Functional analysis of DNA bending and unwinding by the high mobility group domain of LEF-1

LEF-1 (lymphoid enhancer-binding factor 1) is a cell type-specific member of the family of high mobility group (HMG) domain proteins that recognizes a specific nucleotide sequence in the T cell receptor (TCR) α enhancer. In this study, we extend the analysis of the DNA-binding properties of LEF-1 and examine their contributions to the regulation of gene expression. We find that LEF-1, like nonspecific HMG-domain proteins, can interact with irregular DNA structures such as four-way junctions, albeit with lower efficiency than with specific duplex DNA. We also show by a phasing analysis that the LEF-induced DNA bend is directed toward the major groove. In addition, we find that the interaction of LEF-1 with a specific binding site in circular DNA changes the linking number of DNA and unwinds the double helix. Finally, we identified two nucleotides in the LEF-1-binding site that are important for protein-induced DNA bending. Mutations of these nucleotides decrease both the extent of DNA bending and the transactivation of the TCRα enhancer by LEF-1, suggesting a contribution of protein-induced DNA bending to the function of TCRα enhancer.

Multiple domains of repressor activator protein 1 contribute to facilitated binding of glycolysis regulatory protein 1

The function of repressor activator protein 1 (Rap1p) at glycolytic enzyme gene upstream activating sequence (UAS) elements in Saccharomyces cerevisiae is to facilitate binding of glycolysis regulatory protein 1 (Gcr1p) at adjacent sites. Rap1p has a modular domain structure. In its amino terminus there is an asymmetric DNA-bending domain, which is distinct from its DNA-binding domain, which resides in the middle of the protein. In the carboxyl terminus of Rap1p lie its silencing and putative activation domains. We carried out a molecular dissection of Rap1p to identify domains contributing to its ability to facilitate binding of Gcr1p. We prepared full-length and three truncated versions of Rap1p and tested their ability to facilitate binding of Gcr1p by gel shift assay. The ability to detect ternary complexes containing Rap1p⋅DNA⋅Gcr1p depended on the presence of binding sites for both proteins in the probe DNA. The DNA-binding domain of Rap1p, although competent to bind DNA, was unable to facilitate binding of Gcr1p. Full-length Rap1p and the amino- and carboxyl-truncated versions of Rap1p were each able to facilitate binding of Gcr1p at an appropriately spaced binding site. Under these conditions, Gcr1p displayed an approximately 4-fold greater affinity for Rap1p-bound DNA than for otherwise identical free DNA. When spacing between Rap1p- and Gcr1p-binding sites was altered by insertion of five nucleotides, the ability to form ternary Rap1p⋅DNA⋅Gcr1p complexes was inhibited by all but the DNA-binding domain of Rap1p itself; however, the ability of each individual protein to bind the DNA probe was unaffected.

Retinoic acid induces proteasome-dependent degradation of retinoic acid receptor α (RARα) and oncogenic RARα fusion proteins

Analyzing the pathways by which retinoic acid (RA) induces promyelocytic leukemia/retinoic acid receptor α (PML/RARα) catabolism in acute promyelocytic leukemia (APL), we found that, in addition to caspase-mediated PML/RARα cleavage, RA triggers degradation of both PML/RARα and RARα. Similarly, in non-APL cells, RA directly targeted RARα and RARα fusions to the proteasome degradation pathway. Activation of either RARα or RXRα by specific agonists induced degradation of both proteins. Conversely, a mutation in RARα that abolishes heterodimer formation and DNA binding, blocked both RARα and RXRα degradation. Mutations in the RARα DNA-binding domain or AF-2 transcriptional activation region also impaired RARα catabolism. Hence, our results link transcriptional activation to receptor catabolism and suggest that transcriptional up-regulation of nuclear receptors by their ligands may be a feedback mechanism allowing sustained target-gene activation.

Varying the number of telomere-bound proteins does not alter telomere length in tel1Δ cells

Yeast telomere DNA consists of a continuous, ≈330-bp tract of the heterogeneous repeat TG1-3 with irregularly spaced, high affinity sites for the protein Rap1p. Yeast monitor, or count, the number of telomeric Rap1p C termini in a negative feedback mechanism to modulate the length of the terminal TG1-3 repeats, and synthetic telomeres that tether Rap1p molecules adjacent to the TG1-3 tract cause wild-type cells to maintain a shorter TG1-3 tract. To identify trans-acting proteins required to count Rap1p molecules, these same synthetic telomeres were placed in two short telomere mutants: yku70Δ (which lack the yeast Ku70 protein) and tel1Δ (which lack the yeast ortholog of ATM). Although both mutants maintain telomeres with ≈100 bp of TG1-3, only yku70Δ cells maintained shorter TG1-3 repeats in response to internal Rap1p molecules. This distinct response to internal Rap1p molecules was not caused by a variation in Rap1p site density in the TG1-3 repeats as sequencing of tel1Δ and yku70Δ telomeres showed that both strains have only five to six Rap1p sites per 100-bp telomere. In addition, the tel1Δ short telomere phenotype was epistatic to the unregulated telomere length caused by deletion of the Rap1p C-terminal domain. Thus, the length of the TG1-3 repeats in tel1Δ cells was independent of the number of the Rap1p C termini at the telomere. These data indicate that tel1Δ cells use an alternative mechanism to regulate telomere length that is distinct from monitoring the number of telomere binding proteins.

Insulin-like growth factor binding protein 2 is a growth inhibitory protein conserved in zebrafish

Fish serum contains several specific binding proteins for insulin-like growth factors (IGFBPs). The structure and physiological function of these fish IGFBPs are unknown. Here we report the complete primary sequence of a zebrafish IGFBP deduced from cDNA clones isolated by library screening and rapid amplification of cDNA ends. The full-length 1,757-bp cDNA encodes a protein of 276 aa, which contains a putative 22-residue signal peptide and a 254-residue mature protein. The mature zebrafish IGFBP has a predicted molecular size of 28,440 Da and shows high sequence identity with human IGFBP-2 (52%). The sequence identities with other human IGFBPs are <37%. Chinese hamster ovary cells stably transfected with the zebrafish IGFBP-2 cDNA secreted a 31-kDa protein, which bound to IGF-I and IGF-II with high affinity, but did not bind to Des(1–3)IGF-I or insulin. Northern blot analyses revealed that the zebrafish IGFBP-2 transcript is a 1.8-kb band expressed in many embryonic and adult tissues. In adult zebrafish, IGFBP-2 mRNA levels were greatly reduced by growth hormone treatment but increased by prolonged fasting. When overexpressed or added to cultured zebrafish and mammalian cells, the zebrafish IGFBP-2 significantly inhibited IGF-I-stimulated cell proliferation and DNA synthesis. These results indicate that zebrafish IGFBP-2 is a negative growth regulator acting downstream in the growth hormone-IGF-I axis.

Lune/eye gone, a Pax-like protein, uses a partial paired domain and a homeodomain for DNA recognition

Pax proteins, characterized by the presence of a paired domain, play key regulatory roles during development. The paired domain is a bipartite DNA-binding domain that contains two helix–turn–helix domains joined by a linker region. Each of the subdomains, the PAI and RED domains, has been shown to be a distinct DNA-binding domain. The PAI domain is the most critical, but in specific circumstances, the RED domain is involved in DNA recognition. We describe a Pax protein, originally called Lune, that is the product of the Drosophila eye gone gene (eyg). It is unique among Pax proteins, because it contains only the RED domain. eyg seems to play a role both in the organogenesis of the salivary gland during embryogenesis and in the development of the eye. A high-affinity binding site for the Eyg RED domain was identified by using systematic evolution of ligands by exponential enrichment techniques. This binding site is related to a binding site previously identified for the RED domain of the Pax-6 5a isoform. Eyg also contains another DNA-binding domain, a Prd-class homeodomain (HD), whose palindromic binding site is similar to other Prd-class HDs. The ability of Pax proteins to use the PAI, RED, and HD, or combinations thereof, may be one mechanism that allows them to be used at different stages of development to regulate various developmental processes through the activation of specific target genes.

The leucine zipper may induce electrophoretic mobility anomalies without DNA bending

Numerous proteins bend DNA upon binding, a phenomenon of potential significance for regulation of gene expression and chromatin. DNA bending is commonly predicted from the presence of electrophoretic mobility anomalies in protein–DNA complexes. However, as compared with electrophoretic methods, several DNA binding oncoprotein families do not display comparable evidence of DNA bends in x-ray structural studies. Herein, circularization kinetics and affinity measurements with prebent DNA templates were employed to assess bending and DNA structural preferences for Max and other basic helix–loop–helix/leucine zipper proteins. In this way, proteins in the Myc/Max basic helix–loop–helix/leucine zipper family were found not to bend DNA in solution but to actually stabilize DNA in an unbent configuration that resists circularization. The mobility anomaly was found to be induced by the leucine zipper protein motif, rather than structural distortions of DNA. Thus rigid protein domain structures may induce anomalous electrophoretic mobility. Moreover, the energetic preference of non-DNA bending proteins for unbent templates suggests mechanisms whereby chromatin structure may regulate transcription.

Similar DNA recognition properties of alternatively spliced Drosophila POU factors

The POU-IV or “Brn-3” class of POU-domain transcription factors is represented in Drosophila by I-POU and twin-of-I-POU, alternative splice products of the I-POU gene. I-POU has been previously reported to inhibit DNA binding by the POU-III class factor drifter/Cf1a via the formation of heterodimeric complexes. Here we report that expression of the I-POU/tI-POU message is maximal late in the embryonic phase of Drosophila development, and I-POU is the preferred splice variant. Although I-POU lacks two basic amino acid residues in the POU-homeodomain found in tI-POU and Brn-3.0, these three POU-IV class proteins exhibit very similar DNA-binding specificity. In contrast to previously published reports, the results presented here show no effect of I-POU on DNA binding by drifter, and no evidence for I-POU/drifter dimerization. These results suggest that the I-POU/tI-POU gene products function by transcriptional mechanisms similar to those of the homologous POU-IV class factors expressed in other species, not by a unique inhibitory mechanism.

A double-hexamer archaeal minichromosome maintenance protein is an ATP-dependent DNA helicase

The minichromosome maintenance (MCM) proteins are essential for DNA replication in eukaryotes. Thus far, all eukaryotes have been shown to contain six highly related MCMs that apparently function together in DNA replication. Sequencing of the entire genome of the thermophilic archaeon Methanobacterium thermoautotrophicum has allowed us to identify only a single MCM-like gene (ORF Mt1770). This gene is most similar to MCM4 in eukaryotic cells. Here we have expressed and purified the M. thermoautotrophicum MCM protein. The purified protein forms a complex that has a molecular mass of ≈850 kDa, consistent with formation of a double hexamer. The protein has an ATP-independent DNA-binding activity, a DNA-stimulated ATPase activity that discriminates between single- and double-stranded DNA, and a strand-displacement (helicase) activity that can unwind up to 500 base pairs. The 3′ to 5′ helicase activity requires both ATP hydrolysis and a functional nucleotide-binding site. Moreover, the double hexamer form is the active helicase. It is therefore likely that an MCM complex acts as the replicative DNA helicase in eukaryotes and archaea. The simplified replication machinery in archaea may provide a simplified model for assembly of the machinery required for initiation of eukaryotic DNA replication.

Solution structure and peptide binding studies of the C-terminal Src homology 3-like domain of the diphtheria toxin repressor protein

The diphtheria toxin repressor (DtxR) is the best-characterized member of a family of homologous proteins that regulate iron uptake and virulence gene expression in the Gram-positive bacteria. DtxR contains two domains that are separated by a short, unstructured linker. The N-terminal domain is structurally well-defined and is responsible for Fe2+ binding, dimerization, and DNA binding. The C-terminal domain adopts a fold similar to eukaryotic Src homology 3 domains, but the functional role of the C-terminal domain in repressor activity is unknown. The solution structure of the C-terminal domain, consisting of residues N130-L226 plus a 13-residue N-terminal extension, has been determined by using NMR spectroscopy. Residues before A147 are highly mobile and adopt a random coil conformation, but residues A147-L226 form a single structured domain consisting of five β-strands and three helices arranged into a partially orthogonal, two-sheet β-barrel, similar to the structure observed in the crystalline Co2+ complex of full-length DtxR. Chemical shift perturbation studies demonstrate that a proline-rich peptide corresponding to residues R125-G139 of intact DtxR binds to the C-terminal domain in a pocket formed by residues in β-strands 2, 3, and 5, and helix 3. Binding of the proline-rich peptide by the C-terminal domain of DtxR presents an example of peptide binding by a prokaryotic Src homology 3-like protein. The results of this study, combined with previous x-ray studies of intact DtxR, provide insights into a possible biological function of the C-terminal domain in regulating repressor activity.

Proliferation of adult T cell leukemia/lymphoma cells is associated with the constitutive activation of JAK/STAT proteins

Human T cell leukemia/lymphotropic virus type I (HTLV-I) induces adult T cell leukemia/lymphoma (ATLL). The mechanism of HTLV-I oncogenesis in T cells remains partly elusive. In vitro, HTLV-I induces ligand-independent transformation of human CD4+ T cells, an event that correlates with acquisition of constitutive phosphorylation of Janus kinases (JAK) and signal transducers and activators of transcription (STAT) proteins. However, it is unclear whether the in vitro model of HTLV-I transformation has relevance to viral leukemogenesis in vivo. Here we tested the status of JAK/STAT phosphorylation and DNA-binding activity of STAT proteins in cell extracts of uncultured leukemic cells from 12 patients with ATLL by either DNA-binding assays, using DNA oligonucleotides specific for STAT-1 and STAT-3, STAT-5 and STAT-6 or, more directly, by immunoprecipitation and immunoblotting with anti-phosphotyrosine antibody for JAK and STAT proteins. Leukemic cells from 8 of 12 patients studied displayed constitutive DNA-binding activity of one or more STAT proteins, and the constitutive activation of the JAK/STAT pathway was found to persist over time in the 2 patients followed longitudinally. Furthermore, an association between JAK3 and STAT-1, STAT-3, and STAT-5 activation and cell-cycle progression was demonstrated by both propidium iodide staining and bromodeoxyuridine incorporation in cells of four patients tested. These results imply that JAK/STAT activation is associated with replication of leukemic cells and that therapeutic approaches aimed at JAK/STAT inhibition may be considered to halt neoplastic growth.

Maintenance of Epstein–Barr virus (EBV) oriP-based episomes requires EBV-encoded nuclear antigen-1 chromosome-binding domains, which can be replaced by high-mobility group-I or histone H1

EBV-encoded nuclear antigen-1 (EBNA-1) binding to a cis-acting viral DNA element, oriP, enables plasmids to persist in dividing human cells as multicopy episomes that attach to chromosomes during mitosis. In investigating the significance of EBNA-1 binding to mitotic chromosomes, we identified the basic domains of EBNA-1 within amino acids 1–89 and 323–386 as critical for chromosome binding. In contrast, the EBNA-1 C terminus (amino acids 379–641), which includes the nuclear localization signal and DNA-binding domain, does not associate with mitotic chromosomes or retain oriP plasmid DNA in dividing cell nuclei, but does enable the accumulation of replicated oriP-containing plasmid DNA in transient replication assays. The importance of chromosome association in episome maintenance was evaluated by replacing EBNA-1 amino acids 1–378 with cell proteins that have similar chromosome binding characteristics. High-mobility group-I amino acids 1–90 or histone H1–2 could substitute for EBNA-1 amino acids 1–378 in mediating more efficient accumulation of replicated oriP plasmid, association with mitotic chromosomes, nuclear retention, and long-term episome persistence. These data strongly support the hypothesis that mitotic chromosome association is a critical factor for episome maintenance. The replacement of 60% of EBNA-1 with cell protein is a significant step toward eliminating the need for noncellular protein sequences in the maintenance of episomal DNA in human cells.

Arrayed transposase-binding sequences on the ends of transposon Tn5090/Tn402

The transposon Tn5090/Tn402 encodes a 559 amino acid transposase, TniA, with a DDE motif. Gel mobility shifting and cleavage protection analysis with DNase I and hydroxyl radical probes revealed that TniA binds to multiple repeat sequences on either terminus of Tn5090/Tn402. Four of these TniA-binding 19mers occurred on the left-hand (t) end and two on the right-hand (i) end. Hydroxyl radical cleavage protection demonstrated the presence of 3–6 bp contact sequences on one face of the DNA helix. The binding pattern and organisation of repeats suggested parallels between Tn5090/Tn402 and Mu, which controls its transpositional activity in the assembly step of a higher order transpososome complex. The complex terminal structure and genes of transposase and nucleotide-binding proteins in tandem are hallmarks of the handful of Mu-like elements that are known to date.

Polarity of human replication protein A binding to DNA

Replication protein A (RPA), the nuclear single-stranded DNA binding protein is involved in DNA replication, nucleotide excision repair (NER) and homologous recombination. It is a stable heterotrimer consisting of subunits with molecular masses of 70, 32 and 14 kDa (p70, p32 and p14, respectively). Gapped DNA structures are common intermediates during DNA replication and NER. To analyze the interaction of RPA and its subunits with gapped DNA we designed structures containing 9 and 30 nucleotide gaps with a photoreactive arylazido group at the 3′-end of the upstream oligonucleotide or at the 5′-end of the downstream oligonucleotide. UV crosslinking and subsequent analysis showed that the p70 subunit mainly interacts with the 5′-end of DNA irrespective of DNA structure, while the subunit orientation towards the 3′-end of DNA in the gap structures strongly depends on the gap size. The results are compared with the data obtained previously with the primer–template systems containing 5′- or 3′-protruding DNA strands. Our results suggest a model of polar RPA binding to the gapped DNA.

Identification, retinoid binding, and x-ray analysis of a human retinol-binding protein

Two cellular retinol-binding proteins (CRBP I and II) with distinct tissue distributions and retinoid-binding properties have been recognized thus far in mammals. Here, we report the identification of a human retinol-binding protein resembling type I (55.6% identity) and type II (49.6% identity) CRBPs, but with a unique H residue in the retinoid-binding site and a distinctively different tissue distribution. Additionally, this binding protein (CRBP III) exhibits a remarkable sequence identity (62.2%) with the recently identified ι-crystallin/CRBP of the diurnal gecko Lygodactylus picturatus [Werten, P. J. L., Röll, B., van Alten, D. M. F. & de Jong, W. W. (2000) Proc. Natl. Acad. Sci. USA 97, 3282–3287 (First Published March 21, 2000; 10.1073/pnas.050500597)]. CRBP III and all-trans-retinol form a complex (Kd ≈ 60 nM), the absorption spectrum of which is characterized by the peculiar fine structure typical of the spectra of holo-CRBP I and II. As revealed by a 2.3-Å x-ray molecular model of apo-CRBP III, the amino acid residues that line the retinol-binding site in CRBP I and II are positioned nearly identically in the structure of CRBP III. At variance with the human CRBP I and II mRNAs, which are most abundant in ovary and intestine, respectively, the CRBP III mRNA is expressed at the highest levels in kidney and liver thus suggesting a prominent role for human CRBP III as an intracellular mediator of retinol metabolism in these tissues.