The impact of long-interspersed nuclear elements-1 in the mouse genome

Approximately half of the mammalian genome is composed of various types of transposable elements. One class of transposable elements are the LINE-1 elements (long interspersed nucleotide elements) or L1 retrotransposable elements comprising 17% of the human genome and 19% of the mouse genome, respectively. Although most of the LINE-1 retrotransposable elements are in an inactive state, the activation of LINE-1 retrotransposable elements can have an impact on genome stability in various ways, some of which are beneficial and others detrimental, due to their mechanism of retrotransposition. Over evolutionary timescales, LINE-1 retrotransposable elements have effected major structural remodeling of the genome, altered gene expression and influenced the transcriptional activity of neighboring genes. Although transposition of LINE-1 elements have the effect of generating genomic variation and diversification of mammalian genomes, reliable estimates of the numbers of actively transposing LINE-1 elements are mostly nonexistent. While the human and mouse genomes contain comparable numbers of LINE-1 elements, several phylogenetic and LIXplore analyses in the mouse genome suggest that 1,500-3,000 active LINE-1 elements currently exist and that they are still expanding, Conversely, the human genome contains only 150 active LINE-1 elements. In addition, there is a discrepancy between the nature and number of the mouse LINE-1 elements in L1Xplore database and in the UCSC genome browser. Currently, it is not known why the mouse genome contains such a large number of active LINE-1 elements in comparison to the human genome. Moreover, the potential mechanisms that are responsible for transcriptional activation of mouse LINE-1 elements are not known. In this study, to address these questions, the promoter sequences of 1,501 potentially active mouse LINE-1 elements retrieved from the GenBank and L1Xplore databases were analyzed and their transcription factor binding sites and CpG content were evaluated. Our computational analysis indicated that substantial numbers of the mouse LINE-1 elements contained modified YY1 transcription factor binding sites in their promoter sequences, which are required for their transcriptional initiation. This suggests that only about a half of LINE-1 elements are capable of being transcriptionally active. In addition, experimental evidence using luciferase assays showed that previously unreported CpG islands exist in the promoters of the most active (TF) family of mouse LINE-1 elements. The presence of sequence variation and polymorphisms in CpG islands of LINE-1 promoters (arising from transition mutations) indicates that CpG methylation could playa significant role in determining the activity of L1 elements in the mouse genome. We speculate that LINE-1 activation in the mouse genome is associated with specific epigenetic markers located along the promoter regions of the LINE-1 elements. Recent studies in maize, Arabidopsis, yeast and human genomes have revealed that epigenetic mechanisms such as DNA methylation and histone modifications playa significant role in silencing and defending genome stability against the effects of retrotransposable elements. However, the mechanistic pathways by which LINE-1 elements are silenced in the mouse genome are still unknown, even though great numbers of full-length active mouse LINE-1 elements exist in the mouse genome. To elucidate the possible silencing mechanisms, two different mouse LINE-1 promoters, one with, and one lacking CpG islands, were cloned into a Flp-In system by site-specific homologous recornbinanan. which was then transfected into mouse NIH3T3 cell lines. Once the LINE-1 promoter with CpG or without CpG islands was integrated into the euchromatin region of the host genome, promoter activities could then be determined. The transcriptional activity of introduced exogenous LINE-1 promoters was found to be gradually silenced in the mouse NIH3T3 cell line. DNA methylation profiles and histone modifications in the exogenous LINE-1 promoter regions were investigated over the same period of time, using MeDIP, bisulfite DNA sequencing and ChIP analysis. Our results showed that DNA methylation at YY1 binding sites and H3K9Me3 histone methylation play a critical role in the repression of LINE-1 elements, and these repressive markers were enriched in CpG islands of LINE-1 promoters. Interestingly, the H2A.Z variant was found to be predominantly enriched in the LINE-1 promoters lacking CpG islands, whereas LINE-1 elements with CpG islands were bound by H2A histones. This suggests an inverse relationship between H2A.Z occupancy and DNA methylation at CpG islands, and that DNA methylation markers are related to the silencing of LINE-1 elements. LINE-1 elements are capable of mobilizing themselves into new genomic sites by retrotransposition. As a consequence of their mobility, they can potentially act as insertional mutagens and disrupt gene function in the mammalian genome. Although the expression of LINE-1 elements is silenced by epigenetic mechanisms in somatic cells, embryonic stem (ES) cells show high levels of LINE-1 expression due to global hypomethylation and active (or open) chromatin structures. Therefore, insertional mutagenesis resulting from LINE-1 activity in ES cells is a very useful strategy for understanding the effect of LINE-1 integrated sites during ES cell proliferation and differentiation. In order to investigate the mechanisms by which LINE-1 inserts exert their effect on ES cell differentiation pathways, the intact full-length mouse LINE-1 element was isolated and cloned into a scaffold/matrix attachment region (S/MAR)-based vector. This vector system has been previously shown to express a high level of LINE-1 elements in the mammalian cells. The resulting construct was transfected into mouse ES cells and their retrotransposition activity was analyzed. Surprisingly, mouse ES cells transfected with the full-length LINE-1 elements, showed delayed rates of cell differentiation in the absence of LIF. The intrinsic molecular mechanisms that influence the ES cell cycle and cell proliferation is not clearly understood. To elucidate these, the integrated sites of LINE-1 elements were confirmed by inverse PCR. Two key genes, Tollip and USP31, were then found to be disrupted by LINE-1 integration. Using microarray analysis, the regulatory network of genes that are disrupted by Tollip and USP31 were identified. The disruption of the gene regulatory networks that control the cell cycle, cell proliferation and differentiation was further confirmed in mouse ES cells. These studies revealed the important role of LINE-1 element insertions in the mouse genome. In conclusion, this thesis provides three major discoveries. First, CpG islands and YYi transcription factor binding sites are required for transcriptional initiation of mouse LINE-1 elements. Second, the transcriptional activity of LINE-1 elements is modulated by epigenetic mechanisms such as DNA methylation and histone methylation. Third, the full-length intact mouse LINE-1 element can disrupt the function of endogenous genes such as Tollip and USP31, which are essential for ES cell differentiation pathways. The significance of this thesis is that it not only provides us with a deeper understanding of the epigenetic mechanisms involved in the activation or the silencing of LINE-1 elements, but also provides new insights into the roles of LINE-1 elements in ES cell proliferation and differentiation