Profiling genome-wide chromatin methylation with engineered posttranslation apparatus within living cells.

Protein methyltransferases (PMTs) have emerged as important epigenetic regulators in myriad biological processes in both normal physiology and disease conditions. However, elucidating PMT-regulated epigenetic processes has been hampered by ambiguous knowledge about in vivo activities of individual PMTs particularly because of their overlapping but nonredundant functions. To address limitations of conventional approaches in mapping chromatin modification of specific PMTs, we have engineered the chromatin-modifying apparatus and formulated a novel technology, termed clickable chromatin enrichment with parallel DNA sequencing (CliEn-seq), to probe genome-wide chromatin modification within living cells. The three-step approach of CliEn-seq involves in vivo synthesis of S-adenosyl-L-methionine (SAM) analogues from cell-permeable methionine analogues by engineered SAM synthetase (methionine adenosyltransferase or MAT), in situ chromatin modification by engineered PMTs, subsequent enrichment and sequencing of the uniquely modified chromatins. Given critical roles of the chromatin-modifying enzymes in epigenetics and structural similarity among many PMTs, we envision that the CliEn-seq technology is generally applicable in deciphering chromatin methylation events of individual PMTs in diverse biological settings.

Identification in rat liver and serum of water-soluble class I MHC molecules possibly homologous to the murine Q10 gene product.

We have identified large quantities of a water-soluble, non-RT1.A class I MHC molecule in the serum of the DA rat strain, with a similar molecule being found in aqueous extracts of DA liver. The non-RT1.A class I molecules have heavy chains of 41 kD, which is smaller than RT1.A class I molecules isolated from liver membranes (45 kD) but larger than water-soluble RT1.A class I molecules previously identified in serum and aqueous extracts of liver and kidney (40 kD). NH3-terminal amino acid sequencing of bulk-purified RT1.A class I molecules and of this novel non-RT1.A class I molecule revealed two substitutions, in the first 25 amino acids, Tyr----His at position 9, and Ala----Ser at position 24. The non-RT1.A class I molecule did not react with any of the well-characterized polymorphic and monomorphic antibodies directed against RT1.Aa class I molecules, but did react with the MRC OX18 antibody. A similar class I molecule could not be identified on liver membranes. The non-RT1.A class I molecule was found in large quantities (approximately 20 micrograms/ml) in the serum of the DA rat strain, and similarly large quantities appeared to be present in the sera of BN, PVG, and LEW.RT1a rats. WAG and LEW.RT1u rats had readily detectable but lower amounts of this molecule in their serum, while LEW and SHR rats had little if any present. This molecule probably represents the rat homologue of the murine Q10 gene product, and is the major class I product in the serum of the DA rat strain.

Guinea pig immune response-related histocompatibility antigens. Partial characterization and distribution.

We have previously demonstrated that guinea pig alloantisera directed at strain 2 and strain 13 membrane antigens block specific lymphocyte activation in immune response gene-controlled systems. In this communication we describe the partial characterization of the antigens against which these antisera are directed (the 2 and 13 antigens) and, in addition, that of the B antigen which by distribution resembles the human HL-A and mouse H-2 major histocompatibility antigens. Lymphoid cells from strain 2 and strain 13 guinea pigs were surface labeled with 125I by the lactoperoxidase technique. Nonidet P-40 extracts of these labeled cells were precipitated by sandwiches of strain 2 antistrain 13, strain 13 antistrain 2, or outbred anti-B antisera, followed by rabbit antiguinea pig immunoglobulin antisera. Precipitates were dissolved in sodium dodecyl sulfate (SDS) and electrophoresed on SDS polyacrylamide gels. Radioactive peaks representing the 2 and B-cell membrane antigens were obtained from strain 2 lymph node cells, as well as from a B-lymphoid cell population (L2C leukemia cells) and a T-lymphocyte population (STRAIN 2 PERITONEAL EXUDATE LYMPHOCYTES [PELs]). Radioactive peaks representing the 13 and B-cell membrane antigens were obtained from strain 13 lymph node cells and strain 13 PELs. All anti-B precipitates produced two peaks when electrophoresed on SDS polyacrylamide gels; one representing an antigen with a mol wt of approximately 45,000, and one representing an antigen with a mol wt of about 12,000. Both may be components of a single protein. All anti-2 and anti-13 precipitates produced a single peak when electrophoresed on SDS polyacrylamide gels. Both the 2 and 13 antigens were found by this technique to have mol wt of approximately 25,000. By molecular weight criteria, as well as by previously investigated distributional criteria, the B antigen is similar to the human LA and Four antigens, and to the mouse D and K antigens, and the 2 and 13 antigens are similar to the mouse Ia antigens.

Growth-inhibitory activity of lymphoid cell plasma membranes. II. Partial characterization of the inhibitor.

We have shown that plasma membranes from lymphoid cells have inhibitory activity for the growth of normal lymphocytes and lymphoid tumor cells (Stallcup, K. C., A. Dawson, and M. F. Mescher, J. Cell Biol. 99:1221-1226). This growth-inhibitory activity has been found to co-purify with major histocompatibility complex class I antigens (H-2K and D) when these cell surface glycoproteins are isolated from detergent lysates of cells by affinity chromatography on monoclonal antibody columns. When incorporated into liposomes, the affinity-purified H-2 antigens inhibited the growth of both normal lymphocytes and tumor cells at concentrations of 1-3 micrograms/ml. Inhibition was readily reversed upon removal of the liposomes from the cell cultures, even after several days of exposure of cells to the inhibitor. Inhibitory activity was insensitive to protease digestion or heat treatment, indicating that it was not due to the H-2 glycoproteins. This was confirmed by the demonstration that inhibitory activity could be separated from the H-2 protein by gel filtration in the presence of deoxycholate and could be extracted from membranes or H-2 antigen preparations with organic solvents. The results demonstrate that the growth-inhibitory component(s) of the plasma membrane is a minor lipid or lipid-like molecule which retains activity in the absence of other membrane components. The findings reported here and in the preceding article suggest that this novel membrane component may have a role in control of lymphoid cell growth, possibly mediated by cell contacts.

Myogenin is required for late but not early aspects of myogenesis during mouse development.

Mice with a targeted mutation in the myogenic basic helix-loop-helix regulatory protein myogenin have severe muscle defects resulting in perinatal death. In this report, the effect of myogenin's absence on embryonic and fetal development is investigated. The initial events of somite differentiation occurred normally in the myogenin-mutant embryos. During primary myogenesis, muscle masses in mutant embryos developed simultaneously with control siblings, although muscle differentiation within the mutant muscle masses was delayed. More dramatic effects were observed when secondary myofibers form. During this time, very little muscle formation took place in the mutants, suggesting that the absence of myogenin affected secondary myogenesis more severely than primary myogenesis. Monitoring mutant neonates with fiber type-specific myosin isoforms indicated that different fiber types were present in the residual muscle. No evidence was found to indicate that myogenin was required for the formation of muscle in one region of the embryo and not another. The expression patterns of a MyoD-lacZ transgene in myogenin-mutant embryos demonstrated that myogenin was not essential for the activation of the MyoD gene. Together, these results indicate that late stages of embryogenesis are more dependent on myogenin than early stages, and that myogenin is not required for the initial aspects of myogenesis, including myotome formation and the appearance of myoblasts.

Expression, purification and preliminary X-ray crystallographic analysis of the human major histocompatibility antigen HLA-B*1402 in complex with a viral peptide and with a self-peptide.

The product of the human major histocompatibility (HLA) class I allele HLA-B*1402 only differs from that of allele HLA-B*1403 at amino-acid position 156 of the heavy chain (Leu in HLA-B*1402 and Arg in HLA-B*1403). However, both subtypes are known to be differentially associated with the inflammatory rheumatic disease ankylosing spondylitis (AS) in black populations in Cameroon and Togo. HLA-B*1402 is not associated with AS, in contrast to HLA-B*1403, which is associated with this disease in the Togolese population. The products of these alleles can present peptides with Arg at position 2, a feature shared by a small group of other HLA-B antigens, including HLA-B*2705, the prototypical AS-associated subtype. Complexes of HLA-B*1402 with a viral peptide (RRRWRRLTV, termed pLMP2) and a self-peptide (IRAAPPPLF, termed pCatA) were prepared and were crystallized using polyethylene glycol as precipitant. The complexes crystallized in space groups P2(1) (pLMP2) and P2(1)2(1)2(1) (pCatA) and diffracted synchrotron radiation to 2.55 and 1.86 A resolution, respectively. Unambiguous solutions for both data sets were obtained by molecular replacement using a peptide-complexed HLA-B*2705 molecule (PDB code 1jge) as a search model.

Isolation of polyomavirus-induced surface antigens of mouse cells: affinity chromatography and biologic activities.

Tumor-specific transplantation antigen (TSTA) and polyomavirus-induced tumor-associated surface antigen (TASA) were isolated from polyomavirus-transformed BALB/c mouse cells with the use of 3-M KCl solubilization and affinity chromatography on antibody-coated Sepharose 4B. Particular conditions were chosen for removal of the nonspecifically bound proteins from the gel. Under these conditions, the ratio between the amounts of crude extract and specifically bound proteins giving the same TASA activity was about 5,000. Mice inoculated with this material were protected against tumor challenge. We therefore assume that TSTA is a part of TASA. Furthermore, these antigens can be dissociated from the major histocompatibility antigens.

Noncytolytic extraction of murine tumor-specific transplantation antigens with the nonionic detergent octyl-beta-D-glucopyranoside.

The nonionic detergent octyl-beta-D-glucopyranoside (C8Glu) was used to extract immunogenic tumor-specific transplantation antigen (TSTA) from intact cells and purified plasma membranes of the 3-methylcholanthrene-induced fibrosarcoma MCA-F. Pretreatment of syngeneic C3H/HeJ mice with 100 micrograms of C8Glu extracts induced specific immunoprotection such that mice resisted the outgrowth of MCA-F but not the antigenically distinct tumors MCA-D or MCA-2A. Incubation of intact cells with 7 mM (0.2%) C8Glu for 30 minutes at 23 degrees C was judged to be noncytolytic because extracted cells excluded trypan blue. Preparative isoelectric focusing partially purified the MCA-F-specific antigen from crude C8Glu extracts into the pH 6.5-6.8 region of the gradient. Electrofocusing yielded 60% of the applied antigen activity and a fourfold to fivefold increase in specific activity. In addition, immunoprotective activity was obtained in 2% C8Glu extracts of MCA-F plasma membranes, confirming the membrane localization of the MCA-FTSTA. Three properties of C8Glu rendered it an attractive agent for the preparation of cell surface proteins: a nonionic character, large critical micellar concentration, and its capacity to extract antigens without complete membrane solubilization or cell disruption.

Differential extraction of tumor-specific transplantation antigen and embryonic antigen from simian virus 40- and adenovirus 7-induced sarcoma cells of hamsters with 1-butanol and 3 M potassium chloride.

Simian virus 40 (SV40)-induced sarcomas and adenovirus 7-induced sarcomas (Adv-7) exhibit both specific tumor-specific transplantation antigens (TSTA) and cross-protective embryonic antigens at the cell surface in the LAK:LVG(SYR) strain of Syrian golden hamsters. Specific SV40 TSTA could be released from the surfaces of living hamster sarcoma cells in a 2.5% crude 1-butanol extract (CBE) and served as immunogen to protect syngeneic recipients against subsequent homologous but not heterologous tumor cell challenge. The CBE-extracted SV40-induced TSTA (tumor-specific) was observed to be free of detectable, cross-protective embryonic antigens (EA) by tumor transplantation assays. The induction of cytotoxic lymphocyte-mediated immunity with the CBE-released TSTA was dependent on the administration of a single sensitizing injection of 12-20 micrograms antigen protein. Higher concentrations (50-1,000 micrograms) of the CBE tumor cell extract, given in a single injection, enhanced tumor growth as did two injections of 12.5 micrograms CBE-extracted SV40-induced TSTA at 1-week intervals. A cross-protective antigen(s), not detected in the CBE tumor extracts, was retained in the intact, 1-butanol-extracted SV40 and Adv-7-induced tumor cell lines after completion of the CBE extraction procedure and in similarly extracted 10-day hamster fetal cells. Some alterations in the normal immunogenicity of EA extracted with CBE followed by KCI from SV40-induced sarcoma cells could be detected in the transplantation assays and lymphocyte transformation assays, whereas EA extracted from CBE-KCI-treated Adv-7 cells or 10-day hamster fetal cells retained normal immunogenicity in vivo and in vitro. These procedures provide a means for successful separation of immunogenic SV40- and Adv-7-induced TSTA from detectable, biologically active, cross-protective EA from the surfaces of these sarcoma cells.

Isolation of polyoma virus-induced surface antigens in hamster cells: potassium chloride solubilization and differential precipitation.

Surface antigens were extracted in soluble and active form with 3 M KCL from polyoma virus-transformed and embryonic hamster cells. These were tumor-specific transplantation antigens (TSTA), shown by tumor rejection, and a surface (S) antigen, demonstrated by the inhibition of surface fluorescence on living polyoma virus-transformed cells. The extracts were fractionated by salting out with ammonium sulfate. In tumor cell extracts, all TSTA activity and a part of S antigen activity were found in the fraction precipitated with 60% saturation in (NH/)2SO4. Another part of S antigen activity was found in the fraction precipitating at 80% saturation in tumor cell extracts. The precipitate at 60% saturation of embryonic cell extracts also had a part of S antigen activity. Receptor site activity for concanavalin A was also retained after solubilization and was confined to the 40% saturation (NH4)2SO4 precipitate in the case of tumor and embryonic cell extracts.

Biologic and biochemical properties of detergent-solubilized tumor-specific transplantation antigen from a simian virus 40-induced neoplasm: brief communication.

The detergent Nonidet P40 was used to solubilize tumor-specific transplantation antigens (TSTA) from crude membranes obtained from dissociated cells of simian virus 40-induced sarcoma of BALB/c mice. A good recovery of specific tumor rejection activity was observed. One fraction, fraction V, was obtained following polyacrylamide-agarose filtration of the solubilized material, and this fraction contained most of the activity. An increased specific activity followed gel filtration. Preliminary data from lectin column chromatography of the active fraction V indicated a separation of TSTA activity from H-2 activity.

Extraction of a murine tumor-specific transplantation antigen with 1-butanol. I. Partial purification by isoelectric focusing.

Treatment of whole viable MCA-F murine sarcoma cells from syngeneic female C3H/HeJ mice with a single-phase solution of 2.5% (vol/vol) 1-butanol yielded a crude membrane protein extract without loss of cell viability. 1-Butanol-extracted cells were capable of in vitro proliferation. Variations in extraction conditions significantly influenced not only protein yield but also viability. The crude butanol extract (CBE) contained a tumor-specific transplantation antigen (TSTA) more potent than that obtained by 3-M KCl extraction of MCA-F cells: a specific activity of 40 TSTA U/mg protein in the former and 2 U/mg in the latter. The antigen was specific and protected host against challenge with MCA-F cells but not the antigenically different MCA-D cells. The growth of MCA-F cells was not reduced by pretreatment with equivalent doses of CBE obtained from MCA-D cells. Partial purification of the antigenic activity by preparative isoelectric focusing revealed that most activity focused in the pH 6.4 fraction, which increased the specific activity to 1,000 TSTA U/mg protein. Thus 1-butanol was an effective agent for the extraction of certain membrane polypeptides, including an immunogenic TSTA, from whole cells.

A unique tumor rejection antigen from the S91 murine malignant melanoma.

We have identified and described the characteristics of a unique tumor rejection antigen (tumor-specific transplantation antigen) obtained from the murine malignant melanoma S91. This antigen is highly restricted to the autologous melanoma and provides striking inhibition of its growth. Previously, we described common or shared tumor-specific transplantation antigens on the murine malignant melanomas B16 F10, K1735, JB/RH, and JB/MS. No cross-reactivity was obtained in this study between S91 and those four other malignant melanomas. The common tumor-specific transplantation antigen resides on a glycoprotein molecule with a molecular weight of 65,000, termed B700, that shares homology with serum albumin as determined by NH2-terminal amino acid sequencing. B700, however, purified from S91 proved to be ineffective as an immunogen.

Extraction of a murine tumor-specific antigen from cells and plasma membranes using 1-butanol: augmentation of antigen yield by colchicine.

The purpose of this investigation was to examine the ability of single-phase aqueous solutions of 1-butanol to release immunoprotective tumor antigen activity from partially purified plasma membranes of the methylcholanthrene-induced fibrosarcoma, MCA-F. Tumor antigen activity was assessed by s.c. immunization of syngeneic C3H/HeJ mice 10 days prior to supralethal challenge. Although brief incubation of intact MCA-F cells in 2.5% butanol releases potent immunoprotective activity, application of this protocol to plasma membranes did not result in antigen extraction. Modification of the extraction protocol using higher concentrations of butanol and longer extraction times did release measurable tumor antigen activity. However, a significant amount of the membrane-associated activity remained with the insoluble membrane fraction, as demonstrated by the immunoprotective capacity of the extracted membranes. The dramatic difference in the extractability of antigen from intact cells and plasma membranes suggested that membrane architecture may influence antigen release. To investigate this possibility, we extracted with butanol MCA-F cells that had been preincubated in colchicine. Treatment of cells with colchicine significantly potentiated the extraction of tumor antigen activity. Augmentation of antigen yield was also observed when plasma membranes were pretreated with colchicine prior to 2.5% butanol extraction. These results suggest that the tumor-specific transplantation antigen may be directly or indirectly associated with the cytoskeleton underlying the plasma membrane.

Biochemical characterization of 1-butanol-extracted murine tumor-specific transplantation antigens.

This investigation sought to characterize biochemically the tumor-specific transplantation antigens (TSTA) expressed on the cell surface of a panel of chemically induced fibrosarcomas of C3H/HeJ mice. Results suggest a uniform antigenic framework upon which individual specificities are superimposed. The antigens expressed by the 3-methylcholanthrene-induced fibrosarcomas MCA-D, MCA-F, and MCA-2A fulfill the requirements of a TSTA; namely, immunization of syngeneic hosts with irradiated cells or soluble extracts engenders a tumor-specific immune response such that animals resist challenge with the same, but not another, tumor. Brief incubation of intact tumor cells in single-phase aqueous solutions of 2.5% (v/v) 1-butanol extracts an immunoprotective TSTA, but not alloantigenic activity, from MCA-F cells. This extraction protocol was extended to the two other MCA-induced neoplasms. The butanol-extracted TSTA from the three tumors displayed isoelectric pHs of 6.4 to 6.6 following preparative isoelectric focusing. The tumor-specific immunoprotective activity from all three tumors displayed an apparent molecular weight of 150,000 (150 kDa) during high-performance gel permeation chromatography. The chromatographic properties of the 150 kDa antigens were unaffected by reduction using dithiothreitol, but incubation in acetate buffer, pH 3.0, dissociated the 150 kDa complex into at least two components with molecular weights of 70 to 100 kDa and 20 to 40 kDa. Only the smaller component displayed TSTA activity. The presence of two major components in the 150-kDa antigen was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. TSTA activity was sensitive to digestion with pronase, papain, chymotrypsin, and alpha-mannosidase, but resistant to DNase, RNase, neuraminidase, trypsin, endoglycosidase H, and a mixed-function glycosidase. In addition, the TSTA activity was unaffected by heating. These data demonstrate that MCA carcinogenesis results in the expression of immunologically unique epitopes on biochemically related glycoproteins and suggest a unified mechanism for the generation of TSTA polymorphism.

Comparative behavior of simian virus 40 T-antigen and of tumor-specific surface and transplantation antigens during partial purification.

The simian virus 40-specific T-antigen has been extracted from SV AL/N mouse embryo tissue culture cells by treatment with Triton X-100 detergent. The extracts contained tumor-specific transplantation antigen (TSTA) and tumor-specific surface antigen. These extracts were purified by ammonium sulfate precipitation and diethyl-aminoethyl cellulose and phosphocellulose column chromatography and were assayed for the three antigens. We found that T-antigen, TSTA, and much of the tumor-specific surface antigen copurified through all purification steps. This finding is consistent with previous suggestions of the close degree of homology that must exist between the protein species carrying these three antigenic determinants. The antibody-mediated cytolytic assay appears to detect a new type of antigen on the cell surface, different from T-antigen and TSTA; two antigenic fractions were obtained from the phosphocellulose column that had tumor-specific surface antigen activity, but one of these did not have T-antigen or TSTA activities.

Biological and biochemical properties of Nonidet P40-solubilized and partially purified tumor-specific antigens of the transplantation type from plasma membranes of a methylcholanthrene-induced sarcoma.

Tumor-specific transplantation antigen (TSTA) was solubilized from cell membranes of sarcoma Meth-A with non-ionic detergent Nonidet P40. Soluble TSTA was partially characterized by chromatographic separation and electrophoresis. The antigen responsible for tumor rejection activity had a molecular weight of approximately 70,000 daltons in the presence of detergent and an electrophoretic mobility of alpha-globulin. TSTA was well separated from mouse histocompatibility antigen H-2 by a sequence of procedures, including gel filtration, lectin affinity chromatography, column electrophoresis, and rechromatography on agarose, showed only three major bands on polyacrylamide gel electrophoresis. TSTA was specific for sarcoma Meth-A.

Using MHC Molecules to Define a Chlamydia T Cell Vaccine.

Vaccines based on humoral immunity alone are unlikely to protect against infections caused by intracellular pathogens and today's most pressing infectious diseases of public health importance are caused by intracellular infections that include tuberculosis, malaria, HIV/AIDS, and others such as Chlamydia trachomatis. For these infections, vaccines that induce cellular immune responses are essential. Major impediments in developing such vaccines include difficulty in identifying relevant T cell antigens and delivering them in ways that elicit protective cellular immunity. Genomics and proteomics now provide tools to allow unbiased empirical identification of candidate T cell antigens. This approach represents an advance on bioinformatic searches for candidate T cell antigens. This chapter discusses an immunoproteomic approach we have used to identify Chlamydia T cell antigens. We further discuss how these T cell antigens can be developed into a human vaccine.

Single-step purification of human C4b-binding protein (C4BP) by affinity chromatography on a peptide derived from a streptococcal surface protein.

Many Gram-positive bacteria express surface proteins that bind human plasma proteins. These bacterial proteins, and derivatives of them, are of interest for analysis of bacterial pathogenesis and as immunochemical tools. Well-characterized examples include the IgG-binding reagents staphylococcal protein A and streptococcal protein G, and the recently described streptococcal IgA-binding peptide Sap. Here, we show that a peptide derived from the streptococcal M22 protein can be used for single-step affinity purification of the human complement regulator C4b-binding protein (C4BP). Binding of C4BP was strongly enhanced by dimerization of the peptide via a C-terminal cysteine residue not present in the intact M22 protein. The purified C4BP had the expected binding characteristics, and acted as a cofactor for factor I in the degradation of C4b. Passage of serum through a peptide column under non-saturating conditions resulted in binding of >99.5% of serum C4BP, implying that such a column can be used to deplete serum of C4BP. These data indicate that the C4BP-binding peptide is a versatile tool that can be used for simple and rapid purification of biologically active human C4BP or for removal of C4BP from serum.