Volume 3, Issue 2, Pages 207-217 (February 1999) Dynamic Repositioning of Genes in the Nucleus of Lymphocytes Preparing for Cell Division  Karen E Brown, Jonathan Baxter, Daniel Graf, Matthias Merkenschlager, Amanda G Fisher  Molecular Cell  Volume 3, Issue 2, Pages 207-217 (February 1999) DOI: 10.1016/S1097-2765(00)80311-1

Figure 1 Ikaros Proteins Are Redistributed in the Nucleus of Resting Splenic B Lymphocytes following Cellular Activation Confocal images of ex vivo isolated, resting splenic B lymphocytes at sequential times following in vitro stimulation with anti-CD40 and IL-4 (top, left to right, 0, 1/2, and 1 day) (lower left to right, 11/2, 2, and 3 days) are shown in (a), in which immunofluorescence labeling with antibody to Ikaros (yellow/green) and DNA counterstaining (propidium iodide, red) of representative cells are shown. Data were collected at equivalent magnification and laser intensity, and summed Z series are shown to display the brightest regions of each section. Confocal image (b) shows a single optical section through the nucleus of an activated (day 3) splenic B cell, simultaneously labeled with antibody to Ikaros (green) and γ-satellite probe for centromeric DNA (red) using immuno-FISH. Ikaros proteins in noncycling (R) B lymphocytes and following 3-day stimulation with anti-CD40 antibody and IL-4 (A) or PMA and ionomycin (A′) were compared in Western blotting analysis (c). Lysates were prepared from equivalent numbers of cells (1 × 104) and proteins detected using antibody to C-terminal Ikaros, as indicated. Molecular Cell 1999 3, 207-217DOI: (10.1016/S1097-2765(00)80311-1)

Figure 2 Confocal Images Showing the Position of Target Genes Relative to Centromeric DNA Clusters in Resting and Activated Splenic B Lymphocytes Confocal images of multiple summed (a and b) or single optical sections (c–h) of resting (a, c, and e), 1/2 day (g), or 3-day-stimulated (b, d, f, and h) normal splenic B lymphocytes are shown. In (a) and (b), the clustered distribution of γ-satellite sequences (green) within the nucleus (counterstained red with propidium iodide) of noncycling and stimulated cells is shown, respectively. Confocal images of single optical sections show the position of CD2 alleles (green) (c and d) and λ5 alleles (green) (e and f) in nuclei relative to centromeric γ-satellite DNA (red). Sections that contained both (c and d) or a single allele are shown. For scoring purposes, the position of both alleles in each cell was analyzed in serial optical sections spanning each nucleus. Images (g) and (h) illustrate Ikaros distribution (red) relative to CD8α alleles (green) as assessed by immuno-FISH analysis. Molecular Cell 1999 3, 207-217DOI: (10.1016/S1097-2765(00)80311-1)

Figure 3 Expression and Nuclear Position of Rag and TdT Genes in Developing CD4+CD8+ DP Thymocytes Confocal images of single optical sections of ex vivo DP thymocytes and following overnight culture with anti-TcR antibody are shown (a) in which the position of Rag and TdT alleles (green) relative to γ-satellite (red) are illustrated. The numbers in the gray boxes under each image indicate the proportion of total nuclei examined in which neither of the indicated alleles was associated with γ-satellite DNA (upper panels) and, similarly, the proportion of nuclei in which one or both of the alleles indicated was associated with γ-satellite DNA (lower panels). The expression of Rag-1 and TdT genes in developing T cells was analyzed by Northern blotting and RT-PCR as shown in (b) (left and right panels, respectively). RNA from MHC-deficient CD4+CD8+ thymocytes (lane 1) and following 8 (lane 2) and 24 hr (lane 3) culture on anti-TCR antibody-coated plates was analyzed for Rag-1, TdT, and β-actin mRNA as indicated. Lanes 4–6 show RT-PCR analysis of gene expression by MHC-deficient thymocytes following reaggregate organ culture with MHC-deficient stroma (24 hr control, lane 4), MHC-expressing stroma (24 hr, sorted for CD69 expression, lane 5), or following full differentiation to CD4+ and CD8+ SP cells, and restimulation (as described in Experimental Procedures) (day 10, lane 6). Molecular Cell 1999 3, 207-217DOI: (10.1016/S1097-2765(00)80311-1)

Figure 4 The Downregulation of Rag and TdT Genes in VL3-3M2 Cells Is Transient and Fails to Elicit Nuclear Repositioning Immunofluorescence analyses of surface CD69 and CD5 expression by VL3-3M2 cells before (black histogram) and after stimulation with PMA and ionomycin (24 hr, red histogram) are shown in (a). Green histograms show control staining of stimulated cells with second layer reagent only. Rag-1 and TdT RNA expression by VL3-3M2 cells following PMA/ionomycin induction was analyzed in time course experiments using Northern blotting (b). Similar RNA loading (4 μg/lane) was confirmed using a β-actin probe, and samples of RNA derived from cultures treated with actinomycin D (5 μg/ml) at the time of PMA and ionomycin stimulation, or untreated, were compared for TdT mRNA expression (TdT and TdT′, respectively). Confocal images showing single optical sections of VL3-3M2 cells pre- and 19 hr poststimulation are presented in the top and lower panels of (c), respectively. TdT alleles (green) remain unassociated with γ-satellite regions (red) as summarized in additional data shown in Table 2. In the Northern blotting analyses shown in (d), expression of Rag-1 and TdT RNA was compared among clones derived from PMA/ionomycin-induced (24 hr) cultures. Representative results are shown for 20 of 75 subclones isolated (left hand lanes 1–20), 24 hr stimulated cultures (I), and unstimulated controls (C). Approximately 0.5 μg of RNA was loaded per sample, except in the lanes marked C (a titration of 2 μg, 1 μg, and 0.5 μg is shown), and equivalent RNA loading was verified by control hybridizations with β-actin probe ([d], lower panel). Molecular Cell 1999 3, 207-217DOI: (10.1016/S1097-2765(00)80311-1)

Figure 5 A Model for the Nuclear Repositioning of Genes during Lymphocyte Development A hypothetical candidate gene (yellow box), initially present in the nucleus of an immature lymphocyte is transcriptionally active and remains discrete from centromeric heterochromatin (comprising centromeric DNA, surrounded by centromere-associated proteins such as HP-1 and GAGA, depicted as a gray circle surrounded by black rosette) (left hand panel). As differentiation proceeds, transcription at the candidate gene declines (by repression or neglect), and the locus is repositioned in close spatial proximity with a centromeric domain. When a mature lymphocyte exits from the cell cycle (right hand panel), the spatial association between centromeric domains and the inactive gene relaxes but resumes once the cell enters the cell cycle. Molecular Cell 1999 3, 207-217DOI: (10.1016/S1097-2765(00)80311-1)