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The transcription coactivator CBP is a dynamic component of the promyelocytic leukemia nuclear body.

Boisvert FM, Kruhlak MJ, Box AK, Hendzel MJ, Bazett-Jones DP - J. Cell Biol. (2001)

Bottom Line: In cells where CBP does not normally accumulate in PML bodies, it can be induced to accumulate in PML bodies through overexpression of either CBP or Pml, but not Sp100.They possess the characteristics expected of proteins that would play a structural role in the integrity of these subnuclear domains.Our results are consistent with CBP being a dynamic component of PML bodies and that the steady-state level in these structures can be modulated by Pml.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, University of Calgary, Calgary, Alberta T2N 4N1, Canada.

ABSTRACT
The transcription coactivator and histone acetyltransferase CAMP response element-binding protein (CBP) has been demonstrated to accumulate in promyelocytic leukemia (PML) bodies. We show that this accumulation is cell type specific. In cells where CBP does not normally accumulate in PML bodies, it can be induced to accumulate in PML bodies through overexpression of either CBP or Pml, but not Sp100. Using fluorescence recovery after photobleaching, we demonstrate that CBP moves rapidly into and out of PML bodies. In contrast, Pml and Sp100 are relatively immobile in the nucleoplasm and within PML nuclear bodies. They possess the characteristics expected of proteins that would play a structural role in the integrity of these subnuclear domains. Our results are consistent with CBP being a dynamic component of PML bodies and that the steady-state level in these structures can be modulated by Pml.

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Colocalization by deconvolution immunofluorescence microscopy of CBP (NH2 terminus antibody; Santa Cruz Biotechnology, Inc.) and Pml (5E10 antibody) proteins in SK-N-SH cells (A, F, K, and P) but not in 293 cells (B, G, L, and Q). Overexpression of GFP–Pml in 293 cells (C and H) allows proper targeting into PML bodies (C) and concentrates CBP in PML bodies (M and R). Overexpression of GFP–Sp100 in 293 cells (D and I) does not concentrate CBP in PML bodies (N and S). Overexpression of GFP–CBP in 293 cells (E and J) targets this protein to PML bodies (O and T). Bar, 5 μm.
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Figure 1: Colocalization by deconvolution immunofluorescence microscopy of CBP (NH2 terminus antibody; Santa Cruz Biotechnology, Inc.) and Pml (5E10 antibody) proteins in SK-N-SH cells (A, F, K, and P) but not in 293 cells (B, G, L, and Q). Overexpression of GFP–Pml in 293 cells (C and H) allows proper targeting into PML bodies (C) and concentrates CBP in PML bodies (M and R). Overexpression of GFP–Sp100 in 293 cells (D and I) does not concentrate CBP in PML bodies (N and S). Overexpression of GFP–CBP in 293 cells (E and J) targets this protein to PML bodies (O and T). Bar, 5 μm.

Mentions: The transcription coactivator CBP is a component of all PML nuclear bodies in HEp-2 (LaMorte et al. 1998), SK-N-SH, COS-1, and CHO cells as detected by immunofluorescence microscopy (Boisvert et al. 2000; Fig. 1A, Fig. F, Fig. K; data not shown). However, we have found that not all cell lines exhibit this property. For example, HeLa, 293, HS-68, and Hep-G2 (Fig. 1 Q; data not shown) cells do not accumulate CBP in PML nuclear bodies. To determine the subnuclear distribution of overexpression of the GFP–CBP fusion protein, 293 cells were transiently transfected with an expression construct coding for this protein. This fusion protein has histone acetyltransferase activity, since the levels of histone H3 and H4 acetylation detected by immunofluorescence rise in proportion to the levels of GFP–CBP overexpression (not shown). This indicates that the GFP tag does not interfere with at least some of CBP's protein–protein interactions. The fusion protein is targeted to PML nuclear bodies as well as being distributed throughout the nucleoplasm (Fig. 1 J). These cells do not normally concentrate CBP in PML nuclear bodies. Furthermore, expression of GFP–Pml (Fig. 1C, Fig. H, Fig. M, and Fig. R) also leads to the accumulation of the endogenous CBP in PML nuclear bodies of these cells (293) (Fig. 1 R). However, overexpression of Sp100 does not bring the endogenous CBP into PML nuclear bodies (Fig. 1D, Fig. I, Fig. N). This shows that the presence of CBP within PML nuclear bodies depends on Pml but not Sp100. Interestingly, expression of Sp100 and Pml increases the number of PML nuclear bodies (Fig. 1H and Fig. I), whereas expression of CBP does not. This indicates that Pml and Sp100, but not CBP, participate in the formation of this nuclear structure.


The transcription coactivator CBP is a dynamic component of the promyelocytic leukemia nuclear body.

Boisvert FM, Kruhlak MJ, Box AK, Hendzel MJ, Bazett-Jones DP - J. Cell Biol. (2001)

Colocalization by deconvolution immunofluorescence microscopy of CBP (NH2 terminus antibody; Santa Cruz Biotechnology, Inc.) and Pml (5E10 antibody) proteins in SK-N-SH cells (A, F, K, and P) but not in 293 cells (B, G, L, and Q). Overexpression of GFP–Pml in 293 cells (C and H) allows proper targeting into PML bodies (C) and concentrates CBP in PML bodies (M and R). Overexpression of GFP–Sp100 in 293 cells (D and I) does not concentrate CBP in PML bodies (N and S). Overexpression of GFP–CBP in 293 cells (E and J) targets this protein to PML bodies (O and T). Bar, 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2198823&req=5

Figure 1: Colocalization by deconvolution immunofluorescence microscopy of CBP (NH2 terminus antibody; Santa Cruz Biotechnology, Inc.) and Pml (5E10 antibody) proteins in SK-N-SH cells (A, F, K, and P) but not in 293 cells (B, G, L, and Q). Overexpression of GFP–Pml in 293 cells (C and H) allows proper targeting into PML bodies (C) and concentrates CBP in PML bodies (M and R). Overexpression of GFP–Sp100 in 293 cells (D and I) does not concentrate CBP in PML bodies (N and S). Overexpression of GFP–CBP in 293 cells (E and J) targets this protein to PML bodies (O and T). Bar, 5 μm.
Mentions: The transcription coactivator CBP is a component of all PML nuclear bodies in HEp-2 (LaMorte et al. 1998), SK-N-SH, COS-1, and CHO cells as detected by immunofluorescence microscopy (Boisvert et al. 2000; Fig. 1A, Fig. F, Fig. K; data not shown). However, we have found that not all cell lines exhibit this property. For example, HeLa, 293, HS-68, and Hep-G2 (Fig. 1 Q; data not shown) cells do not accumulate CBP in PML nuclear bodies. To determine the subnuclear distribution of overexpression of the GFP–CBP fusion protein, 293 cells were transiently transfected with an expression construct coding for this protein. This fusion protein has histone acetyltransferase activity, since the levels of histone H3 and H4 acetylation detected by immunofluorescence rise in proportion to the levels of GFP–CBP overexpression (not shown). This indicates that the GFP tag does not interfere with at least some of CBP's protein–protein interactions. The fusion protein is targeted to PML nuclear bodies as well as being distributed throughout the nucleoplasm (Fig. 1 J). These cells do not normally concentrate CBP in PML nuclear bodies. Furthermore, expression of GFP–Pml (Fig. 1C, Fig. H, Fig. M, and Fig. R) also leads to the accumulation of the endogenous CBP in PML nuclear bodies of these cells (293) (Fig. 1 R). However, overexpression of Sp100 does not bring the endogenous CBP into PML nuclear bodies (Fig. 1D, Fig. I, Fig. N). This shows that the presence of CBP within PML nuclear bodies depends on Pml but not Sp100. Interestingly, expression of Sp100 and Pml increases the number of PML nuclear bodies (Fig. 1H and Fig. I), whereas expression of CBP does not. This indicates that Pml and Sp100, but not CBP, participate in the formation of this nuclear structure.

Bottom Line: In cells where CBP does not normally accumulate in PML bodies, it can be induced to accumulate in PML bodies through overexpression of either CBP or Pml, but not Sp100.They possess the characteristics expected of proteins that would play a structural role in the integrity of these subnuclear domains.Our results are consistent with CBP being a dynamic component of PML bodies and that the steady-state level in these structures can be modulated by Pml.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, University of Calgary, Calgary, Alberta T2N 4N1, Canada.

ABSTRACT
The transcription coactivator and histone acetyltransferase CAMP response element-binding protein (CBP) has been demonstrated to accumulate in promyelocytic leukemia (PML) bodies. We show that this accumulation is cell type specific. In cells where CBP does not normally accumulate in PML bodies, it can be induced to accumulate in PML bodies through overexpression of either CBP or Pml, but not Sp100. Using fluorescence recovery after photobleaching, we demonstrate that CBP moves rapidly into and out of PML bodies. In contrast, Pml and Sp100 are relatively immobile in the nucleoplasm and within PML nuclear bodies. They possess the characteristics expected of proteins that would play a structural role in the integrity of these subnuclear domains. Our results are consistent with CBP being a dynamic component of PML bodies and that the steady-state level in these structures can be modulated by Pml.

Show MeSH
Related in: MedlinePlus