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. 2000 Nov;20(22):8560-70.
doi: 10.1128/MCB.20.22.8560-8570.2000.

DNA replication progresses on the periphery of nuclear aggregates formed by the BCL6 transcription factor

O Albagli  1 , C Lindon, D Lantoine, S Quief, E Puvion, C Pinset, F Puvion-Dutilleul
Affiliations

DNA replication progresses on the periphery of nuclear aggregates formed by the BCL6 transcription factor

O Albagli et al. Mol Cell Biol. 2000 Nov.

Abstract

The BCL6 proto-oncogene, frequently alterated in non-Hodgkin lymphoma, encodes a POZ/zinc finger protein that localizes into discrete nuclear subdomains. Upon prolonged BCL6 overexpression in cells bearing an inducible BCL6 allele (UTA-L cells), these subdomains apparently coincide with sites of DNA synthesis. Here, we explore the relationship between BCL6 and replication by both electron and confocal laser scanning microscopy. First, by electron microscope analyses, we found that endogenous BCL6 is associated with replication foci. Moreover, we show that a relatively low expression level of BCL6 reached after a brief induction in UTA-L cells is sufficient to observe its targeting to mid, late, and at least certain early replication foci visualized by a pulse-labeling with bromodeoxyuridine (BrdU). In addition, when UTA-L cells are simultaneously induced for BCL6 expression and exposed to BrdU for a few hours just after the release from a block in mitosis, a nuclear diffuse BCL6 staining indicates cells in G(1), while cells in S show a more punctate nuclear BCL6 distribution associated with replication foci. Finally, ultrastructural analyses in UTA-L cells exposed to BrdU for various times reveal that replication progresses just around, but not within, BCL6 subdomains. Thus, nascent DNA is localized near, but not colocalized with, BCL6 subdomains, suggesting that they play an architectural role influencing positioning and/or assembly of replication foci. Together with its previously function as transcription repressor recruiting a histone deacetylase complex, BCL6 may therefore contribute to link nuclear organization, replication, and chromatin-mediated regulation.

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Figures

FIG. 1
FIG. 1
Endogenous BCL6 is associated with replication foci in lymphoid Karpas 422 cells. Images show simultaneous visualization at the ultrastructural level of BCL6 and BrdU-containing DNA following a 15-min pulse. The arrow points to a cluster of BCL6 (polyclonal antibody N3 and 5-nm gold particles) located among the BrdU-containing DNA molecules (monoclonal anti-BrdU antibodies and 10-nm gold particles). c, cytoplasm; ch, perinuclear condensed chromatin. Bar, 0.5 μm.
FIG. 2
FIG. 2
BCL6 is associated with replication foci throughout most of S phase. Vertebrate cells undergoing replication show a typical sequence of replication foci patterns. Unsynchronized UTA-L cells were induced for BCL6 expression during 4 h, then pulse-labeled (15 min) with BrdU, and submitted to laser scanning confocal microscopy to detect both BCL6 (red) and BrdU (green) by immunofluorescence. The pattern of replication was assigned to early (A), mid (B and C), and late (D) S phase according to reference (see text). BCL6 appears to be associated with replication foci throughout most of S phase, as its staining concentrates in certain early replication foci (A, arrow), and decorates some mid (B and C, arrow) and late (D) replication foci. Bar, 5 μm.
FIG. 3
FIG. 3
Synchronization of UTA-L cells. UTA-L cells were synchronized in mitosis by a double thymidine block followed by overnight exposure to nocodazole (see Materials and Methods). Poorly attached cells were then collected and stained for relative DNA content with propidium iodide. FACS analyses demonstrated that the vast majority (>95%) of them were in mitosis (A). They were next simultaneously released from the nocodazole block and plated in BrdU-supplemented, tetracycline-free medium to induce BCL6. After 6 h, 50 to 70% of the cells had entered S phase, while almost all of the remaining cells were in G1, as indicated by FACS analyses upon propidium iodide staining (B).
FIG. 4
FIG. 4
BCL6 localization in G1- and S-phase cells. UTA-L cells were synchronized in mitosis, then simultaneously induced for BCL6, and exposed to BrdU as described for Fig. 3. After 6 h, they were submitted to immunofluorescence to detect both BCL6 (red) and BrdU (green) by laser scanning confocal microscopy. We observed that (i) cells displaying a diffuse BCL6 nuclear staining are almost systematically negative for BrdU (top row) and therefore very likely in G1 (see Fig. 3) and (ii) cells positive for both BCL6 and BrdU almost systematically display a punctate BCL6 nuclear distribution showing some coincidence with the BrdU staining (middle row). The two patterns are shown in neighboring cells, indicating that the BCL6 punctate staining does not solely result from higher expression levels. Indeed, the diffuse (presumably in an G1-phase cell [left]), BCL6 staining can be stronger than the punctated, replication-associated distribution observed in an S-phase cell (right) (bottom row). Note that a given nucleus shows some nuclear dots containing both BrdU and BCL6 together, as well as BrdU foci with little or no BCL6 staining and vice versa. Bars, 10 μm.
FIG. 5
FIG. 5
Blocking cell cycle progression alters the subnuclear distribution of BCL6. UTA-L cells were synchronized in mitosis and then both induced for BCL6 and exposed to BrdU for 6 h as described for Fig. 3 in the absence (A) or presence (B) of 0.5 mM l-mimosine, which blocks the cells in late G1 (37). Untreated cells expressing BCL6 typically show either nuclear diffuse or nuclear punctate BCL6 staining (green), corresponding to BrdU-negative (presumably in G1) or BrdU-positive (in S; detected in red) cells, as shown in Fig. 4, top or middle row, respectively (A). In contrast, l-mimosine-treated cells are almost completely devoid of BrdU staining, as expected, and most show nuclear diffuse BCL6 staining (B). Bar, 10 μm.
FIG. 6
FIG. 6
Intranuclear BCL6-containing aggregates are not preferentially associated with condensed chromatin. Images show ultrastructural localization of BCL6 in UTA-L cells induced for 20 h using monoclonal antibody M2. Gold particles which localize BCL6 accumulate over newly formed, electron-dense structures in the nucleoplasm. (A) Three labeled structures (BCL6 macroaggregates; arrows). Two structures exhibit a ring-shaped configuration. (B) BCL6 macroaggregate juxtaposed to the nucleolus. (C) Smaller aggregate (BCL6 microaggregate) located at the border of the nucleus but not in contact with the perinuclear layer of condensed chromatin. Identical results were obtained with either the N3 or C19 polyclonal anti-BCL6 antibody (data not shown). c, cytoplasm; ch, perinuclear condensed chromatin; ig, cluster of interchromatin granules; nu, nucleolus. Bars, 0.5 μm.
FIG. 7
FIG. 7
Newly synthesized DNA progressively wraps around the intranuclear BCL6-containing aggregates. Images show ultrastructural distribution of BrdU-containing DNA in UTA-L cells induced for 20 h. Following short (5-min [A] and 15-min [B]) pulse-labelings, gold particles which localize BrdU are restricted to limited portions of the fibrillar clear halo which surrounds the BCL6 macroaggregates. No labeling occurs over the macroaggregates themselves. Following a 90-min pulse, (C and D), a crown of gold particles entirely surrounds certain BCL6 macroaggregates, while others are only partially surrounded (arrow in panel C). In all cases, however, the BCL6 aggregates themselves are entirely devoid of BrdU labeling, as shown at low magnification in panel C. Bars, 0.5 μm.
FIG. 8
FIG. 8
Newly synthesized DNA and BCL6 are located in two juxtaposed, concentric domains. Images show simultaneous visualization at the ultrastructural level of BCL6 (polyclonal antibodies: N3 [A and B] and C19 [C]) and BrdU-containing DNA (monoclonal anti-BrdU antibody) in UTA-L cells induced for 20 h. (A and B) Gold particles, 5 nm in diameter, label the BCL6 protein, whereas the 10-nm gold particles label the BrdU. (A) Following a short (5-min) pulse, a small cluster of 10-nm gold particles only (arrow) and a cluster of mixed 5- and 10-nm gold particles (double arrow) are close to a BCL6 macroaggregate decorated only by 5-nm gold particles. (B) Following a 90-min pulse, the BCL6 macroaggregate is stained only with anti-BCL6 antibodies (5-nm gold particles), whereas it is surrounded by a crown of 10-nm gold particles only. c, cytoplasm; ch, perinuclear condensed chromatin. (C) In this nucleus, BCL6 constitutes microaggregates (arrows) labeled with the 10-nm gold particles. Following a 15-min pulse, the clumps of BrdU (5-nm gold particles) are closely juxtaposed to the BCL6 microaggregates. Bars, 0.5 μm.
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