracking of ancestral histone proteins over multiple generations of genome replication in yeast reveals that old histones move along genes from 3′ toward 5′ over time, and that maternal histones move up to around 400 bp during genomic replication

Drug-Class Specific Impact of Antivirals on the Reproductive Capacity of HIV

Predictive markers linking drug efficacy to clinical outcome are a key component in the drug discovery and development process. In HIV infection, two different measures, viral load decay and phenotypic assays, are used to assess drug efficacy in vivo and in vitro. For the newly introduced class of integrase inhibitors, a huge discrepancy between these two measures of efficacy was observed. Hence, a thorough understanding of the relation between these two measures of drug efficacy is imperative for guiding future drug discovery and development activities in HIV. In this article, we developed a novel viral dynamics model, which allows for a mechanistic integration of the mode of action of all approved drugs and drugs in late clinical trials. Subsequently, we established a link between in vivo and in vitro measures of drug efficacy, and extract important determinants of drug efficacy in vivo. The analysis is based on a new quantity—the reproductive capacity—that represents in mathematical terms the in vivo analog of the read-out of a phenotypic assay. Our results suggest a drug-class specific impact of antivirals on the total amount of viral replication. Moreover, we showed that the (drug-)target half life, dominated by immune-system related clearance processes, is a key characteristic that affects both the emergence of resistance as well as the in vitro–in vivo correlation of efficacy measures in HIV treatment. We found that protease- and maturation inhibitors, due to their target half-life, decrease the total amount of viral replication and the emergence of resistance most efficiently

Proteomic Interrogation of Human Chromatin

Chromatin proteins provide a scaffold for DNA packaging and a basis for epigenetic regulation and genomic maintenance. Despite understanding its functional roles, mapping the chromatin proteome (i.e. the “Chromatome”) is still a continuing process. Here, we assess the biological specificity and proteomic extent of three distinct chromatin preparations by identifying proteins in selected chromatin-enriched fractions using mass spectrometry-based proteomics. These experiments allowed us to produce a chromatin catalog, including several proteins ranging from highly abundant histone proteins to less abundant members of different chromatin machinery complexes. Using a Normalized Spectral Abundance Factor approach, we quantified relative abundances of the proteins across the chromatin enriched fractions giving a glimpse into their chromosomal abundance. The large-scale data sets also allowed for the discovery of a variety of novel post-translational modifications on the identified chromatin proteins. With these comparisons, we find one of the probed methods to be qualitatively superior in specificity for chromatin proteins, but inferior in proteomic extent, evidencing a compromise that must be made between biological specificity and broadness of characterization. Additionally, we attempt to identify proteins in eu- and heterochromatin, verifying the enrichments by characterizing the post-translational modifications detected on histone proteins from these chromatin regions. In summary, our results provide insights into the value of different methods to extract chromatin-associated proteins and provide starting points to study the factors that may be involved in directing gene expression and other chromatin-related processes

The NSL Complex Regulates Housekeeping Genes in Drosophila

MOF is the major histone H4 lysine 16-specific (H4K16) acetyltransferase in mammals and Drosophila. In flies, it is involved in the regulation of X-chromosomal and autosomal genes as part of the MSL and the NSL complexes, respectively. While the function of the MSL complex as a dosage compensation regulator is fairly well understood, the role of the NSL complex in gene regulation is still poorly characterized. Here we report a comprehensive ChIP–seq analysis of four NSL complex members (NSL1, NSL3, MBD-R2, and MCRS2) throughout the Drosophila melanogaster genome. Strikingly, the majority (85.5%) of NSL-bound genes are constitutively expressed across different cell types. We find that an increased abundance of the histone modifications H4K16ac, H3K4me2, H3K4me3, and H3K9ac in gene promoter regions is characteristic of NSL-targeted genes. Furthermore, we show that these genes have a well-defined nucleosome free region and broad transcription initiation patterns. Finally, by performing ChIP–seq analyses of RNA polymerase II (Pol II) in NSL1- and NSL3-depleted cells, we demonstrate that both NSL proteins are required for efficient recruitment of Pol II to NSL target gene promoters. The observed Pol II reduction coincides with compromised binding of TBP and TFIIB to target promoters, indicating that the NSL complex is required for optimal recruitment of the pre-initiation complex on target genes. Moreover, genes that undergo the most dramatic loss of Pol II upon NSL knockdowns tend to be enriched in DNA Replication–related Element (DRE). Taken together, our findings show that the MOF-containing NSL complex acts as a major regulator of housekeeping genes in flies by modulating initiation of Pol II transcription

The Yin and Yang of Yeast Transcription: Elements of a Global Feedback System between Metabolism and Chromatin

When grown in continuous culture, budding yeast cells tend to synchronize their respiratory activity to form a stable oscillation that percolates throughout cellular physiology and involves the majority of the protein-coding transcriptome. Oscillations in batch culture and at single cell level support the idea that these dynamics constitute a general growth principle. The precise molecular mechanisms and biological functions of the oscillation remain elusive. Fourier analysis of transcriptome time series datasets from two different oscillation periods (0.7 h and 5 h) reveals seven distinct co-expression clusters common to both systems (34% of all yeast ORF), which consolidate into two superclusters when correlated with a compilation of 1,327 unrelated transcriptome datasets. These superclusters encode for cell growth and anabolism during the phase of high, and mitochondrial growth, catabolism and stress response during the phase of low oxygen uptake. The promoters of each cluster are characterized by different nucleotide contents, promoter nucleosome configurations, and dependence on ATP-dependent nucleosome remodeling complexes. We show that the ATP:ADP ratio oscillates, compatible with alternating metabolic activity of the two superclusters and differential feedback on their transcription via activating (RSC) and repressive (Isw2) types of promoter structure remodeling. We propose a novel feedback mechanism, where the energetic state of the cell, reflected in the ATP:ADP ratio, gates the transcription of large, but functionally coherent groups of genes via differential effects of ATP-dependent nucleosome remodeling machineries. Besides providing a mechanistic hypothesis for the delayed negative feedback that results in the oscillatory phenotype, this mechanism may underpin the continuous adaptation of growth to environmental conditions

Advancing our understanding of functional genome organisation through studies in the fission yeast

Significant progress has been made in understanding the functional organisation of the cell nucleus. Still many questions remain to be answered about the relationship between the spatial organisation of the nucleus and the regulation of the genome function. There are many conflicting data in the field making it very difficult to merge published results on mammalian cells into one model on subnuclear chromatin organisation. The fission yeast, Schizosaccharomyces pombe, over the last decades has emerged as a valuable model organism in understanding basic biological mechanisms, especially the cell cycle and chromosome biology. In this review we describe and compare the nuclear organisation in mammalian and fission yeast cells. We believe that fission yeast is a good tool to resolve at least some of the contradictions and unanswered questions concerning functional nuclear architecture, since S. pombe has chromosomes structurally similar to that of human. S. pombe also has the advantage over higher eukaryotes in that the genome can easily be manipulated via homologous recombination making it possible to integrate the tools needed for visualisation of chromosomes using live-cell microscopy. Classical genetic experiments can be used to elucidate what factors are involved in a certain mechanism. The knowledge we have gained during the last few years indicates similarities between the genome organisation in fission yeast and mammalian cells. We therefore propose the use of fission yeast for further advancement of our understanding of functional nuclear organisation