DNA Affinity Precipitation Assay Research Articles

Vitamin D3 receptor/Sp1 complex is required for the induction of p27Kip1 expression by vitamin D3.

1alpha,25-dihydroxyvitamin D3 (vitamin D3) has been shown to upregulate p27Kip1 expression via Sp1 and NF-Y binding sites in the p27Kip1 promoter. However, whether vitamin D3 receptor (VDR) involves in this process is unclear. In this study, we demonstrated that expression of VDR in SW620 cells, which exhibited low level of endogenous VDR, increased vitamin D3-stimulated p27Kip1 promoter activity. On the contrary, suppression of Sp1 expression by small interference RNA reduced the stimulation of p27Kip1 promoter activity by vitamin D3 in LNCaP cells. DNA affinity precipitation assay and chromatin immunoprecipitation assay showed that VDR bound to the p27Kip1 promoter in vitro and in vivo. In addition, we also demonstrated that VDR interacted with Sp1 in vitro and in cells. Collectively, our results suggest that VDR is involved in the induction of p27Kip1 by vitamin D3 and may interact with Sp1 to modulate the expression of target genes that lack VDR response element (VDRE) in their promoters.

Tenascin-C upregulation by transforming growth factor-beta in human dermal fibroblasts involves Smad3, Sp1, and Ets1.

In cultured human dermal fibroblasts, transforming growth factor (TGF)-beta induced the mRNA expression of tenascin-C (TN-C). The molecular mechanism(s) underlying this process is not presently understood. In this study, we performed serial 5' deletion and a transient transfection analysis to define a region in the TN-C promoter mediating the inducible responsiveness to TGF-beta. This region contains an atypical nucleotide recognition element for the Smad family of transcriptional regulators. A DNA affinity precipitation assay revealed that Smad2/Smad3 bound to this site in a transient and specific manner. Overexpression of Smad3 or Smad4 activated the TN-C promoter activity and superinduced the TN-C promoter activity stimulated by TGF-beta. Moreover, simultaneous cotransfection of Smad3 and Smad4 activated the TN-C promoter activity in a synergistic manner. Mutation of the Smad-binding sites, the Ets-binding sites, or Sp1/3-binding sites in the TN-C promoter abrogated the TGF-beta/Smad-inducible promoter activity. Immunoprecipitation analysis revealed that Smad3, Sp1, and Ets1 form a transcriptionally active complex. Furthermore, the interaction between Smads and CBP/p300 in TGF-beta signaling was confirmed. These findings demonstrate the existence of a novel, functional binding element in the proximal region of the TN-C promoter mediating responsiveness to TGF-beta involving Smad3/4, Sp1, Ets1, and CBP/p300.

Involvement of histone deacetylation in ras-induced down-regulation of the metastasis suppressor RECK

RECK is a membrane-anchored glycoprotein that may negatively regulate matrix metalloproteinase (MMP) activity and inhibit tumor metastasis. Previous study demonstrated that oncogenic ras inhibited RECK expression via an Sp1 binding site in the RECK promoter. In this study, we investigated the molecular mechanism by which ras inhibited RECK expression. Co-transfection assay showed that Sp1 and Sp3 are transactivators, rather than repressors, for RECK gene. So, we tested whether ras activation induced the binding of histone deacetylases (HDACs) to Sp1 to repress RECK expression. Our data showed Sp1-associated HDAC1 in cells was increased after ras induction. By using DNA affinity precipitation assay, we found that induction of oncogenic ras enhanced the binding of HDAC1 to the DNA probe corresponding to the Sp1 site in the RECK promoter. Additionally, a HDAC inhibitor trichostatin A (TSA) potently antagonized the inhibitory action of ras on RECK. The signaling pathway by which ras suppresses RECK was also addressed. Induction of oncogenic ras activated extracellular signal-regulated kinase (ERK), but not c-Jun N-terminal kinase (JNK) and p38 HOG kinase in 2–12 cells. Addition of PD98059 or overexpression of dominant-negative mutant of ERK2 indeed reversed ras-mediated inhibition of RECK promoter activity. Taken together, our results suggest that oncogenic ras represses RECK expression via a histone deacetylation mechanism.

Induction of Disease-associated Keratin 16 Gene Expression by Epidermal Growth Factor Is Regulated through Cooperation of Transcription Factors Sp1 and c-Jun

Overexpression of keratin 16 has been observed in keratinocytes in those skin diseases characterized by hyperproliferation such as psoriasis. Therefore, keratin 16 is usually referred to as a disease-associated keratin. In the present study, we found that epidermal growth factor (EGF) increased the expression of keratin 16 mRNA and protein synthesis in a time-dependent manner in HaCaT cells. Reporter assays revealed that the EGF response region was in the range of -162 to -114 bp. Disruption of the Sp1 site (-127 to -122 bp) and the AP1 site (-148 to -142 bp) of the keratin 16 promoter by site-directed mutagenesis significantly inhibited keratin 16 promoter activity induced by EGF. Furthermore, keratin 16 gene expression induced by Ras activation was also regulated in the same manner as the EGF response. By using the DNA affinity precipitation assay in HaCaT and SL2 cells, Sp1 directly interacted with the Sp1 site of the promoter, and c-Jun and c-Fos precipitated with the Sp1 oligonucleotide was attributable to the interaction between the Sp1 and AP1 proteins. Moreover, cotransfection assays revealed that Sp1 acted synergistically with c-Jun to activate keratin 16. The coactivators p300/CBP could collaborate with Sp1 and c-Jun in the activation of keratin 16 promoter, and EGF-induced promoter activation was blocked by the viral oncoprotein E1A. Taken together, these results suggest that Sp1 and AP1 sites in the essential promoter region are critical for EGF response, and Sp1 showed a functional cooperation with c-Jun and coactivators p300/CBP in driving the transcriptional regulation of EGF-induced keratin 16 gene expression.

CO2 Response Element and Corresponding trans-acting Factor of the Promoter for Ribulose-1,5-bisphosphate Carboxylase/oxygenase Genes in Synechococcus sp. PCC7002 Found by an Improved Electrophoretic Mobility Shift Assay

We analyzed the promoter of the genes encoding the ribulose-1,5-bisphosphate carboxylase/oxygenase (rbc) in the cyanobacterium Synechococcus sp. PCC7002 and localized the CO(2)-regulatory element. Cyanobacterial transformants were constructed with several DNA segments of the rbc promoter fused to the chloramphenicol acetyltransferase (CAT) gene, and their acetyltransferase activities were analyzed under 0.03% and 1% CO(2) conditions. We found that the AT-rich element localized from -262 to -291 relative to the rbc translation-starting site was required for CO(2)-dependent repression. Fluorescent-labeled oligonucleotide probes of identical sequence to the AT-rich element were reacted with protein extracts from cells cultured under conditions of low and high CO(2) atmospheric content. We detected a gel retardation complex of a strong signal intensity in extracts from cells cultured under 15% CO(2), but only a weak signal from cells cultured under 1% CO(2). Moreover, a DNA affinity precipitation assay identified a 16-kDa protein that bound to nucleotide sequences within the AT-rich element. The partial amino acid sequence of the protein was similar to the deduced protein sequences of ORF129 and ORF155 from Synechocystis 6803. Our findings suggest that the AT-rich element plays a role as a negative CO(2)-regulatory element and its trans-acting factor possibly regulates the rbc transcription in response to CO(2) levels.

Constitutive expression of the Id-1 promoter in human metastatic breast cancer cells is linked with the loss of NF-1/Rb/HDAC-1 transcription repressor complex.

The helix-loop-helix protein Id-1 is a dominant negative regulator of basic helix-loop-helix transcription factors, and plays a key role in the control of breast epithelial cell growth, invasion and differentiation. Previous investigations in our laboratory have shown that Id-1 mRNA was constitutively expressed in highly aggressive and invasive human breast cancer cells in comparison to non-transformed or non-aggressive cancerous cells, and that this loss of regulation is mediated by a 2.2-kb region of the human Id-1 promoter. Here we show that a 31 bp sequence within this 2.2-kb promoter, located 200 bp upstream of the initiation of transcription, is responsible for the constitutive expression of Id-1 in metastatic human breast cancer cells. Using gel shift experiments, we identified a high molecular weight complex present only in non-aggressive breast cancer cells cultured in serum-free medium and which appear to be necessary for proper Id-1 repression. In contrast, nuclear extracts from highly aggressive and metastatic cell lines do not contain this large molecular weight complex. Using DNA affinity precipitation assays (DAPA), we show that this complex contains SP-1, NF-1, Rb and HDAC-1 proteins. On the basis of these findings, we propose a mechanism for the loss of regulation of Id-1 promoter in invasive and metastatic human breast cancer cells.

Interaction of Gli2 with CREB protein on DNA elements in the long terminal repeat of human T-cell leukemia virus type 1 is responsible for transcriptional activation by tax protein.

The long terminal repeat (LTR) of human T-cell leukemia virus type 1 (HTLV-1) has two distinct DNA elements, one copy of TRE2S and three copies of a 21-bp sequence that respond to the viral trans-activator protein, Tax. Either multiple copies of the 21-bp sequence or a combination of one copy each of TRE2S and 21-bp sequence is required for efficient trans activation by Tax. In the trans activation of multiple copies of 21-bp sequence, CREB/ATF protein plays an essential role in forming a complex with Tax. To understand the role of TRE2S in trans activation of one copy of 21-bp sequence, we examined protein binding to the DNA elements by DNA affinity precipitation assay including Gli2 protein binding to TRE2S and CREB protein binding to 21-bp sequence. Binding of CREB to a DNA probe containing both elements, TRE2S-21bp probe, was dependent on Gli2 protein under restricted conditions and was enhanced in a dose-dependent fashion by the binding of Gli2 protein to the same probe. Mutation in either element abolished the efficient binding of CREB. A glutathione S-transferase fusion protein of a fragment of Gli2 was able to bind to CREB. Therefore, Gli2-CREB interaction on the DNA probe is proposed to stabilize CREB binding to DNA. Tax can bind to CREB protein on the DNA; therefore, stabilization of DNA binding of CREB results in more recruitment of Tax onto DNA. Conversely, Tax increased the DNA binding of CREB, although it had almost no effect on the binding of Gli2. These results suggest that Gli2 binds to the DNA element and interacts with CREB, resulting in more recruitment of Tax, which in turn stabilizes DNA binding of CREB. Similar cooperation of the protein binding to TRE2S-21bp probe was also observed in nuclear extract of an HTLV-1-infected T-cell line. Consistent with the Gli2-CREB interaction on the DNA elements, Tax-mediated trans activation was dependent on the size of the spacer between TRE2S and 21-bp sequence. The effective sizes of the spacer suggest that TRE2S in the LTR would cooperate with the second and third copies of the 21-bp sequence and contribute to trans activation of the viral gene transcription.

The trans-activator tax of human T-cell leukemia virus type 1 (HTLV-1) interacts with cAMP-responsive element (CRE) binding and CRE modulator proteins that bind to the 21-base-pair enhancer of HTLV-1.

The trans-activator protein Tax of human T-cell leukemia virus type 1 (HTLV-1) activates the viral 21-base-pair (bp) enhancer in the long terminal repeat and has been suggested to associate indirectly with the enhancer DNA. To demonstrate this, we used DNA-affinity precipitation assay and detected the Tax protein in 21-bp DNA-protein complexes isolated from HTLV-1-infected cells. To identify cellular components in the complexes, we tested various 21-bp DNA-binding proteins by gel electrophoretic mobility-shift assay. Each binding protein gave a shifted band of each 21-bp DNA-protein complex, and exogenously added Tax protein further shifted these bands of cAMP-responsive element (CRE) binding protein (CREB) and CRE modulator but did not shift other bands. Anti-Tax antibodies blocked formation of the complex, indicating complex formations of [Tax-CREB(or CRE modulator)-21-bp DNA]. The formations of these complexes paralleled the functional activities of Tax mutants. Furthermore, the Tax-CREB complex was detected in a nuclear extract of HTLV-1-infected cells, and the Tax-CREB-21-bp-DNA complex was demonstrated as a major component of Tax complexes containing the 21-bp DNA probe. These observations indicate that Tax protein binds to CREB and CRE modulator and the complexes then bind to the 21-bp enhancer, suggesting that the complex binding to the enhancer mediates trans-activation of transcription.

The kappa B enhancer motifs in human immunodeficiency virus type 1 and simian virus 40 recognize different binding activities in human Jurkat and H9 T cells: evidence for NF-kappa B-independent activation of the kappa B motif.

The kappa B transcriptional enhancer motif, present in many viruses, is broadly active in many cell types. It is recognized by c-Rel/HIVEN86A in DNA affinity precipitation (DNAP) assays and by the Rel-related p50 and p65 subunits of the nuclear factor NF-kappa B in electrophoretic mobility shift assays (EMSA). We have analyzed activities that bind the human immunodeficiency virus type 1 and simian virus 40 kappa B motifs in two human leukemia cell lines, Jurkat and H9. In both DNAP and EMSA analyses of Jurkat cell extracts, we detected multiple kappa B motif-binding activities in addition to c-Rel/HIVEN86A and p50-p65 NF-kappa B. In Jurkat cell nuclear extracts, EMSA analysis revealed at least six specific DNA-protein complexes, of which one comigrated with the p50-p65 NF-kappa B complex. Formation of all six complexes was enhanced by stimulation of the cells with phorbol 12-myristate-13-acetate and phytohemagglutinin but was differentially affected by the salt concentration in the binding reaction and by the conditions of Jurkat cell growth. Nuclear extracts from both unstimulated and stimulated H9 cells revealed similar levels of five kappa B motif-specific complexes, all of which displayed mobilities distinct from those of the Jurkat cell complexes. Indeed, a complex corresponding to p50-p65 NF-kappa B was not detectable in nuclear extracts from unstimulated H9 cells although such a complex was apparent in nuclear extracts from stimulated H9 cells. In contrast to the inducibility of a p50-p65 NF-kappa B-like complex, transcriptional enhancers composed of multimerized kappa B motifs displayed similar high levels of activity in both the unstimulated and stimulated H9 cells. Thus, the activity of the kappa B motif in H9 cells corresponded to the abundance of the H9 cell-specific kappa B motif complexes and not to the levels of p50-p65 NF-kappa B complex. These results suggest that the broad activity of the kappa B enhancer element is not only due to the broadly distributed NF-kappa B activator but also to cell type-specific kappa B motif-binding activities.

Binding of the HTLV-I Tax1 transactivator to the inducible 21 bp enhancer is mediated by the cellular factor HEB1.

Transcription driven by the HTLV-I promoter is strongly activated by the viral transactivator protein Tax1. This effect is mediated via a 21 bp sequence which is imperfectly repeated three times in the viral promoter. We showed previously that a single 21 bp copy exhibits a strong Tax1-inducible enhancer activity and is able to bind different cellular proteins, namely ATF, HEB1 and HEB2. We have further investigated the molecular mechanism involved in the Tax1 induction of the 21 bp motif's enhancer activity by analysing Tax1 interaction with this DNA sequence. For this purpose a HeLa cell line constitutively expressing a functional Tax1 protein was established and nuclear extracts of these cells were used to perform a DNA affinity precipitation assay. This experimental approach allowed us to show that Tax1 specifically binds to the 21 bp motif. The same sequence elements of the 21 bp motif are required both for Tax1 binding and for Tax1-induced enhancer activity. Chromatographic fractionation of the HeLa tax nuclear extract showed that the binding is indirect and is mediated by the cellular factor HEB 1.

Interaction of the v-rel protein with an NF-kappa B DNA binding site.

The avian reticuloendotheliosis virus T contains within its genome the oncogene rel. The expression of this gene is responsible for the induction of lymphoid tumors in birds. Recently, the rel gene was shown to be related to the p50 DNA binding subunit of the transcription factor complex NF-kappa B. Binding sites for the NF-kappa B complex are found in the enhancer regions of a number of genes, including the immunoglobulin kappa gene and the human immunodeficiency virus long terminal repeat. In this communication we identify an activity from avian reticuloendotheliosis virus T-transformed avian lymphoid cells that binds in an electrophoretic-mobility-shift assay to an NF-kappa B binding site from the kappa enhancer. This activity contains proteins immunologically related to rel, as detected by polyclonal and monoclonal antibodies directed against v-rel. In a DNA affinity precipitation assay using the NF-kappa B site from the human immunodeficiency virus long terminal repeat, v-rel and several other proteins were identified. These data suggest that oncogenic transformation by v-rel is the result of an altered pattern of gene expression.

Stimulation of the human immunodeficiency virus type 1 enhancer by the human T-cell leukemia virus type I tax gene product involves the action of inducible cellular proteins

The human immunodeficiency virus type 1 (HIV-1) preferentially infects CD4+ T lymphocytes and may exist as a latent provirus within these cells for extended periods. The transition to a productive retroviral infection results in T-cell death and clinically may lead to the acquired immune deficiency syndrome. Accelerated production of infectious HIV-1 virions appears to be closely linked to a heightened state of T-cell activation. The transactivator (Tax) protein of the type I human T-cell leukemia virus (HTLV-I) can produce such an activated T-cell phenotype and augments activity of the HIV-1 long terminal repeat. One Tax-responsive region within the HIV-1 long terminal repeat has been mapped to a locus composed of two 10-base-pair direct repeats sharing homology with the binding site for the eucaryotic transcription factor NF-kappaB (GGGACTTTCC). Tax-expressing Jurkat T cells contain one or more inducible cellular proteins that specifically associate with the HIV-1 enhancer at these binding sites. Microscale DNA affinity precipitation assays identified a Tax-inducible 86-kilodalton protein, HIVEN86A, as one of these HIV-1 enhancer-binding factors. The interaction of HIVEN86A, and presumably other cellular proteins, with the HIV-1 enhancer appears functionally important as oligonucleotides corresponding to this enhancer were sufficient to impart Tax inducibility to an unresponsive heterologous promoter. These findings suggest that the Tax-inducible cellular protein HIVEN86A plays an important role in the transcriptional activation of the HIV-1 enhancer. These specific protein-DNA interactions may also be important for the transition of HIV-1 from a latent to a productive mode of infection. Furthermore, these findings highlight an intriguing biological interplay between HTLV-1 and HIV-1 through a cellular transcriptional pathway that is normally involved in T-cell activation and growth.

HTLV-I Tax Induces Cellular Proteins that Activate the κB Element in the IL-2 Receptor α Gene

Jurkat T cell lines constitutively expressing Tax, the 40-kilodalton transactivator protein of human T lymphotropic virus type I (HTLV-I), were used to investigate the mechanism by which this viral product deregulates the expression of the interleukin-2 receptor alpha gene (IL-2R alpha, Tac). Transfection of deleted forms of the IL-2R alpha promoter and in vitro DNA-binding studies revealed that a 12-base pair promoter segment, which has homology with the binding site for NF-kappa B, was required for Tax-induced activation of the IL-2R alpha promoter in vivo. An 18-base pair oligonucleotide containing this kappa B-like regulatory element proved sufficient to confer Tax inducibility upon a heterologous promoter. DNA affinity precipitation assays showed that Tax, like mitogenic stimuli, induced the expression of the 86-kilodalton cellular protein HIVEN86A, which specifically binds to the IL-2R alpha kappa B element in vitro. Furthermore, DNA/protein cross-linking studies revealed that several polypeptides interact with this sequence motif. Thus, the deregulation of IL-2R alpha gene expression encountered in HTLV-I leukemias appears to involve Tax activation of one or more cellular proteins that are normally induced by mitogens and that directly contribute to transcriptional activation of this receptor gene.

HTLV-I tax induces cellular proteins that activate the kappa B element in the IL-2 receptor alpha gene.

Jurkat T cell lines constitutively expressing Tax, the 40-kilodalton transactivator protein of human T lymphotropic virus type I (HTLV-I), were used to investigate the mechanism by which this viral product deregulates the expression of the interleukin-2 receptor alpha gene (IL-2R alpha, Tac). Transfection of deleted forms of the IL-2R alpha promoter and in vitro DNA-binding studies revealed that a 12-base pair promoter segment, which has homology with the binding site for NF-kappa B, was required for Tax-induced activation of the IL-2R alpha promoter in vivo. An 18-base pair oligonucleotide containing this kappa B-like regulatory element proved sufficient to confer Tax inducibility upon a heterologous promoter. DNA affinity precipitation assays showed that Tax, like mitogenic stimuli, induced the expression of the 86-kilodalton cellular protein HIVEN86A, which specifically binds to the IL-2R alpha kappa B element in vitro. Furthermore, DNA/protein cross-linking studies revealed that several polypeptides interact with this sequence motif. Thus, the deregulation of IL-2R alpha gene expression encountered in HTLV-I leukemias appears to involve Tax activation of one or more cellular proteins that are normally induced by mitogens and that directly contribute to transcriptional activation of this receptor gene.

Characterization of cellular proteins recognizing the HIV enhancer using a microscale DNA-affinity precipitation assay.

Numerous nucleic acid sequence motifs have been identified as transcriptional regulatory elements, and proteins involved in the control of gene replication and transcription have been successfully purified using nucleic acid affinity procedures. We have developed an assay that allows direct characterization of cellular proteins binding to the enhancer element of the human immunodeficiency virus (HIV), which is analogous to immunoprecipitation techniques. In extracts of H9 cells, a human CD4+ lymphoblast line clonally selected for its ability to support HIV replication, we find a class of proteins that interact specifically with the HIV enhancer. Reproducible high-resolution, two-dimensional gel electrophoresis has allowed us to resolve these proteins into two major sets. One set is common to H9 cells, two human B-lymphoblast lines, and a phytohaemagglutinin (PHA)-stimulated human CD4+ lymphoblast line (Jurkat). A second set of proteins is found only in H9 cells. Thus, with this assay we have identified HIV enhancer-binding proteins that are constitutive or inducible and that are cell-type specific.