Major protein domains of the SP100 variant proteins generated by alternative splicing. Schematic representation of the SP100A, SP100B, SP100-HMG, and SP100C protein variants derived from alternatively spliced mRNAs (the corresponding cDNA sequences are deposited in the GenEMBL database under accession numbers M60618 [52], U36501 [9], AF056322 [18, 48], and AF255565 [this study], respectively). Brackets indicate the extent of the major domains: A, B, C′, HMG(SP), and C. Note that domains A, B, and C′ are shared by SP100-HMG and SP100C. The positions of the HSR dimerization, HP1-binding, and SAND domains, as well as of the PHD and bromodomains, are indicated.

(A) Nucleotide and deduced amino acid sequence of the SP100C variant. The nucleotide numbering of the original SP100A sequence (GenEMBL accession number M60618) has been retained, but only the C termini of the SP100B-specific domain (B domain), the C′ domain and the C domain are shown here, as indicated above the sequence. Bold slashes in the cDNA sequence indicate exon boundaries deduced from analysis of HTGS sequences (GenEMBL accession numbers AC010149 and AC022295). (B) Expression of SP100C mRNA. RT-PCR was performed on total RNA extracted from HeLa and NB4 cells untreated (−) or treated with 200 U of IFN-α per ml for 16 h (I) or 1 μM all-trans-retinoic acid for 48 hrs (R) as indicated, using a primer pair combination spanning nucleotides 69 to 2879 of the SP100C cDNA (upper panel). Subcloning and sequencing of reaction products confirmed the specific amplification of SP100C cDNA and furthermore, the existence of minor splice variants (data not shown), as found previously by Guldner et al. (18). Control amplification (C) was performed on cDNA reaction products in which RT had been omitted. The amount of template cDNA was normalized by PCR with primers directing the synthesis of vinculin (lower panel). Interestingly, IFN-α treatment of NB4 cells does not lead to a marked increase in SP100C expression. (C) Amino acid alignment of the SP100C domain with homologous domains from LYSP100B (GenEMBL accession number U36500) and mouse TIF1α (GenEMBL accession number S78219). Amino acid identities are shaded grey. The PHD finger and bromodomains as defined by Aasland et al. (1) and Jeanmougin et al. (21), respectively, are boxed.

The SP100 variants display distinct subnuclear localization behavior. HeLa cells grown on coverslips were transfected with expression plasmids encoding HA-tagged SP100A (A), SP100-HMG (B), or SP100C (C), immunolabeled with anti-HA monoclonal antibody 12CA5 (left panels) or rabbit polyclonal antibodies against PML (middle panels), and revealed with anti-mouse immunoglobulin G–fluorescein isothiocyanate (green) or anti-rabbit immunoglobulin G–Texas red (red) secondary antibodies, respectively, for confocal immunofluorescence microscopy. Colocalization appears yellow in the merged images (right panels). Four representative nuclei (i to iv) are shown in each panel, arranged in order of increasing apparent expression level. Arrow, densely packed aggregate at high expression levels.

(A) SP100-HMG and SP100C are covalently modified by SUMO-1 in vivo. Whole-cell extracts (WCE) of HeLa cells transfected with vectors expressing HA-tagged SP100-HMG or SP100C or His-tagged SUMO-1, as indicated, were either immunoblotted with anti-HA antibody directly (lanes 1 to 6) or subjected to Ni affinity chromatography prior to blotting (Ni ppte.) (lanes 7 to 12). The arrowhead marks the position of the SUMO-1-conjugated species. Note the presence of conjugates modified by the endogenous SUMO protein(s) (lanes 3 and 5) which are not precipitated by Ni affinity beads. (B) SUMO modification in vitro. [³⁵S]methionine-labeled, in vitro-translated SP100-HMG and SP100C and their K297R point mutant derivatives, as indicated, were subjected to the in vitro modification reaction in the presence (even lanes) or absence (odd lanes) of recombinant SUMO-1 protein. Arrowheads mark the position of SUMO-1 conjugates.

(A) TIF1α is covalently modified by SUMO-1 in vivo. HeLa cells were transfected with vectors expressing TIF1α, SUMO-1, or His–SUMO-1, as indicated (lanes 1 to 12). Whole-cell extracts (WCE) prepared under denaturing conditions were either trichloroacetic acid (TCA) precipitated (lanes 1 to 6) or subjected to Ni-agarose chromatography (Ni Ppt.) (lanes 7 to 12) prior to electrophoresis and Western blotting with the anti-TIF1α monoclonal antibody 5T. Arrowheads indicate the positions of the TIF1α–SUMO-1 conjugates. TCA precipitates of extracts from NIH 3T3 cells (lanes 13 and 14) were Western blotted with two different mouse TIF1α-specific monoclonal antibodies, 5T (lane 13) and 6T (lanes 14). Arrowheads indicate the positions of the two TIF1α-SUMO conjugates. (B) Lysines at positions 690 and 708 of TIF1α are the SUMO-1 conjugation sites. HeLa cells were transfected with vectors expressing wild-type (WT) TIF1α or Lys-to-Arg point mutations at position 690, 708, or both, either in the presence (+) or in the absence (−) of a His–SUMO-1 expression vector, as indicated. Whole-cell extracts were either TCA precipitated (lanes 1 to 10) or purified on nickel-agarose (lanes 11 to 20) before being subjected to Western blotting with anti-TIF1α monoclonal antibody 5T. The positions of the TIF1α–SUMO-1 conjugates are indicated by arrows. (C) Mutation of SUMO target lysines does not affect nuclear localization of TIF1α. HeLa cells were transfected with plasmids for wild-type (left panel) or mutant (K690/708R; right panel) TIF1α and treated for immunofluorescence analysis with anti-TIF1α monoclonal antibody 5T and fluorescein isothiocyanate-conjugated anti-mouse secondary antibodies. Phase-contrast (PhC) images confirm the nucleoplasmic (extranucleolar) distribution of both proteins.

(A) SUMO-1 modification of SP100A enhances binding to HP1α in vitro. [³⁵S]methionine-labeled, in vitro-translated SP100A protein was incubated with the SUMO-1 conjugation reaction mixture in the absence (lane 1) or presence (lane 2) of recombinant SUMO-1 protein. This mixture of unmodified and SUMO-1-modified (arrowhead) SP100A protein was then used in a pull-down assay with GST (negative control [lane 3]), GST-SP100A (positive control [lane 4]), or GST-HP1α (lane 5) immobilized on glutathione-Sepharose beads. Quantitative PhosphorImager analysis was used to determine the percentage of SUMO-1-modified SP100A protein in the input (lane 2) as well as the proteins eluted from the beads (lanes 4 and 5). To rule out the possibility that the enhancement of HP1α binding on SUMO modification requires dimerization of SP100A, a similar experiment (lanes 6 to 10) was carried out with the mutant SP100AΔN protein in which the N-terminal 129 aa (the HSR dimerization domain) are deleted. (B) SUMO-1 modification of TIF1α in vitro does not significantly affect HP1α -binding. In vitro-translated wild-type TIF1α (lanes 1 and 2) and mutants with the Lys-to-Arg point mutations at position 690, 708, or both, as indicated, were subjected to the modification reaction in the presence (+) or absence (−) of recombinant SUMO-1 protein. In vitro-modified, wild-type TIF1α was used in a pull-down assay with GST (lane 10, negative control) and GST-HP1α (lane 11) as in panel A; the modification led to the appearance of two slower-migrating bands (arrowheads).