Biochemical and Functional Characterisation of the Novel Non Specific Lethal Complex

Genomic DNA is not freely accessible but it is compacted into chromatin by wrapping DNA around a histone octamer. Basic unit of chromatin is a nucleosome. Accessibility of nucleosomal DNA highly regulated and is orchestrated by many proteins that combinatorially alter the positional phasing of nucleosomes by chromatin remodeling enzymes, substitution of variant histones, post-translational modification of nucleosomes and the partitioning of chromatin into specific nuclear locations. X chromosomal regulation by the process of dosage compensation provides an ideal model system to study the effect of chromatin and epigenetic factors on gene expression. In mammals, genes on the active X (Xa) chromosome are upregulated about twofold, with a corresponding inactivation of one of the two X chromosomes (Xi) ensuring equivalent sex chromosome expression in males and females. In Drosophila, dosage compensation is accomplished by the work of the MSL complex, which provokes a two-fold increase in the expression of genes on the male X chromosome. The MSL complex specifically binds to genes that require to be unregulated and, through the action of MOF, a histone acetyltransferase subunit within the complex, induces acetylation of H4K16, which is associated with an increase in the rate of transcription of genes. In contradiction to the classic view that MOF was restricted to the male X chromosome, it has been found recently by our lab that MOF binds to multiple sites on the autosomes in both sexes. This suggests that MOF has a role in transcriptional regulation beyond dosage compensation. The work presented in this thesis shows the purification of a novel complex of evolutionary conserved proteins, which contains MOF. We termed the complex the NSL complex (Non-Specific Lethal), as mutation of proteins of the complex is lethal to both sexes. The NSL complex is composed of the evolutionary conserved proteins MOF, NSL1, NSL2, NSL3, MCRS2, MBDR2, WDS, Z4 and Chromator. These components of the NSL complex broadly decorate all chromosomes, and overlap with MOF on the X chromosome(s), as well as on all autosomes in males and females. Colocalization of NSL complex members with MOF occurs at the level of individual genes, with NSL associated with the promoters of MOF-bound genes. Analysis of total RNA from fly lines expressing RNAi against NSLs specifically in salivary glands demonstrates that the binding of the NSL complex to promoters is functional, as there is a strong correlation between the absence of NSL and a decrease in transcription in males and females. Taken together, work performed in this thesis demonstrates that the NSL complex functions as a novel transcription regulator in Drosophila

Contrasting effects of Elg1–RFC and Ctf18–RFC inactivation in the absence of fully functional RFC in fission yeast

Proliferating cell nuclear antigen loading onto DNA by replication factor C (RFC) is a key step in eukaryotic DNA replication and repair processes. In this study, the C-terminal domain (CTD) of the large subunit of fission yeast RFC is shown to be essential for its function in vivo. Cells carrying a temperature-sensitive mutation in the CTD, rfc1-44, arrest with incompletely replicated chromosomes, are sensitive to DNA damaging agents, are synthetically lethal with other DNA replication mutants, and can be suppressed by mutations in rfc5. To assess the contribution of the RFC-like complexes Elg1–RFC and Ctf18–RFC to the viability of rfc1-44, genes encoding the large subunits of these complexes have been deleted and overexpressed. Inactivation of Ctf18–RFC by the deletion of ctf18(+), dcc1(+) or ctf8(+) is lethal in an rfc1-44 background showing that full Ctf18–RFC function is required in the absence of fully functional RFC. In contrast, rfc1-44 elg1Δ cells are viable and overproduction of Elg1 in rfc1-44 is lethal, suggesting that Elg1–RFC plays a negative role when RFC function is inhibited. Consistent with this, the deletion of elg1(+) is shown to restore viability to rfc1-44 ctf18Δ cells

Predicting stimulation-dependent enhancer-promoter interactions from ChIP-Seq time course data

We have developed a machine learning approach to predict stimulation-dependent enhancer-promoter interactions using evidence from changes in genomic protein occupancy over time. The occupancy of estrogen receptor alpha (ER), RNA poly- merase (Pol II) and histone marks H2AZ and H3K4me3 were measured over time using ChIP-Seq experiments in MCF7 cells stimulated with estrogen. A Bayesian classifier was developed which uses the correlation of temporal binding patterns at enhancers and promoters and genomic proximity as features to predict interactions. This method was trained using experimentally determined interactions from the same system and was shown to achieve much higher precision than predictions based on the genomic proximity of nearest ER binding. We use the method to identify a genome-wide confident set of ER target genes and their regulatory enhancers genome- wide. Validation with publicly available GRO-Seq data demonstrates that our predicted targets are much more likely to show early nascent transcription than predictions based on genomic ER binding proximity alone.Peer reviewe

Genome-wide modeling of transcription kinetics reveals patterns of RNA production delays

Genes with similar transcriptional activation kinetics can display very different temporal mRNA profiles because of differences in transcription time, degradation rate, and RNA-processing kinetics. Recent studies have shown that a splicing-associated RNA production delay can be significant. To investigate this issue more generally, it is useful to develop methods applicable to genome-wide datasets. We introduce a joint model of transcriptional activation and mRNA accumulation that can be used for inference of transcription rate, RNA production delay, and degradation rate given data from high-throughput sequencing time course experiments. We combine a mechanistic differential equation model with a nonparametric statistical modeling approach allowing us to capture a broad range of activation kinetics, and we use Bayesian parameter estimation to quantify the uncertainty in estimates of the kinetic parameters. We apply the model to data from estrogen receptor alpha activation in the MCF-7 breast cancer cell line. We use RNA polymerase II ChIP-Seq time course data to characterize transcriptional activation and mRNA-Seq time course data to quantify mature transcripts. We find that 11% of genes with a good signal in the data display a delay of more than 20 min between completing transcription and mature mRNA production. The genes displaying these long delays are significantly more likely to be short. We also find a statistical association between high delay and late intron retention in pre-mRNA data, indicating significant splicing-associated production delays in many genes.Peer reviewe

Integrative Analysis of Deep Sequencing Data Identifies Estrogen Receptor Early Response Genes and Links ATAD3B to Poor Survival in Breast Cancer

Peer reviewe

CRISPR/Cas9: Vom Fachwissen zum Bewerten: Eine Unterrichtseinheit für die Jahrgangstufe 12/13

Das CRISPR/Cas9-System hat aufgrund der vielfältigen Anwendungsmöglichkeiten zu einer breiten öffentlichen Debatte geführt und ist aus dem heutigen Dialog von Biologen, Biotechnologen, Medizinern und Agrarwissenschaftlern nicht mehr wegzudenken. Um Schülerinnen und Schülern höherer Jahrgangsstufen näherzubringen, was sich hinter dieser scheinbar genialen Methode der Gentechnik verbirgt, wird in diesem Artikel eine erprobte Unterrichtseinheit vorgestellt. In dieser Einheit soll nicht nur die Funktionsweise des CRSIRP/Cas9-Systems erarbeitet werden. Sie soll auch den aktuellen Forschungsstand sowie eine eigene ethische Bewertung dieses Verfahrens vermitteln

Data from: A transient ischemic environment induces reversible compaction of chromatin

Background: Cells detect and adapt to hypoxic and nutritional stress through immediate transcriptional, translational and metabolic responses. The environmental effects of ischemia on chromatin nanostructure were investigated using single molecule localization microscopy of DNA binding dyes and of acetylated histones, by the sensitivity of chromatin to digestion with DNAseI, and by fluorescence recovery after photobleaching (FRAP) of core and linker histones. Results: Short-term oxygen and nutrient deprivation of the cardiomyocyte cell line HL-1 induces a previously undescribed chromatin architecture, consisting of large, chromatin-sparse voids interspersed between DNA-dense hollow helicoid structures 40–700 nm in dimension. The chromatin compaction is reversible, and upon restitution of normoxia and nutrients, chromatin transiently adopts a more open structure than in untreated cells. The compacted state of chromatin reduces transcription, while the open chromatin structure induced upon recovery provokes a transitory increase in transcription. Digestion of chromatin with DNAseI confirms that oxygen and nutrient deprivation induces compaction of chromatin. Chromatin compaction is associated with depletion of ATP and redistribution of the polyamine pool into the nucleus. FRAP demonstrates that core histones are not displaced from compacted chromatin; however, the mobility of linker histone H1 is considerably reduced, to an extent that far exceeds the difference in histone H1 mobility between heterochromatin and euchromatin. Conclusions: These studies exemplify the dynamic capacity of chromatin architecture to physically respond to environmental conditions, directly link cellular energy status to chromatin compaction and provide insight into the effect ischemia has on the nuclear architecture of cells