Genes are activated and repressed within the cell nucleus, an organelle with a complex three dimensional structure. Along with changes in gene expression, changes to gene positions within the nucleus also occur during normal development and in some genetic diseases, particularly in cancers. To better understand the relationship between gene expression and nuclear organization, we are carefully mapping the nuclear locations of several genes that are activated during very early stages of normal mammalian development and in embryonic stem cells. These include genes in a region of mouse chromosome 14 that shows great similarity to human chromosome 13. These genes are organized near the nuclear periphery, a nuclear compartment that plays a role in chromosome structure and gene regulation. We are investigating how gene positioning at the nuclear periphery is related to gene regulation during development. This compartment also is implicated in a number of human genetic diseases, including muscular dystrophies, lipodystrophies, and premature aging. In addition, we are testing how the nuclear organization of genes goes awry in cancer (lymphoma) cells, where chromosome sequences have been rearranged. These studies will help to elucidate the ways in which genes work together during normal development, as well as how gene regulation can go wrong in cancer and other human genetic diseases.
Three-Dimensional Organization of Chromosomes During Development and Tumorigenesis
Gene organization in the nucleus
We investigate the relationships between mammalian gene expression, chromosome sequence organization and chromosome structure in the nucleus. We are particularly interested in characterizing chromosome architecture during gene expression program changes in normal development and in tumorigenesis. Our toolbox includes high-resolution imaging and numerous mouse models with changes in chromosome and nuclear structure.
The expression status of a gene correlates with its three-dimensional (3-D) organization in the nucleus. In fact, a gene's activity can be influenced by its location near different sub-nuclear compartments and by physical interactions with other genes. While these structure-function relationships are known to exist for certain genes, the extent of physical interactions among genes across the genome, the role of nuclear compartments and the underlying mechanisms of genome 3-D organization are poorly understood.
We have found that genes across large chromosomal regions aggregate together within the nucleus. This work began with a 4 Mb region on distal mouse Chromosome 14 (Mmu14) that contains several genes expressed during mouse embryogenesis. These genes are organized in the primary sequence into discrete clusters separated by long (>500 kb) "gene deserts" depleted of coding sequence. In nuclei where the genes are expressed, the Mmu14 region folds into multiple but non-random 3-D structures. Remarkably, these structures contain multiple gene clusters, from as far away as 4 Mb, aggregated together at one nuclear site. These findings suggest dynamic chromosome 3-D folding where genes transiently interact in nuclear "hubs." Moreover, the Mmu14 region localizes to the nuclear periphery, a compartment implicated in chromatin folding and gene regulation.
Chromatin at the nuclear periphery
The mammalian nuclear periphery has long been thought of as a silencing compartment. However, we found that active genes on Mmu14 associate with this compartment. Thus, we are further investigating the chromatin constituents of the nuclear periphery to determine whether this compartment plays a role in gene expression as well as gene repression. This work focuses on the nuclear periphery of mouse embryonic stem cells, which undergo dramatic chromatin rearrangements upon differentiation. We are examining the nuclear distributions of chromosomal loci with different epigenetic signatures using quantitative, high resolution imaging, including the 4Pi confocal microscope, which can resolve objects at the nuclear periphery that are within 100 nanometers of each other.
The nuclear periphery contains the nuclear lamina, a network of intermediate filaments thought to serve as a scaffold for chromatin. In addition, the nuclear periphery has been implicated in gene regulation by association with transcription factors (e.g. RB, SMADs) and by promoting heterochromatin formation. We are currently investigating the role of the nuclear periphery in chromatin folding and gene expression using a number of mouse models with mutations in different nuclear periphery components. We are testing, for example, whether these mutations alter the formation of gene hubs on Mmu14.
Chromosomal rearrangements in lymphoma cells
Our studies of Mmu14 indicate that chromosome 3-D structure is closely linked to primary sequence organization. This finding is particularly relevant to cancer, since chromosomal rearrangements are present in virtually every tumor cell. We are testing the effects of chromosomal rearrangements on chromosome 3-D folding and gene expression using a mouse model of progenitor B-cell lymphoma, in collaboration with Dr. Kevin Mills. These cells carry a recurrent translocation and a complex amplification of the Myc oncogene. Our goal is to determine whether these rearrangements affect the nuclear organization, folding, epigenetic state and expression of genes beyond those disrupted at chromosomal breakpoints. These studies will provide novel insights into how gene expression programs are controlled during tumorigenesis and will point to new targets for cancer therapy.
Principal Investigator: Lindsay S. Shopland, Ph.D.
Research Assistant I: Nali Jia, B.S., Jacob Bolewski B.S., Lihua Wang, B.S.
Research Administrative Assistant: Maxine Friend
Pratt CH, Curtain M, Donahue LR, Shopland LS. 2011. Mitotic defects lead to pervasive aneuploidy and accompany loss of RB1 activity in mouse LmnaDhe dermal fibroblasts. PLoS One. 6:e18065. PMC3064591.
Snow KJ, Wright S, Woo Y, Titus L, Mills K, Shopland LS. 2010. Nuclear positioning, higher-order folding, and gene expression of Mmu15 sequences are refactory to chromosomal translocation. Chromosoma. 120:61-71. PMC3057431
Odgren PR, Pratt CH, MacKay CA, Mason-Savas A, Curtain M, Shopland LS, Ichicki T, Sundberg J, Donahue LR. 2010. Disheveled hair and ear (Dhe), a spontaneous mouse Lmna mutation modeling human laminopathies. PLoS One. 5: e9959. PMC2848607
Luo L, Gassman KL, Petell LM, Wilson CL, Bewersdorf J, Shopland LS. 2009. The nuclear periphery of embryonic stem cells is a transcriptionally permissive and repressive compartment. J Cell Sci 122:3729-3737. PMC275884
Shopland LS, Bewersdorf J. 2008. Seeing the world through a new set of glasses: Emerging technologies for the study of nuclei and chromosomes. Chromosome Research 16:350-353.
Shopland LS, Lynch CR, Peterson K, Thorton K, Kepper N, Stein S, Vincent S, Molloy K, Kreth G, Cremer C, Bult CJ, OIbrien TP. 2006. Folding and organization of a contiguous chromosome region according to the gene distribution pattern in primary genomis sequence. J Cell Biol 174: 27-38.
Moen PT, Johnson CV, Byron M, Shopland LS, de la Serna I, Imbalzano A, Lawrence JB. 2004. Repositioning of muscle-specific genes to the periphery of SC-35 domains during skeletal myogenesis. Mol Biol Cell 15:197-206.
Shopland LS, Johnson CV, Byron M, McNeil J, Lawrence JB. 2003. Clustering of multiple specific genes and gene-rich R-bands around SC-35 domains: Evidence for local euchromatic neighborhoods. J Cell Biol 162:981-990.