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The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these epigenetic marks is accomplished by sequential oxidation by ten-eleven translocation dioxygenases (TET1-3), followed by the thymine-DNA glycosylase-dependent base excision repair. Inactivation of the TET2 gene due to genetic mutations or by other epigenetic mechanisms is associated with a poor prognosis in patients with diverse cancers, especially hematopoietic malignancies. Here, we describe an efficient single step purification of enzymatically active untagged human TET2 dioxygenase using cation exchange chromatography. We further provide a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach that can separate and quantify the four normal DNA bases (A, T, G, and C), as well as the four modified cytosine bases (5-methyl, 5-hydroxymethyl, 5-formyl, and 5-carboxyl). This assay can be used to evaluate the activity of wild type and mutant TET2 dioxygenases.

5-Methylcytosine within CpG islands in DNA plays a crucial role in epigenetic transcriptional regulation during metazoan development. Recently, it has been established that the Ten-Eleven Translocation (TET) family, Fe(II)- and 2-oxoglutarate (2OG/αKG)-dependent oxygenases initiate 5-methylcytosine demethylation by iterative oxidation reactions. Mutations in the TET2 gene are frequently detected in patients with myeloid malignancies. Here, we describe the cloning of untagged human TET2 demethylase using Gateway technology and its efficient expression in E. coli. The untagged TET2 enzyme was purified using cation exchange and heparin sepharose chromatography. In addition, a reliable quantitative liquid chromatography-tandem mass spectrometry-based assay was utilized to analyze the activity of TET2 oxygenase. This assay was further used to analyze the activity of a number of clinical TET2 variants with mutations in the 2OG binding sites. Our results demonstrate that the activity of one TET2 mutant, TET2-R1896S, can be restored using an excess of 2OG in the reaction mixture. These studies suggest that dietary 2OG supplements, which are commonly used for several other conditions, may be used to treat some patients with myeloid malignancies harboring TET2-R1896S mutation. Results described in this paper serve as a foundation for better characterization of wild type as well as mutant TET2 demethylases., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Methylation of lysine 4 on histone H3 (H3K4) at promoters is tightly linked to transcriptional regulation in human cells. At least six different COMPASS-like multisubunit (SET1/MLL) complexes that contain methyltransferase activity for H3K4 have been described, but a comprehensive and quantitative analysis of these SET1/MLL complexes is lacking. We applied label-free quantitative mass spectrometry to determine the subunit composition and stoichiometry of the human SET1/MLL complexes. We identified both known and novel, unique and shared interactors and determined their distribution and stoichiometry over the different SET1/MLL complexes. In addition to being a core COMPASS subunit, the Dpy30 protein is a genuine subunit of the NURF chromatin remodeling complex. Furthermore, we identified the Bod1 protein as a discriminator between the SET1B and SET1A complexes, and we show that the H3K36me-interactor Psip1 preferentially binds to the MLL2 complex. Finally, absolute protein quantification in crude lysates mirrors many of the observed SET1/MLL complex stoichiometries. Our findings provide a molecular framework for understanding the diversity and abundance of the different SET1/MLL complexes, which together establish the H3K4 methylation landscape in human cells.

O-linked N-acetylglucosamine (O-GlcNAc) transferase (Ogt) activity is essential for embryonic stem cell (ESC) viability and mouse development. Ogt is present both in the cytoplasm and the nucleus of different cell types and catalyzes serine and threonine glycosylation. We have characterized the biochemical features of nuclear Ogt and identified the ten-eleven translocation (TET) proteins Tet1 and Tet2 as stable partners of Ogt in the nucleus of ESCs. We show at a genome-wide level that Ogt preferentially associates with Tet1 to genes promoters in close proximity of CpG-rich transcription start sites. These regions are characterized by low levels of DNA modification, suggesting a link between Tet1 and Ogt activities in regulating CpG island methylation. Finally, we show that Tet1 is required for binding of Ogt to chromatin affecting Tet1 activity. Taken together, our data characterize how O-GlcNAcylation is recruited to chromatin and interacts with the activity of 5-methylcytosine hydroxylases., (Copyright © 2013 Elsevier Inc. All rights reserved.)

One of the most exciting recent advances in the epigenetic field is the discovery that 5-methylcytosine (5mC) in DNA can be iteratively oxidized by a family of proteins known as Tet proteins to generate 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). These 5mC derivatives can be further processed by thymine-DNA glycosylase (TDG) followed by base excision repair or by replication-dependent dilution leading to DNA demethylation. Given the similarity between 5mC and its oxidation derivatives, many of the conventional techniques used for 5mC analysis cannot distinguish between 5mC and 5hmC/5fC/5caC. Here, we describe 2D-TLC and mass spectrometry methods that we have successfully used in differentiating 5mC from its oxidative derivatives as well as in characterizing the enzymatic activity of Tet proteins both in vitro and in vivo., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Ski is an avian sarcoma virus oncogene homolog best known for inhibiting TGF beta signaling through its association with the SMAD proteins. Anti-Ski antibodies (MAbs) of high titer were prepared by immunizing BALB/c mice with multifocal intradermal injections and fusing high titer antibody producing spleen cells with myeloma cells of SP2/0 origin. Three MAbs were selected for further characterization as classes and subclasses. Antibodies were produced by these three clones with high affinities ranging from 10(9) to 10(11)/m. These clones were found to be of the immunoglobulin IgG1 and IgG2b subclass with kappa light chain. They could recognize Ski as determined by Western blot analysis. The produced MAbs will be a useful tool for further investigation of Ski functions in organisms.

The products encoded by ski and its related gene, sno, (Ski and Sno) act as transcriptional co-repressors and interact with other co-repressors such as N-CoR/SMRT and mSin3A. Ski and Sno mediate transcriptional repression by various repressors, including Mad, Rb and Gli3. Ski/Sno also suppress transcription induced by multiple activators, such as Smads and c-Myb. In particular, the inhibition of TGF-beta-induced transcription by binding to Smads is correlated with the oncogenic activity of Ski and Sno. However, the molecular mechanism by which Ski and Sno mediate transcriptional repression remains unknown. In this study, we report the purification and characterization of Ski complexes. The Ski complexes purified from HeLa cells contained histone deacetylase 3 (HDAC3) and protein arginine methyltransferase 5 (PRMT5), in addition to multiple Smad proteins (Smad2, Smad3 and Smad4). Chromatin immunoprecipitation assays indicated that these components of the Ski complexes were localized on the SMAD7 gene promoter, which is the TGF-beta target gene, in TGF-beta-untreated HepG2 cells. Knockdown of these components using siRNA led to up-regulation of SMAD7 mRNA. These results indicate that Ski complexes serve to maintain a TGF-beta-responsive promoter at a repressed basal level via the activities of histone deacetylase and histone arginine methyltransferase.

Immunoglobulin class switch recombination joins a new constant (C) region to the rearranged and expressed heavy chain variable (VDJ) region in antigen-activated B cells (Figure 1A) (reviewed in [1, 2]). Switch recombination is activated by transcription of intronic, G-rich and repetitive switch (S) regions and produces junctions that are heterogeneous in sequence and position in the S regions. Switch recombination depends upon the B cell-specific cytidine deaminase, AID, and conserved DNA repair factors, including the mismatch repair heterodimer, MutSalpha (MSH2/MSH6). In mice, ablation of Msh2 or Msh6, but not Msh3, decreases levels of switch recombination and diminishes heterogeneity of switch junctions [3-7]. Here, we demonstrate that MSH2 associates with transcribed S regions in primary murine B cells activated for switch recombination. Electron microscopic imaging reveals that MutSalpha binds in vitro to DNA structures formed within transcribed S regions and mediates their synapsis. MutSalpha binds with high affinity to G4 DNA formed upon transcription of the S regions and also binds to U.G mismatches, initial products of DNA deamination by AID. These results suggest that MutSalpha interacts with the S regions in switching B cells to promote DNA synapsis and recombination.

In B cells, the Igh gene locus contains several DNase I-hypersensitive (hs) sites with enhancer activity. These include the 3' Igh enhancers, which are located downstream of the Calpha gene(s) in both mouse and human. In vivo experiments have implicated murine 3' enhancers, hs3B and/or hs4, in class switching and somatic hypermutation. We previously reported that murine hs4 was regulated by NF-kappaB, octamer binding proteins, and Pax5 (B cell-specific activator protein). In this study we report that human hs4 is regulated differently. EMSAs and Western analysis of normal B cells before and after stimulation with anti-IgM plus anti-CD40 showed the same complex binding pattern formed by NF-kappaB, Oct-1, and Oct-2 (but not by Pax5). A similar EMSA pattern was detected in mature human B cell lines (BL-2, Ramos, and HS-Sultan) and in diffuse large B cell lymphoma cell lines, although yin yang 1 protein (YY1) binding was also observed. We have confirmed the in vivo association of these transcription factors with hs4 in B cells by chromatin immunoprecipitation assays. The diffuse large B cell lymphoma cell lines had a distinctive slow-migrating complex containing YY1 associated with Rel-B. We have confirmed by endogenous coimmunoprecipitation an association of YY1 with Rel-B, but not with other NF-kappaB family members. Transient transfection assays showed robust hs4 enhancer activity in the mature B cell lines, which was dependent on synergistic interactions between NF-kappaB and octamer binding proteins. In addition, human hs4 enhancer activity required Oct-2 and correlated with expression of Oct coactivator from B cells (OCA-B).

The regulation of PBC protein function through subcellular distribution is a crucial evolutionarily conserved mechanism for appendage patterning. We investigated the processes controlling PBX1 nuclear export. Here we show that in the absence of MEINOX proteins nuclear export is not a default pathway for PBX1 subcellular localization. In different cell backgrounds, PBX1 can be imported or exported from the nucleus independently of its capacity to interact with MEINOX proteins. The cell context-specific balance between nuclear export and import of PBX1 is controlled by the PBC-B domain, which contains several conserved serine residues corresponding to phosphorylation sites for Ser/Thr kinases. PBX1 subcellular localization correlates with the phosphorylation state of these residues whose dephosphorylation induces nuclear export. Protein kinase A (PKA) specifically phosphorylates PBX1 at these serines, and stimulation of endogenous PKA activity in vivo blocks PBX1 nuclear export in distal limb mesenchymal cells. Our results reveal a novel mechanism for the control of PBX1 nuclear export in addition to the absence of MEINOX protein, which involves the inhibition of PKA-mediated phosphorylation at specific sites within the PBC-B domain.

The acute myeloid leukaemia 1 (AML1) protein belongs to the Runx family of transcription factors and is crucial for haematopoietic development. The genes encoding Runx1 and its associated factor CBF beta are the most frequent targets for chromosomal rearrangements in acute human leukaemias. In addition, point mutations of Runx1 in acute leukaemias and in the familial platelet disorder FPD/AML cluster within the evolutionary conserved runt domain that binds both DNA and CBF beta. Here, the crystallization of the Runx1 runt domain is reported. Crystals belong to space groups C2 and R32 and diffract to 1.7 and 2.0 A resolution, respectively.

Deficiencies in DNA mismatch repair (MMR) have been found in hereditary colon cancers (hereditary non-polyposis colon cancer, HNPCC) as well as in sporadic cancers, illustrating the importance of MMR in maintaining genomic integrity. We have examined the interactions of specific mismatch repair proteins in human nuclear extracts. Western blot and co-immunoprecipitation studies indicate two complexes as follows: one consisting of hMSH2, hMSH6, hMLH1, and hPMS2 and the other consisting of hMSH2, hMSH6, hMLH1, and hPMS1. These interactions occur without the addition of ATP. Furthermore, the protein complexes specifically bind to mismatched DNA and not to a similar homoduplex oligonucleotide. The protein complex-DNA interactions occur primarily through hMSH6, although hMSH2 can also become cross-linked to the mismatched substrate when not participating in the MMR protein complex. In the presence of ATP the binding of hMSH6 to mismatched DNA is decreased. In addition, hMLH1, hPMS2, and hPMS1 no longer interact with each other or with the hMutSalpha complex (hMSH2 and hMSH6). However, the ability of hMLH1 to co-immunoprecipitate mismatched DNA increases in the presence of ATP. This interaction is dependent on the presence of the mismatch and does not appear to involve a direct binding of hMLH1 to the DNA.

Cas-Br-M murine leukemia virus (MuLV) is a slow-transforming retrovirus that potently induces leukemias in mice and therefore is well suited for retroviral insertional mutagenesis. We used Cas-Br-M MuLV in NIH/Swiss mice to establish a new panel of mainly myeloid leukemias. All tumors found in leukemic animals were classified by gross pathology, morphology, and immunophenotype, as well as the incidence of known common virus integration sites (VISs) in MuLV-induced myeloid malignancies (i.e., Evi1, Evi11/Cb2, Evi12, Fli1, and c-Myb). Interestingly, male mice were more susceptible than females to the induction of leukemia by Cas-Br-M MuLV. Seventy-four of the Cas-Br-M MuLV-inoculated mice developed a severe splenomegaly, sometimes in association with a thymoma. Although most of the immunophenotyped Cas-Br-M MuLV tumors were of myeloid origin (58%), numerous T-cell leukemias (21%) and mixed myeloid/T-cell leukemias (21%) were found. The myeloid leukemias and myeloid compartment of the mixed leukemias were further characterized by immunophenotyping with stem cell-, myeloid-, and erythroid-specific antibodies. The known Cas-Br-M MuLV common VISs (Evi1, Evi11/Cb2, and Evi12) were demonstrated in 19%, 12%, and 20% of the cases, respectively, whereas no Fli1 and c-Myb rearrangements were found. Integrations into Evi1 were restricted to myeloid leukemias, whereas those in Evi11/Cb2 and Evi12 were identified in myeloid as well as T-lymphoid leukemias. This panel of well characterized Cas-Br-M MuLV-induced hematopoietic tumors may be useful for the isolation and characterization of new proto-oncogenes involved in myeloid or T-cell leukemias., (Copyright 2000 Academic Press.)

Growth and patterning during Drosophila wing development are mediated by signaling from its dorsoventral (D/V) organizer. In the metathorax, wing development is essentially suppressed by the homeotic selector gene Ultrabithorax (Ubx) to mediate development of a pair of tiny balancing organs, the halteres. Here we show that expression of Ubx in the haltere D/V boundary down-regulates its D/V organizer signaling compared to that of the wing D/V boundary. Somatic loss of Ubx from the haltere D/V boundary thus results in the formation of a wing-type D/V organizer in the haltere field. Long-distance signaling from this organizer was analyzed by assaying the ability of a Ubx(-) clone induced in the haltere D/V boundary to effect homeotic transformation of capitellum cells away from the boundary. The clonally restored wing D/V organizer in mosaic halteres not only enhanced the homeotic transformation of Ubx(-) cells in the capitellum but also caused homeotic transformation of even Ubx(+) cells in a genetic background known to induce excessive cell proliferation in the imaginal discs. In addition to demonstrating a non-cell-autonomous role for Ubx during haltere development, these results reveal distinct spatial roles of Ubx during maintenance of cell fate and patterning in the halteres., (Copyright 1999 Academic Press.)

Marked osteopetrosis is observed only in mi/mi mutant mice although normal osteoclastgenesis is observed in other mutant mice including null mutants at the mi locus. Mutant microphthalmia-associated transcription factor (mi-MITF) has defective nuclear localization potential. We found normal MITF (+-MITF)-c-Fos and mi-MITF-c-Fos complexes in the cytoplasm by immunoblotting, and showed that PU.1 bound with both +-MITF and mi-MITF using an electrophoretic mobility shift assay. Furthermore, the nuclear localization of PU.1 and c-Fos was inhibited by over-expressed mi-MITF in WEHI-3 cells. These results indicate that mi-MITF expressing in osteoclasts specifically binds to c-Fos and PU.1 which are essential transcription factors of osteoclastgenesis and that mi-MITF blocks the nuclear localization of these other transcription factors, which may result in osteopetrosis in mi/mi mutant mice., (Copyright 1999 Academic Press.)

DNA mismatch binding by an extensively purified hMutS alpha mismatch recognition complex was investigated using a bandshift assay. The complex bound to G. T mispairs and to looped structures containing an unpaired single or two adjacent bases. A CA loop was preferentially recognised if the unpaired bases formed part of a repeated sequence. In general, single base loops were also more favourably recognised by hMutS alpha when present in monotonic runs of two to five. In one series of substrates, based on a known hotspot for frameshift mutations in the hypoxanthine-guanine phosphoribosyltransferase gene, in which different length G, A, C or T tracts were flanked by closely similar sequences, an A loop was bound preferentially in the absence of adjacent As and a C loop in the absence of adjacent Cs. This preferential binding was influenced by the base immediately 5' to the loop. Thus, while repeated regions generally favour recognition of single base loops by hMutS alpha, other factors related to local sequence may influence this interaction.

DNA mismatch repair ensures genomic stability by correcting biosynthetic errors and by blocking homologous recombination. MutS-like and MutL-like proteins play important roles in these processes. In Escherichia coli and yeast these two types of proteins form a repair initiation complex that binds to mismatched DNA. However, whether human MutS and MutL homologs interact to form a complex has not been elucidated. Using immunoprecipitation and Western blot analysis we show here that human MSH2, MLH1, PMS2 and proliferating cell nuclear antigen (PCNA) can be co-immunoprecipitated, suggesting formation of a repair initiation complex among these proteins. Formation of the initiation complex is dependent on ATP hydrolysis and at least functional MSH2 and MLH1 proteins, because the complex could not be detected in tumor cells that produce truncated MLH1 or MSH2 protein. We also demonstrate that PCNA is required in human mismatch repair not only at the step of repair initiation, but also at the step of repair DNA re-synthesis.

Background: The interplay between growth factors and transcription factors in vertebrate neurogenesis is poorly understood. MASH1 is a basic helix-loop-helix (bHLH) transcription factor that is essential for autonomic neurogenesis. Bone morphogenetic protein (BMP) 2, and its relative BMP4, have been shown to induce expression of MASH1 and to promote autonomic neuronal differentiation in neural crest stem cells. The relationship between expression of MASH1 and the neurogenic competence of neural crest cells has not been investigated, however., Results: We have examined the function of MASH1 in neurogenic competence using a population of immuno-isolated neural-crest-derived progenitor cells. Post-migratory neural crest cells isolated from fetal rat gut expressed Mash1, yet comprised a mixture of committed neuronal precursors and non-neuronal cells. The non-neuronal cells remained competent to differentiate to neurons, however, if challenged with BMP2. Such competence declines with time and is paralleled by a decline in Mash1 expression in the cells. Expression of endogenous Mash1 can be maintained by BMP2; in turn, constitutive expression of Mash1 from a retroviral vector maintains competence for neuronal differentiation in response to late addition of BMP2., Conclusions: These data suggest that MASH1 promotes competence for neurogenesis, in a manner similar to its homologs, the proneural genes achaete-scute in Drosophila. They also reveal an unexpected feedback interaction between BMP2 and MASH1 during neuronal differentiation. MASH1 may play multiple roles at successive stages of development within a neurogenic lineage, only one of which is revealed by a loss-of-function mutation.

Interleukin-12 (IL-12) is a proinflammatory cytokine produced by antigen-presenting cells in response to many microbial infections. IL-12 plays an important role in the generation of T helper type-1 cells, which favor cell-mediated immune response. IL-12 is composed of two different subunits, p40 and p35, whose expression can be regulated concomitantly or differentially. Monocytic cells, the major producers of IL-12, can be primed by interferon-gamma (IFN-gamma) to produce optimal amounts of IL-12 in response to LPS stimulation as a consequence of bacterial infection. The priming effect is exerted primarily at the transcriptional level on the p40 promoter in conjunction with the effects of LPS, possibly by inducing specific transcription factors, which individually have no direct effect but which cooperatively can activate the promoter. We examined in detail one of these DNA-protein interactions observed around an Ets-2 element situated at -211/-207 of the p40 promoter, which is known to be a functionally critical site. This region interacts with a nuclear complex termed F1 that appears to be highly inducible by either IFN-gamma treatment for 16 h or lipopolysaccharide stimulation for 8 h. F1 binding to the Ets-2 site requires a considerable amount of spacing around the Ets-2 site, as revealed by gel mobility shift and in vitro methylation assays. Supershift experiments and DNA affinity purification indicated that both Ets-2 and a novel, antigenically related protein with an approximate molecular mass of 109 kDa are part of the F1 complex, together with additional components including IRF-1 and c-Rel. This novel protein is designated GLp109 for its inducibility by IFN-gamma or lipopolysaccharide. Its possible role in the activation of the IL-12 p40 promoter is discussed.

We have used a functional assay to identify a putative T-box transcription factor (Xombi) that has the ability to induce sites of invagination in the ectoderm of Xenopus embryos that resemble the blastopore lip. Maternal Xombi transcript is first localized to the oocyte's vegetal cortex and cytoplasm, early sources of mesoderm and endoderm-inducing signals. Soon after zygotic transcription begins, there is a wave of Xombi expression, beginning in dorsal mesoderm and then extending to lateral and ventral mesoderm, that precedes and parallels blastopore lip formation at the border between the mesoderm and endoderm. Transcripts encoding brachyury, Xwnt8 and goosecoid colocalize with Xombi transcripts within the marginal zone; ectopic expression of Xombi induces expression of all three mesodermal genes. In ectodermal explants, Xombi expression is induced by the secreted mesoderm inducers activinA, activinB, Xnrl and eFGF, and by brachyury, another Xenopus T-box containing gene. The time course and location of Xombi expression, its biological activities and the partial dependence of Xombi expression and blastopore lip formation on fibroblast growth factor (FGF) signaling suggest that Xombi contributes to a traveling wave of morphogenesis and differentiation during Xenopus gastrulation.

c-Myb is the prototype of a family of transcription factors characterised by a unique DNA binding domain. Previous analyses have concentrated on truncated versions of c-Myb as it has been very difficult to produce full length c-Myb. To overcome these difficulties we expressed full length c-Myb in HeLa cells using a recombinant vaccinia virus. Partially purified native full length c-Myb bound efficiently and specifically to DNA with a dissociation constant similar to that obtained with bacterially expressed DNA binding domains. No evidence was found for a negative effect of the leucine zipper on DNA binding. Furthermore the DNA binding domain was protease resistant in contrast to the transactivation and negative regulatory domains. Phosphorylation had no apparent effect on this differential protease sensitivity. The increased sensitivity of the C-terminal domain suggests a more open conformation, which may be relevant in the integration of signals and/or in protein-protein interactions.

The ETS domain family of transcription factors is comprised of several important proteins that are involved in controlling key cellular events such as proliferation, differentiation, and development. One such protein, Elk-1, regulates the activity of the c-fos promoter in response to extracellular stimuli. Elk-1 is representative of a subgroup of ETS domain proteins that utilize a bipartite recognition mechanism that is mediated by both protein-DNA and protein-protein interactions. In this study, we have overexpressed, purified, and characterized the ETS DNA-binding domain of Elk-1 (Elk-93). Elk-93 was expressed in Escherichia coli as a fusion protein with glutathione S-transferase and purified to homogeneity from both the soluble and insoluble fractions using a two-column protocol. A combination of CD, NMR, and fluorescence spectroscopy demonstrates that Elk-93 represents an independently folded domain of mixed alpha/beta structure in which the three conserved tryptophans appear to contribute to the hydrophobic core of the protein. Moreover, DNA binding studies demonstrate that Elk-93 binds DNA with both high affinity (Kd approximately 0.85 x 10(-10)M) and specificity. Circular permutation analysis indicates that DNA binding by Elk-93 does not induce significant bending of the DNA. Our results are discussed with respect to predictive models for the structure of the ETS DNA-binding domain.

The 5' half of the EWS gene has recently been described to be fused to the 3' regions of genes encoding the DNA-binding domain of several transcriptional regulators, including ATF1, FLI-1, and ERG, in several human tumors. The most frequent occurrence of this situation results from the t(11;22)(q24;q12) chromosome translocation specific for Ewing sarcoma (ES) and related tumors which joins EWS sequences to the 3' half of FLI-1, which encodes a member of the Ets family of transcriptional regulators. We show here that this chimeric gene encodes an EWS-FLI-1 nuclear protein which binds DNA with the same sequence specificity as the wild-type parental FLI-1 protein. We further show that EWS-FLI-1 is an efficient sequence-specific transcriptional activator of model promoters containing FLI-1 (Ets)-binding sites, a property which is strictly dependent on the presence of its EWS domain. Comparison of the properties of the N-terminal activation domain of FLI-1 to those of the EWS domain of the fusion protein indicates that EWS-FLI-1 has altered transcriptional activation properties compared with FLI-1. These results suggest that EWS-FLI-1 contributes to the transformed phenotype of ES tumor cells by inducing the deregulated and/or unscheduled activation of genes normally responsive to FLI-1 or to other close members of the Ets family. ES and related tumors are characterized by an elevated level of c-myc expression. We show that EWS-FLI-1 is a transactivator of the c-myc promoter, suggesting that upregulation of c-myc expression is under control of EWS-FLI-1.

The c-Myb protein is a transcription factor with an apparent but poorly defined role in hematopoietic cell growth and differentiation. The DNA binding and several transcriptional regulatory domains of the c-Myb protein have been defined by transient transfections into nonhematopoietic cell lines. Although the relationship between these domains and transformation has been studied, little is known about the function of these domains during hematopoietic maturation. Up-regulation of stably transfected c-myb in murine erythroleukemia (MEL) cells blocks terminal differentiation when MEL cells are induced to differentiate with N,N'-hexamethylene bisacetamide. To determine which functional domains of c-Myb are necessary and sufficient to block differentiation, mutated c-myb constructs under the control of a murine metallothionein promoter were transfected into C19 MEL cells, and stable clonal cell lines were established. The ability of Myb mutants to block differentiation paralleled their ability to transactivate transcription of a reporter gene containing Myb-responsive elements, by transient transfection into a lymphoid cell line. The smallest c-Myb mutant able to block differentiation consisted of the DNA binding domain juxtaposed to the transactivation domain. Therefore, the DNA binding domain and the transactivation domain are necessary and sufficient for c-Myb to block differentiation in MEL cells.

The maize C1 gene is a regulatory gene of the anthocyanin pathway. It is a part of a multigene family whose predicted protein products contain a region with homology to the DNA-binding domain of myb proto-oncogene products. Using as a probe an oligonucleotide mixture encoding a highly conserved region in plant MYB proteins, cDNAs encoding three related polypeptides expressed in the flower of Petunia hybrida have been isolated. Predicted proteins are 280-517 amino acids long and show sequence similarities among themselves as well as with other MYB proteins (45-80% amino acid identity) in the MYB DNA-binding domain. Outside this domain no significant sequence similarity was found. However, in all cases regions with structural characteristics of activator domains were found. Analysis with Southern blots allowed an estimation of at least 20-30 myb genes in P. hybrida. Expression patterns were specific for each gene as shown by Northern blotting analysis. Subcellular location studies of the largest MYB product, by both immunoblotting of the protein in subcellular fractions and by 'in situ' immunolocation experiments, showed that it is a nuclear protein. The role of MYB proteins as transcriptional activators regulating the synthesis of different anthocyanin(flavonoid)-related compounds is discussed.

The cDNA encoding adenovirus E1A enhancer-binding protein E1A-F was isolated by screening a HeLa cell lambda gt11 expression library for E1A-F site-specific DNA binding. One cDNA clone produced recombinant E1A-F protein with the same DNA binding specificity as that endogenous to HeLa cells. Sequence analysis of the cDNA showed homology with the ETS-domain, a region required for sequence-specific DNA binding and common to all ets oncogene members. Analysis of the longest cDNA revealed about a 94% identity in amino acids between human E1A-F and mouse PEA3 (polyomavirus enhancer activator 3), a recently characterized ets oncogene member. E1A-F was encoded by a 2.5kb mRNA in HeLa cells, which was found to increase during the early period of adenovirus infection. In contrast, ets-2 mRNA was significantly reduced in infected HeLa cells. The results indicate that E1A enhancer binding protein E1A-F is a member of the ets oncogene family and is probably a human homologue of mouse PEA3.

The human ETS1 proto-oncogene proteins have been isolated from the T-cell leukemia line, CEM, by immunoaffinity chromatography and their identity confirmed by NH2-terminal amino acid sequencing. Incubation of CEM cells with N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) indicates that ETS proteins can be modified in their cellular context and that pretreatment of the cells with N-ethylmaleimide (NEM) protects ETS1 proteins from TLCK modification. These data show that ETS1 proteins can exist in at least two different states, -SH-available and -SH-protected. Renatured human ETS1 has DNA sequence-specific binding to the PEA3 (CAGGAAGT) motif. The ETS1.PEA3 complex can be observed by electrophoretic mobility shift assays (EMSA). Purified ETS1 retards a band which is exactly the same size as a complex that is retarded from nuclear extracts prepared from CEM cells. Reduced ETS1 is required to form the ETS1.PEA3 complex, however; modification of the ETS1 -SH groups by either NEM or by TLCk does not inhibit formation of the complex. The ETS1.PEA3 complex formed with TLCK-modified ETS1 has a slower mobility than the complex formed with unmodified ETS1. Zone sedimentation analysis of purified ETS1 indicates that it is the monomer of ETS1 which binds to the PEA3 oligonucleotide.

The proteins encoded by the proto-oncogenes c-fos and c-jun (Fos and Jun, respectively) form a heterodimeric complex that regulates transcription by interacting with the DNA-regulatory element known as the activator protein 1 (AP-1) binding site. Fos and Jun are members of a family of related transcription factors that dimerize via a leucine zipper structure and interact with DNA through a bipartite domain formed between regions of each protein that are rich in basic amino acids. Here we have defined other domains in the Fos-Jun heterodimer that contribute to transcriptional function in vitro. Although DNA-binding specificity is mediated by the leucine zipper and basic regions, Jun also contains a proline- and glutamine-rich region that functions as an ancillary DNA-binding domain but does not contribute directly to transcriptional activation. Transcriptional stimulation in vitro was associated with two regions in Fos and a single N-terminal activation domain in Jun. These activator regions were capable of operating independently; however, they appear to function cooperatively in the heterodimeric complex. The activity of these domains was modulated by inhibitory regions in Fos and Jun that repressed transcription in vitro. In the context of the heterodimer, the Jun activation domain was the major contributor to transcriptional stimulation and the inhibitory regions in Fos were the major contributors to transcriptional repression in vitro. Potentially, the inhibitory domains could serve a regulatory function in vivo. Thus, transcriptional regulation by the Fos-Jun heterodimer results from a complex integration of multiple activator and regulatory domains.

We have characterized the expression of c-Jun, JunB, JunD, c-Fos, and FosB proteins following serum stimulation of quiescent Swiss 3T3 cells by immunoprecipitation analyses. The synthesis of the three Jun proteins rapidly increases following stimulation, remaining at a significant level for at least 8 h. JunB protein presents the highest expression of all. FosB, like c-Fos, is transiently induced. Pulse-chase experiments show that all of the proteins except JunD are short-lived. We have shown that c-Fos and FosB form complexes in vivo with the different Jun proteins and that JunB complexes are predominant. In vitro association and competition experiments show that the affinities between the different Fos and Jun proteins are similar. This finding, together with the in vivo observations described above, suggests that the proportion of the different Jun/Fos heterodimers is governed by the concentration of the different components. The Fos and Jun proteins are phosphoproteins, and some remain relatively highly phosphorylated in their heterodimeric form.

The avian acute leukemia virus (E26) induces a mixed erythroid-myeloid leukemia in chickens and carries two distinct oncogenes, v-myb and v-ets. The viral protein responsible for transformation is a gag-myb-ets fusion protein that is located in the nucleus of the transformed cells. The cellular homologue of v-ets (c-ets-1) is highly expressed in lymphoid cells and differs from the v-ets gene at its carboxy terminal region. Here, we show that both the c-ets-1 and v-ets gene products are DNA-binding proteins and their DNA-binding activity is located in the carboxy terminal (46 amino acid residues) region. It appears that this DNA-binding activity is modulated by the extreme carboxy terminal region. The amino acid sequences of the putative ets DNA-binding domain at its carboxy terminal region showed a helix-turn-helix secondary structure. Exchanging the nonhomologous extreme carboxy terminal regions of c-ets-1 with v-ets gene sequences showed differences in DNA-binding affinity, indicating that these differences may be partly responsible for the activation of the ets oncogene.

We constructed replication-competent avian retrovirus vectors that contain two of the three known types of chicken c-ski cDNAs and a third vector that contains a truncated c-ski cDNA. We developed antisera that recognize the c-ski proteins made by the three transforming c-ski viruses. All three proteins (apparent molecular masses, 50, 60, and 90 kilodaltons) are localized primarily in the nucleus. The proteins are differentially phosphorylated; immunofluorescence also suggests that there are differences in subnuclear localization of the c-ski proteins and that c-ski protein is associated with condensed chromatin in dividing cells.

We previously showed that thymocytes express high levels of c-ets-1 protein (Ets1) that can be rapidly phosphorylated following mitogenic stimulation using lectins. We demonstrate here that T cell receptor (TCR) specific stimulation with monoclonal antibodies of mature CD8+ or CD4+ T cells also results in the rapid phosphorylation of Ets1, reinforcing the hypothesis of a possible role for Ets1 in T cell activation. In addition to the major Ets1 product (mu-p63c-ets-1), we identify in mouse thymocytes and mature T cells a distinct 52 Kd Ets1 related protein (mu-p52c-ets-1). In contrast to the major Ets1 protein, mu-p52c-ets-1 is poorly phosphorylated in unstimulated cells. Furthermore, mitogenic stimulation of thymocytes and T cells failed to induce in mu-p52c-ets-1 the Ca2(+)-dependent phosphorylation events which are known to drastically affect the migration of the major Ets1 protein in SDS polyacrylamide gels. Mu-p52c-ets-1, like mu-p63c-ets-1, is a nuclear-chromatin associated protein which exhibits DNA binding activity in vitro. However, in contrast to the major Ets1 protein, the association of mu-p53c-ets-1 with chromatin and its ability to bind to DNA in vitro are unaffected by activation stimuli resulting in an increase in [Ca2+]i. Finally, we present indications suggesting that mu-p52c-ets-1 might be the murine equivalent of the translation product of an alternatively c-ets-1 spliced mRNA described in human cells by others.

We identified the human c-ski gene product (c-Ski) as a protein with the apparent molecular weight of 100,000, p100c-ski, by using a c-Ski-specific polyclonal antibody. p100c-ski was a nuclear protein and p100c-ski in nuclear extracts of Molt4 cells bound to calf thymus DNA cellulose, but the bacterially synthesized c-Ski did not, suggesting that Ski was associated with another protein(s) and that the Ski complex had DNA-binding activity. This hypothesis was supported by the finding that the bacterially synthesized Ski bounds to DNA cellulose after being mixed with a nuclear extract of Molt4 cells. By use of a series of deletion mutants of Ski synthesized in an in vitro translation system, two portions in Ski were found to be necessary for the DNA binding of the Ski complex: the N-proximal portion containing a cystein/histidine-rich domain and the C-terminal portion including a region rich in basic amino acids.

A homolog of mammalian enhancer binding factor AP-1 was detected in Drosophila and was purified from embryo nuclear extracts by sequence-specific DNA affinity chromatography. The purified fraction, dAP-1, displays the sequence specificity as well as transcriptional activation properties of mammalian AP-1 and consists of two major proteins of mol. wts 40 and 70 kd. Antibody cross-reactivity experiments suggest that these proteins are Drosophila homologs of proto-oncogene products, Jun and Fos. The Drosophila Jun- and Fos-related antigens, when separated, are individually capable of sequence-specific DNA binding, and the Jun-related antigen activates transcription in vitro.

The bacterially expressed v-myb protein served as antigen for the isolation of several monoclonal antibodies, one of which recognized the human cellular myb protein (p75hu-c-myb) indicating a conserved epitope. The epitope was mapped to amino acid positions 208-232 by the use of several bacterially expressed v-myb proteins with various deletions. Furthermore, a synthetic oligopeptide which had been selected on the basis of its hydrophilicity by computer analysis of the v-myb oncogene (amino acids 213-231) blocked the action of this monoclonal antibody, indicating the immunological significance of this region. The monoclonal antibody allowed efficient purification of the p75hu-c-myb protein by immunoaffinity chromatography. The purified protein binds to double-stranded DNA in vitro in a filter-binding assay. Since the monoclonal antibody does not interfere with DNA binding it allowed analysis of DNA-protein interaction in a modified McKay assay using the purified p75hu-c-myb protein. Specific binding was observed predominantly to one of 12 lambda DNA fragments in vitro in the presence of high molar excess of competing co-polymer poly [d(I:C)]. Enhancer/promoter-like sequences of SV40 were not preferentially recognized.

Lymph node T cells of MRL-lpr/lpr mice are characterized by the production of very large amounts of c-myb mRNA. To study the control of c-myb expression, a search was made for sites on the 5' c-myb gene which could bind regulatory proteins. DNase I digestion of nuclear chromatin uncovered four DNase I hypersensitive sites in the first intron of the c-myb gene, and a single site approximately 300 bp 5' to the initiation codon. Lambda exonuclease digestion of a 5'-myb fragment in the presence of nuclear extracts from either MRL-lpr/lpr PLN or EL-4 thymoma revealed stop sites approximately 300 bp 5' (-271 to -322) to the ATG initiation codon. DNase I footprint analysis demonstrated a guanine-cytosine enriched region of potential binding sites (-274 to -319) in the region of the stop sites and a fifth potential binding site closer to the initiation codon (-163 to -168). Specific gel shift bands were detected by a 5'-myb fragment (-346 to -155) with extracts from a number of different lymphoid cell lines and the appropriate specific and non-specific competitor DNA. The DNA giving rise to these gel shift bands encompassed the region defined by the stop site and footprinting studies. To determine whether or not the protein binding to the 5' c-myb gene at -274 to -319 was associated with increased c-myb mRNA, we studied nuclear extracts of several cell lines and compared the amount of binding to the amount of c-myb mRNA found on Northern analyses. Among the cell lines, there was a correlation between c-myb expression and the amount of the 5'-myb DNA binding protein. In addition, MRL-lpr/lpr lymph node cells had high c-myb expression and large amounts of the 5'-myb binding protein. This result suggests that the binding may play some role in the c-myb expression. Moreover, the most immature cell lines had the greatest amount of the binding factor, suggesting that its regulatory effect on c-myb expression might be important in early differentiation events.

As a first step in the analysis of the molecular function of the nuclear c-fos proto-oncogene product we have studied its subnuclear localization in serum-stimulated mouse fibroblasts where it forms a non-covalent, apparently monodisperse complex with another nuclear protein, p39. The c-fos/p39 complex is almost quantitatively released from intact nuclei by DNasel or micrococcus nuclease treatment under conditions where only a minor fraction of DNA and nuclear proteins is released. In gel filtration experiments, c-fos/p39 comigrates with chromatin and seems to be associated with regions of increased DNasel accessibility. c-fos/p39 is bound to chromatin by electrostatic forces of moderate strength since greater than 90% of the complex can be eluted from nuclei at 0.4 M NaCl. In vitro, the c-fos/p39 complex in nuclear extracts binds to double- and single-stranded calf thymus DNA, suggesting that the association of c-fos/p39 with chromatin is at least in part due to its interaction with DNA. In agreement with this conclusion, c-fos/p39 is released from nuclei by incubation with tRNA, presumably due to competition for binding sites. Our observations are compatible with the hypothesis that c-fos may play a role in the regulation of gene expression.

c-erbA cDNAs encoding thyroid hormone-binding receptor proteins have been previously isolated from several species and tissues. We have isolated a novel rat c-erbA cDNA, r-erbA alpha-2, identical to the rat brain c-erbA alpha cDNA (which we refer to as r-erbA alpha-1) except at the 3'-end of the cDNA, which encodes an altered carboxy-terminal region of the predicted amino acid sequence. The r-erbA alpha-2 cDNA is most closely related to the human testicular c-erbA alpha cDNA. The apparent molecular weight of the in vitro protein product of this cDNA is 55 K, close to that predicted from the nucleotide sequence. The r-erbA alpha-2 protein binds a DNA fragment containing a putative T3-responsive sequence from the rat GH gene. However, the r-erbA alpha-2 protein product does not bind T3. RNA isolated from rat GH3 cells and various rat tissues contains at least three mRNAs hybridizing to c-erbA alpha probes. A predominant 2.6 kilobase mRNA hybridizes specifically to r-erbA alpha-2, while a 5.0 kb mRNA hybridizes to r-erbA alpha-1-specific cDNA probes. The r-erbA alpha-1-related 5.0 kb mRNA is down-regulated by T3 treatment in GH3 cells as has previously been described for the 2.6 kb mRNA. The r-erbA alpha-2 mRNA is most abundant in brain, whereas the r-erbA alpha-1 mRNA is found in highest concentration in brown fat and skeletal muscle. r-erbA alpha-1 mRNA, unlike r-erbA alpha-2 mRNA, is undetectable in testis. Southern analyses suggest that the r-erbA alpha-1 and r-erbA alpha-2 mRNAs are derived from a single gene transcript.

In the present study, we examined the expression of various protooncogenes and the specific binding of nuclear factor to c-myc gene in peripheral blood mononuclear cells (PBMC) from progressive systemic sclerosis (PSS) patients. We demonstrated first amplified expression of c-myc and c-myb gene and the existence of a nuclear protein specifically bound to 5'-non-coding fragment of c-myc gene. We then found a positive correlation between the degree of expression and/or affinity for the c-myc gene fragment of the nuclear protein and clinical disease activity. The amount of the factor was significantly reduced along with or prior to the amelioration of clinical symptoms and laboratory abnormalities with treatment. The significance of c-myc and c-myb proto-oncogene expression and nuclear factor specifically bound to c-myc gene is discussed.