Functional nuclear topography of transcriptionally inducible extra-chromosomal transgene clusters.

A new experimental approach was designed to test different predictions of current models of the nuclear architecture with respect to the topography of transcription. We constructed a plasmid, termed pIndi, which carries a reporter gene coding for a red cytoplasmic fluorescent reporter protein. Transcription of the reporter gene is regulated by the inducible promoter of the human immunodeficiency virus (HIV) and is strongly dependent on the HIV-1 Tat protein. Expressing the red fluorescent reporter protein allowed us to distinguish between cells with active and silent reporter genes. Importantly, transient transfection resulted in the clustering of plasmids, forming one or several extra-chromosomal pIndi bodies. Repetitive lac operator sequences in pIndi allowed us to visualize these bodies in living cells by the binding of LacI proteins tagged with a fluorescent protein. Using this model, we analyzed the three-dimensional nuclear topography of pIndi bodies with active or silent reporter genes. Our results are compatible with predictions of the chromosome territory-interchromatin compartment (CT-IC) model. We demonstrate that pIndi bodies localize in the IC, both in the silent and active state. Activation of transgene transcription resulted in the recruitment of RNA polymerase II and NFkappaB and a closer positioning to splicing speckles.

Analysis of the influence of subcellular localization of the HIV Rev protein on Rev-dependent gene expression by multi-fluorescence live-cell imaging.

The human immunodeficiency virus Rev protein is a post-transcriptional activator of HIV gene expression. Rev is a nucleocytoplasmic shuttle protein that displays characteristic nuclear/nucleolar subcellular localization in various cell lines. Cytoplasmic localization of Rev occurs under various conditions disrupting Rev function. The goal of this study was to investigate the relationship between localization of Rev and its functional activity in living cells. A triple-fluorescent imaging assay, called AQ-FIND, was established for automatic quantitative evaluation of nucleocytoplasmic distribution of fluorescently tagged proteins. This assay was used to screen 500 rev genes generated by error-prone PCR for Rev mutants with different localization phenotypes. Activities of the Rev mutants were determined with a second quantitative, dual-fluorescent reporter assay. In HeLa cells, the majority of nuclear Rev mutants had activities similar to wild-type Rev. The activities of Rev mutants with abnormal cytoplasmic localization ranged from moderately impaired to nonfunctional. There was no linear correlation between subcellular distribution and levels of Rev activity. In astrocytes, nuclear Rev mutants showed similar impaired activities as the cytoplasmic wild-type Rev. Our data suggest that steady-state subcellular localization is not a primary regulator of Rev activity but may change as a secondary consequence of altered Rev function. The methodologies described here have potential for studying the significance of subcellular localization for functions of other regulatory factors.

Three-dimensional arrangements of centromeres and telomeres in nuclei of human and murine lymphocytes.

The location of centromeres and telomeres was studied in human and mouse lymphocyte nuclei (G0) employing 3D-FISH, confocal microscopy, and quantitative image analysis. In both human and murine lymphocytes, most centromeres were found in clusters at the nuclear periphery. The distribution of telomere clusters, however, differed: in mouse nuclei, most clusters were detected at the nuclear periphery, while, in human nuclei, most clusters were located in the nuclear interior. In human cell nuclei we further studied the nuclear location of individual centromeres and their respective chromosome territories (CTs) for chromosomes 1, 11, 12, 15, 17, 18, 20, and X. We found a peripheral location of both centromeres and CTs for 1, 11, 12, 18, X. A mostly interior nuclear location was observed for CTs 17 and 20 and the CTs of the NOR-bearing acrocentric 15 but the corresponding centromeres were still positioned in the nuclear periphery. Autosomal centromeres, as well as the centromere of the active X, were typically located at the periphery of the respective CTs. In contrast, in about half of the inactive X-CTs, the centromere was located in the territory interior. While the centromere of the active X often participated in the formation of centromere clusters, such a participation was never observed for the centromere of the inactive X.