SATB1 cleavage by caspase 6 disrupts PDZ domain-mediated dimerization, causing detachment from chromatin early in T-cell apoptosis.

SATB1 is expressed primarily in thymocytes and orchestrates temporal and spatial expression of a large number of genes in the T-cell lineage. SATB1 binds to the bases of chromatin loop domains in vivo, recognizing a special DNA context with strong base-unpairing propensity. The majority of thymocytes are eliminated by apoptosis due to selection processes in the thymus. We investigated the fate of SATB1 during thymocyte and T-cell apoptosis. Here we show that SATB1 is specifically cleaved by a caspase 6-like protease at amino acid position 254 to produce a 65-kDa major fragment containing both a base-unpairing region (BUR)-binding domain and a homeodomain. We found that this cleavage separates the DNA-binding domains from amino acids 90 to 204, a region which we show to be a dimerization domain. The resulting SATB1 monomer loses its BUR-binding activity, despite containing both its DNA-binding domains, and rapidly dissociates from chromatin in vivo. We found this dimerization region to have sequence similarity to PDZ domains, which have been previously shown to be involved in signaling by conferring protein-protein interactions. SATB1 cleavage during Jurkat T-cell apoptosis induced by an anti-Fas antibody occurs concomitantly with the high-molecular-weight fragmentation of chromatin of ~50-kb fragments. Our results suggest that mechanisms of nuclear degradation early in apoptotic T cells involve efficient removal of SATB1 by disrupting its dimerization and cleavage of genomic DNA into loop domains to ensure rapid and efficient disassembly of higher-order chromatin structure.

Anti-repression of RNA polymerase II transcription by pyrrole-imidazole polyamides.

Pyrrole-imidazole polyamides are ligands that bind in the minor groove of DNA with high affinity and sequence selectivity. Molecules of this class have been shown to disrupt specific transcription factor-DNA interactions and to inhibit basal and activated transcription from various RNA polymerase II and III promoters. A set of eight-ring hairpin-motif pyrrole-imidazole polyamides has been designed to bind within the binding site for the human cytomegalovirus (CMV) UL122 immediate early protein 2 (IE86). IE86 represses transcription of the CMV major immediate early promoter (MIEP) through its cognate cis recognition sequence (crs) located between the TATA box and the transcription initiation site. The designed polyamides bind to their target DNA sequence with nanomolar affinities and with a high degree of sequence selectivity. The polyamides effectively block binding of IE86 protein to the crs in DNase I footprinting experiments. A mismatch polyamide, containing a single imidazole to pyrrole substitution, and also a polyamide binding to a site located 14 base pairs upstream of the repressor binding site, do not prevent IE86 binding to the crs. IE86-mediated transcriptional repression in vitro is relieved by a match polyamide but not by a mismatch polyamide. Transcription from a DNA template harboring a mutation in the crs is not affected either by IE86 protein or by the match polyamides. These results demonstrate that this new class of small molecules, the pyrrole-imidazole polyamides, are not only effective inhibitors of basal and activated transcription, but also can be used to activate transcription by blocking the DNA-binding activity of a repressor protein.

Inhibition of Ets-1 DNA binding and ternary complex formation between Ets-1, NF-kappaB, and DNA by a designed DNA-binding ligand.

Sequence-specific pyrrole-imidazole polyamides can be designed to interfere with transcription factor binding and to regulate gene expression, both in vitro and in living cells. Polyamides bound adjacent to the recognition sites for TBP, Ets-1, and LEF-1 in the human immunodeficiency virus, type 1 (HIV-1), long terminal repeat inhibited transcription in cell-free assays and viral replication in human peripheral blood lymphocytes. The DNA binding activity of the transcription factor Ets-1 is specifically inhibited by a polyamide bound in the minor groove. Ets-1 is a member of the winged-helix-turn-helix family of transcription factors and binds DNA through a recognition helix bound in the major groove with additional phosphate contacts on either side of this major groove interaction. The inhibitory polyamide possibly interferes with phosphate contacts made by Ets-1, by occupying the adjacent minor groove. Full-length Ets-1 binds the HIV-1 enhancer through cooperative interactions with the p50 subunit of NF-kappaB, and the Ets-inhibitory polyamide also blocks formation of ternary Ets-1. NF-kappaB.DNA complexes on the HIV-1 enhancer. A polyamide bound adjacent to the recognition site for NF-kappaB also inhibits NF-kappaB binding and ternary complex formation. These results broaden the application range of minor groove-binding polyamides and demonstrate that these DNA ligands are powerful inhibitors of DNA-binding proteins that predominantly use major groove contacts and of cooperative protein-DNA ternary complexes.

Inhibition of RNA polymerase II transcription in human cells by synthetic DNA-binding ligands.

Sequence-specific DNA-binding small molecules that can permeate human cells potentially could regulate transcription of specific genes. Multiple cellular DNA-binding transcription factors are required by HIV type 1 for RNA synthesis. Two pyrrole-imidazole polyamides were designed to bind DNA sequences immediately adjacent to binding sites for the transcription factors Ets-1, lymphoid-enhancer binding factor 1, and TATA-box binding protein. These synthetic ligands specifically inhibit DNA-binding of each transcription factor and HIV type 1 transcription in cell-free assays. When used in combination, the polyamides inhibit virus replication by >99% in isolated human peripheral blood lymphocytes, with no detectable cell toxicity. The ability of small molecules to target predetermined DNA sequences located within RNA polymerase II promoters suggests a general approach for regulation of gene expression, as well as a mechanism for the inhibition of viral replication.

An atypical homeodomain in SATB1 promotes specific recognition of the key structural element in a matrix attachment region.

SATB1 is a cell type-specific nuclear matrix attachment region (MAR) DNA-binding protein, predominantly expressed in thymocytes. We identified an atypical homeodomain and two Cut-like repeats in SATB1, in addition to the known MAR-binding domain. The isolated MAR-binding domain recognizes a certain DNA sequence context within MARs that is highly potentiated for base unpairing. Unlike the MAR-binding domain, the homeodomain when isolated binds poorly and with low specificity to DNA. However, the combined action of the MAR-binding domain and the homeodomain allows SATB1 to specifically recognize the core unwinding element within the base-unpairing region. The core unwinding element is critical for MAR structure, since point mutations within this core abolish the unwinding propensity of the MAR. The contribution of the homeodomain is abolished by alanine substitutions of arginine 3 and arginine 5 in the N-terminal arm of the homeodomain. Site-directed mutagenesis of the core unwinding element in the 3' MAR of the immunoglobulin heavy chain gene enhancer revealed the sequence 5'-(C/A)TAATA-3' to be essential for the increase in affinity mediated by the homeodomain. SATB1 may regulate T-cell development and function at the level of higher order chromatin structure through the critical DNA structural elements within MARs.

A novel matrix attachment region DNA binding motif identified using a random phage peptide library.

SATB1 is a nuclear matrix attachment DNA (MAR)-binding protein which is predominantly expressed in thymocytes. This protein binds to the minor groove specifically recognizing an unusual DNA context exhibited by a specific MAR region with strong base-unpairing propensity. A phage library displaying nonamer random peptides without any built-in structure was used to identify a MAR binding motif of SATB1. One predominant cyclic peptide C1 of CRQNWGLEGC selected by a MAR-affinity column showed 50% identity with a segment in SATB1 (amino acids 355-363). Replacement of the C1 similarity segment in SATB1 by a random amino acid sequence or its truncation resulted in more than 80% reduction in MAR binding. In contrast, replacement of the same SATB1 segment with the C1 peptide restored full MAR binding activity and specificity as the wild-type protein. Single amino acid mutation of the conserved Arg or Glu residue to Ala greatly reduced MAR binding. Taken together our data show that a nine amino acid sequence in SATB1 represents a key MAR binding motif. Phage display may provide a general tool for rapid identification of DNA binding peptide motifs.

Nucleolin is a matrix attachment region DNA-binding protein that specifically recognizes a region with high base-unpairing potential.

A DNA affinity column containing a synthetic double-stranded nuclear matrix attachment region (MAR) was used to purify a 100-kDa protein from human erythroleukemia K562 cells. This protein was identified as nucleolin, the key nucleolar protein of dividing cells, which is thought to control rRNA gene transcription and ribosome assembly. Nucleolin is known to bind RNA and single-stranded DNA. We report here that nucleolin is also a MAR-binding protein. It binds double-stranded MARs from different species with high affinity. Nucleolin effectively distinguishes between a double-stranded wild-type synthetic MAR sequence with a high base-unpairing potential and its mutated version that has lost the unpairing capability but is still A+T rich. Thus, nucleolin is not merely an A+T-rich sequence-binding protein but specifically binds the base-unpairing region of MARs. This binding specificity is similar to that of the previously cloned tissue-specific MAR-binding protein SATB1. Unlike SATB1, which binds only double-stranded MARs, nucleolin binds the single-stranded T-rich strand of the synthetic MAR probe approximately 45-fold more efficiently than its complementary A-rich strand, which has an affinity comparable to that of the double-stranded form of the MAR. In contrast to the high selectivity of binding to double-stranded MARs, nucleolin shows only a small but distinct sequence preference for the T-rich strand of the wild-type synthetic MAR over the T-rich strand of its mutated version. The affinity to the T-rich synthetic MAR is severalfold higher than to its corresponding RNA and human telomere DNA. Quantitative cellular fractionation and extraction experiments indicate that nucleolin is present both as a soluble protein and tightly bound to the matrix, similar to other known MAR-binding proteins.

A novel DNA-binding motif in the nuclear matrix attachment DNA-binding protein SATB1.

The nuclear matrix attachment DNA (MAR) binding protein SATB1 is a sequence context-specific binding protein that binds in the minor groove, making virtually no contact with the DNA bases. The SATB1 binding sites consist of a special AT-rich sequence context in which one strand is well-mixed A's, T's, and C's, excluding G's (ATC sequences), which is typically found in clusters within different MARs. To determine the extent of conservation of the SATB1 gene among different species, we cloned a mouse homolog of the human STAB1 cDNA from a cDNA expression library of the mouse thymus, the tissue in which this protein is predominantly expressed. This mouse cDNA encodes a 764-amino-acid protein with a 98% homology in amino acid sequence to the human SATB1 originally cloned from testis. To characterize the DNA binding domain of this novel class of protein, we used the mouse SATB1 cDNA and delineated a 150-amino-acid polypeptide as the binding domain. This region confers full DNA binding activity, recognizes the specific sequence context, and makes direct contact with DNA at the same nucleotides as the whole protein. This DNA binding domain contains a novel DNA binding motif: when no more than 21 amino acids at either the N- or C-terminal end of the binding domain are deleted, the majority of the DNA binding activity is lost. The concomitant presence of both terminal sequences is mandatory for binding. These two terminal regions consist of hydrophilic amino acids and share homologous sequences that are different from those of any known DNA binding motifs. We propose that the DNA binding region of SATB1 extends its two terminal regions toward DNA to make direct contact with DNA.

A tissue-specific MAR/SAR DNA-binding protein with unusual binding site recognition.

A human cDNA was cloned that encodes a DNA-binding protein (SATB1) that is expressed predominantly in thymus and binds selectively to the nuclear matrix/scaffold-associating DNAs (MARs/SARs). Missing nucleoside experiments showed that SATB1 selectively binds in a special AT-rich sequence context where one strand consists of mixed A's, T's, and C's, excluding G's (ATC sequences). When this feature is destroyed by mutation, SATB1 binding is greatly reduced even if the direct contact sequence remains intact. Conjunctional SATB1-binding sequences become stably unpaired in supercoiled DNA. Specific mutations that diminish the unwinding potential greatly reduce SATB1 binding. However, SATB1 does not bind single-stranded DNA. Chemical interference assays show that SATB1 binds along the minor groove with very little contact with the bases. This suggests that SATB1 recognizes the ATC sequence indirectly through the altered sugar-phosphate backbone structure present in the double-stranded DNA.

Synthesis and assembly of soybean beta-conglycinin in vitro.

The construction of SP6-derived expression plasmids that encode normal and modified beta-conglycinin subunits is described. With the exception of an additional methionine at their NH2-terminal ends and the lack of glycans, the normal subunits synthesized at the direction of these plasmids corresponded to mature alpha and beta subunits isolated from soybean seeds. The subunits assembled into trimers in vitro that were equivalent in size to those formed in vivo. This result shows that the glycans are not required either for protein folding or oligomer assembly. Subunits produced from other plasmids, which had modifications in a highly conserved hydrophobic region in the COOH-terminal end of the subunits, either did not assemble or assembled at an extremely low rate compared to unmodified subunits. Structural changes at the more hydrophilic NH2-terminal end had mixed effects. Several subunits modified in this region assembled into trimers at rates that were either equal or greater than those for normal alpha subunits. Others assembled less completely than the normal subunits. Our results indicate that the in vitro synthesis and assembly assay will be useful in evaluating structure-function relationships in modified beta-conglycinin subunits. The results also show that structural changes at the NH2-terminal end of the subunits are tolerated to a greater extent than modifications in the hydrophobic conserved region in the COOH-terminal half of the subunits, and this information will be useful in efforts to improve soybean quality.

The coherence of synthetic telomeres.

The chromosomal telomeres of Oxytricha were synthesized and their ability to cohere examined on non-denaturing acrylamide gels containing the stabilizing cation K+. At least 5 different mobility species were observed, in addition to that of the monomeric telomere. By cohering synthetic telomeres containing different lengths of subtelomeric DNA, we showed that each of the different mobility species was a dimer of two telomeres. Since the different mobility species did not differ in numbers or sequences of nucleotides, they must correspond to different molecular shapes probably caused by different degrees of bending of the dimer. Paradoxically, telomeres with longer subtelomeric stems cohered more efficiently. In the presence of K+, solutions had to be heated to over 90 degrees before the telomeres separated. Various synthetic constructs, restriction endonuclease and dimethyl sulfate protection experiments showed that the only nucleotides involved in the cohered structures were the 16 base 'tails' of sequence 3'G4T4G4T4. Extension of this motif was actually inimical to coherence. Oligomers containing 2 G4T4 motifs protected their GN7 positions by forming dimers, those with 5 G4T4 could do so by internal folding, but the 3' terminal group of G4 was left unprotected. This suggests that only four groups of G4 are necessary for the cohered structure. Single-chain specific nuclease, S1, as well as osmium tetroxide, which oxidizes the thymine residues of single chains, reacted less efficiently with the cohered structures. Synthetic telomeres containing inosine replacing guanosine were not observed to cohere, indicating that the C2-NH2 is strongly stabilizing. The cohered structures appear to be unusually compact and sturdy units in which four G4 blocks form quadruplexes stabilized by K+. A new model for the cohered structure is presented.