Specific acetylation of chromosomal protein HMG-17 by PCAF alters its interaction with nucleosomes.

Nonhistone chromosomal proteins HMG-14 and HMG-17 are closely related nucleosomal binding proteins that unfold the higher-order chromatin structure, thereby enhancing the transcription and replication potential of chromatin. Here we report that PCAF, a transcription coactivator with intrinsic histone acetyltransferase activity, specifically acetylates HMG-17 but not HMG-14. Using mass spectrum sequence analysis, we identified the lysine at position 2 as the predominant site acetylated by PCAF. Lysine 2 is a prominent acetylation site in vivo, suggesting that this PCAF-mediated acetylation is physiologically relevant. Experiments with HMG-17 deletion mutants and competition studies with various protein fragments indicate that the specific acetylation of HMG-17 is not determined solely by the primary sequence near the acetylation site. By equilibrium dialysis we demonstrated that acetylation reduces the affinity of HMG-17 to nucleosome cores. In addition, we found that the binding of HMG-14 and HMG-17 to nucleosome cores inhibits the PCAF-mediated acetylation of histone H3. Thus, the presence of HMG-14 and HMG-17 affects the ability of PCAF to acetylate chromatin, while the acetylation of HMG-17 reduces its binding affinity to chromatin. Conceivably, in HMG-17-containing chromatin, acetylation of HMG-17 precedes the acetylation of histones.

Monoclonal antibodies to immunogenic lymphoma cell variants displaying impaired neoplastic properties: characterization and applications.

Immunogenic, nontumorigenic cell variants derived from the highly tumorigenic mouse lymphoma cell line S-49 were used to raise monoclonal antibodies (MAbs) in syngeneic BALB/c mice. MAbs of the following specifications were derived: (a) MAbs that interacted preferentially with the immunogenic variants, (b) MAbs that interacted with both immunogenic variants and parental tumorigenic cells, and (c) a MAb that interacted with both immunogenic and tumorigenic S-49 cells and the normal BALB/c splenocytes. Six MAbs raised in this way were found to recognize at least five different cell-surface epitopes. Functional analysis of the different MAbs suggested their potential usefulness in passive immunization against parental tumorigenic cells as well as in enrichment of immunogenic cells from a mixed population containing a preponderance of tumorigenic cells.

Interferon regulatory factor-1 is a major regulator of epidermal growth factor receptor gene expression.

Overexpression of the epidermal growth factor receptor (EGFR) occurs in many tumors and in breast cancer correlates with poor prognosis for treatment. Here, we report that interferon regulatory factor-1 (IRF-1) induces EGFR promoter activity up to 200-fold compared to 3-10-fold induction by other regulators. The region of the promoter that is required for this induction was defined using deletion mutants. In addition, we found that IRF-1 and tricostatin A, a deacetylase inhibitor, have a synergistic effect on EGFR promoter activity. This indicates that the increase in EGFR promoter activity by IRF-1 may also involve changes in chromatin structure. These results identify IRF-1 as a major regulator of EGFR gene expression.

Generation of monospecific peptide antibodies to the DNA binding domain of p53.

The DNA binding domain (DBD) is the most mutated region of p53 in tumors and has proven to be relatively resistant to the generation of specific antibodies. Template assembled synthetic peptide (TASP) synthesis of a peptide derived from the DBD creates a highly immunogenic molecule without the need for large carriers such as keyhole limpet hemocyanin (KLH). In addition, a rapid means of generating monoclonal antibodies can be achieved through immunization in conjunction with ABL/MYC retrovirus injection into recipient mice. In this paper, we demonstrate that an antibody generated by this means, KH2, reacts specifically with the DBD of p53. To date, this is the first example of a peptide immunogen used successfully in ABL/MYC monoclonal antibody production. KH2 is also the first example of a monospecific antibody that directly binds to and, by definition, assumes the conformation of the DNA binding region of p53.

Association of tumorigenic and nontumorigenic (immunogenic) variants in a mouse T-cell lymphoma with two distinct p53 mutations.

An in vitro model system for xenogenization has been developed in which an immunogenic, nonmalignant phenotype was selected from a highly malignant T-cell line (S49). We showed by single-strand conformation polymorphism and DNA sequence analysis that specific point mutations in the p53 tumor suppressor gene correlated with a change from a tumorigenic to a nontumorigenic (immunogenic) phenotype. Specifically, we found that the highly malignant S49 cell line T-60 contains an Arg-->Gln substitution at residue 246 in exon 7 of p53. In contrast, nontumorigenic (immunogenic) variants (T-25-Adh and Rev-1) exhibited a Gly-->Ser substitution at residue 242 of p53. In two subsequent tumorigenic revertants derived from Rev-1, we again found the Arg-->Gln substitution at residue 246 that was found initially in the T-60 cells. Thus, mutation at residue 246 of p53 was associated with a highly malignant phenotype, whereas a novel mutation at residue 242 of p53 appeared to be associated with a nonmalignant phenotype and may have actually protected the host through immunization. We conclude that mutation of residue 242 may represent a new class of permissive (nonmalignant) mutations in the mouse that are analogous to the Li-Fraumeni mutation in humans.

Immunomagnetic separation and analysis of non-malignant variants and parental malignant mouse lymphoma cells.

We have devised conditions whereby non-tumorigenic, immunogenic cell variants of S49 mouse lymphoma were analyzed and separated from parental tumorigenic lymphoma cells. This was carried out using polyclonal antibodies (raised against the immunogenic variants) and immunomagnetic beads. The efficacy of the procedure depended on the amount of polyclonal antiserum, the immunobead to cell ratio, incubation time and the number of repetitions of the procedure. Experiments with mixed tumorigenic and non-tumorigenic cells have resulted in an enrichment of up to 200-fold of the non-tumorigenic, immunogenic cells in the population. These findings indicate the potential use of this procedure (in conjunction with other approaches) to isolate from a population of tumorigenic cells those variant cells that might be used to immunize against the parental tumor.

Variant mouse lymphoma cells with modified response to interferon demonstrate enhanced immunogenicity.

We have previously developed an experimental model for the xenogenization of malignant lymphoma. From highly tumorigenic S49 mouse lymphoma cells that proliferate in suspension culture (designated T-25), we selected variant clones that grew as an adherent monolayer (designated T-25-Adh) and were non-tumorigenic in syngeneic mice. Furthermore, priming of syngeneic hosts with T-25-Adh cells protected them against subsequent challenges with the tumorigenic T-25 cells. Several lines of evidence have indicated that antigens of an endogenous mouse mammary tumor virus (MMTV) are involved in the immunogenicity of T-25-Adh cells. Since interferon (IFN) is known to affect retroviral assembly and maturation on the cell membrane, we have studied the effects of IFN on endogenous MMTV-related structures, as well as on the immunogenicity of T-25-Adh cells. We observed that mouse alpha and beta interferons affect the morphogenesis of intracellular MMTV-related precursors in the immunogenic T-25-Adh cells, but not in tumorigenic T-25 cells. From T-25-Adh cells we selected variants that were either high responders or low responders to the above-mentioned interferon effect. The high-response variants were significantly more protective against tumorigenic T-25 cells than the low-response variants. Involvement of MMTV-related antigens in the immune response of the host to T-25-Adh cells was further suggested by immunoelectron-microscopical analysis, demonstrating that antisera from mice, immunized with T-25-Adh cells, interacted specifically with cell-surface MMTV budding particles. These findings indicate a novel method for xenogenization of lymphoma cells by IFN. Since endogenous retroviruses are present in all tissues of the mouse, this approach might be applicable to a wide variety of tumors.

The histone acetyltransferase activity of human GCN5 and PCAF is stabilized by coenzymes.

Here we report that PCAF and human GCN5, two related type A histone acetyltransferases, are unstable enzymes that under the commonly used assay conditions are rapidly and irreversibly inactivated. In addition, we report that free histone H1, although not acetylated in vivo, is a preferred and convenient in vitro substrate for the study of PCAF, human GCN5, and possibly other type A histone acetyltransferases. Using either histone H1 or histone H3 as substrates, we find that preincubation with either acetyl-CoA or CoA stabilizes the acetyltransferase activities of PCAF, human GCN5 and an enzymatically active PCAF deletion mutant containing the C-terminal half of the protein. The stabilization requires the continuous presence of coenzyme, suggesting that the acetyltransferase-coenzyme complexes are stable, while the isolated apoenzymes are not. Human GCN5 and the N-terminal deletion mutant of PCAF are stabilized equally well by preincubation with either CoA or acetyl-CoA, while intact PCAF is better stabilized by acetyl-CoA than by CoA. Intact PCAF, but not the N-terminal truncation mutant or human GCN5, is autoacetylated. These findings raise the possibility that the intracellular concentrations of the coenzymes affect the stability and therefore the nuclear activity of these acetyltransferases.

Acetylation of novel sites in the nucleosomal binding domain of chromosomal protein HMG-14 by p300 alters its interaction with nucleosomes.

The reversible acetylation of histones is associated with structural alterations in the chromatin fiber that affect various DNA-related activities. Here we show that the histone acetyltransferase p300 specifically acetylates HMG-14, a nonhistone structural protein that binds to nucleosomes and reduces the compactness of the chromatin fiber. We identify 7 major acetylation sites, 6 of which are novel and have not been known to be acetylated in either HMG-14 or the closely related HMG-17 protein. All the acetylation sites involve evolutionarily conserved residues: 3 within the HMG-14/-17 nucleosomal binding domain and 4 in or near the bipartite nuclear localization domains of the proteins. In tissue culture cells the acetylation pattern is indicative of a selective process in which a subfraction of HMG-14 is preferentially acetylated. We find that the nucleosomal binding domain is a major target for acetylation in vivo and that the specific acetylation of HMG-14 by p300 weakens its interaction with nucleosome cores. Our results suggest that p300 modulates the interaction of HMG-14 with nucleosomes. Thus, p300 may affect chromatin-related activities not only by modifying histones or transcription factors but also by targeting structural nonhistone proteins.