The N-terminal domains of SAP30L and SAP30 bind DNA in a sequence-independent manner. (A) A ³²P-labeled DNA probe was incubated with GST fusion proteins, and the DNA-protein complexes were analyzed by an EMSA. (B) Median fluorescence intensities of all microarray probe oligonucleotides containing a particular 8-mer sequence, from PBM experiments performed with GST, GST-SAP30L, GST-SAP30, and GST-Cbf1.

The N-terminal domains of SAP30L and SAP30 contain an evolutionarily conserved zinc-binding module which is needed for DNA binding. (A) Clustal V alignment of the N-terminal amino acid sequences of SAP30L and SAP30 from various (selected) animals. Conserved cysteine (positions 29, 30, 38, and 74) and histidine (positions 70 and 77) residues in SAP30L are boxed, and the previously identified NLS is shaded in gray. Identical amino acids are marked by asterisks, and conservative substitutions are indicated by punctuation marks (colons and dots). (B) Charge-deconvoluted ESI Q-FT-ICR mass spectra of wild-type SAP30L in denaturing (upper) and nondenaturing (lower) solutions. The most abundant isotopic masses for the detected peptide variants are indicated. The insets show an expanded view of the isotopic distributions for the variant comprising residues 1 to 94 (this construct contains aa 1 to 92 of SAP30L and two amino acids derived from the thrombin cleavage site of the vector). The theoretical isotopic distributions were calculated from the sequence-derived elemental compositions (Table 1). The small arrow indicates the most abundant isotopic peak. (C) The SAP30L C29S, C38S, C74S, and H77A mutant constructs are degraded within 16 h after cleavage from GST, as analyzed by SDS-PAGE and Coomassie blue staining. wt, wild type. (D) L-EMSA with the GST-SAP30/SAP30L fusion proteins. (E) L-EMSA with the GST-SAP30L 1-92 fusion protein in the presence of 50 mM 1, 10-o-phenanthroline, a zinc-chelating agent.

SAP30L bends DNA. (A) Probes of various lengths (170, 150, 130, 110, 90, and 70 bp), labeled internally with ³²P and containing EcoRI “sticky ends,” were incubated with GST only (lanes 4, 9, 14, 19, 24, and 29) or with GST-SAP30L (lanes 5, 10, 15, 20, 25, and 30). The reaction mixtures for lanes 2 to 5, 7 to 10, 12 to 15, 17 to 20, 22 to 25, and 27 to 30 were incubated in the presence of T4 DNA ligase at 30°C for 20 min. The reaction mixtures for lanes 3 to 5, 8 to 10, 13 to 15, 18 to 20, 23 to 25, and 28 to 30 were subsequently treated with exonuclease (exo) III to remove any linear ligation products. The reaction products were electrophoresed on a 7% polyacrylamide gel, which was dried and subjected to autoradiography. Mono-, di-, and tricircular DNA ligation products are indicated by the numbered circles. nt, nucleotides or base pairs. (B) Increasing amounts of GST-SAP30L 1-92 were used in the ligation reactions, which were performed as described above.

SAP30L and SAP30 bind histones and nucleosomes. (A) Schematic representation of the domain architecture of the HMG proteins, SAP30L, and SAP30. The charge average of SAP30L is presented as a sliding window of 10 aa. Surface probability predictions were performed using the approach of Emini (11), and values greater than 2 are shown as black lines. Zn, zinc-binding module; DNAbd & bending, DNA-binding and -bending domain. (B) GST fusion protein pulldowns of calf thymus histones (Roche) were analyzed by SDS-PAGE and stained with Coomassie blue. The asterisks mark the GST fusion proteins, and the arrows indicate interaction with histones 2A/2B. (C) GST fusion protein pulldowns of intact nucleosomes, of nucleosomes from which the tails had been removed with trypsin (see Fig. S4 in the supplemental material), and of calf thymus histones. WB, Western blot. (D) HEK293T cells were cotransfected with an H2B-GFP fusion protein and either wild-type SAP30L or SAP30Ldel109-113 containing a myc-His tag. The cells were stained with the anti-myc antibody, and nuclei positive for both H2B and GFP were scored from 50 cells. The results are illustrated in the histogram. (E) GST fusion pulldowns of intact nucleosomes were analyzed using a Western blot probed with the anti-H2B antibody.

The zinc-binding structure and the PBR in SAP30L and SAP30 bind monophosphoinositides. (A) Manual alignment of the sequences of the PBRs following the zinc-binding modules in SAP30L, SAP30, and Pf1. The last zinc-coordinating residue is boxed, and the basic residues are indicated with bold letters. (B) Schematic diagram of a lipid blot membrane (PIP strip) containing 20-pmol spots from samples of the following: lysophosphatidic acid (LPA), lysophosphocholine (LPC), PI (PtdIns), PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, phosphatidylethanolamine (PE), phosphatidylcholine (PC), sphingosine 1-phosphate (S1P), PtdIns(3,4)P₂, PtdIns(3,5)P₂, PtdIns(4,5P)₂, PtdIns(3,4,5)P₃, phosphatidic acid (PA), phosphatidylserine (PS), and blank. (C, D and E) The indicated GST fusion proteins (0.5 μg/ml) were incubated with PIP strips or with the PIP array as described in Materials and Methods. The lipids which bound most strongly are indicated. Arrows indicate the specificity differences from the 1-92 construct.

Chromatin association, subcellular localization, and transcriptional repression activities of SAP30L are regulated by its interactions with DNA and monophosphoinositides. (A) The SAP30L-GFP fusion protein colocalizes with interphase chromatin. (B) HEK293T cells were transfected with the indicated constructs, fractionated into subcellular fractions, and immunoblotted as indicated. Data from three independent experiments are illustrated as histograms, in which the bars represent the ranges of band intensities as measured by a densitometer. wt, wild type. (C) L-EMSA with GST-SAP30L 1-92 in the presence of equivalent molar quantities of PI (PtdIns) or PtdIns(5)P. (D) Cells were transfected with wild-type SAP30L, treated with H₂O₂, and subjected to subcellular fractionation as described for panel B. (E) Confocal images of cells transfected with SAP30L-GFP and treated with H₂O₂. (F) (Upper) In vitro-translated, ³⁵S-methionine-labeled Sin3A1-855 was subjected to a pulldown experiment with GST-SAP30L and a GST-only control. PtdIns(5)P was added as indicated, and the results from the experiment were analyzed by SDS-PAGE and autoradiography. (Lower) Pulldown of nucleosomes with GST-SAP30L in the presence of PIs. (G) HEK293T cells were cotransfected with a 5× Gal4-14D luciferase reporter vector, GAL4DBD fusions, and a LacZ vector, as indicated. At 24 h posttransfection, the cells were treated with 500 μM H₂O₂ for 15 min, washed, and lysed after 4 h of incubation. Luciferase and β-galactosidase activities were measured, and the histograms illustrate the average repressions of the GAL4DBD fusions relative to the level for GAL4 alone. The measurements were done in duplicate in two independent experiments, and the bars represent the ranges of observed values.

Domain structure and a proposed mode of action for SAP30L and SAP30. (A) Schematic representation of the N-terminal zinc-coordinating motif of SAP30L. (B) Various domains of SAP30L identified in this and other studies (35, 57). Zn, zinc-coordinating motif; DNAbd, DNA-binding domain; PIPbd, PIP binding domain; protein bd, protein binding domain; acidic region, a central region contributing to histone binding. (C) Proposed model. (Panel 1) When the histones are acetylated, the DNA is loosely packed and therefore accessible to RNA polymerase II. (Panel 2) A sequence-specific transcriptional repressor (TF) recruits the Sin3A complex to its target promoter. SAP30 or SAP30L stabilizes the complex through interactions with DNA and histones 2A/2B. The interaction of SAP30/SAP30L with DNA induces bending of the DNA, as a result of which the nucleosomes are more accessible to HDAC enzymes, and the repressome is fully formed. (Panel 3) Nuclear PIPs (PtdInsPs) interact with the N-terminal domain of SAP30/SAP30L, displacing the DNA, which leads to relocalization of SAP30/SAP30L to the cytoplasm.