LETTERS Transcriptional repression mediated by repositioning of genes to the nuclear lamina K. L. Reddy 1,2 , J. M. Zullo 1,2 , E. Bertolino 2 & H. Singh 1,2 Nuclear compartmentalization seems to have an important role in regulating metazoan genes 1,2 . Although studies on immuno- globulin and other loci have shown a correlation between posi- tioning at the nuclear lamina and gene repression, the functional consequences of this compartmentalization remain untested 2,3 . We devised an approach for inducible tethering of genes to the inner nuclear membrane (INM), and tested the consequences of such repositioning on gene activity in mouse fibroblasts. Here, using three-dimensional DNA-immunoFISH, we demonstrate repositioning of chromosomal regions to the nuclear lamina that is dependent on breakdown and reformation of the nuclear enve- lope during mitosis. Moreover, tethering leads to the accumulation of lamin and INM proteins, but not to association with pericentro- meric heterochromatin or nuclear pore complexes. Recruitment of genes to the INM can result in their transcriptional repression. Finally, we use targeted adenine methylation (DamID) to show that, as is the case for our model system, inactive immunoglobulin loci at the nuclear periphery are contacted by INM and lamina proteins. We propose that these molecular interactions may be used to compartmentalize and to limit the accessibility of immuno- globulin loci to transcription and recombination factors. In mammalian nuclei, chromatin is organized into structural domains by association with distinct nuclear compartments 2 . Several studies have shown a correlation between the transcriptional repression of mammalian genes and their positioning at the nuclear periphery 3–7 . In yeast, the nuclear periphery is comprised of at least two sub-compartments: a repressive compartment consisting of foci of silencing factors, and a permissive compartment involving nuclear pore complexes (NPCs) that facilitates gene expression 8–10 . However, metazoan systems exhibit a greater complexity of nuclear compart- ments and chromosome organization. The nuclear periphery in mammalian cells is constituted by a distinct set of INM proteins, such as LBR, LAP2 and emerin (EMD), as well as an underlying nuclear lamina, which have been proposed to interact with transcrip- tional repressors 11–14 . The ability of this nuclear compartment to regulate gene activity has not been functionally tested in metazoan cells 2 . We designed a two-component inducible system that would relo- calize an integrated reporter gene from the interior of a mammalian nucleus to the INM (Fig. 1a). The reporter construct is comprised of the herpes simplex virus thymidine kinase promoter and the hygro- mycin resistance gene (Tk-hyg) as well as a nearby array of Lac opera- tors (lacO) that constitute binding sites for the Escherichia coli Lac repressor (LacI) (Fig. 1 and Supplementary Fig. 1a) 15 . The second component is either a nucleoplasmic green fluorescent protein (GFP)–LacI that binds lacO sites and enables visualization of the reporter gene or a tethering protein GFP–LacI–DEMD that is tar- geted to the INM by means of a carboxy-terminal segment of EMD 16 . The GFP fusion proteins were stably expressed in NIH3T3 fibroblast clones harbouring the reporter gene(s) integrated at single (S) or multiple (M) chromosomal sites. Reporter gene visualization and/or repositioning were controlled using the allosteric inhibitor IPTG (isopropyl b-D-1-thiogalactopyranoside), which regulates LacI bind- ing to lacO sites. The initial disposition of the integrated reporter genes was analysed in cells stably expressing GFP–LacI. Up to four bright GFP foci were visible in clone-M nuclei because these cells have four integration sites, each containing multiple copies of the reporter gene (Supplementary Fig. 1d, e). In contrast, clone-S nuclei exhibited dimmer single GFP foci owing to a single site of insertion with fewer copies (1–2) of the reporter (Supplementary Fig. 1c, d). We next generated clone-M and clone-S derivatives stably expressing GFP– LacI–DEMD. As anticipated, this tethering protein localized to the INM. On removal of IPTG, large GFP foci were observed at the nuclear periphery in clone-M but not in clone-S cells expressing GFP–LacI–DEMD (Supplementary Fig. 1e). This suggested that the reporter genes were being repositioned to the nuclear membrane in clone-M cells. Not all tethered reporter genes were expected to accumulate the fusion protein at levels that are discernable as fluorescent signals above the distribution in the INM. This was probably the case for clone-S cells. Therefore, we undertook fluorescent DNA in situ hybridization on three-dimensional preserved nuclei (3D DNA- immunoFISH) to assess quantitatively the disposition of all Tk-hyg integrations (Fig. 1b, c and Supplementary Fig. 2). Under control conditions, the integrated reporter genes were distributed through- out the nucleoplasm, with approximately 25–30% being positioned near the nuclear periphery (Fig. 1d). This frequency represents the initial sub-nuclear distribution and is similar to that observed for endogenous genes that are not associated with the nuclear peri- phery 17 . On withdrawal of IPTG, most Tk-hyg insertions were found to be associated with the nuclear lamina in clone-M (70%) and clone- S (90%) cells expressing GFP–LacI–DEMD. Moreover, in clone-M cells, reporter genes residing on different chromosomes were repositioned to distinct regions of the INM in a single nucleus (Fig. 1b). In clone-S cells, repositioning was mediated by fewer copies of the lacO segments (1–2) compared with in clone-M cells (,25 copies per integration site, Supplementary Fig. 1d). We note that repositioning requires breakdown and reformation of the nuclear envelope during mitosis (Supplementary Fig. 3 and Supplementary Discussion). These data provide the first demonstration of directed repositioning of chromosomal segments to the INM–lamina com- partment, and suggest that an intervening cell cycle may be necessary for such re-configuration. We next analysed the consequences of accumulating GFP–LacI– DEMD at sites of tethering on the disposition of other proteins at the INM. Lamin A and B1, key components of the lamina, and the INM protein LAP2 accumulated at sites of tethering (Fig. 2a and Supple- mentary Fig. 4). No such interactions were observed on non-tethered 1 Howard Hughes Medical Institute, 2 Department of Molecular Genetics and Cell Biology, The University of Chicago, GCIS W522, 929 East 57th Street, Chicago, Illinois 60637, USA. Vol 452 | 13 March 2008 | doi:10.1038/nature06727 243 Nature Publishing Group ©2008