Genome-wide Identification of Non-coding Transcription by RNA Polymerase V and Its Involvement in Transcriptional Gene Silencing

RNA-mediated transcriptional gene silencing is a conserved process where non-coding RNAs target transposons and other sequences for repression by establishing repressive chromatin modifications. A central element of this process is long non-coding RNAs (lncRNAs), which in Arabidopsis thaliana are produced by a specialized RNA polymerase known as Pol V. These lncRNAs recruit small interfering RNAs (siRNAs) and a series of proteins that lead to the establishment of RNA-directed DNA methylation (RdDM) on transposable elements. Transposable elements extant in eukaryotic genomes pose a constant risk of disrupting the integrity of the genome via random integration events and are targeted for silencing by the RdDM machinery. The RdDM pathway results in de novo DNA methylation and it has been quite extensively researched, however, questions about the mechanism of recruitment of Pol V to RdDM loci and subsequent interplay between chromatin modifications and the downstream mechanism of gene silencing are still less understood. In this work, I have utilized high-throughput molecular sequencing data to expand our understanding of the transcriptional gene silencing pathway. First, I addressed and expanded our understanding of Pol V transcription at RdDM loci. I have successfully identified and annotated Pol V transcribed RdDM loci throughout the genome. I have further shown how Pol V transcription is controlled by preexisting chromatin modifications located within the transcribed regions. I observed that Pol V transcribes into transposons in a non-strand specific manner and the DNA methylation targeted to these transposons also occur on both strands and is tightly restricted to the Pol V transcribed regions. I further show that the preferential enrichment of Pol V transcription and downstream DNA methylation at the edges of transposons depicts a possible role of Pol V in determining heterochromatin boundaries. Second, my research helped us better understand the mechanism of Pol V transcription. I have shown that Pol V transcription is not restricted to RdDM loci but is much more pervasive. Through my research, I show how at already established RdDM targets, Pol V and siRNA work together to maintain silencing. In contrast, some euchromatic sequences do not give rise to siRNA but are covered by low levels of Pol V transcription, which is needed to establish RdDM de novo, if a transposon is reactivated. Through this study, I show that Pol V surveils the genome to make it competent to silence newly activated transposons, making it essential for maintaining the integrity of the genome. Third, I address the effect of Pol V transcription on downstream repressive chromatin modifications and gene silencing. I show that RdDM affects nucleosomes through recruitment of the SWI/SNF chromatin remodeling complex. Next, I address the relationship between the two chromatin modifications showing that despite DNA methylation being predominantly enriched at linkers, RdDM target loci show an enrichment of both nucleosomes and DNA methylation. My data further depicts that nucleosome placement by RdDM has no detectable effects on the pattern of DNA methylation. Instead, I show that DNA methylation by RdDM affects nucleosome positioning, suggesting that DNA methylation directs nucleosomes and they both coordinately bring about gene and transposon silencing at the RdDM loci.PHDBioinformaticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/167911/1/shriyas_1.pd

PRC2 represses transcribed genes on the imprinted inactive X chromosome in mice

Abstract Background Polycomb repressive complex 2 (PRC2) catalyzes histone H3K27me3, which marks many transcriptionally silent genes throughout the mammalian genome. Although H3K27me3 is associated with silenced gene expression broadly, it remains unclear why some but not other PRC2 target genes require PRC2 and H3K27me3 for silencing. Results Here we define the transcriptional and chromatin features that predict which PRC2 target genes require PRC2/H3K27me3 for silencing by interrogating imprinted mouse X-chromosome inactivation. H3K27me3 is enriched at promoters of silenced genes across the inactive X chromosome. To abrogate PRC2 function, we delete the core PRC2 protein EED in F1 hybrid trophoblast stem cells (TSCs), which undergo imprinted inactivation of the paternally inherited X chromosome. Eed –/– TSCs lack H3K27me3 and Xist lncRNA enrichment on the inactive X chromosome. Despite the absence of H3K27me3 and Xist RNA, only a subset of the inactivated X-linked genes is derepressed in Eed –/– TSCs. Unexpectedly, in wild-type (WT) TSCs these genes are transcribed and are enriched for active chromatin hallmarks on the inactive-X, including RNA PolII, H3K27ac, and H3K36me3, but not the bivalent mark H3K4me2. By contrast, PRC2 targets that remain repressed in Eed –/– TSCs are depleted for active chromatin characteristics in WT TSCs. Conclusions A comparative analysis of transcriptional and chromatin features of inactive X-linked genes in WT and Eed –/– TSCs suggests that PRC2 acts as a brake to prevent induction of transcribed genes on the inactive X chromosome, a mode of PRC2 function that may apply broadly.https://deepblue.lib.umich.edu/bitstream/2027.42/136651/1/13059_2017_Article_1211.pd

Data Throughput of Wireless Network for Fire Alarms

Import 22/07/2015Tato bakalářská práce se zabývá ostravskou hasičskou sítí, propustností, rušením a návrhem na vylepšení sítě z hlediska datové propustnosti. Analýza datové propustnosti byla provedena pomoci vlastního programu napsaného v C#. Pomocí USB tuneru Rafael Micro R820T s čipsetem RTL2832U a počítačem s operačním systémem Ubuntu 14.04, na kterém byly nainstalován software Librtlsdr, GNU radio GQRX , Teamviewer a Kazam. Těmito programy byly sledovány vstupní kmitočty převaděčů, které neodhalily žádné rušení. Dále byly vypsány možné vlivy teoretického rušení. Následně byly vymyšleny dvě teoreticky zlepšené varianty systému. První se zabývá obousměrným přenosem, kdy koncové vysílače přijímají zprávu o potvrzení přijetí z převaděče a druhá přidáním dalšího převaděče, který by se při správném umístění, které by bylo na výškové budově domova sester. Hlavní výhodou tohoto řešení je větší pokrytí oblasti. Oba tyto návrhy mají lepší vlastnosti v oblasti datové propustnosti.The bachelor thesis deals with the fire-fighting net in Ostrava, it's permeability, disturbance and improvement proposal for this net from the point of view of data permeability. Analysis of data permeability was made by own programme wrote in C#. Disturbance was watched by USB tuner Rafael Micro R820T with chipset RTL2832U and with computer with operating system Ubuntu 14.04. On Ubuntu was install a software Librtlsdr, GNU radio GQRX, Teamviewer and Kazam. But the disturbance was not found. The list of the theroretical influences on disturbance was made. Two theoretical better options were invented. The first one deals with two-way transfer and the second one proposes additional convertor. These suggestions have better properties in the field of data permeability.440 - Katedra telekomunikační technikydobř