Expression and chromatin association of noncoding RNAs

Paulo Amaral (2011). Expression and chromatin association of noncoding RNAs PhD Thesis, Institute for Molecular Bioscience, The University of Queensland.

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Author Paulo Amaral
Thesis Title Expression and chromatin association of noncoding RNAs
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
Publication date 2011-08
Thesis type PhD Thesis
Supervisor John Mattick
Andrew Perkins
Total pages 393
Total colour pages 51
Total black and white pages 342
Language eng
Subjects 06 Biological Sciences
Abstract/Summary Although only a small fraction of the mammalian genome comprises annotated protein-coding sequences, transcriptomic studies have demonstrated that a large proportion of its length is transcribed in different cell types. Such pervasive transcription generates hundreds of thousands of different RNAs that lack substantial open reading frames, and are broadly defined as “noncoding RNAs” (ncRNAs). Biological roles have been assigned to an increasing number of individually characterized ncRNAs, notably in the regulation of gene expression at the level of chromatin structure. However, the expression patterns and functional properties of the vast majority of the novel transcripts are still unexplored, and it is likely that many more RNAs remain to be discovered. This thesis aimed to identify and characterize the expression of ncRNAs and to investigate the global association of RNAs with chromatin using mouse embryonic stem (ES) cells as the model. In the first part of this work, a custom microarray platform was used to survey long ncRNA (lncRNA) expression during mouse ES cell differentiation as embryoid bodies (EBs) into mesoderm lineages over a 16-day time-course. The expression profiling allowed the identification of 945 expressed ncRNAs, of which 174 were significantly differentially expressed. Expressed RNAs were characterized by combining available genome-wide chromatin state maps, genomic context and promoter transcription factor binding sites. Many of the expressed RNAs were found genomically associated with protein-coding loci that have important roles in ES cell pluripotency or differentiation. Selected lncRNAs were chosen for further characterization, including novel spliced ncRNAs that are transcribed antisense (‘as’) to homeobox genes, such as Evx1, Hoxb5/6 and all three Dlx gene clusters. Some of these sense-antisense RNA pairs are spatially co-expressed in the mouse embryo and this co-regulation is maintained in adult tissues, indicating functional relationships. In addition, using a novel RNA-chromatin immunoprecipitation (RNA-ChIP) technique, evidence was found that Evx1as and Hoxb5/6Aas ncRNAs are associated with histone H3 trimethylated at lysine 4 (H3K4me3) and histone methyltransferase MLL1 chromatin fractions, which are involved in homeotic gene activation. One of the ncRNAs expressed in mouse ES cells, Sox2 overlapping transcript (Sox2ot), in whose intron is embedded the pluripotency regulator Sox2 gene, was analyzed in more detail. We observed that this spliced RNA is part of a complex locus with several regulated isoforms associated with the Sox2 gene throughout vertebrates. Sox2 and Sox2ot expression were found to be differentially regulated in a number of analyzed systems, including during mouse ES and neural stem cell differentiation. Sox2ot was also found to have highly conserved promoters and to be dynamically regulated in fish, chicken and mammalian development, with a consistent pattern of expression in the central nervous system, including in several regions of the human brain. The second part of this work aimed to identify transcripts with potential roles in chromatin regulation in ES cells. In a pilot study, I used RNA-ChIP to co-purify RNAs directly or indirectly associated with histone H3 and with H3K4me3 chromatin domains, which specifically mark RNA Polymerase II promoters. These RNAs were subsequently identified by deep sequencing (“RNA-ChIP-seq”). Analysis of these transcripts revealed a number of different RNAs potentially associated with chromatin, as well as ~6 thousand discrete short RNA clusters (averaging <200 nt) that co-localized with mapped H3K4me3 domains in a genome-wide manner. These results indicate that RNA-ChIP is a useful method to isolate RNAs associated with specific chromatin domains and allowed the identification of RNAs potentially involved in transcriptional regulation. Finally, I describe the preparation of a database for annotation of long noncoding RNAs (“lncRNAdb”). The goal of lncRNAdb is to organize and to increase the accessibility of the data resulting from the rapid discovery and characterization of lncRNAs. The database is an online resource that compiles information for long regulatory RNAs described in the literature, which currently contains over one hundred fifty manually curated lncRNAs in unicellular eukaryotes, plants, and animals. LncRNAdb highlights the extensive array of functional mechanisms and biological processes in which ncRNAs have been involved. Collectively, the results obtained in this thesis show that many ncRNAs, which increasingly appear to represent the primary output of the mammalian genome, are specifically regulated and likely play different roles in ES cell biology and developmental processes. These include one of the first characterized ncRNAs whose structure and expression patterns is conserved in vertebrates (Sox2ot), as well as RNAs with potential roles in epigenetic regulation in ES cells. The establishment of the RNA-ChIP-seq methodology introduced here may also pave the way for the targeted identification of regulatory RNAs involved in different molecular processes that are influenced by chromatin structure.
Keyword noncoding RNA, antisense, transcriptome, RNA-ChIP-seq, embryonic stem cell, pluripotency, epigenetic regulation, mouse development, regulatory evolution
Additional Notes *In colour: 27, 32, 37, 48, 55-56, 58-59, 66-70, 72-73, 79, 81-82, 84, 94-97, 107, 110-111, 113, 115-117, 119-121, 141-142, 149, 256, 291, 301, 313, 315, 320, 341, 353, 357-358, 360, 368, 370, 389-390. *Additional special printing request: please also print page 96 as landscape (this is a colour page).

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Created: Thu, 24 Nov 2011, 21:15:19 EST by Mr Paulo Amaral on behalf of Library - Information Access Service