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. 1998 Jun 23;95(13):7316-21.
doi: 10.1073/pnas.95.13.7316.

Interaction of SP100 with HP1 proteins: a link between the promyelocytic leukemia-associated nuclear bodies and the chromatin compartment

J S Seeler  1 , A Marchio, D Sitterlin, C Transy, A Dejean
Affiliations

Interaction of SP100 with HP1 proteins: a link between the promyelocytic leukemia-associated nuclear bodies and the chromatin compartment

J S Seeler et al. Proc Natl Acad Sci U S A. .

Abstract

The PML/SP100 nuclear bodies (NBs) were first described as discrete subnuclear structures containing the SP100 protein. Subsequently, they were shown to contain the PML protein which is part of the oncogenic PML-RARalpha hybrid produced by the t(15;17) chromosomal translocation characteristic of acute promyelocytic leukemia. Yet, the physiological role of these nuclear bodies remains unknown. Here, we show that SP100 binds to members of the heterochromatin protein 1 (HP1) families of non-histone chromosomal proteins. Further, we demonstrate that a naturally occurring splice variant of SP100, here called SP100-HMG, is a member of the high mobility group-1 (HMG-1) protein family and may thus possess DNA-binding potential. Both HP1 and SP100-HMG concentrate in the PML/SP100 NBs, and overexpression of SP100 leads to enhanced accumulation of endogenous HP1 in these structures. When bound to a promoter, SP100, SP100-HMG and HP1 behave as transcriptional repressors in transfected mammalian cells. These observations present molecular evidence for an association between the PML/SP100 NBs and the chromatin nuclear compartment. They support a model in which the NBs may play a role in certain aspects of chromatin dynamics.

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Figures

Figure 1
Figure 1
Yeast two-hybrid interaction experiments. (A) A LexA DBD fusion of SP100A interacts with GAL4 AD fusions of human (HP1α, −β, −γ) and Drosophila (dHP1) heterochromatin 1 proteins. The indicated expression plasmids were cotransformed into yeast strain L40 and assayed for β-galactosidase reporter activity. (B) SP100A and the indicated deletion derivatives, fused to the GAL4 DBD, were expressed in yeast strain Y-190 together with the GAL4 AD (negative control) or a GAL4 AD fusion of HP1α. (C) GAL4 AD fusions of HP1α and deletions were transformed into yeast with the GAL4 DBD alone (control) or a GAL4 DBD fusion of SP100A. Interactions were quantified by using β-galactosidase reporter activity, such that “−” corresponds to <1, “+” to 1–10, “++” to 10–100, and “+++” to >100 β-galactosidase units. The deduced interaction domains of each protein are shaded grey. All interactions also were verified against GAL4 DBD or −AD fusions of unrelated proteins (data not shown).
Figure 2
Figure 2
(A) Schematic diagram of SP100A (GenBank accession no. M60618), SP100B (accession no. U36501), and SP100-HMG (accession no. AF056322) proteins discussed in this study. The dimerization and HP1-binding domains as well as the position of the HMG-1-like domain containing the conserved A and B boxes and acidic domains (E) are indicated. (B) Amino acid alignment of the HMG-1 domain of SP100-HMG with the two human homologs, HMG-1 and HMG-2. Only residues differing from SP100-HMG are given. Sequence in lower case corresponds to SP100B-derived residues; sequence in bold is neither SP100B-derived nor homologous to HMG-1. Asterisks indicate the extent of conserved α-helices in the HMG-1 and -2 proteins.
Figure 3
Figure 3
SP100 homodimerizes and interacts with HP1α in vitro. (A) The indicated GST fusion proteins coupled to glutathione Sepharose beads were incubated with 35S-labeled in vitro translated SP100A (lanes 2–6), SP100-HMG (lanes 8–12), and HP1α (lanes 14–17) and washed, eluted, and subjected to electropheresis and autoradiography to reveal the bound radiolabeled proteins. Twenty percent of the input for each radiolabeled protein is shown in lanes 1, 7, and 13. (B) Amino acid alignment of the human [SP100(287–334)] and murine [mSP100(233–280)] HP1-binding domains of SP100. Identical residues are joined by lines, conservative substitutions by dots, and periods indicate alignment gaps.
Figure 4
Figure 4
SP100-HMG and HP1α are targeted to the PML/SP100 NBs. HeLa cells were transfected with plasmid constructs expressing HA-tagged SP100-HMG (A–C), HP1α(1–191) (D–F), the CSD-containing HP1α(58–191) (G–I), and the CD-containing HP1α(1–158) (J–L) or SP100A (P–R) proteins, or were left untransfected (M–O), or were treated with 200 units/ml interferonα for 16 hr (S–U), as indicated. Cells in A–L were permeabilized with TritonX-100 after fixation with formaldehyde, whereas cells in M–U were briefly extracted with TritonX-100 before formaldehyde fixation to remove excess soluble proteins. The overexpressed proteins were revealed with antibodies against the HA tag (HA; 12CA5 mAb in A, D, G, and J or the SC802 rabbit polyclonal serum in Q). Native NB components were visualized with rabbit sera against PML (B) or SP100 (E, H, K, N, and T). A murine polyclonal serum (“HP1” in M, P, and S) was used to stain endogenous HP1α. The secondary antibodies used were conjugated to fluorescein isothiocyanate- (Left, green) or to TexasRed (Center, red). Confocal overlay of red and green panels yields yellow in the Right panels (C, F, I, L, O, R, and U). (Scale bar, in U, = 10 μm.)
Figure 5
Figure 5
HP1α, SP100A, and SP100-HMG behave as transcriptional repressors when tethered to DNA. The GAL4 DBD alone, or GAL4 DBD fusions of HP1α, SP100A, or SP100-HMG were transfected into HeLa cells together with a plasmid containing five GAL4-binding sites fused to the β-globin (A) or the thymidylate kinase (B) minimal promoters upstream of the CAT reporter gene. CAT reporter activity was normalized for transfection efficiency to an internal β-galactosidase control and expressed as a percentage of the activity obtained with the GAL4 DBD alone. Transfections were carried out with 3 μg each of reporter and effector plasmids.
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References

    1. Weis K, Rambaud S, Lavau C, Jansen J, Carvalho T, Carmo-Fonseca M, Lamond A, Dejean A. Cell. 1994;76:345–356. - PubMed
    1. Maul G G, Yu E, Ishov A M, Epstein A L. J Cell Biochem. 1995;59:498–513. - PubMed
    1. Dyck J A, Maul G G, Miller W H, Jr, Chen J D, Kakizuka A, Evans R M. Cell. 1994;76:333–343. - PubMed
    1. Ascoli C A, Maul G G. J Cell Biol. 1991;112:785–795. - PMC - PubMed
    1. Xie K, Lambie E J, Snyder M. Mol Cell Biol. 1993;13:6170–6179. - PMC - PubMed

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