Research
Nuclear Architecture -- PML Nuclear Bodies
Our hypothesis is that in mammalian cells the activity of a given gene is a function of its three-dimensional location and organization within its chromosome territory and with respect to other "subnuclear organelles" or sub-compartments in the nucleus. Therefore, the transcriptional program of each cell type should correlate with an ultrastructural signature of chromatin domains within the nuclei of cells of a given cell/tissue type.
Promyelocytic leukemia nuclear bodies (PML NBs or NBs) are a class of subnuclear organelles that contribute to the compartmentalization of nuclear proteins. This process must be important because PML NBs play a central role in cellular differentiation and tumour suppression. Indeed, a number of human cancers are characterized by the down regulation or mutations of PML, including acute promyelocytic leukemia, prostate and colon. Though implicated in processes such as transcription, DNA damage repair, and apoptosis, the exact function of PML NBs is not known.
A prevailing model is that they are storage sites of nuclear proteins, serving as platforms from which they move into the nucleoplasm to carry out their functions. By studying the structure, biochemical composition and dynamics of PML NBs, studies from my laboratory support another model. Though our model does not exclude the storage or platform concepts, we propose that PML NBs interact directly with the surrounding chromatin, and thereby perform a more active role in servicing the regulation of genes on their periphery.
Nuclear Architecture – Chromatin Organization
Besides nuclear bodies, chromatin itself demonstrates compartmentalization, in that each chromosome occupies a discrete location in the nucleus, determined by chromatin painting based on fluorescence in situ hybridization (FISH) techniques. The location of chromosome territories relative to each and to the interior vs. the periphery of the nucleus is non-random. Moreover, the location of specific genes appears to be a function of their activity in particular cell types. Many lines of evidence support the idea that the control of gene activity can be described as location-dependent.
Our laboratory is developing approaches to define the internal structure of chromosome territories and their relationship to other subnuclear domains at high spatial resolution. We would like to be able to map transcriptionally active chromatin as well as transcriptionally inert heterochromatin within and between territories using electron spectroscopic imaging. With this technique we can resolve nucleosomes in 10 nm chromatin fibres, we can delineate protein- from nucleic acid-based structures, and we can combine this information with FISH, immuno-detection of important factors and proteins in an individual cell. The technical developments are permitting us to determine the signature chromatin organization in cells in their natural tissue environment, not deprived of all of the inter-cellular contacts. We will also be able to study gene organization and functional nuclear architecture in situ murine stem cells, in both their undifferentiated state and following programming towards defined specialized cell lineages.