Protein Recognition Sequences Research Articles

Rolling Circle Amplification-Assisted Flow Cytometry Approach for Simultaneous Profiling of Exosomal Surface Proteins.

Exosomes that carry multiple proteins from the originating cells are known as emerging biomarkers for tumor diagnostics. However, it is still technically challenging to accurately evaluate subtle differences of exosomal membrane proteins. Here, we developed a rolling circle amplification (RCA)-assisted flow cytometry approach (FCA) to simultaneously profile surface proteins and quantify exosomes. In this work, specific anti-CD63 antibody-conjugated magnetic beads were first utilized to capture exosomes. Then, the captured exosomes were bound with DNA primers, which comprise exosomal surface protein-specific recognition aptamers. The RCA reaction generates repeat DNA sequences for fluorescent probe hybridization. Finally, a conventional flow cytometer was introduced to phenotype exosomal protein markers. Such a sensitive RCA-assisted FCA displays an excellent detection limit of 1.3 × 105 exosome/mL. The variable composition of four protein markers on different cell-derived exosomes was sensitively detected through changing the protein-recognition sequence of the DNA primer, which reveals a heterogeneous pattern. Exosomes from different cell sources could be distinguished by the abundance difference of multiple surface proteins. Furthermore, the developed RCA-assisted FCA enabled quantitative analysis of blood samples from lung cancer patients, indicating its potential for early clinical diagnosis and prognosis of cancer.

Nanoparticles: Untying the Gordian Knot in Conventional Computed Tomography Imaging

X-ray computed tomography (CT) imaging plays an essential role in disease diagnosis due to its noninvasive, painless mode and superior penetration depth. However, the resolution of the soft tissue ...

Metformin inhibits hepatocellular glucose, lipid and cholesterol biosynthetic pathways by transcriptionally suppressing steroid receptor coactivator 2 (SRC-2).

The ability of the anti-diabetic drug metformin to inhibit anabolic processes including gluconeogenesis and lipogenesis is partly attributable to activation of the AMP-activated protein kinase (AMPK) pathway. The p160 steroid receptor coactivator 2 (SRC-2) is a key regulator of cellular metabolism and drives expression of the gluconeogenic enzyme glucose-6-phosphatase (G6Pc). Here, we uncovered a role for SRC-2 in the metabolic reprogramming imposed by metformin. In FaO cells, metformin dose-dependently reduced mRNA expression of SRC-2. Microarray analysis of metformin-treated cells revealed an overrepresentation of downregulated genes involved in biosynthesis of lipids and cholesterol. Several metformin-regulated genes including fatty acid synthase (FASN) were validated as transcriptional targets of SRC-2 with promoters characterized by sterol regulatory element (SRE) binding protein (SREBP) recognition sequences. Transactivation assays of the FASN promoter confirmed that SRC-2 is a coactivator of SREBP-1. By suppressing SRC-2 at the transcriptional level, metformin impeded recruitment of SRC-2 and RNA polymerase II to the G6Pc promoter and to SREs of mutual SRC-2/SREBP-1 target gene promoters. Hepatocellular fat accretion was reduced by metformin or knock-down of both SRC-2 and SREBP-1. Accordingly we propose that metformin inhibits glucose and lipid biosynthesis partly by downregulating SRC-2 gene expression.

Automatic polymerase chain reaction product detection system for food safety monitoring using zinc finger protein fused to luciferase

An automatic polymerase chain reaction (PCR) product detection system for food safety monitoring using zinc finger (ZF) protein fused to luciferase was developed. ZF protein fused to luciferase specifically binds to target double stranded DNA sequence and has luciferase enzymatic activity. Therefore, PCR products that comprise ZF protein recognition sequence can be detected by measuring the luciferase activity of the fusion protein. We previously reported that PCR products from Legionella pneumophila and Escherichia coli (E. coli) O157 genomic DNA were detected by Zif268, a natural ZF protein, fused to luciferase. In this study, Zif268–luciferase was applied to detect the presence of Salmonella and coliforms. Moreover, an artificial zinc finger protein (B2) fused to luciferase was constructed for a Norovirus detection system. In the luciferase activity detection assay, several bound/free separation process is required. Therefore, an analyzer that automatically performed the bound/free separation process was developed to detect PCR products using the ZF–luciferase fusion protein. By means of the automatic analyzer with ZF–luciferase fusion protein, target pathogenic genomes were specifically detected in the presence of other pathogenic genomes. Moreover, we succeeded in the detection of 10 copies of E. coli BL21 without extraction of genomic DNA by the automatic analyzer and E. coli was detected with a logarithmic dependency in the range of 1.0×10 to 1.0×106 copies.

Presenting ENCODE

The Encyclopedia of DNA Elements (ENCODE) project has systematically mapped regions of transcription, transcription-factor association, chromatin structure and histone modification. In this overview, the Consortium guides the readers through the project itself, the data and their integrated analyses. Eighty per cent of the human genome now has at least one biochemical function assigned to it. In addition to expanding our understanding of how gene expression is regulated on a genome-wide scale, the newly identified functional elements should help researchers to interpret the results of genome-wide associated studies because many correspond to sites associated with human disease. This paper describes the first extensive map of human DNaseI hypersensitive sites — markers of regulatory DNA — in 125 diverse cell and tissue types. Integration of this information with other data sets generated by ENCODE (Encyclopedia of DNA Elements) identified new relationships between chromatin accessibility, transcription, DNA methylation and regulatory-factor occupancy patterns. Evolutionary-conservation analysis revealed signatures of recent functional constraint within DNaseI hypersensitive sites. DNaseI footprinting detects DNA sequences that are protected from cleavage by DNaseI because they are bound by regulatory factors. Studying these footprints in 41 diverse cell and tissue types, the authors describe millions of short sequence elements that are conserved recognition sequences for DNA-binding proteins. The effort nearly doubles the size of the human cis-regulatory lexicon and provides insight into chromatin states and levels of evolutionary conservation. A large collection of novel regulatory-factor recognition motifs that closely parallel major regulators of development, differentiation and pluripotency is also described. This manuscript describes the effort of the ENCODE (Encyclopedia of DNA Elements) Consortium to examine the principles of human transcriptional regulatory networks, using a subset of 119 transcription factors. The results are integrated with other genomic information to form a multi-level meta-network in which different levels have distinct properties. The findings will aid future interpretations of human genomics and help us to understand the basic principles of human biology and disease. These authors describe the ENCODE (Encyclopedia of DNA Elements) effort to provide a complete catalogue of primary and processed RNAs found either in specific sub-cellular compartments or throughout the cell. They show that three-quarters of the human genome can be transcribed, and provide a wealth of information about the range and levels of expression, localization, processing fates and modifications of both known and previously unannotated RNAs. Collectively, these observations suggest that the current concept of a gene should be revisited. In this ENCODE (Encyclopedia of DNA Elements) manuscript, the authors use chromosome conformation capture carbon copy (5C2) to look at the relationships between functional elements and distal target genes in 1% of the human genome in three dimensions. They describe numerous long-range interactions between promoters and distal sites that include elements resembling enhancers, promoters and CTCF-bound sites, their genomic distribution and complex interactions. Because only ∼7% of looping interactions are with the nearest gene, genomic proximity is not a simple predictor for long-range interactions.

An expansive human regulatory lexicon encoded in transcription factor footprints.

Regulatory factor binding to genomic DNA protects the underlying sequence from cleavage by DNaseI, leaving nucleotide-resolution footprints. Using genomic DNaseI footprinting across 41 diverse cell and tissue types, we detected 45 million factor occupancy events within regulatory regions, representing differential binding to 8.4 million distinct short sequence elements. Here we show that this small genomic sequence compartment, roughly twice the size of the exome, encodes an expansive repertoire of conserved recognition sequences for DNA-binding proteins that nearly doubles the size of the human cis-regulatory lexicon. We find that genetic variants affecting allelic chromatin states are concentrated in footprints, and that these elements are preferentially sheltered from DNA methylation. High-resolution DNaseI cleavage patterns mirror nucleotide-level evolutionary conservation and track the crystallographic topography of protein-DNA interfaces, indicating that transcription factor structure has been evolutionarily imprinted on the human genome sequence. We identify a stereotyped 50 base-pair footprint that precisely defines the site of transcript origination within thousands of human promoters. Finally, we describe a large collection of novel regulatory factor recognition motifs that are highly conserved in both sequence and function, and exhibit cell-selective occupancy patterns that closely parallel major regulators of development, differentiation, and pluripotency.

Single-Molecule In Vitro and Live-Cell Studies of Allostery through DNA

Often in nature, a macromolecule undergoes conformational changes upon binding of a ligand in order to modify its affinity for another ligand at a distant binding site. This phenomenon, called “allostery”, is a fundamental mechanism for dynamic regulation of macromolecular properties. Although allostery is well-documented in proteins, it is less recognized for DNA-protein interactions, in which DNA has been often considered a mere template providing recognition sequences for proteins. Here we investigate the allosteric interactions through DNA both in single molecule experiments in vitro and in live cells. In the in vitro experiments, we demonstrate that when two proteins specifically bind to DNA within tens of base pairs, the binding affinity of one protein is altered by the other. We prove that this is not due to protein-protein interactions but to allostery through DNA. As the distance between the two proteins is varied, this allosteric coupling oscillates between positive and negative cooperativity with a periodicity of ∼10 base pairs, the helical pitch of the B-form DNA. The allostery through DNA is explained in terms of the free energy associated with the overall conformation of the ternary complex. We also demonstrate that such allostery affects gene expression in live E. coli cells, suggesting its physiological relevance.

DNA Nuclear Targeting Sequences for Non-Viral Gene Delivery

PurposeTo evaluate if introduction of DNA nuclear Targeting Sequences (DTS; i.e. recognition sequences for endogenous DNA-binding proteins) in plasmid DNA (pDNA) leads to increased transfection efficiency of non-viral gene delivery by virtue of enhanced nuclear import of the pDNA.MethodsA set of DTS was identified and cloned into EGFP-reporter plasmids controlled by the CMV-promoter. These pDNA constructs were delivered into A431 and HeLa cells using standard electroporation, pEI-based polyfection or lipofection methods. The amount of pDNA delivered into the nucleus was determined by qPCR; transfection efficiency was determined by flow cytometry.ResultsNeither of these DTS increased transgene expression. We varied several parameters (mitotic activity, applied dose and delivery strategy), but without effect. Although upregulated transgene expression was observed after stimulation with TNF-α, this effect could be ascribed to non-specific upregulation of transcription rather than enhanced nuclear import. Nuclear copy numbers of plasmids containing or lacking a DTS did not differ significantly after lipofectamine-based transfection in dividing and non-dividing cells.ConclusionNo beneficial effects of DTS on gene expression or nuclear uptake were observed in this study.

Characterization of the site-specific recombination system of phage ΦD145, and its capacity to promote recombination in human cells

Phage integrases have the potential of becoming tools for safe site-specific integration of genes into unmodified human genomes. The P2-like phages have been found to have different bacterial host integration sites and consequently they have related integrases with different sequence specificities. In this work the site-specific recombination system of the P2-like phage ΦD145 is characterized. The minimal attB site is determined to 22 nt with 18 nt identity to the core region of attP. A non-coding sequence on the human chromosome 13 is shown to be a rather good substrate for recombination in vivo in bacteria as well as in a plasmid system in HeLa cells when HMG protein recognition sequences are inserted between the left arm-binding site and the core in the complex phage attachment site attP. Thus ΦD145 integrase that belongs to the tyrosine family shows potential as a tool for site-specific integration into the human genome.

A Conserved Molecular Mechanism Is Responsible for the Auto-Up-Regulation of Glucocorticoid Receptor Gene Promoters

Glucocorticoid (GC) hormones are widely used in the treatment of acute lymphoblastic leukemia (ALL). Whereas a high level of GC receptor (GR) protein is associated with the sensitivity of ALL cells to steroid-mediated apoptosis, the auto-up-regulation of human (h)GR mRNA and protein is also found in hormone-sensitive ALL cell lines. We have characterized the hGR gene-proximal promoters for DNA sequences and transcription factors required for hormone responsiveness in T lymphoblasts. Sequences at -4559/-4525 and -2956/-2916, relative to the translation start site, function as strong composite GC response units (GRUs). Both GRUs include adjacent protein recognition sequences for the c-Myb transcription factor and the GR as a DNA cassette. An Ets-binding sequence overlaps the GR-binding site in the -4559/-4525 GRU, whereas an Ets-binding site present in the -2956/-2916 GRU does not overlap the GR/c-Myb-binding cassette. The Ets protein family member, PU.1, blocks hormonal activation of the -4559/-4525 GR/c-Myb-binding cassette but does not interfere with the responsiveness of the -2956/-2916 GRU. Thus, the hGR 1A GRU (described previously), the -4559/-4525 GRU, and the -2956/-2916 GRU have a similar structure and can mediate cell type-specific hormonal auto-up-regulation of hGR promoter activity in steroid-sensitive ALL cells. However, subtle differences in the GRU architecture result in differential sensitivity of the promoters to Ets family members such as PU.1. The architecture of the GRU and the spectrum of specific transcription factors present in different types of ALL might allow the development of a tailored therapy to enhance steroid sensitivity in ALL patients.

Probing the Structure and Function of Human Glutaminase-Interacting Protein: A Possible Target for Drug Design

PDZ domains are one of the most ubiquitous protein-protein interaction modules found in living systems. Glutaminase interacting protein (GIP), also known as Tax interacting protein 1 (TIP-1), is a PDZ domain-containing protein, which plays pivotal roles in many aspects of cellular signaling, protein scaffolding and modulation of tumor growth. We report here the overexpression, efficient refolding, single-step purification, and biophysical characterization of recombinant human GIP with three different C-terminal target protein recognition sequence motifs by CD, fluorescence, and high-resolution solution NMR methods. It is clear from our NMR analysis that GIP contains 2 alpha-helices and 6 beta-strands. The three target protein C-terminal recognition motifs employed in our interaction studies are glutaminase, beta-catenin and FAS. This is the first report of GIP recognition of the cell surface protein FAS, which belongs to the tumor necrosis factor (TNF) receptor family and mediates cell apoptosis. The dissociation constant ( K D) values for the binding of GIP with different interacting partners as measured by fluorescence spectroscopy range from 1.66 to 2.64 microM. Significant chemical shift perturbations were observed upon titration of GIP with above three ligands as monitored by 2D {(1)H, (15)N}-HSQC NMR spectroscopy. GIP undergoes a conformational change upon ligand binding.

Engineered Holliday Junctions as Single-Molecule Reporters for Protein−DNA Interactions with Application to a MerR-Family Regulator

Protein-DNA interactions are essential for gene maintenance, replication, and expression. Characterizing how proteins interact with and change the structure of DNA is crucial in elucidating the mechanisms of protein function. Here, we present a novel and generalizable method of using engineered DNA Holliday junctions (HJs) that contain specific protein-recognition sequences to report protein-DNA interactions in single-molecule FRET measurements, utilizing the intrinsic structural dynamics of HJs. Because the effects of protein binding are converted to the changes in the structure and dynamics of HJs, protein-DNA interactions that involve small structural changes of DNA can be studied. We apply this method to investigate how the MerR-family regulator PbrR691 interacts with DNA for transcriptional regulation. Both apo- and holo-PbrR691 bind the stacked conformers of the engineered HJ, change their structures, constrain their conformational distributions, alter the kinetics, and shift the equilibrium of their structural dynamics. The information obtained maps the potential energy surfaces of HJ before and after PbrR691 binding and reveals the protein actions that force DNA structural changes for transcriptional regulation. The ability of PbrR691 to bind both HJ conformers and still allow HJ structural dynamics also informs about its conformational flexibility that may have significance for its regulatory function. This method of using engineered HJs offers quantification of the changes both in structure and in dynamics of DNA upon protein binding and thus provides a new tool to elucidate the correlation of structure, dynamics, and function of DNA-binding proteins.

Induction of Macrophage Chemotaxis by Aortic Extracts of the mgR Marfan Mouse Model and a GxxPG-Containing Fibrillin-1 Fragment

The primary cause of early death in untreated Marfan syndrome (MFS) patients is aortic dilatation and dissection. We investigated whether ascending aortic samples from the fibrillin-1-underexpressing mgR mouse model for MFS or a recombinant fibrillin-1 fragment containing an elastin-binding protein (EBP) recognition sequence can act as chemotactic stimuli for macrophages. Both the aortic extracts from the mgR/mgR mice and the fibrillin-1 fragment significantly increased macrophage chemotaxis compared with extracts from wild-type mice or buffer controls. The chemotactic response was significantly diminished by pretreatment of macrophages with lactose or with the elastin-derived peptide VGVAPG and by pretreatment of samples with a monoclonal antibody directed against an EBP recognition sequence. Mutation of the EBP recognition sequence in the fibrillin-1 fragment also abolished the chemotactic response. These results indicate the involvement of EBP in mediating the effects. Additionally, investigation of macrophages in aortic specimens of MFS patients demonstrated macrophage infiltration in the tunica media. Our findings demonstrate that aortic extracts from mgR/mgR mice can stimulate macrophage chemotaxis by interaction with EBP and show that a fibrillin-1 fragment possesses chemotactic stimulatory activity similar to that of elastin degradation peptides. They provide a plausible molecular mechanism for the inflammatory infiltrates observed in the mgR mouse model and suggest that inflammation may represent a component of the complex pathogenesis of MFS.

Comparison of the structure and regulation of the udp gene of Vibrio cholerae, Yersinia pseudotuberculosis, Salmonella typhimurium, and Escherichia coli

The nucleotide sequences of the udp gene encoding uridine phosphorylase of Yersinia pseudotuberculosis and Vibrio cholerae are presented and compared with the udp sequences of Salmonella typhimurium and Escherichia coli. Both genes contain 759 bases and encode a 253 amino acid polypeptide, which is the same as for E. coli and S. typhimurium. The amino acid sequence derived from S. typhimurium gene was more similar to the derived E. coli sequence, with only a 7 amino acid difference. The Y. pseudotuberculosis and V. cholerae uridine phosphorylases presented a higher degree of divergence in their amino acid sequence as compared to the corresponding E. coli amino acid sequence, with 20 and 64 changes, respectively. The promoter regions of the udp gene for S. typhimurium ( udpP St ), Y. pseudotuberculosis ( udpP Yp ) and V. cholerae ( udpP Vc ) were identified by primer extension analysis. Comparative analysis of the udpP promoter region from Y. pseudotuberculosis, V. cholerae, S. typhimurium and E. coli revealed that location, spacing and orientation of putative binding sites for CRP protein are highly conserved, whereas CytR protein recognition sequences of udpP Yp and udpP Vc deviate markedly from the E. coli and S. typhimurium CytR binding site. In vitro studies demonstrated that the CytR protein from E. coli shows different affinity for each promoter region analyzed. According to this, the degree of CytR derepression after introduction of heterologous promoters into E. coli cells is different.

Multiprotein complexes present at the MIF motifs flanking the promoter of the human c-myc gene

The activated c-myc allele in Burkitt’s lymphoma is associated with a clustering of somatic mutations within a discrete domain of intron I that define protein recognition sequences, designated as myc intron factors (MIF-1, MIF-2 and MIF-3). We have previously shown that MIF-1 binding activity consists of two polypeptides, myc intron binding polypeptide (MIBP1) and RFX1. In the present study we identified two polypeptides, p105 and p115, and showed that these proteins give rise to a DNA–protein complex at the MIF-2 as well as the adjacent MIF-1 site. In addition, we demonstrated that all four proteins interact with a novel MIF-1 like motif upstream from the c-myc promoter region, designated 5′MIF. These data suggest a model, where the interactions of MIBP1/RFX1 and p105/p115 with the MIF-like sites may play a role in the promoter topology of the c-myc gene.

Tyrosine kinase inhibitors targeted to the epidermal growth factor receptor subfamily: role as anticancer agents.

Abnormal cell signal transduction arising from protein tyrosine kinases has been implicated in the initiation and progression of a variety of human cancers. Over the past 2 decades pharmaceutical and university laboratories have been involved in a tremendous effort to develop compounds that can selectively modulate these abnormal signalling pathways. Targeting receptor tyrosine kinases, especially the epidermal growth factor receptor subfamily, has been at the forefront of this effort as a result of strong clinical data correlating over-expression of these receptors with more aggressive cancers. There are a variety of strategies under development for inhibiting the kinase activity of these receptors, targeting both the extracellular and intracellular domains. Antibody-based approaches, immunotoxins and ligand-binding cytotoxic agents use the extracellular domain for targeted tumour therapy. Small molecule inhibitors target the intracellular catalytic region by interfering with ATP binding, while nonphosphorylatable peptides are aimed at the intracellular substrate binding region. Compounds that inhibit subsequent downstream signals from the receptor by interrupting intracellular protein recognition sequences are also being investigated. In the past 5 years enormous progress has been made in developing tyrosine kinase inhibitor compounds with sufficient potency, bioavailability and selectivity against this subfamily of receptor tyrosine kinases. The anti-HER2 monoclonal antibody, trastuzumab, for patients with metastatic breast cancer is the first of these inhibitor compounds to gain FDA approval. However, preclinical and clinical trials are ongoing with a variety of other monoclonal antibodies, immunotoxins, and small molecule quinazoline and pyrimidine-based inhibitors. Although their cytotoxic and cytostatic potential has been proven, they are not likely to replace standard chemotherapy regimens as single-agent, first-line therapeutics. Instead, their promising additive and synergistic antitumour effects in combination with standard chemotherapeutics suggest that these novel agents will find their greatest utility and efficacy in conjunction with existing anticancer agents.

The dnaA gene region of Mycobacterium avium and the autonomous replication activities of its 5′ and 3′ flanking regions

A 3.9 kb DNA fragment containing the dnaA gene region of Mycobacterium avium was cloned and its nucleotide sequence was determined. Nucleotide sequence analyses indicated that this region encodes three genes in the order rpmH (ribosomal protein L34), dnaA (the putative initiator protein) and dnaN (the beta subunit of DNA polymerase III). The intergenic regions between the rpmH-dnaA and dnaA-dnaN genes were found to contain several putative DnaA boxes, 9 nt long DnaA protein recognition sequences. A DNA fragment containing the 3' but not the 5' flanking region of the M. avium dnaA gene when cloned in Escherichia coli plasmids, which are otherwise non-replicative in mycobacteria, exhibited autonomous replication activity in M. avium but not in Mycobacterium bovis BCG and Mycobacterium smegmatis. The 5' flanking region of dnaA, on the other hand, exhibited autonomous replication activity in M. bovis BCG but not in M. avium and M. smegmatis. The implications of these results for the understanding of the M. avium oriC replication initiation process are discussed.

The exocyclic groups of DNA modulate the affinity and positioning of the histone octamer.

To investigate the nature of the chemical determinants in DNA required for nonspecific binding and bending by proteins we have created a novel DNA in which inosine-5-methylcytosine and 2, 6-diaminopurine-uracil base pairs are substituted for normal base pairs in a defined DNA sequence. This procedure completely switches the patterns of the base pair H bonding and attachment of exocyclic groups. We show that this DNA binds a histone octamer more tightly than normal DNA but, surprisingly, does not alter the orientation of the sequence on the surface of the protein. However, in general, the addition or removal of DNA exocyclic groups reduces or increases, respectively, the affinity for the histone octamer. The average incremental change in binding energy for a single exocyclic group is approximately 40 J/mol. The orientation of the DNA in core nucleosomes also is sensitive to the number and nature of the exocyclic groups present. Notably, substitution with the naturally occurring cytosine analogue, 5-methylcytosine, shifts the preferred rotational position by 3 bp, whereas incorporating 2,6-diaminopurine shifts it 2 bp in the opposite direction. These manipulations potentially would alter the accessibility of a protein recognition sequence on the surface of the histone octamer. We propose that exocyclic groups impose steric constraints on protein-induced DNA wrapping and are also important in determining the orientation of DNA on a protein surface. In addition, we consider the implications of the selection of A-T and G-C base pairs in natural DNA.

An adipogenic basic helix-loop-helix-leucine zipper type transcription factor (ADD1) mRNA is expressed and regulated by retinoic acid in osteoblastic cells.

ADD1 is a recently identified basic helix-loop-helix leucine zipper-type transcription factor that acts as a positive regulator of adipocyte-specific gene expression. Since adipocytes may share their precursor with osteoblasts, we examined the expression of ADD1 mRNA in osteoblast-like cells. In osteoblastic MC3T3-E1 cells, the level of the ADD1 mRNA expression was low at the early period of cultures while it subsequently increased with time up to more than 10-fold in the later period of cultures along with the expression of alkaline phosphatase, a differentiation marker of these cells. In ROS17/2.8 cells, which represent mature osteoblasts, ADD1 mRNA was expressed constitutively. Treatment with retinoic acid (RA) enhanced the ADD1 mRNA expression several fold in these cells within 4 h in a dose-dependent manner. This RA effect on the ADD1 mRNA expression was blocked by dichloro-D-ribofuranosylbenzimidazole but not by cycloheximide. RA treatment did not affect the ADD1 mRNA stability, suggesting the involvement of transcriptional control. Electrophoretic mobility shift assay revealed that proteins in the crude nuclear extracts prepared from ROS17/2.8 cells were bound to the E box-containing ADD1 recognition DNA sequence, E/C, and that this binding activity was enhanced by the RA treatment. Neither the E2A protein recognition sequence nor the Myo-D/E12 recognition sequence competed against the E/C sequence for the binding, indicating the sequence specificity of the binding activity. Furthermore, RA treatment enhanced the transactivation activity of the chloramphenicol acetyltransferase construct containing the E/C sequence in the transient transfection assay in ROS17/2.8 cells. RA treatment also enhanced the ADD1 mRNA expression in another rat calvaria-derived cell line, RCT1, and in the primary cultures of newborn rat calvaria cells. Overexpression of ADD1 in ROS17/2.8 enhanced the level of the osteocalcin mRNA expression. These results indicated that the adipogenic basic helix-loop-helix leucine zipper-type transcription factor (ADD1) mRNA was expressed in osteoblastic cells and that its expression was associated with the expression of an osteoblastic phenotype-related gene.

Somatic mutations in c-myc intron I cluster in discrete domains that define protein binding sequences.

The activated c-myc allele in Burkitt's lymphoma tumor cells is associated with a clustering of somatic mutations within intron I near the exon I boundary. We have identified several discrete protein binding sites within this region of c-myc intron I designated as myc intron factor-1 (MIF-1), MIF-2, and MIF-3. In addition to our previous characterization of a 20-nucleotide binding site for MIF-1, we now have identified adjacent 20-nucleotide and 34-nucleotide binding sites for MIF-2 and MIF-3, respectively. All three elements are protected from exonuclease digestion by nuclear protein extracts, and each gives rise to a distinct migration pattern on mobility shift assays. In addition, MIF-1, 2, and 3 share a 5-nucleotide (TTATG) internal sequence, which may account for cross-competition of these binding sites in the exonuclease protection experiment. Deletion mutant analyses showed that selective removal of the MIF-3 binding site alone was sufficient to enhance chloramphenicol acetyltransferase reporter activity similar to that observed with larger deletions of myc intron I. We have demonstrated that somatic mutations in activated c-myc alleles are frequently clustered in discrete domains that define protein recognition sequences.