Intracellular redistribution of Ku immunoreactivity in response to cell-cell contact and growth modulating components in the medium.

Ku is a heterodimeric protein first recognized as a human autoantigen but now known to be widely distributed in mammalian cells. Analysis of repair-deficient mutant cells has shown that Ku is required for DNA repair, and roles in DNA replication and transcription have also been suggested on the basis of in vitro observations. Ku is generally regarded as a nuclear component. However, in the present paper, we show that a quantitatively significant fraction (half or more) of Ku is located in the cytoplasm of cultured primate cells, and that major changes in epitope accessibility of both nuclear and cytoplasmic Ku components are associated with the transition from sparse to confluent cell densities. The same changes in immunoreactivity were seen in HeLa, 293, CV-1 (monkey) and HPV-transformed keratinocyte cell lines, and in primary cultures of human keratinocytes. The immunostaining pattern of sparsely grown cells could be converted to the 'confluent' configuration by re-plating them at the same low density on a monolayer of mouse 3T3 cells. The confluent antigen pattern could also be induced in sparse cells within 15-30 minutes by exposure of the cells to serum- or Ca(2+)-free medium or overnight with 2 mM hydroxyurea. Somatostatin at 0.12 mM blocked the effects of serum/Ca2+ deprivation of Ku p70 antigen distribution in sparse CV-1 cells, and in confluent cultures reversed the usual nuclear concentration of p70 immunoreactivity. However, somatostatin did not alter the expected immunostaining patterns of p86. Preliminary studies indicate that sparse CV-1 cells, but not HeLa cells, respond to as little as 1 pM of TGF-beta 1 in the culture medium by the rapid appearance of nuclear immunoreactivity. TGF-alpha had no apparent effect. These findings are consistent with the participation of Ku in a signal transduction system responsive to the inhibitory effect of cell-cell contact on the one hand and to cytokines and growth-supportive components of the culture medium on the other.

Interacisternal A-particle (IAP) genes show similar patterns of hypomethylation in established and primary mouse plasmacytomas.

Alterations in cell programming associated with neoplastic transformation may involve widespread changes in patterns of DNA methylation. Increased expression of IAP elements in plasmacytomas compared with LPS-stimulated normal B-cells is accompanied by extensive hypomethylation of IAP sequences (Mietz and Kuff 1990), subsets of which are revealed with the LS2, LS3 and T1 probes. Multiple common LS- and PC-specific IAP loci are hypomethylated in established plasmacytomas, showing that hypomethylation does not occur entirely randomly. Many of the same IAP loci are hypomethylated in primary plasmacytomas induced by two different methods, as soon as recognizable tumor tissue can be isolated. In primary tumors hypomethylation frequently appears to occur in DNA flanking the IAP elements. In the established tumors the hypomethylated sites occur primarily in the IAP LTR, suggesting that for these loci hypomethylation begins in the flanking DNA and is extended into the IAP LTRs during progression of the tumors. The newly hypomethylated IAP LTRs in primary plasmacytomas (as compared to normal B cells) may provide a set of reporter genes for chromosomal regions that are characteristically hypomethylated in these transformed cells and that may contain cellular genes whose activation is related to the transformation process.

DNA-dependent protein kinase is activated by nicks and larger single-stranded gaps.

DNA-PK is a DNA-activated serine/threonine protein kinase capable of phosphorylating a number of nuclear DNA-binding proteins. Purified human DNA-PK has two subunits, a 350-kDa polypeptide, Prkdc, which binds ATP and is presumed to contain the catalytic site, and the Ku autoantigen which mediates DNA binding and activation. Previous studies have shown that DNA-PK is activated in vitro by linear double-stranded DNA fragments; however, the Ku subunit binds a broader range of DNA structures. Here we show that EBP-80, a protein originally isolated as a transcription factor for a retroviral long terminal repeat element and subsequently found to be similar to if not identical with Ku, also mediates kinase activation. The EBP-80-Prkdc complex is activated by nanomolar concentrations of DNA constructs containing single-to-double strand transitions, including a closed stem-loop structure and single strand gaps of 0 (nick), 6, and 30 nucleotides. Kinase activation parallels the ability of EBP-80 to bind these and other constructs. Our results extend the range of DNA configurations known to activate DNA-PK and are consistent with the participation of this enzyme complex in several nuclear functions.

Selective expression of intracisternal A-particle genes in established mouse plasmacytomas.

Mouse plasmacytomas generally express higher levels of RNA transcripts from endogenous intracisternal A-particle (IAP) proviral elements than do lipopolysaccharide-stimulated normal lymphocytes. Lymphocytes express a limited and highly characteristic set of IAP elements (lymphocyte-specific [LS] elements). In this study, we examined whether LS elements are expressed at higher levels after transformation of the cells and/or whether new IAP elements are activated. The IAP elements expressed in plasmacytoma MPC11 were characterized by sequence analysis of 22 cDNA clones. The long terminal repeats (LTRs) of the tumor cDNAs proved to be highly related in sequence. None of the clones was of the LS cDNA type. The MPC11 LTRs were five- to sixfold more active than an LS cDNA LTR when tested for promoter activity by transfection into plasmacytoma cells. The LTRs of the tumor-derived cDNAs contained a canonical ATF core sequence (ATF-PC), while the LS cDNAs contained an altered sequence (ATF-LS). An ATF-PC oligonucleotide probe detected multiple IAP transcripts on Northern (RNA) blots of RNA from several plasmacytoma but gave no reaction with RNA from stimulated B lymphocytes. In contrast, an ATF-LS probe detected higher levels of RNA in lymphocyte than in tumor RNAs. Thus, expression of IAP elements in transformed B cells is selective for a different set of regulatory sequence variants than those expressed in normal B cells. Other oligonucleotide probes representing LS- and PC-specific sequence variants detected multiple common hypomethylated IAP proviral loci in three independently derived plasmacytomas. Overall, the results show that established plasmacytomas exhibit a characteristic pattern of IAP proviral hypomethylation and regulatory sequence selection.

EBP-80, a transcription factor closely resembling the human autoantigen Ku, recognizes single- to double-strand transitions in DNA.

We have previously reported the purification and characterization of the transcription factor EBP-80 (Falzon, M., and Kuff, E. L. (1989) J. Biol. Chem. 264, 21915-21922). EBP-80 mediates the DNA methylation effect on transcription from an endogenous proviral long terminal repeat. Here we show that EBP-80 is very similar if not identical to the Ku autoantigen, a heterodimeric nuclear protein first detected by antibodies from autoimmune patients (Mimori, T., Akizuki, M., Yamagata, H., Inada, S., Yoshida, S., and Homma, M. (1981) J. Clin. Invest. 68, 611-620). A number of laboratories have shown that the Ku protein complex binds to free double-stranded DNA ends. In this study, we have examined the binding properties of EBP-80. EBP-80 binds single-stranded DNA with low affinity. Binding to random sequence double-stranded DNA depends on the length of the duplex and is optimal with oligomers of 30 and 32 base pairs; the protein complexes formed with these oligomers have Kd values of 15-20 pM. It binds with comparable high affinities to blunt-ended duplex DNA, to duplex DNA ending in hairpin loops, and to constructs in which an internal segment of duplex DNA is flanked by single-strand extensions. EBP-80 also interacts effectively with circular duplex molecules containing a 30-nucleotide single-stranded region (gap) or a double-stranded segment of nonhomology (bubble), but only weakly with the corresponding closed circular construct made up entirely of duplex DNA. EBP-80 prefers A/T to G/C ends. The binding properties of EBP-80 are consistent with the hypothesis that is recognizes single- to double-strand transitions in DNA. A model is presented for the interaction of EBP-80 with its target sequence in the proviral long terminal repeat.

Genomic mapping of intracisternal A-particle proviral elements.

Intracisternal A-particle (IAP) proviral elements are moderately reiterated and widely dispersed in the mouse genome. Oligonucleotide probes have been derived from three distinctive IAP element subfamilies (LS elements) that are transcriptionally active in normal mouse B- and T-cells. In HindIII digests, LS element-specific oligonucleotides each react with a limited number of restriction fragments that represent junctions between proviral and flanking DNA. These fragments have characteristic strain distribution patterns (SDPs) which are polymorphic in the DNAs of different mouse strains. We have established chromosomal assignments for 44 LS proviral loci by comparing their SDPs with those of known genetic markers in the BXD set of RI mouse strains. Some of the loci have also been scored in the CXB RI set. The IAP LS loci can provide a significant number of markers with a recognized genetic organization to the mouse genome map.

Selective activation of a discrete family of endogenous proviral elements in normal BALB/c lymphocytes.

Intracisternal A-particle (IAP) proviral elements are abundant and widely dispersed in the mouse genome. IAP-related transcripts have been detected in normal mouse tissues where expression is under genetic control. In this study, we sought to determine whether IAP expression in BALB/c thymus and lipopolysaccharide-stimulated B cells was due to selective or indiscriminate activation of IAP elements. cDNA libraries were prepared from each source. A total of 86 IAP cDNA clones were isolated from both libraries, and 37 of these were sequenced over a common 0.7- to 1.0-kb region of the IAP genome that included the 3' long terminal repeat (LTR). Three highly related families of elements were found to be expressed in the two cell types examined. All of the related elements had a distinctive U3 regulatory region. Thirteen individual IAP proviral elements were distinguished on the basis of sequence differences within the R region of the LTR. Hybridization of genomic DNA with element-specific oligonucleotide probes confirmed the presence of a restricted number of proviral copies in the lymphocyte-specific family of elements. Most of these copies were found to be methylated in the lymphocyte DNA, but at least seven were hypomethylated in their 5' LTRs. This study shows that activation of IAP elements in normal normal mouse lymphocytes is highly selective. Activation is probably a function of both sequence specificity and methylation status of the proviral LTR.

Intracisternal A-particle-specific oligonucleotides provide multilocus probes for genetic linkage studies in the mouse.

Oligonucleotide probes representing distinct intracisternal A-particle (IAP) subfamilies were derived from the long terminal repeats (LTRs) of transcriptionally active IAP genes in normal mouse cells. These probes were used to examine the distribution of IAP proviral elements in the genomic DNA of several inbred mouse strains. Each oligonucleotide probe identified multiple polymorphisms between the different strains. The distribution of polymorphic restriction fragments among the CXB set of recombinant inbred (RI) strains demonstrates the feasibility of using these probes for chromosome mapping. These and other subset-specific IAP probes can provide a useful series of multilocus markers for genomic mapping and genetic analysis in the mouse.

Binding of the transcription factor EBP-80 mediates the methylation response of an intracisternal A-particle long terminal repeat promoter.

Intracisternal A-particle (IAP) expression in mouse cells has been correlated with hypomethylation of HhaI and HpaII sites in proviral long terminal repeats (LTRs). In a previous study, in vitro methylation of three HhaI sites in the U3 region of the LTR from the cloned genomic IAP element, MIA14, was shown to inhibit promoter activity in vivo. In this study, we found by site-directed mutagenesis that the two more downstream HhaI sites within this LTR were responsible for the methylation effects on promoter activity in vivo; methylation of the other (5') HhaI site, which lies within a putative SP1 binding domain, did not affect promoter activity. Methylation of the HhaI sites also inhibited promoter activity of the LTR in a cell-free transcription system. Exonuclease III footprinting demonstrated methylation-induced changes in protein binding over the region encompassing the downstream HhaI site, designated the Enh2 domain. The protein that interacts specifically with this domain, EBP-80, was characterized in a previous study (M. Falzon and E. L. Kuff, J. Biol. Chem. 264:21915-21922, 1989). We show here that the presence of methylcytosine in the HhaI site within the Enh2 domain inhibited binding of EBP-80 in vitro. The methylated MIA14 LTR construct was much less responsive to added EBP-80 in an in vitro transcription system than was the unmethylated construct. These data suggest that CpG methylation within the Enh2 domain may exert its effect on transcription in vivo by altering the interaction between EBP-80 and its cognate DNA sequence.

A variant binding sequence for transcription factor EBP-80 confers increased promoter activity on a retroviral long terminal repeat.

The cloned long terminal repeats (LTRs) of mouse intracisternal A-particle (IAP) proviral elements differ in their promoter activity. In this study, the LTR from a recently transposed IAP element (rc-mos) is shown to be a more effective promoter both in vivo and in vitro than the LTR from a randomly cloned genomic element (MIA14). These LTRs differ in nucleotide sequence in certain previously defined protein-binding domains. In particular, the MIA14 LTR contains two domains, designated Enh1 and Enh2, with sequence homology to the SV40 enhancer core motif, while in rc-mos the Enh2 position is occupied by a variant sequence which lacks core homology. EBP-80 is a general enhancer core-binding protein originally isolated by virtue of its affinity for the MIA14 Enh2 sequence (Falzon, M., and Kuff, E.L. (1989) J. Biol. Chem. 264, 21915-21922). We now find by quantitative binding studies, binding competition, and UV cross-linking that EBP-80 from both human and mouse cells binds to the "Enh2" motif of rc-mos more strongly than to the Enh2 of MIA14. In vitro transcription from both LTRs is strongly enhanced by addition of EBP-80 showing that binding is related to function. The rc-mos LTR remains the more effective promoter in the presence of added EBP-80. Reciprocal substitution of the Enh2 domains in the two LTRs by site-directed mutagenesis shows that the rc-mos variant confers a 3-fold increment in in vivo promotor activity. The rc-mos motif or a closely related sequence is found in the cloned LTRs of many expressed and/or recently transposed IAP elements. EBP-80 is identified as a cellular transcription factor whose heightened levels in certain mouse cells might result in preferential expression of IAP elements containing this sequence motif.

Tissue and strain-specific patterns of endogenous proviral hypomethylation analyzed by two-dimensional gel electrophoresis.

Proviral sequences related to the intracisternal A particle (IAP) are amplified and dispersed in the mouse genome. Their expression is associated with hypomethylation at CpG sites in the 5' long terminal repeat. We have used two-dimensional agarose gel electrophoresis to examine patterns of IAP hypomethylation in mouse DNA. The method is sensitive to both the methylation status of a conserved Hae II site in the 5' long terminal repeat and the location of the closest BamHI site in the flanking DNA upstream of each hypomethylated long terminal repeat. The method also defects restriction fragments derived from IAP elements that are themselves methylated but have an unmethylated Hae II site in their 5' adjacent DNA. DNAs from each of four inbred mouse strains (BALB/c, C3H/He, C57BL/6, and DBA/2) gave distinctive two-dimensional patterns of BamHI/Hae II restriction fragments detected by hybridization with an IAP probe. This constitutive pattern was largely conserved among several tissues of each strain, but some tissue-specific variations were observed. The site-specific hypomethylations reflected in the two-dimensional patterns were heritable properties, since DNA from progeny of an interstrain cross contained both parental sets of fragments. IAP elements may be useful endogenous reporters of genomic methylation patterns.

Isolation and characterization of a protein fraction that binds to enhancer core sequences in intracisternal A-particle long terminal repeats.

The U3 region of mouse intracisternal A-particle (IAP) long terminal repeats (LTRs) contains several nuclear protein-binding domains. Two of these contain sequences with homology to the SV40 enhancer core. We refer to these two domains as Enh1 and Enh2. The Enh2 domain is an important determinant of promoter activity in vivo. We report here the isolation of nuclear fractions from human 293 and mouse MOPC-315 cells which interact with Enh1 and Enh2. Purification was achieved via DNA-affinity chromatography on a multimerized oligonucleotide representing the Enh2 region from the LTR of the mouse genomic IAP element, MIA14. Glycerol gradient sedimentation suggested a native Mr of approximately 80-100 for the binding component(s) in both crude and affinity-purified fractions. UV cross-linking showed that the binding activity involved two polypeptides within this size range. The affinity-isolated fraction from each cell line was highly purified, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in vitro binding analysis. Exonuclease III footprinting showed that the two polypeptides interacted preferentially with the Enh1 and Enh2 domains within a 139-base pair segment from the MIA14 LTR. The polypeptides interacted in a sequence-specific manner with oligonucleotides representing these domains within the IAP LTR and with oligonucleotides containing the enhancer core sequence from SV40 and polyoma virus. Equilibrium binding studies indicated that the apparent dissociation constants for the polypeptides binding to the enhancer core sequence from MIA14, SV40, and polyoma virus were similar. Therefore, this affinity-purified fraction may represent a novel enhancer core-binding component which is distinct from the previously characterized rat CCAAT/enhancer-binding protein, C/EBP.

Multiple protein-binding sites in an intracisternal A particle long terminal repeat.

The long terminal repeats (LTRs) of cloned intracisternal A particles (IAPs) can function as effective promoters in heterologous and homologous cell types (K. K. Lueders, J. W. Fewell, E. L. Kuff, and T. Koch, Mol. Cell. Biol. 4:2128-2135, 1984) and respond to transcriptional factors induced by various nuclear oncogene products (S. Luria and M. Horowitz, J. Virol. 57:998-1003, 1986). Using the first 139 base pairs of the U3 region of a cloned mouse IAP LTR as probe, we demonstrated multiple exonuclease III stop sites which appeared specifically in the presence of nuclear extract protein. Various extracts gave similar footprints, but the amount of nuclear protein required varied up to 10-fold. Cell lines transformed with known nuclear oncogenes, such as adenovirus E1a and E1b (293 cells), simian virus 40 large T antigen (COS7 cells), and c-myc (MOPC-315 cells) had more and/or higher-affinity factors for the IAP LTR than extracts from HeLa, CV1, and NIH 3T3 cells did. DNase I footprinting revealed at least five distinct protein-binding domains within the 139-base-pair region. These domains correspond to segments of highly conserved nucleotide sequence among a number of IAP LTRs. Gel retardation studies with oligonucleotides encompassing the DNase I footprint sites showed that the nuclear factors are present in different proportions and different absolute levels in extracts from different cell types. Moreover, the oligonucleotide probes indicate that individual motifs can be occupied independently of one another. Three of the DNase I footprints include a sequence with homology to the simian virus 40 core enhancer and sequence motifs that closely resemble the binding sites for transcription factors SP1 and AP-1. The other two binding sites are not obviously related to previously recognized motifs. The multiple protein-binding sites in close proximity indicate the complex regulatory mechanism for IAP transcription.

Molecular mimicry between insulin and retroviral antigen p73. Development of cross-reactive autoantibodies in sera of NOD and C57BL/KsJ db/db mice.

Enzyme-linked immunosorbent assay (ELISA) was used to study temporal development of murine autoantibodies against insulin and both type C and intracisternal type A retroviral antigens. The nonobese diabetic (NOD) mouse, a model for autoimmune, insulin-dependent diabetes, was compared with a related, but diabetes-resistant, strain, nonobese normal (NON). Similarly, C57BL/KsJ db/db mice (insulin-resistant model of insulin-dependent diabetes and obesity) were compared with diabetes-resistant C57BL/6 db/db mice. NOD mice developed much higher autoantibody titers than did NON mice. Whereas type C autoantibodies in NOD developed to peak titer shortly after mice were weaned, autoantibodies against insulin and p73 (group-specific antigen of the intracisternal type A particle) did not develop until shortly before, or concomitant with, the development of hyperglycemia. Two NOD mice not developing hyperglycemia during the 40-wk study period were distinguished from the mice developing diabetes by a delayed onset of insulin (but not p73) autoantibodies. Our findings suggest that in NOD mice, the appearance of insulin and p73 autoantibodies signifies that extensive underlying necrosis of beta-cells occurred. C57BL/KsJ db/db mice (with extensive beta-cell necrosis and early hyperglycemia) developed much higher autoantibody titers to insulin and p73 than did the diabetes-resistant C57BL/6 db/db mice. However, the presence of autoantibodies in normoglycemic C57BL/KsJ +/db controls demonstrated that elevated autoantibody titers alone were insufficient to produce diabetes in this model. Absorption studies indicated that autoantibodies against p73 recognized a common epitope on insulin and IgE-binding factor. The potential significance of this molecular mimicry is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies from patients and mice with autoimmune diseases react with recombinant hnRNP core protein A1.

Sera from autoimmune patients and normal volunteers were tested for antibodies to the A1 core protein of heteronuclear ribonucleoprotein (hnRNP) particles by ELISA and Western blot assays. The A1 protein used in these studies was produced by recombinant DNA technology. Thirty-seven per cent of patients with systemic lupus erythematosus produced anti-A1 antibodies, compared to 7% of normal controls. Several strains of autoimmune mice were also analysed. They spontaneously made high-titered responses to A1. Normal strains showed very low anti-A1 response unless they were specifically immunized with the antigen. Following immunization, normal mice also made high-titered responses to the A1 protein. These studies demonstrated that the A1 protein itself is an autoantigenic component of hnRNP particles. Our studies uncovered no evidence of linkage between the production of anti-A1 and of anti-DNA antibodies.