Overexpression of the Transcriptional Repressor Complex BCL-6/BCoR Leads to Nuclear Aggregates Distinct from Classical Aggresomes

<div><p>Nuclear inclusions of aggregated proteins have primarily been characterized for molecules with aberrant poly-glutamine repeats and for mutated or structurally altered proteins. They were termed “nuclear aggresomes” and misfolding was shown to promote association with molecular chaperones and proteasomes. Here, we report that two components of a transcriptional repressor complex (BCL-6 and BCoR) of wildtype amino acid sequence can independently or jointly induce the formation of nuclear aggregates when overexpressed. The observation that the majority of cells rapidly downregulate BCL-6/BCoR levels, supports the notion that expression of these proteins is under tight control. The inclusions occur when BCL-6/BCoR expression exceeds 150-fold of endogenous levels. They preferentially develop in the nucleus by a gradual increase in aggregate size to form large, spheroid structures which are not associated with heat shock proteins or marked by ubiquitin. In contrast, we find the close association of BCL-6/BCoR inclusions with PML bodies and a reduction in aggregation upon the concomitant overexpression of histone deacetylases or heat shock protein 70. In summary, our data offer a perspective on nuclear aggregates distinct from classical “nuclear aggresomes”: Large complexes of spheroid structure can evolve in the nucleus without being marked by the cellular machinery for protein refolding and degradation. However, nuclear proteostasis can be restored by balancing the levels of chaperones.</p> </div

BCL-6 aggregate formation upon co-expression of exogenous HSP70 and HSP90.

<p>ECs were transfected with EFp-BCL‑6 in combination with HSP70-1A or HSP90-HA expression plasmid or with EFp-Link control vector. After 12 and 24 hours cells were stained for CLSM imaging with antibodies against BCL-6 and HSP70 or with antibodies against BCL-6 and HA-tag for HSP90 detection. Hoechst 33342 was applied to counterstain nuclear DNA. (A) CLSM images demonstrate the concomitant overexpression of BCL-6 with HSP70 or HSP90 at 24 hours after transfection (scale bars: 10 µM). (B) The percentage of BCL-6 positive cells was determined from four randomly acquired tile scans (40x objective) covering approximately 700 cells. Mean values and standard deviations from two independent experiments are given; T-test revealed no statistically significant differences. (C) 35 randomly selected nuclei with BCL-6 aggregations were analyzed (per condition) for the diameter of their aggregates. The boxplot illustrates the distribution of acquired values with a statistically significant reduction in aggregate diameter upon co-expression of HSP70 after 12 h (T-test; p < 0.001) and 24 h (T-test; p = 0.001). (D) 300 BCL-6 positive cells (per condition) were classified according to their nuclear BCL-6 expression pattern into diffuse (no aggregation), small punctate, large spherical and additional cytosolic aggregates. Mean values and standard deviations were calculated from 2 independent experiments. The frequency of large, spherical aggregates was significantly reduced upon co-expression of BCL-6 and HSP70 after 24 h (T-test; p = 0.031). *, p < 0.05; **, p < 0.01; ***, p < 0.001.</p

Relation of BCL-6/BCoR aggregates to components of the nuclear pore complex.

<p>ECs were transfected with EFpBCL-6 or EFp-BCoR-A (A) or with a combination of both plasmids (B). Cells were processed after 12 hours with Hoechst 33342 DNA stain and with antibodies against NUPs, BCL-6 and BCoR for CLSM imaging. Scale bars: 5 µm (applying to all panels). The last lane shows magnifications of the indicated regions (white rectangles).</p

Endogenous HSP70 expression in cells with BCL-6/BCoR aggregates.

<p>ECs were transfected with EFp-BCL‑6, EFp-BCoR-A, EGFP-BCL-6 or EGFP-C3 plasmid and processed for CLSM imaging 12 hours after transfection. All cells were stained with α-HSP70 antibody and Hoechst 33342. EFp-BCL-6 and EFp-BCoR-A transfected cells were further immunostained with antibodies against BCL-6 or BCoR. BCL‑6/BCoR aggregates without HSP70 association are indicated by arrow heads. Regions of co-localization are indicated by arrows and marked in yellow. Scale bar: 10 µm (applying to all panels).</p

BCL-6/BCoR aggregates during cell cycle progression.

<p>(A) Confocal images of ECs transfected with EFp-BCL-6 or EFp-BCoR-A, cultured for 18 h and subsequently loaded with BrdU for 1 h. Cells were immunostained with antibodies against BCL-6 or BCoR, and with antibodies against BrdU and H3(pS28) to detect ECs in S-phase or mitosis, respectively. Cells in G0/G1 phase are reflected by the absence of BrdU and H3(pS28) staining. (B) Confocal images of EFp-BCL-6 or EFp-BCoR-A transfected ECs, processed with antibodies against BCL-6 or BCoR and with DNA stain Hoechst 33342 to illustrate the appearance of cytosolic aggregates in neighboring cells. (C) Based on confocal images exemplified in (A), 2000 cells were counted and classified in G0/G1, S or M/G2 phase. The BCL-6 positive and negative (non-transfected) cell populations were compared for their cell cycle distribution. (D) Cell cycle distribution as established by PI stain and flow cytometry of ECs at 2 to 24 h after transfection with EGFP-BCL-6 or EGFP-C3 control plasmid. Only EGFP positive cells were evaluated. Scale bars: 5 µm.</p

BCL-6/BCoR protein distribution upon overexpression in primary ECs.

<p>ECs were transfected with EFp-BCL-6 (A), EFp-BCoR-A (B) or a combination of the two expression plasmids (C). 24 hours after transfection cells were fixed and stained for CLSM imaging with antibodies against BCL-6 and/or BCoR. Hoechst 33342 was applied to counterstain nuclear DNA. Regions of co-localization of BCL-6 and BCoR protein are indicated in yellow; non-transfected cells are labeled by an asterisk. Scale bars: 5 µm. (D and E) ECs were transfected with 0, 2, 10 or 20 µg of EFp-BCL-6. The total amount of DNA was adjusted to 20 µg by the addition of EFp-Link control vector to standardize transfection conditions. Twelve hours later cells were subjected to immunostaining with BCL-6 antibody and Hoechst 33342 DNA stain and were analyzed with a TissueFAXS fluorescence detection system suited for the automated analysis of staining intensities in adherent cell cultures. The mock-treated control was used to determine the level of endogenous protein. BCL-6 expression in transfected cells is given in fold of mean control and divided in categories of low (> 2 fold), medium (> 4 fold) and high (> 6 fold) intensity. The scatter plot of DNA and BCL-6 fluorescence as recorded in 340 msec of detection is shown in (D) for the transfection with 10 µg EFp-BCL-6. For better resolution of high expressors, a second scan of 25 msec was performed (E). The frequency of transfected cells, the percentage of low, medium and high expressors and the occurrence of nuclear aggregates are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076845#pone-0076845-t001" target="_blank">Table 1</a>.</p

Time course of EGFP-BCL-6 expression in relation to cell death.

<p>ECs were transfected with EGFP-BCL-6 or EGFP-C3 control plasmid. Discrimination of dead and live cells was based on the endothelial characteristic that dying cells detach in culture, and detached cells do not survive in suspension. Thus, live (adherent) cells as well as dead (non-adherent) cells were harvested and analyzed by flow cytometry for EGFP fluorescence. The percentage of EGFP-BCL-6 or EGFP positive cells was compared in the live (A) and dead (B) cell populations over a time period of 24 h post transfection. (C) PI staining of the adherent cell population was applied to detect cells with hallmarks of necrosis or late apoptosis (sub G1 DNA content). Only EGFP positive cells were evaluated. Mean values and standard deviations were calculated from two independent experiments.</p

Association of overexpressed BCL-6/BCoR protein with HDACs.

<p>ECs were transfected with FLAG-tagged expression constructs of HDAC1 (A), HDAC3 (B), HDAC4 (C), HDAC5 (D), HDAC6 (E) or HDAC7 (F) and processed for CLSM imaging 12 hours after transfection with an antibody directed against the FLAG-tag and with DNA stain (Hoechst 33342). The first lane illustrates HDAC distribution without concomitant BCL-6/BCoR overexpression. All other images were derived from ECs exposed to co-transfection of HDAC expression constructs and EFp-BCL-6 or EFp-BCoR-A plasmid. Cells were additionally stained with antibodies against BCL‑6 or BCoR. The last panel shows magnifications of indicated regions (white rectangles). Arrows and arrowheads in the merged pictures point to regions of co-localization of HDACs with BCL-6 or BCoR. Scale bars: 5 µm (applying to all panels).</p

Structure and nuclear reorganization of BCL-6/BCoR inclusions.

<p>ECs were transfected with EFp-BCL-6 (A) or EFp-BCoR-A (B and C) expression plasmid and processed with Hoechst 33342 DNA stain and antibodies against BCL-6 or BCoR for CLSM imaging after 24 hours. (A and B) Compilation of Z-stack series into orthogonal projections. (C) Comparison between fluorescence and light transmission (gray scale) images of BCoR-A transfected cells. Scale bars: 5 µm.</p

Impact of BCL-6/BCoR aggregates on the nuclear distribution of PML bodies.

<p>ECs were transfected with EFp-BCL-6 or EFp-BCoR-A DNA and processed for CLSM imaging after 24 hours. Antibodies against BCL-6 or BCoR were combined with α-PML antibody for immunostaining. Association of PML bodies with small punctate BCL-6/BCoR aggregates is marked by arrowheads. Arrows indicate incorporation of PML protein in the rim of large spheroid inclusions. Scale bars: 5 µm.</p