Abstract
Differentiation signaling results in reprogramming of cellular gene expression that leads to morphological changes and functional specialization of a precursor cell. This global change in gene expression involves temporal regulation of differentiation-specific genes that are located throughout the genome, raising the idea that genome structure may also be re-organized during cell differentiation to facilitate regulated gene expression. Using in vitro adipocyte differentiation as a model, we explored whether gene organization within the nucleus is altered upon exposure of precursor cells to signaling molecules that induce adipogenesis. The peroxisome proliferator-activated receptor gamma (PPARγ) nuclear hormone receptor is a master determinant of adipogenesis and is required for adipose differentiation. We utilized the chromosome conformation capture (3C) assay to determine whether the position of the PPARγ locus relative to other adipogenic genes is changed during differentiation. We report that the PPARγ2 promoter is transiently positioned in proximity to the promoters of genes encoding adipokines and lipid droplet associated proteins at 6 hours post-differentiation, a time that precedes expression of any of these genes. In contrast, the PPARγ2 promoter was not in proximity to the EF1α promoter, which drives expression of a constitutively active, housekeeping gene that encodes a translation elongation factor, nor was the PPARγ2 promoter in proximity to the promoter driving the expression of the C/EBPα regulatory protein. The formation of the long-range, intergenic interactions involving the PPARγ2 promoter required the regulatory factor C/EBPβ, elevated cyclic AMP (cAMP) levels, and protein kinase A (PKA) signaling. We conclude that genome organization is dynamically remodeled in response to adipogenic signaling, and we speculate that these transient inter-genic interactions may be formed for the purposes of selecting some of the transcriptionally silent tissue-specific loci for subsequent transcriptional activation.
Highlights
Maturation of a pre-adipocyte into a mature adipocyte involves significant changes in cellular structure and organization
Expression of the lamin associated polypeptide 2a (LAP2a) or reduction in the expression of lamins or of the lamin associated protein emerin modulates adipocyte differentiation [5,6,7], while mutations in nuclear lamina proteins have been associated with lipodystrophy syndromes [8,9]
Control western blots demonstrated that C/EBPb protein levels were reduced in cells expressing the shRNA against C/EBPb (Fig. 3F). These results suggest that C/EBPb and the initial events that occur in the first few hours of PPARc2-driven gene activation during adipogenesis are required for subsequent formation of long-range chromatin interactions involving the PPARc2 locus
Summary
Maturation of a pre-adipocyte into a mature adipocyte involves significant changes in cellular structure and organization. Recent studies report structural alterations that occur in the nuclear lamina and in the connections between the nucleus and cytoplasmic filaments as a function of adipogenic differentiation [4]. Other reports document organizational changes within the nucleus. These include reports of differentiation-dependent changes in chromosome territories [10], relocalization of adipogenic gene positioning within the nucleus relative to the nuclear lamina during differentiation [11,12], and preferential association with SC-35 domains [13], which are nuclear structures that are enriched in factors involved in pre-mRNA metabolism and that may act to spatially link gene expression and mRNA processing [14,15]
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