Dynamic transcriptome analysis (DTA)

So far, much attention has been paid to regulation of transcription. However, it has been realized that controlled mRNA decay is an equally important process. To understand the contributions of mRNA synthesis and mRNA degradation to gene regulation, we developed Dynamic Transcriptome Analysis (DTA). DTA allows to monitor these contributions for both processes and for all mRNAs in the cell without perturbation of the cellular system. DTA works by non-perturbing metabolic RNA labeling that supersedes conventional methods for mRNA turnover analysis. It is accomplished with dynamic kinetic modeling to derive the gene-specific synthesis and decay parameters. DTA reveals that most mRNA synthesis rates result in several transcripts per cell and cell cycle, and most mRNA half-lives range around a median of 11 min. DTA can monitor the cellular response to osmotic stress with higher sensitivity and temporal resolution than standard transcriptomics. In contrast to monotonically increasing total mRNA levels, DTA reveals three phases of the stress response. In the initial shock phase, mRNA synthesis and decay rates decrease globally, resulting in mRNA storage. During the subsequent induction phase, both rates increase for a subset of genes, resulting in production and rapid removal of stress-responsive mRNAs. In the following recovery phase, decay rates are largely restored, whereas synthesis rates remain altered, apparently enabling growth at high salt concentration. Stress-induced changes in mRNA synthesis rates are predicted from gene occupancy with RNA polymerase II. Thus, DTA realistically monitors the dynamics in mRNA metabolism that underlie gene regulatory systems. One of the technical obstacles of standard transcriptomics is the unknown normalization factor between samples, i.e. wild-type and mutant cells. Variations in RNA extraction efficiencies, amplification steps and scanner calibration introduce differences in the global intensity levels. The required normalization limits the precision of DTA. We have extended DTA to comparative DTA (cDTA), to eliminate this obstacle. cDTA provides absolute rates of mRNA synthesis and decay in Saccharomyces cerevisiae (Sc) cells with the use of Schizosaccharomyces pombe (Sp) as an internal standard. It therefore allows for direct comparison of RNA synthesis and decay rates between samples. cDTA reveals that Sc and Sp transcripts that encode orthologous proteins have similar synthesis rates, whereas decay rates are five fold lower in Sp, resulting in similar mRNA concentrations despite the larger Sp cell volume. cDTA of Sc mutants reveals that a eukaryote can buffer mRNA levels. Impairing transcription with a point mutation in RNA polymerase (Pol) II causes decreased mRNA synthesis rates as expected, but also decreased decay rates. Impairing mRNA degradation by deleting deadenylase subunits of the Ccr4–Not complex causes decreased decay rates as expected, but also decreased synthesis rates. In this thesis, we provide a novel tool to estimate RNA synthesis and decay rates: a quantitative dynamic model to describe mRNA metabolism in growing cells to complement the biochemical protocol of DTA/cDTA. It can be applied to reveal rate changes for all kinds of perturbations, e.g. in knock-out or point mutation strains, in responses to stress stimuli or in small molecule interfering assays like treatments with miRNA or siRNA inhibitors. In doing so, we show that DTA is a valuable tool for miRNA target validation. The DTA/cDTA approach is in principle applicable to virtually every organism. The bioinformatic workflow of DTA/cDTA is implemented in the open source R/Bioconductor package DTA

On the existence of intermediate groups in multi-level inclusions of triangle groups

Diese Diplomarbeit behandelt eine Fragestellung aus dem Gebiet der Fuchsschen Gruppen. Das Problem, das hier geklärt werden soll, entspringt einer im Jahre 2005 erschienenen Arbeit über Konjugatoren Fuchsscher Gruppen und quasiplatonische Riemannsche Flächen von Jürgen Wolfart und Ernesto Girondo [GW05]. Es ist dort ein alternativer geometrischer Weg gewählt worden, um es zu umgehen, und es soll nun hier mit den bereits 1970 bereitgestellten Methoden zur Fragestellung der Existenz von Untergruppen Fuchsscher Gruppen von David Singerman [SIN70] gelöst werden. Betrachtet man eine Dreiecksgruppe $Delta_{1}$ als Untergruppe einer Dreiecksgruppe $Delta_{2}$, so kann es vorkommen, dass diese Inklusion eine Verfeinerung durch eine dazwischenliegende Dreiecksgruppe $Delta$ gestattet. In den Fällen, in denen zu einer gegebenen Dreiecksgruppe $Delta_{2}$ voneinander verschiedene Untergruppen gleicher Signatur $Delta_{1},Delta_{1}Apostroph,...$ existieren, ist es nicht a priori klar, dass es eine dazwischenliegende Dreiecksgruppe $Delta,DeltaApostroph,...$ gleicher Signatur zu jeder dieser verschiedenen Untergruppen gibt. Das Ziel dieser Arbeit ist es nun, dies zu klären, d.h. zu zeigen, dass es für jedes Paar Dreiecksgruppen $Delta_{1}subseteqDelta_{2},Delta_{1}ApostrophsubseteqDelta_{2},...$ eine dazwischenliegende Dreiecksgruppe $Delta,DeltaApostroph,...$ gibt. Im ersten Teil dieser Arbeit wird eine grobe Einleitung in die Theorie der Diskontinuierlichen Gruppen gegeben, die sehr stark auf Fuchssche Gruppen abzielt und mit deren Verbindung zu Riemannschen Flächen abschließt. Sie orientiert sich sehr stark an einem Standardwerk über Diskontinuierliche Gruppen von Joseph Lehner [LEH64]. Im zweiten Teil dieser Arbeit widmen wir uns ganz den Untergruppen Fuchsscher Gruppen und dem von David Singerman [SIN70] bereitgestellten Apparat, der eine notwendige und hinreichende Bedingung hierfür aufzeigt. Wie David Singerman auch zeigt, lassen sich diese Methoden für Normalteiler und Dreiecksgruppen spezialisieren. Wir werden uns dem auch annehmen. Abschließend erarbeiten wir dann eine ausführliche Methode und somit auch einen Beweis zur Erlangung der kompletten Liste von Inklusionen Fuchsscher Dreiecksgruppen, wie sie sich in einer weiteren Arbeit David Singermans befindet [SIN72]. Dies geschieht mit Hilfe zweier Maple-Programme deren Quellcode und Ausgabe sich im Anhang bzw. vierten Teil dieser Arbeit zur Einsicht bendet. Im dritten Teil dieser Arbeit wird schließlich die oben erläuterte Fragestellung geklärt werden. Sie wird zunächst anhand vieler Beispiele und Erläuterungen verdeutlicht, und im Anschluss dessen eine mögliche Verallgemeinerung auf Fuchssche Gruppen diskutiert.This diploma thesis covers a problem in the field of Fuchsian groups. The problem that is to be solved here originates from a paper about conjugators of Fuchsian groups and quasiplatonic surfaces by Jürgen Wolfart and Ernesto Girondo [GW05]. There, an alternative geometrical way has been chosen in order to avoid it. In this thesis, the problem is to be solved by applying a method from 1970 which deals with the existence of subgroups of Fuchsian groups by David Singerman [SIN70]. Let $Delta_{1}$ be a triangle subgroup of a triangle group $Delta_{2}$. It can happen that this inclusion allows a refinement by an intermediate triangle group $Delta$. In cases where there are different subgroups $Delta_{1},Delta_{1}apostrophe,...$ of the same signature in a given triangle group $Delta_{2}$ it is not a priori clear that there are intermediate triangle groups $Delta,Deltaapostrophe,...$ of the same signature to all those different subgroups. The aim of this thesis is to show, that for every pair of triangle groups $Delta_{1}subseteqDelta_{2},Delta_{1}apostrophesubseteqDelta_{2},...$, there is an intermediate triangle group $Delta,Deltaapostrophe,...$. A short introduction to the theory of discontinuous groups, will be given in the first part of this thesis, with a strong focus on Fuchsian groups and with their connection to Riemann surfaces at its end. It is very much geared to a standard work about discontinuous groups by Joseph Lehner [LEH64]. The second part of this thesis is dedicated to subgroups of Fuchsian groups and to a method developed by David Singerman [SIN70], giving a necessary and sufficient constraint for them. David Singerman also shows that these methods can be specialized when applied to normal subgroups and triangle groups. We will attend to these too. Finally we will develop a detailed method and hence a proof to obtain the complete list of inclusions of Fuchsian triangle groups, which can be found in another work of David Singerman [SIN72]. This is done by means of two maple-programs, whose source code and display is provided in the appendix respectively the fourth part of this thesis. In the third part of this thesis the central issue will eventually be solved. At first it will be clarified on the basis of examples and explanations, and finally a possible generalization on Fuchsian groups will be discussed

Rock glacier inventory of the western Nyainqêntanglha Range, Tibetan Plateau, supported by InSAR time series and automated classification

The western Nyainqêntanglha Range on the Tibetan Plateau reaches an elevation of 7,162 m and is characterized by an extensive periglacial environment under semi-arid climatic conditions. Rock glaciers play an important part of the water budget in high mountain areas and recent studies suggest that they may even act as climate-resistant water storages. In this study we present the first rock glacier inventory of this region containing 1,433 rock glaciers over an area of 4,622 km. To create the most reliable inventory we combine manually created rock glacier outlines with an automated classification approach. The manual outlines were generated based on surface elevation data, optical satellite imagery and a surface velocity estimation. This estimation was generated via InSAR time series analysis with Sentinel-1 data from 2016 to 2019. Our pixel-based automated classification was able to correctly identify 87.8% of all rock glaciers in the study area at a true positive rate of 69.5%. In total, 65.9% of rock glaciers are classified as transitional with surface velocities of 1–10 cm/yr. In total, 18.5% are classified as active with higher velocities of up to 87 cm/yr. The southern windward side of the mountain range contains more numerous and more active rock glaciers. We attribute this to higher moisture availability supplied by the Indian Monsoon

Efficient RNA polymerase II pause release requires U2 snRNP function

Transcription by RNA polymerase II (Pol II) is coupled to pre-mRNA splicing, but the underlying mechanisms remain poorly understood. Co-transcriptional splicing requires assembly of a functional spliceosome on nascent pre-mRNA, but whether and how this influences Pol II transcription remains unclear. Here we show that inhibition of pre-mRNA branch site recognition by the spliceosome component U2 snRNP leads to a widespread and strong decrease in new RNA synthesis from human genes. Multiomics analysis reveals that inhibition of U2 snRNP function increases the duration of Pol II pausing in the promoter-proximal region, impairs recruitment of the pause release factor P-TEFb, and reduces Pol II elongation velocity at the beginning of genes. Our results indicate that efficient release of paused Pol II into active transcription elongation requires the formation of functional spliceosomes and that eukaryotic mRNA biogenesis relies on positive feedback from the splicing machinery to the transcription machinery

Accurate Promoter and Enhancer Identification in 127 ENCODE and Roadmap Epigenomics Cell Types and Tissues by GenoSTAN

Accurate maps of promoters and enhancers are required for understanding transcriptional regulation. Promoters and enhancers are usually mapped by integration of chromatin assays charting histone modifications, DNA accessibility, and transcription factor binding. However, current algorithms are limited by unrealistic data distribution assumptions. Here we propose GenoSTAN (Genomic STate ANnotation), a hidden Markov model overcoming these limitations. We map promoters and enhancers for 127 cell types and tissues from the ENCODE and Roadmap Epigenomics projects, today's largest compendium of chromatin assays. Extensive benchmarks demonstrate that GenoSTAN generally identifies promoters and enhancers with significantly higher accuracy than previous methods. Moreover, GenoSTAN-derived promoters and enhancers showed significantly higher enrichment of complex trait-associated genetic variants than current annotations. Altogether, GenoSTAN provides an easy-to-use tool to define promoters and enhancers in any system, and our annotation of human transcriptional cis-regulatory elements constitutes a rich resource for future research in biology and medicine

The Implication of Early Chromatin Changes in X Chromosome Inactivation.

During development, the precise relationships between transcription and chromatin modifications often remain unclear. We use the X chromosome inactivation (XCI) paradigm to explore the implication of chromatin changes in gene silencing. Using female mouse embryonic stem cells, we initiate XCI by inducing Xist and then monitor the temporal changes in transcription and chromatin by allele-specific profiling. This reveals histone deacetylation and H2AK119 ubiquitination as the earliest chromatin alterations during XCI. We show that HDAC3 is pre-bound on the X chromosome and that, upon Xist coating, its activity is required for efficient gene silencing. We also reveal that first PRC1-associated H2AK119Ub and then PRC2-associated H3K27me3 accumulate initially at large intergenic domains that can then spread into genes only in the context of histone deacetylation and gene silencing. Our results reveal the hierarchy of chromatin events during the initiation of XCI and identify key roles for chromatin in the early steps of transcriptional silencing

Transcription activation depends on the length of the RNA polymerase II C-terminal domain.

Eukaryotic RNA polymerase II (Pol II) contains a tail-like, intrinsically disordered carboxy-terminal domain (CTD) comprised of heptad-repeats, that functions in coordination of the transcription cycle and in coupling transcription to co-transcriptional processes. The CTD repeat number varies between species and generally increases with genome size, but the reasons for this are unclear. Here, we show that shortening the CTD in human cells to half of its length does not generally change pre-mRNA synthesis or processing in cells. However, CTD shortening decreases the duration of promoter-proximal Pol II pausing, alters transcription of putative enhancer elements, and delays transcription activation after stimulation of the MAP kinase pathway. We suggest that a long CTD is required for efficient enhancer-dependent recruitment of Pol II to target genes for their rapid activation

Transcription activation depends on the length of the RNA polymerase II C-terminal domain.

Eukaryotic RNA polymerase II (Pol II) contains a tail-like, intrinsically disordered carboxy-terminal domain (CTD) comprised of heptad-repeats, that functions in coordination of the transcription cycle and in coupling transcription to co-transcriptional processes. The CTD repeat number varies between species and generally increases with genome size, but the reasons for this are unclear. Here, we show that shortening the CTD in human cells to half of its length does not generally change pre-mRNA synthesis or processing in cells. However, CTD shortening decreases the duration of promoter-proximal Pol II pausing, alters transcription of putative enhancer elements, and delays transcription activation after stimulation of the MAP kinase pathway. We suggest that a long CTD is required for efficient enhancer-dependent recruitment of Pol II to target genes for their rapid activation

Insights into a remote cryosphere: a multi-method approach to assess permafrost occurrence at the Qugaqie basin, western Nyainqêntanglha Range, Tibetan Plateau

Permafrost as a climate-sensitive parameter and its occurrence and distribution play an important role in the observation of global warming. However, field-based permafrost distribution data and information on the subsurface ice content in the large area of the southern mountainous Tibetan Plateau (TP) are very sparse. Existing models based on boreholes and remote sensing approaches suggest permafrost probabilities for most of the Tibetan mountain ranges. Field data to validate permafrost models are generally lacking because access to the mountain regions in extreme altitudes is limited. The study provides geomorphological and geophysical field data from a north-orientated high-altitude catchment in the western Nyainqêntanglha Range. A multi-method approach combines (A) geomorphological mapping, (B) electrical resistivity tomography (ERT) to identify subsurface ice occurrence and (C) interferometric synthetic aperture radar (InSAR) analysis to derive multi-annual creeping rates. The combination of the resulting data allows an assessment of the lower occurrence of permafrost in a range of 5350 and 5500 m above sea level (a.s.l.) in the Qugaqie basin. Periglacial landforms such as rock glaciers and protalus ramparts are located in the periglacial zone from 5300–5600 m a.s.l. The altitudinal periglacial landform distribution is supported by ERT data detecting ice-rich permafrost in a rock glacier at 5500 m a.s.l. and ice lenses around the rock glacier (5450 m a.s.l.). The highest multiannual creeping rates up to 150 mm yr−1 are typically observed on these rock glaciers. This study closes the gap of unknown state of periglacial features and potential permafrost occurrence in a high-elevated basin in the western Nyainqêntanglha Range (Tibetan Plateau)

RNA transcription and degradation of Alu retrotransposons depends on sequence features and evolutionary history

AbstractAlu elements are one of the most successful groups of RNA retrotransposons and make up 11% of the human genome with over 1 million individual loci. They are linked to genetic defects, increases in sequence diversity, and influence transcriptional activity. Still, their RNA metabolism is poorly understood yet. It is even unclear whether Alu elements are mostly transcribed by RNA Polymerase II or III. We have conducted a transcription shutoff experiment by α-amanitin and metabolic RNA labeling by 4-thiouridine combined with RNA fragmentation (TT-seq) and RNA-seq to shed further light on the origin and life cycle of Alu transcripts. We find that Alu RNAs are more stable than previously thought and seem to originate in part from RNA Polymerase II activity, as previous reports suggest. Their expression however seems to be independent of the transcriptional activity of adjacent genes. Furthermore, we have developed a novel statistical test for detecting the expression of quantitative trait loci in Alu elements that relies on the de Bruijn graph representation of all Alu sequences. It controls for both statistical significance and biological relevance using a tuned