doi: 10.1128/MCB.20.20.7480-7489.2000.
SAF-Box, a conserved protein domain that specifically recognizes scaffold attachment region DNA
Affiliations
- PMID:11003645
- PMCID: PMC86301
- DOI: 10.1128/MCB.20.20.7480-7489.2000
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SAF-Box, a conserved protein domain that specifically recognizes scaffold attachment region DNA
M Kipp et al. Mol Cell Biol.2000 Oct.
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Abstract
SARs (scaffold attachment regions) are candidate DNA elements for partitioning eukaryotic genomes into independent chromatin loops by attaching DNA to proteins of a nuclear scaffold or matrix. The interaction of SARs with the nuclear scaffold is evolutionarily conserved and appears to be due to specific DNA binding proteins that recognize SARs by a mechanism not yet understood. We describe a novel, evolutionarily conserved protein domain that specifically binds to SARs but is not related to SAR binding motifs of other proteins. This domain was first identified in human scaffold attachment factor A (SAF-A) and was thus designated SAF-Box. The SAF-Box is present in many different proteins ranging from yeast to human in origin and appears to be structurally related to a homeodomain. We show here that SAF-Boxes from four different origins, as well as a synthetic SAF-Box peptide, bind to natural and artificial SARs with high specificity. Specific SAR binding of the novel domain is achieved by an unusual mass binding mode, is sensitive to distamycin but not to chromomycin, and displays a clear preference for long DNA fragments. This is the first characterization of a specific SAR binding domain that is conserved throughout evolution and has DNA binding properties that closely resemble that of the unfractionated nuclear scaffold.
Figures
A SAR binding domain maps to the extreme amino terminus of human SAF-A. (A) Schematic representation of complete SAF-A and the protein fragments used for DNA binding assays. The RNA binding RGG-Box (25) and regions rich in leucine (L), acidic residues (D, E), glutamine (Q), and glycine (G) are indicated. (B) Pull-down DNA binding assays with recombinant constructs of human SAF-A. Proteins ZZ-N247 and ZZ-N45 were overexpressed, purified, immobilized on IgG-Sepharose, and incubated with the human SAR element MII (filled squares), non-SAR pUC18 (filled circles), or an equimolar mixture of both DNAs (filled triangles) in the presence of increasing amounts ofE. coli competitor DNA. Bound DNA was quantified by scintillation counting and expressed as percentage of input. Note that the amount of immobilized protein (1 μg) was chosen to be saturating up to a 100-fold excess of competitor DNA. A control experiment with ZZ-N247 lacking the amino-terminal 45 residues (ZZ-N247ΔN45) is shown in the left panel (open circles). (C) A pull-down DNA binding assay was performed with an equimolar mixture of a SAR (MII) and non-SAR DNA (pUC18) and increasing amounts of unspecific competitor DNA. Bound DNA was eluted from the beads, and aliquots of identical radioactivity were analyzed on agarose gels. Note the high specificity for SAR DNA under stringent conditions.
A synthetic SAR binding peptide. (A) A 46-residue peptide with the SAF-Box from human SAF-A was synthesized and purified by chromatography. The last purification step, reverse-phase HPLC on a C18 column, shows the peptide elutes as a symmetric peak in a water-acetonitrile gradient. (B) MALDI-TOF mass spectrometry demonstrates the integrity of the peptide. The calculated molecular weight is 5,245. (C) The purified peptide (100 ng) was immobilized on Sepharose beads and tested for DNA binding to the MII-pUC18 mixture in the presence of increasing amounts of competitor DNA.
The SAF-Box targets SAF-A to mitotic chromosomes in transient transfection experiments. (A) COS7 cells were transfected with expression vectors encoding fusion proteins of wild-type SAF-A or a SAF-Box deletion mutant with EGFP. Cells were analyzed 24 h posttransfection by SDS-polyacrylamide gel electrophoresis and immunoblotting of total cell extracts with SAF-A-specific antibodies. Control cells were not transfected. (B) COS7 cells cultivated on coverslips were transfected as above and analyzed microscopically. Typical images of interphase cells and mitotic cells are shown for both protein constructs. (C) Mitotic cells transfected with wild-type SAF-A–EGFP, ΔN45 mutant-EGFP, or EGFP alone were scored for localization of green fluorescence on chromosomes (yes or no) or homogeneous cellular staining (homo). In all cases, more than 100 mitotic cells were scored (n=).
Expression of a mutant SAF-A lacking the SAF-Box exerts a dominant negative effect on proliferation. (A) MCF-7 cells were transfected with 12 μg of expression vectors for wild-type SAF-A–EGFP or the ΔN45 mutant-EGFP, and absolute cell numbers were determined 48 h posttransfection by using a Coulter cell counter. (B) MCF-7 cells were transfected with 12 μg of the empty pEGFP-N1 vector (control) or 3, 6, or 12 μg of the expression vectors described in panel A. After 30 h, cells were labeled by exchanging the medium with fresh medium containing 0.5 μCi of [3H]thymidine/ml. After 12 h, cells were harvested and the amount of radioactive thymidine incorporated into genomic DNA was determined by precipitation with TCA, filtration over GF/C fiberglass filters, and scintillation counting. All assays were done in triplicate; error bars indicate the standard deviations. The results are presented as absolute values in counts per minute (cpm), and relative values are normalized to the vector control. In all assays, transfection efficiency and cell death were approximately 60 and 4%, respectively.
The SAF-Box is a conserved protein domain. (A) Alignment of 17 SAF-Boxes from proteins originating in human (hs), mouse (mm),Xenopus laevis (xl), zebra fish (dr),A. thaliana (at),C. elegans (ce),Bombyx mori (bm),S. pombe (sp),Drosophila melanogaster (dm), andS. cerevisiae (sc). The two SAF-Boxes from theA. thaliana PARP are indicated as PARP-N and PARP-C for the amino-terminal or carboxy-terminal box, respectively. Homologies to the homeodomain of Hox-C12(3F) are shown below. Asterisks denote proteins used for further studies. (B) Comparison of the putative structure of the SAF-Box as derived from secondary structure predictions and computer-assisted modeling with known structures of fushi tarazu (42) and engrailed (7, 27) homeoboxes. Helix 3 of a homeobox (light gray) is not present in the SAF-Box. NMR, nuclear magnetic resonance.
SAR binding is a conserved activity of the SAF-Box. (A) Proteins containing the SAF-Boxes from four different proteins originating in humans,C. elegans,S. pombe, andA. thaliana, each with an amino-terminal ZZ-tag, were bacterially overexpressed, purified, and analyzed by SDS-polyacrylamide gel electrophoresis. (B) DNA binding assays. Identical amounts of the four proteins were immobilized on IgG-Sepharose and incubated with two different SAR–non-SAR mixtures in the absence (−) or presence (+) of a 500-fold excess of competitor DNA. Note that all proteins specifically bind to the SARs MII (human) and GN1.5 (petunia) in the presence of competitor DNA but not to plasmid controls (pUC18, pGEM3), although slight differences in specificity are apparent.
The DNA binding mode of the SAF-Box. (A) Increasing amounts of ZZ-N247 and ZZ-N45 from human SAF-A were immobilized on Sepharose beads and tested for binding to the isolated human MII SAR element. Note that both proteins have identical binding curves when expressed in molar terms (lower panel). (B) An experiment similar to that shown in panel A was performed with an equimolar mixture of a SAR (MII) and non-SAR (pUC18) DNA and the SAF-Box proteins ZZ-N247 and ZZ-N45 from human SAF-A and the SAF-Box fromS. pombe mlo1+. Bound DNA was eluted from the beads and analyzed by agarose gel electrophoresis. (C) The human SAF-Box protein ZZ-N45 was immobilized on Sepharose beads under two different sets of conditions that result in identical absolute amounts of protein but different surface densities (upper panel). DNA binding assays with the isolated MII SAR demonstrate that the stoichiometry of bound DNA to protein is dependent on the density of coupled protein but not on the absolute amount of protein. Filled circles, constant density; open circles, decreasing density (lower panel). Note the reverse orientation of thex axis.
The SAR binding peptide prefers to bind to long DNA fragments. (A) Synthetic oligonucleotides containing the MRS sequence (50) were multimerized by ligation, radioactively end labeled, and used as the substrate in pull-down DNA binding assays with the immobilized synthetic peptide. Labeled MII-pUC18 mixture was added as internal specificity control. DNA bound in assays with increasing amounts of competitor DNA was recovered from the beads, split into two equal parts, and analyzed by gel electrophoresis through 3% Resophor agarose (upper panel) to visualize the multimers or 1% agarose to resolve the internal control (lower panel). Samples were normalized on the basis of scintillation counting, and identical radioactivity was applied to each lane. Controls: MII-pUC18 mixture alone, multimers alone, and the input mixture. (B) The gel shown in the upper part of panel A was scanned to quantify bound DNA by densitometry. Lane 3 (input) and lane 9 (bound in the presence of a 500-fold excess of competitor DNA) (shown in panel A, from the left) were used to calculate the bound-to-input ratio separately for each multimer; ratios exceeding 1 result from the application of the same radioactivity to each lane, demonstrating an overrepresentation of higher multimers. Note the clear preference for fragments that are >200 bp.
SAR binding of the synthetic peptide is sensitive to distamycin. (A) Pull-down DNA binding assays with the MII-pUC18 mixture and a 500-fold excess of unspecific competitor DNA in the presence of increasing amounts of distamycin or chromomycin. Bound DNA was quantified by scintillation counting (upper panel) and analyzed by agarose gel electrophoresis (lower panel, samples 3 to 8 from the upper panel). (B) Preexistent binding of SARs to the peptide are stable in the presence of distamycin. Pull-down DNA binding assays were performed as shown in panel A but in the absence of drugs. Distamycin (stippled bars) or chromomycin (hatched bars) was added after 1 h and was incubated with the DNA-peptide complexes for 30 min, before washing and quantification by scintillation counting.
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