Identification and characterization of biallelically cis-silenced genes by trans-complementation

One of the most enduring questions in developmental biology, which has preoccupied scientists for over a century, is how a single fertilized egg can give rise to the vast array of different cell types found in a multi-cellular organism. In order to generate tissue-specific phenotypes, the plasticity and developmental potential of this early pluripotent cell must become increasingly restricted as it divides and progresses through embryogenesis. Thus, this process is inherently dependent upon the progressive restriction of cell fate. On a genetic level, this lineage restriction is the result of alterations in the gene expression profile of a cell. As such, cell fate restriction could be explained by selective gene activation or, alternatively, through progressive gene silencing. Characterizing the mechanisms underlying the changes in gene expression during development remains a major challenge.;The general aim of this project is to examine the role and underlying mechanisms of epigenetic silencing in lineage restriction during mammalian development. This work has utilized the trans-complementation assay, cell fusion, to determine the relative contribution of cis versus trans mechanisms to transcriptional potential. Cell fusion between disparate cell types generates a heterokaryon with a homogenized transcriptional milieu where transcriptional potential of genes can be assessed using the active copy in the opposite genome as a positive control. In particular, this work has identified a mode of silencing termed gene occlusion. Gene occlusion is defined as transcriptional repression by cis-acting mechanisms in a manner that blocks the affected genes from responding to the trans-acting milieu of the cells. Opposite to the occluded state is the competent state whereby a gene is capable of responding to the trans-acting milieu of the cell such that it is active if appropriate transcription activators are present while it is silent if activators are absent or repressors are present.;In chapter 2, the trans-complementation assay is used to identify a set of occluded and competent (transactivated) genes in the human lung fibroblast. This work was extended further by interrogating the conservation and stability of the human lung fibroblast occluded and competent genes in the context of different cell types and across different species. This work shows that the occluded state is extremely stable under a variety of physiological conditions, that it is conserved within similar cell types across species and remains stable through cell division. Moreover, this work suggests that this mode of silencing is particularly enriched in genes known to be upstream master regulators and may therefore be a mechanism involved in maintaining the stability of the differentiated cell type.;In chapter 3, the chromatin state of regulatory regions of both occluded and competent genes is interrogated. Extensive analysis of DNA methylation across regulatory regions using bisulfite sequencing and global demethyaltion revealed a positive correlation between DNA methylation and the occluded state. Although significant differential methylation was not observed around the transcriptional start sites for the majority of occluded genes, genes transactivated after global demethylation were enriched for those that have significantly higher methylation levels in this region. This suggests that for a subset of genes DNA methylation is likely involved in establishing the occluded state. Analysis of chromatin modifications, histone variants and chromatin binding proteins did not reveal a significant enrichment for any of the marks analyzed in the occluded set of genes. The data in this work argue that DNA methylation outside the transcriptional start site is likely involved in establishing the occluded state and provides a candidate epigenetic modification that should be explored further in additional occluded genes across different species and cell types.;In chapter 4, differentiation of pluripotent rat mesenchymal stem cells was used as model system to assess the stability of occluded state during development. Transcriptional competency of occluded genes was assessed in the three mesenchymal stem cell derived cell types: osteoblast, adipocyte and chondrocyte. This work argues that occluded genes are stable during differentiation and development. Furthermore, the transcriptional competency of genes expressed in mesenchymal stem cell derived lineages was assessed in the undifferentiated precursor cell. This work argues that genes expressed in differentiated cells, including major lineage regulators, are not subject to occlusion in their precursor cell type. These case studies are likely to shed light on the general role of gene silencing in determining cell fate; provide insight into the types of genes that are regulated in this manner, and further elucidate the mechanisms by which silencing-mediated cell differentiation occurs.