Retrotransposon mobilization is a major source of genome evolution. However, the functional consequences of these events, and particularly their influence upon transcriptional activity, are poorly defined. The extent of retrotransposon transcription, as well as that of other repetitive elements, has eluded systematic study due to difficulties in discriminating elements copied in multiple genomic loci. Moreover, the potential regulatory effects of retrotransposon transcription upon the expression of neighbouring protein-coding genes are also largely unknown. This thesis develops methods to survey repetitive element expression and assess the functions of retrotransposons in the mouse and human genomes. Chapter 1 summarises the complex transcriptional output of the mammalian genome, the functional annotation of this expression and the genomic and bioinformatic tools available for its detection. Chapter 2 explores the capacity of short sequence tags to discern transcription from individual repetitive elements, as well as from protein-coding genes. It is based upon a publication that critiqued the bioinformatics associated with Cap Analysis Gene Expression (CAGE) and developed novel methodologies to resolve repetitive element transcription. Chapter 3 describes the development of an updated CAGE mapping pipeline for the fourth stage of the international Functional Annotation of Mouse (FANTOM) project, which lead to the generation of a research article and a book chapter. These works demonstrated the enhanced utility of CAGE when coupled with next-generation sequencing, highlighted the benefits of CAGE when applied to systems biology and profiled the temporal expression of human repetitive elements. Chapter 4 presents an in-depth analysis of repetitive element transcription in the mouse and human genomes. Using CAGE, approximately 250,000 retrotransposon associated transcription start sites were defined, many of which were tissue-specific. Retrotransposons were found to frequently function as alternative promoters for protein-coding genes and/or express non-coding RNAs. Furthermore, when retrotransposons were found within the 3’UTR of protein-coding genes, there was strong evidence for the reduced expression of the corresponding transcripts. A genome-wide screen for strong expression correlation between repetitive elements and neighbouring protein-coding genes identified approximately 23,000 candidate regulatory regions derived from retrotransposons, including several hundred putative boundary elements. These were in addition to more than two thousand examples of bidirectional transcription found in retrotransposons, which are known to be a source of double stranded RNAs involved in RNA interference. Chapter 5 explores the proportion of the mouse embryonic stem cell transcriptome comprised of repeat-derived transcripts, using next-generation RNA sequencing. This study defined the dynamic expression of repetitive elements at the greatest resolution achieved to date and demonstrated that repetitive elements are an intrinsic part of the mammalian transcriptional landscape.

It has been suggested that organismal complexity correlates with the complexity of gene regulation. Transcriptional control of gene expression is mediated by binding of regulatory proteins to cis-acting sequences on the genome. Hence, it is crucial to identify the chromosomal targets of transcription factors (TFs) to delineate transcriptional regulatory networks underlying gene expression programs. The development of ChIP-chip technology has enabled high throughput mapping of TF binding sites across the genome. However, there are many limitations to the technology including the availability of whole genome arrays for complex organisms such human or mouse. To circumvent these limitations, we developed the Sequence Tag Analysis of Genomic Enrichment (STAGE) methodology that is based on extracting short DNA sequences or “tags” from ChIP-enriched DNA. With improvements in sequencing technologies, we applied the recently developed ChIP-Seq technique i.e. ChIP followed by ultra high throughput sequencing, to identify binding sites for the TF E2F4 across the human genome. We identified previously uncharacterized E2F4 binding sites in intergenic regions and found that several microRNAs are potential E2F4 targets. Binding of TFs to their respective chromosomal targets requires access of the TF to its regulatory element, which is strongly influenced by nucleosomal remodeling. In order to understand nucleosome remodeling in response to transcriptional perturbation, we used ultra high throughput sequencing to map nucleosome positions in yeast that were subjected to heat shock or were grown normally. We generated nucleosome remodeling profiles across yeast promoters and found that specific remodeling patterns correlate with specific TFs active during the transcriptional reprogramming. Another important aspect of gene regulation operates at the post-transcriptional level. MicroRNAs (miRNAs) are ~22 nucleotide non-coding RNAs that suppress translation or mark mRNAs for degradation. MiRNAs regulate TFs and in turn can be regulated by TFs. We characterized a TF-miRNA network involving the oncofactor Myc and the miRNA miR-22 that suppresses the interferon pathway as primary fibroblasts enter a stage of rapid proliferation. We found that miR-22 suppresses the interferon pathway by inhibiting nuclear translocation of the TF NF-kappaB. Our results show how the oncogenic TF Myc cross-talks with other TF regulatory pathways via a miRNA intermediary. / text