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Functional interaction of chromatin with nucleoporins in Drosophila

ABSTRACT: Nuclear pore complexes (NPCs) mediate transport across the nuclear envelope. In yeast, they have also been proposed to interact with active genes, attracting or retaining them at the nuclear periphery. However, some NPC components (nucleoporins) in higher eukaryotes are also found in the nucleoplasm, with so far unknown function. Therefore, we have functionally distinguished between nucleoporin-chromatin interactions at the NPC and within the nucleoplasm in Drosophila. For this we analyzed genomic interactions of full-length nucleoporins Nup98, Nup50 and Nup62 and nucleoplasmic and NPC-tethered forms of Nup98. We found that nucleoporins predominantly interacted with transcriptionally active genes inside the nucleoplasm. A smaller set of non-active genes interacted with the NPC. We identified a direct role for nucleoplasmic Nup98 in stimulating gene expression, as genes downregulated upon Nup98 depletion were activated upon nucleoplasmic Nup98 overexpression and showed strong nucleoplasmic Nup98 interaction. Thus, nucleoporins stimulate gene expression away from the NPC by interacting with genes inside the nucleoplasm.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE19307 | GEO | 2010/03/01

SECONDARY ACCESSION(S): PRJNA120901

REPOSITORIES: GEO

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Project description:In Saccharomyces cerevisiae, specific genes physically relocate from the nucleoplasm to the nuclear periphery concomitant with transcriptional activation, where they associate with the nuclear pore complex (NPC). We took a genomics approach in order to gain insight into the universality and physiological relevance of the interaction between active genes and the NPC. Using synthetic genetic array (SGA) approach, we identified interactions between components of the SAGA histone acetyltransferase complex and the Mlp and Nup60 subunits of the NPC. Cells lacking these SAGA and NPC components display growth defects under optimal growth conditions, in which cells are grown on rich medium containing the preferred sugar glucose as a carbon source and incubated at their optimal temperature. That growth defects were observed under these non-stress conditions suggests that these interactions are indicative of defects in normal cell physiology. These results are consistent with a model where physical interactions between the NPC and SAGA are important for transcription of constitutively expressed genes. To test this hypothesis, we used microarray analysis to assess changes global transcript levels in the absence of Nup60, Ada2, or Nup60 and Ada2. Microarray analysis reveals that the growth defect in these double mutants is correlated with a synthetic reduction in steady-state transcript levels for numerous genes that are strongly expressed in wildtype cells. These results suggest that SAGA and Nup60 cooperate in transcriptional regulation, and are consistent with a model for global regulation of SAGA-dependent transcription at the NPC. Wildype, single deletion, and double deletion cells were inoculated to equal starting ODs in 50 mL YEPD media and grown at 30C with shaking. Cells were collected at mid-log phase, washed with chilled water, and stored at -80C for later processing. RNA was isolated using TRIzol reagent according to the manufacturer's protocol, with modification for yeast cells.

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Functional interaction of chromatin with nucleoporins in Drosophila

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