Oligonucleotide Affinity Chromatography Research Articles

Transcription factor ICBP90 regulates the MIF promoter and immune susceptibility locus.

The immunoregulatory cytokine macrophage migration inhibitory factor (MIF) is encoded in a functionally polymorphic locus that is linked to the susceptibility of autoimmune and infectious diseases. The MIF promoter contains a 4-nucleotide microsatellite polymorphism (-794 CATT) that repeats 5 to 8 times in the locus, with greater numbers of repeats associated with higher mRNA levels. Because there is no information about the transcriptional regulation of these common alleles, we used oligonucleotide affinity chromatography and liquid chromatography-mass spectrometry to identify nuclear proteins that interact with the -794 CATT5-8 site. An analysis of monocyte nuclear lysates revealed that the transcription factor ICBP90 (also known as UHRF1) is the major protein interacting with the MIF microsatellite. We found that ICBP90 is essential for MIF transcription from monocytes/macrophages, B and T lymphocytes, and synovial fibroblasts, and TLR-induced MIF transcription is regulated in an ICBP90- and -794 CATT5-8 length-dependent manner. Whole-genome transcription analysis of ICBP90 shRNA-treated rheumatoid synoviocytes uncovered a subset of proinflammatory and immune response genes that overlapped with those regulated by MIF shRNA. In addition, the expression levels of ICBP90 and MIF were correlated in joint synovia from patients with rheumatoid arthritis. These findings identify ICBP90 as a key regulator of MIF transcription and provide functional insight into the regulation of the polymorphic MIF locus.

Dystonia type 6 gene product Thap1: identification of a 50kDa DNA-binding species in neuronal nuclear fractions.

Mutations in THAP1 result in dystonia type 6, with partial penetrance and variable phenotype. The goal of this study was to examine the nature and expression pattern of the protein product(s) of the Thap1 transcription factor (DYT6 gene) in mouse neurons, and to study the regional and developmental distribution, and subcellular localization of Thap1 protein. The goal was accomplished via overexpression and knock-down of Thap1 in the HEK293T cell line and in mouse striatal primary cultures and western blotting of embryonic Thap1-null tissue. The endogenous and transduced Thap1 isoforms were characterized using three different commercially available anti-Thap1 antibodies and validated by immunoprecipitation and DNA oligonucleotide affinity chromatography. We identified multiple, novel Thap1 species of apparent Mr 32 kDa, 47 kDa, and 50–52 kDa in vitro and in vivo, and verified the previously identified species at 29–30 kDa in neurons. The Thap1 species at the 50 kDa size range was exclusively detected in murine brain and testes and were located in the nuclear compartment. Thus, in addition to the predicted 25 kDa apparent Mr, we identified Thap1 species with greater apparent Mr that we speculate may be a result of posttranslational modifications. The neural localization of the 50 kDa species and its nuclear compartmentalization suggests that these may be key Thap1 species controlling neuronal gene transcription. Dysfunction of the neuronal 50 kDa species may therefore be implicated in the pathogenesis of DYT6.

Proteomic Analysis Yields an Unexpected Trans -Acting Point in Control of the Human Sympathochromaffin Phenotype

The secretory protein chromogranin A (CHGA) plays a necessary role in formation of catecholamine storage vesicles and gives rise to a catecholamine release-inhibitory fragment. Because genetic variation in the proximal human CHGA promoter predicts autonomic function and blood pressure, we explored how a common genetic variant alters transcription of the gene. Bioinformatic analysis suggested that the common G-462A promoter variant (rs9658634) may disrupt as many as 3 transcriptional control motifs: LEF1, COUP-TF, and PPARγ-RXRα. During electrophoretic mobility shifts, chromaffin cell nuclear proteins bound specifically to the A (though not G) allele of CHGA promoter G-462A. On oligonucleotide affinity chromatography followed by electrospray ionization followed by 2-dimensional (tandem) mass spectrometry analysis of A allele eluates, the transcription factor LEF1 (lymphoid enhancer-binding factor-1) was identified. Interaction of LEF1 with the A allele at G-462A was confirmed by supershift. On cotransfection, LEF1 discriminated between the allelic variants, especially in chromaffin cells. Allele specificity of trans-activation by LEF1 was transferable to an isolated G-462A element fused to a heterologous (SV40) promoter. Because β-catenin (CTNNB1) can heterodimerize with LEF1, we tested the effect of cotransfection of this factor and again found A allele-specific perturbation of CHGA transcription. Common genetic variation within the human CHGA promoter alters the interaction of specific factors in trans with the promoter, with LEF1 identified by proteomic analysis and confirmed by supershift. Coexpression experiments show functional effects of LEF1 and CTNNB1 on CHGA promoter. The findings document a novel role for components of the immune and WNT pathways in control of human sympathochromaffin phenotypes.

An ancestral variant of Secretogranin II confers regulation by PHOX2 transcription factors and association with hypertension

Granins regulate secretory vesicle formation in neuroendocrine cells and granin-derived peptides are co-released with neurotransmitters as modulatory signals at sympathetic sites. We report evidence for association between a regulatory polymorphism in Secretogranin II (SCG2) and hypertension in African-American subjects. The minor allele is ancestral in the human lineage and is associated with disease risk in two case-control studies and with elevated blood pressure in a separate familial study. Mechanistically, the ancestral allele acts as a transcriptional enhancer in cells that express endogenous Scg2, whereas the derived allele does not. ARIX (PHOX2A) and PHOX2B are identified as potential transactivating factors by oligonucleotide affinity chromatography and mass spectrometry and confirmed by chromatin immunoprecipitation. Each of these transcription factors preferentially binds the risk allele, both in vitro and in vivo. Population genetic considerations suggest positive selection of the protective allele within the human lineage. These results identify a common regulatory variation in SCG2 and implicate granin gene expression in the control of human blood pressure and susceptibility to hypertension.

Purification and Identification of Proteins That Bind to the Hereditary Persistence of Fetal Hemoglobin –198 Mutation in the γ-Globin Gene Promoter

Expression of the gamma-globin gene is silenced in adult humans. However, certain point mutations in the gamma-globin gene promoter are capable of maintaining expression of this gene during adult erythropoiesis, a condition called non-deletion hereditary persistence of fetal hemoglobin (HPFH). Among these, the British form of HPFH carrying a T-->C point mutation at position -198 of the Agamma-globin gene promoter results in 4-10% fetal hemoglobin in heterozygotes. In this study, we used nuclear extracts from murine erythroleukemia cells to purify a protein complex that binds the HPFH -198 gamma-globin gene promoter. Members of this protein complex were identified by mass spectrometry and include DNMT1, the transcriptional coactivator p52, the protein SNEV, and RAP74 (the largest subunit of the general transcription factor IIF). Sp1, which was previously considered responsible for HPFH -198 gamma-globin gene activation, was not identified. The potential role of these proteins in the reactivation and/or maintenance of gamma-globin gene expression in the adult transcriptional environment is discussed.

A Role for Poly(ADP-ribose) Polymerase in the Transcriptional Regulation of the Melanoma Growth Stimulatory Activity (CXCL1) Gene Expression

The melanoma growth stimulatory activity/growth-regulated protein, CXCL1, is constitutively expressed at high levels during inflammation and progression of melanocytes into malignant melanoma. It has been shown previously that CXCL1 overexpression in melanoma cells is due to increased transcription as well as stability of the CXCL1 message. The transcription of CXCL1 is regulated through several cis-acting elements including Sp1, NF-kappaB, HMGI(Y), and the immediate upstream region (IUR) element (nucleotides -94 to -78), which lies immediately upstream to the nuclear factor kappaB (NF-kappaB) element. Previously, it has been shown that the IUR is necessary for basal and cytokine-induced transcription of the CXCL1 gene. UV cross-linking and Southwestern blot analyses indicate that the IUR oligonucleotide probe selectively binds a 115-kDa protein. In this study, the IUR element has been further characterized. We show here that proximity of the IUR element to the adjacent NF-kappaB element is critical to its function as a positive regulatory element. Using binding site oligonucleotide affinity chromatography, we have selectively purified the 115-kDa IUR-F. Mass spectrometry/mass spectrometry/matrix-assisted laser desorption ionization/time of flight spectroscopy and amino acid analysis as well as microcapillary reverse phase chromatography electrospray ionization tandem mass spectrometry identified this protein as the 114-kDa poly(ADP-ribose) polymerase (PARP1). Furthermore, 3-aminobenzamide, an inhibitor of PARP-specific ADP-ribosylation, inhibits CXCL1 promoter activity and reduces levels of CXCL1 mRNA. The data point to the possibility that PARP may be a coactivator of CXCL1 transcription.

The role of HMG I(Y) in the assembly and function of the IFN-beta enhanceosome.

Transcriptional activation of the virus inducible enhancer of the human interferon-beta (IFN-beta) gene in response to virus infection requires the assembly of an enhanceosome, consisting of the transcriptional activators NF-kappaB, ATF-2/c-Jun, IRFs and the architectural protein of the mammalian high mobility group I(Y) [HMG I(Y)]. Here, we demonstrate that the first step in enhanceosome assembly, i.e. HMG I(Y)-dependent recruitment of NF-kappaB and ATF-2/c-Jun to the enhancer, is facilitated by discrete regions of HMG I and is mediated by allosteric changes induced in the DNA by HMG I(Y) and not by protein-protein interactions between HMG I(Y) and these proteins. However, we show that completion of the enhanceosome assembly process requires protein-protein interactions between HMG I(Y) and the activators. Finally, we demonstrate that once assembled, the IFN-beta enhanceosome is an unusually stable nucleoprotein structure that can activate transcription at high levels by promoting multiple rounds of reinitiation of transcription.

Expression of Human Glucocorticoid Receptor Gene and Interaction of Nuclear Proteins with the Transcriptional Control Element

We have identified sequences responsible for the expression of the human glucocorticoid receptor gene (GR gene) using a set of 5' promoter deletion mutants in HeLa, human placenta, and human breast tumor (MCF-7) cells. The chimeric gene construct -892 5'-GAAGTGACACACTTC3' -878-CAT was sufficient for high level of expression in HeLa and placenta cells in culture. Deletion of palindromic sequences decreased levels of GR expression in these cells. By oligonucleotide-affinity chromatography with the palindromic glucocorticoid receptor enhancing factor-binding element (GREFE), we have isolated from human placenta nuclear extract two novel proteins glucocorticoid receptor enhancing factors 1 and 2 (GREF1 and GREF2), with apparent molecular masses of 80 and 62 kDa, respectively. These proteins, similar to the DNA-binding autoantigen Ku are, like Ku, heterodimers of polypeptide subunits p80 and p62, immunologically related to factors binding to proximal sequence element 1 in the promoter of small nuclear RNA (PSE1) and transferrin receptor enhancing factors. Both Ku80 and Ku70 polypeptides were present in high concentrations in human placenta and HeLa cells. In MCF-7 cells, however, only a high level of p62 was detected. While cotransfection of pcDNA-Ku80 with pHGR(-892 to -878)-CAT potentiated the expression of CAT, introduction of pcDNA-Ku70 did not affect the expression of CAT in transfected MCF-7 cells. UV cross-linking analysis showed that only GREF1 contacted DNA directly. Supershift assays with monoclonal antibodies Ab 111 (Ku80) or Ab N3H10 (Ku70) showed a direct interaction of GREF1 and GREF2 heterodimers with the palindrome. Partial peptide fingerprinting of GREF1 and GREF2 using alpha-chymotrypsin and immunoblotting with Ab 111 and Ab N3H10 confirmed their identities as Ku80 and Ku70, respectively.

Characterization of an upstream regulatory element of the human apolipoprotein E gene, and purification of its binding protein from the human placenta.

We have previously demonstrated that expression of the human apolipoprotein (apo) E gene is controlled by multiple regulatory elements in the promoter [Paik et al. (1988) J. Biol. Chem. 263, 13340-13349; Chang et al. (1990) J. Biol. Chem. 265, 9496-9504]. To extend these studies, we have characterized an element in the apoE gene promoter that spans nucleotides -101 to -89, upstream regulatory element 3 (URE3). Transcription of promoter/marker gene constructs in vitro showed that URE3 modulates gene expression. Gel mobility shift assays of URE3 using human placental nuclear extracts detected a specific binding protein whose activity can be modulated by micromolar amounts of divalent copper and zinc. Competitive binding and gel shift assays with mutant oligonucleotides revealed critical nucleotides within URE3 required for its specific nuclear protein-binding activity. Gel filtration and oligonucleotide affinity chromatography were employed to isolate a URE3-binding protein (URE3BP) from human placental nuclear extracts. Purified URE3BP appears to be a M(r) = 300,000 protein that is composed of four equally-sized basic subunits of M(r) = 67,000. These studies indicate that URE3 is an active regulatory component of the apoE gene.

The Epstein-Barr virus nuclear antigen 2 transactivator is directed to response elements by the J kappa recombination signal binding protein.

Epstein-Barr virus nuclear antigen 2 (EBNA-2) plays an essential role in primary B-lymphocyte growth transformation. EBNA-2 is an acidic transcriptional transactivator that is brought to virus and cell EBNA-2 response elements by interaction with a factor that recognizes the double-stranded sequence MNYYGTGGGAA, where M is A or C, N is any nucleotide, and Y is a pyrimidine. A 63-kDa protein that recognizes this DNA sequence has now been purified by S-Sepharose and oligonucleotide affinity chromatography. p63 peptide sequence is identical to the predicted amino acid sequence for the human J kappa immunoglobulin recombination signal binding protein. Purified or recombinant in vitro-translated J kappa binds to the MNYYGTGGGAA EBNA-2 response element sequence and interacts with EBNA-2. Surprisingly, J kappa does not bind to the J kappa 1 heptamer recombination signal sequence (CACTGTG), and its prior identification as a heptamer binding protein was most likely due to the addition of a BamHI restriction site to the native heptamer creating a near EBNA-2 response element consensus (CACTGTGGGAT).

Hormone- and DNA-binding mechanisms of the recombinant human estrogen receptor.

We have investigated the hormone- and DNA-binding mechanisms of the wild-type human estrogen receptor (hER) overproduced in insect cells using a baculovirus expression system. The recombinant hER was indistinguishable in size (67 kDa) and immunogenically from the native human estrogen receptor in MCF-7 breast carcinoma cells. The recombinant hER was purified to 70-80% homogeneity with a two-step procedure that included ammonium sulfate precipitation and oligonucleotide affinity chromatography using a unique Teflon affinity matrix. The recombinant hER bound estradiol with a positively cooperative mechanism. At hER concentrations in excess of 13 nM the Hill coefficient reached a maximal value of 1.6, whereas, at lower hER concentrations, the Hill coefficient approached 1.0, suggesting that the hER was dissociated to the monomeric species and site-site interactions were diminished. The hER specifically bound an estrogen responsive element (ERE) from chicken vitellogenin II gene as measured by the gel mobility assay, ethylation, and thymine interference footprinting. Specific interference patterns suggest a two-fold symmetry of the hER binding to the ERE with each monomer of the hER bound in the major groove of the DNA. These data indicate that the recombinant hER is valuable to define the biochemical and structural properties of the native estrogen receptor.

Identification and purification of DBF-A, a double-stranded DNA-binding protein from Saccharomyces cerevisiae.

Using oligonucleotide affinity chromatography with DNase I footprinting as an assay we have looked for proteins that interact with sequence elements within the yeast origin of replication, autonomously replicating sequence 1 (ARS1). In this work we describe a protein that binds with high affinity to DNA but displays only moderate sequence specificity. It is eluted at 0.7 M salt from an ARS1 oligonucleotide column. Footprinting analysis on ARS1 at a high protein concentration revealed at least three sites of protection flanking element A and its repeats. Element A itself is rendered hypersensitive to DNase I digestion upon protein binding. This pattern is also observed for the H4 and HMR-E ARSs, suggesting that the protein alters the DNA conformation at element A and its repeats. The affinity-purified fraction is also capable of supercoiling a relaxed, covalently closed plasmid in the presence of topoisomerase. Highly purified preparations of the protein are enriched in an 18-kDa polypeptide which can be renatured from a denaturing gel and shown to bind ARS1 DNA. We have designated this protein DBF-A, DNA-binding factor A.

Characterization of a cis-acting element required for efficient transcriptional activation of the collagen IV enhancer.

Two regulatory regions in the murine collagen IV enhancer were identified. Transient transfection assays delimited a 210-base pair fragment within the first intron of the alpha 1(IV) collagen gene that had significant transcriptional enhancer activity. DNase I protection and gel mobility shift confirmed that two regions, designated footprints A and B, within this fragment bound nuclear factors. Gel shift studies suggested that the CCTTATCTCTGATGG motif (A-34) in the footprint A region was important for specific nuclear factor binding. Mutations in the A-34 motif abolished factor binding as detected by gel shift and resulted in a significant decrease in enhancer activity in transient transfection assays of F9 teratocarcinoma cells. Two putative transcription factors of Mr = 37,000 and Mr = 94,000, which interact with the A-34 motif, were purified from Engelbreth-Holm-Swarm tumor tissue using DEAE-Sephacel, heparin-Sepharose, salmon sperm DNA-Sepharose, and specific A-34 oligonucleotide affinity chromatography. Southwestern analysis revealed that both of these factors were capable of binding the A-34 oligonucleotide directly and did not require additional subunits for binding. These data suggest that positively acting transcription factor(s) interact with the A-34 site in the enhancer and are required for efficient transcription of the alpha 1 and alpha 2(IV) collagen chain genes.

Identification and purification of a factor that binds to the Mlu I cell cycle box of yeast DNA replication genes.

In Saccharomyces cerevisiae, the genes encoding at least 10 enzymes involved in DNA replication are periodically expressed in the late G1 and S phases of the cell cycle. All of these genes have one copy or more of the sequence ACGCGT, which conforms to the recognition site for the Mlu I restriction endonuclease. For the CDC21, CDC9, and POL1 genes, the Mlu I site has been shown to be absolutely required for periodic transcription. Using nuclear extracts fractionated by conventional and oligonucleotide affinity chromatography, we have purified a 17-kDa protein that recognizes the Mlu I motif. Synthetic oligonucleotides containing mutated Mlu I sites do not bind the protein. In contrast, synthetic oligonucleotides derived from the CDC2, CDC6, and CDC21 genes, which are expressed with the same timing as POL1, bind purified protein efficiently.

Mammalian .beta.-polymerase promoter: large-scale purification and properties of ATF/CREB palindrome binding protein from bovine testes

The mammalian DNA repair enzyme beta-polymerase is encoded by a single-copy gene that is expressed in all tissues and cell lines studied to date. A protein fraction with high binding affinity for an ATF/CREB-like binding element, GTGACGTCAC, at -49 to -40 in the core beta-polymerase promoter has been purified to near-homogeneity from a nuclear extract of bovine testes. The major binding activity, as monitored by gel mobility shift assay, is recovered in 20% yield by a procedure involving oligonucleotide affinity chromatography. The purified protein yields DNase I footprinting and gel shift binding patterns indistinguishable from the activity in crude extracts. The final fraction activates transcription in an in vitro transcription reaction. The native molecular weight of the purified binding activity is about 100-120K as measured by gel filtration. SDS-PAGE of the purified fraction revealed that it contains several polypeptides in the molecular weight range of 30-52K, yet two of these peptides (Mr 49K and 52K) are predominant. Specific binding to the palindrome is salt-sensitive and is consistent with the formation of nine ion pairs (from log KA vs log KCl plots) and has a KA at 200 mM KCl of 5.8 X 10(11) M-1. Kinetic studies with synthetic oligonucleotides as binding ligands indicate that the purified protein can bind tighter to or discriminate between the beta-polymerase ATF/CREB element and similar elements derived from somatostatin and chorionic gonadotropin genes.

Functional analysis of a liver-specific enhancer of the hepatitis B virus.

The liver-specific enhancer I of the human hepatitis B virus contains several regions of DNA-protein interaction. Located within this element are also the domains of a promoter controlling the synthesis of the X open reading frame. Functional domains of the enhancer I and the X gene promoter were identified using DNase I protection analysis, deletion mutagenesis, and cell transfections. A unique liver-specific interaction was identified within this element whose binding site includes a direct sequence repeat, 5'-AGTAAACAGTA-3'. The factor(s) binding to this sequence motif was purified by oligonucleotide-affinity chromatography. Binding of this factor appears to play a key role in determining the overall enhancer function. Additionally, the interaction of several purified factors is presented. Cotransfection of liver cells with expression vectors encoding transcriptional factors resulted in trans-activation of the promoter/enhancer function. Based on the results of genetic analysis a model outlining the functional domains of the enhancer/promoter region is presented.

Properties and partial characterization of the heat-shock factor from Tetrahymena pyriformis

A heat-shock-factor-binding activity was identified in Tetrahymena pyriformis whole-cell extracts and was further purified by sequential heparin-agarose and sequence-specific oligonucleotide affinity chromatography. Tetrahymena heat-shock factor (HSF) was able to bind to the heat-shock elements (HSE) both before and after thermal stress, although heat shock altered both the HSE-binding affinity and the protein.DNA-complex mobility on polyacrylamide gels. The mobility difference was significantly reduced by treatment of the proteins with phosphatase. The HSE-binding proteins, isolated by oligonucleotide-affinity chromatography, migrated on SDS/polyacrylamide gels as a closely spaced doublet to about 70 kDa. Polypeptides with similar molecular mass were recovered from preparative band-shift gels indicating that both are components of the protein.DNA complex.

Nuclear proteins TREF1 and TREF2 bind to the transcriptional control element of the transferrin receptor gene and appear to be associated as a heterodimer.

Two novel proteins that bind specifically to the transferrin receptor (TR) promoter, have been isolated from HeLa cell nuclear extract using a combination of ion exchange and oligonucleotide-affinity chromatography. TREF1 and TREF2, which have apparent molecular weights of 82 and 62 kDa, respectively, appear to be associated as a heterocomplex (TREF), and both proteins are able to contact target DNA directly. TREF interacts specifically with a region of the TR promoter which contains the TR transcriptional control element. This region is similar in sequence to the cAMP-responsive and phorbol ester-responsive elements found in several viral and cellular genes. Binding of TREF to the TR promoter results in modification of DNA topology over multiple helical turns, including a sequence revealed by a helical periodicity map as having an unusual structure.

Identification of multiple protein binding domains in the promoter-regulatory region of the phosphoenolpyruvate carboxykinase (GTP) gene

Transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) from the rat is acutely regulated by a number of hormones, including glucagon (acting via cAMP), glucocorticoids, and insulin. In this study we demonstrate by DNase I footprinting that a region of the PEPCK promoter, extending from -460 to +73, contained eight protein binding domains. Two nuclear proteins protected adjacent sites from -121 to -99 and -96 to -77, which have been previously shown to be involved in maintaining the level of basal gene transcription and conferring cAMP responsiveness, respectively. Oligonucleotide competition studies suggested that the protein(s) binding to the cAMP-responsive element (CRE) occupies a second site at -147 to -130, which has a high degree of sequence homology to the CRE, and also binds to two other elements that show partial sequence homologies. The protein(s) which bound to these four elements copurified through oligonucleotide affinity chromatography, suggesting that the PEPCK promoter has four binding sites for the CRE-binding protein(s). Potential tissue-specific elements in the PEPCK promoter were identified by footprinting with nuclear extracts prepared from rat liver, kidney, brain, and spleen. The multiple protein-binding sites in this promoter-regulatory region reflect the complex transcriptional regulation that is characteristic of this gene.

Identification and purification of the enhancer-binding factor of human immunodeficiency virus-1: Multiple proteins and binding to other enhancers

Abstract The enhancer-binding factor of human immunodeficiency virus (HIV)-1 was purified from human B cells by sequence-specific duplex oligonucleotide affinity chromatography. Gel retardation assay and footprint analysis showed that the purified factor bound specifically to the HIV-1 enhancer sequence, and protected both direct repeats in the HIV-1 enhancer. The purified factor consisted of four main polypeptides of the molecular weight of 36,000-42,000. At least three of them had enhancer-binding activity after elution from sodium dodecyl sulfate-polyacrylamide gel and renaturation. UV cross-linking analysis also showed that at least two of the polypeptides in purified fraction had a binding activity specific for the HIV-1 enhancer. The purified factor activated transcription from the HIV-1 promoter in vitro, confirming that it was indeed a transcription factor for HIV-1. The purified factor also recognized sequences in the immunoglobulin kappa gene enhancer and the major histocompatibility complex class I gene enhancer with almost the same affinity as the HIV-1 enhancer. These results suggested the existence of multiple proteins which recognize the kappa B-related sequences. This regulatory factor should help in the study of the biochemical pathway underlying HIV production from latently infected cells.