Mi-2 complex couples DNA methylation to chromatin remodelling and histone deacetylation.

Methylation of DNA at the dinucleotide CpG is essential for mammalian development and is correlated with stable transcriptional silencing. This transcriptional silencing has recently been linked at a molecular level to histone deacetylation through the demonstration of a physical association between histone deacetylases and the methyl CpG-binding protein MeCP2 (refs 4,5). We previously purified a histone deacetylase complex from Xenopus laevis egg extracts that consists of six subunits, including an Rpd3-like deacetylase, the RbA p48/p46 histone-binding protein and the nucleosome-stimulated ATPase Mi-2 (ref. 6). Similar species were subsequently isolated from human cell lines, implying functional conservation across evolution. This complex represents the most abundant form of deacetylase in amphibian eggs and cultured mammalian cells. Here we identify the remaining three subunits of this enzyme complex. One of them binds specifically to methylated DNA in vitro and molecular cloning reveals a similarity to a known methyl CpG-binding protein. Our data substantiate the mechanistic link between DNA methylation, histone deacetylation and transcriptional silencing.

Multiple domains for the chicken cellular sequences homologous to the v-ets oncogene of the E26 retrovirus.

We have investigated the structure of chicken genomic DNA homologous to v-ets, the second cell-derived oncogene of avian retrovirus E26. We isolated a c-ets locus spanning ca. 30.0 kilobase pairs (kbp) in the chicken genome with homologies to 1,202 nucleotides (nt) of v-ets (total length, 1,508 nt) distributed in six clusters along 18.0 kbp of the cloned DNA. The 5'-distal part of v-ets (224 nt) was homologous to chicken cellular sequences contained upstream within a single 16.0-kbp EcoRI fragment as two typical exons but not found transcribed into the major 7.5-kb c-ets (or 4.0-kb c-myb) RNA species. Between these two v-ets-related cellular sequences we found ca 40.0 kbp of v-ets-unrelated DNA. Finally, the most 3' region of homology to v-ets in the cloned DNA was shown to consist of a truncated exon lacking the nucleotides coding for the 16 carboxy-terminal amino acids of the viral protein but colinear to one of the two human c-ets loci, c-ets-2.

Cellular and viral trans-acting factors modulate N-myc2 promoter activity in woodchuck liver tumors.

Activation of the N-myc2 oncogene by integration of woodchuck hepatitis virus (WHV) DNA is a central event in woodchuck liver oncogenesis. In this study, we have evaluated the influence of several cellular and viral trans-acting factors and mediators of inflammation on N-myc2 promoter activity in hepatoma cell lines. Ets oncoproteins, including Ets1, Ets2 and PEA3 efficiently activated a chimeric N-myc2 promoter/luciferase reporter gene. By electrophoretic mobility shift assays, we show that Etsl and Ets2 proteins can efficiently bind two consensus Ets sites located within a 59 bp sequence upstream of the N-myc2 transcription start site. Site-directed mutagenesis of these Ets-binding motifs abolished transactivation of the N-myc2 promoter by Ets proteins. Addition of interleukin-6 (IL-6) induced a weak but reproducible activation of the N-myc2 promoter, while IL-1 was ineffective. We further show that the N-myc2 promoter can be transactivated by the hepadna-virus X protein, and that distal promoter sequences are required for both IL-6 and X responsiveness. Similar effects of these factors were observed in the context of the N-myc2 promoter activated by WHV cis-regulatory elements. In view of the high-level expression of the N-myc2 oncogene in most woodchuck liver tumors, the Ets oncoproteins, inflammation-associated cytokine IL-6 and the viral X transactivator might play important roles in hepadnavirus-associated tumorigenesis.

Expression of ETS proto-oncogenes in astrocytes in human cortex.

In order to investigate a possible function of ETS proto-oncogenes in human brain, we incubated a polyclonal antibody raised against the viral region of E26 homologous to ETS1 and ETS2 with human brain frontal cortex sections. Our results show that this antibody decorates astrocytes but not neurons. By using astrocytomas of different grades as a source of astrocytes, we demonstrate the presence of ETS1 and ETS2 messenger RNAs and proteins. This leads to the idea that ETS genes are expressed in cells with dividing potentialities in human cortex and that they could provide a new marker for glial cells. Recently, a microduplication on chromosome 21 including ETS2 locus was described in karyotypically 'normal' Down's syndrome and suspected in Alzheimer's disease; when testing Alzheimer's disease-affected brain cortex sections, no obvious difference was observed with the technique used.

Synergistic activation of the HTLV1 LTR Ets-responsive region by transcription factors Ets1 and Sp1.

Ets1 is the prototype of a family of transcriptional activators whose activity depends on the binding to specific DNA sequences characterized by an invariant GGA core sequence. We have previously demonstrated that transcriptional activation by Ets1 of the long terminal repeat (LTR) of human T cell lymphotropic virus type 1 is strictly dependent on the binding of Ets1 to two sites, ERE-A and ERE-B, localized in a 44 bp long Ets-responsive region (ERR1). We report here that the activity of ERR1 as an efficient Ets1 response element in HeLa cells also depends on the integrity of an Sp1 binding site localized immediately upstream of ERE-A. The response to Ets1 of an element restricted to the SP1/ERE-A binding sites is also strictly dependent on both the Ets1 and Sp1 binding sites. In vitro, Sp1 and Ets1 are shown to cooperate to form a ternary complex with the SP1/ERE-A element. Reconstitution experiments in Drosophila melanogaster Schneider cells show that Ets1 and Sp1 act synergistically to activate transcription from either the ERR1 or the SP1/ERE-A elements and that synergy requires the binding of both Sp1 and Ets1 to their cognate sites. SP1/ERE-A elements are found in the enhancer/promoter region of several cellular genes, suggesting that synergy between Ets1 and Sp1 is not restricted to the ERR1 region of the HTLV1 LTR. These results strengthen the notion that Ets1 as well as other members of the Ets family usually function as components of larger transcription complexes to regulate the activity of a variety of viral and cellular genes.

[Abnormal localization of proto-oncogene c-ets 1 in acute leukemia with translocation t(1: 11)(q21: q23)]. / Localisation anormale du proto-oncogène c-ets 1 dans une leucémie aiguë avec translocation t(1; 11)(q21; q23).

The chromosome localization of a 5.4 kb DNA genomic probe of proto-oncogene c-ets 1 has been analysed in an acute monocytic leukemia with t (1; 11) (q21; q23) translocation. The c-ets probe has been translocated onto the rearranged chromosome 1, suggesting the involvement of the proto-oncogene in leukemias with chromosome rearrangements at band 11 q23.

TAFII55 binding to TAFII250 inhibits its acetyltransferase activity.

The general transcription factor, TFIID, consists of the TATA-binding protein (TBP) associated with a series of TBP-associated factors (TAFs) that together participate in the assembly of the transcription preinitiation complex. One of the TAFs, TAF(II)250, has acetyltransferase (AT) activity that is necessary for transcription of MHC class I genes: inhibition of the AT activity represses transcription. To identify potential cellular factors that might regulate the AT activity of TAF(II)250, a yeast two-hybrid library was screened with a TAF(II)250 segment (amino acids 848-1279) that spanned part of its AT domain and it's the domain that binds to the protein, RAP74. The TFIID component, TAF(II)55, was isolated and found to interact predominantly with the RAP74-binding domain. TAF(II)55 binding to TAF(II)250 inhibits its AT activity. Importantly, the addition of recombinant TAF(II)55 to in vitro transcription assays inhibits TAF(II)250-dependent MHC class I transcription. Thus, TAF(II)55 is capable of regulating TAF(II)250 function by modulating its AT activity.

Determinants of vitellogenin B1 promoter architecture. HNF3 and estrogen responsive transcription within chromatin.

The liver-specific vitellogenin B1 promoter is efficiently activated by estrogen within a nucleosomal environment after microinjection into Xenopus laevis oocytes, consistent with the hypothesis that significant nucleosome remodeling over this promoter is not a prerequisite for the activation by the estrogen receptor (ERalpha). This observation lead us to investigate determinants other than ERalpha of chromatin structure and transcriptional activation of the vitellogenin B1 promoter in this system and in vitro. We find that the liver-enriched transcription factor HNF3 has an important organizational role for chromatin structure as demonstrated by DNase I-hypersensitive site mapping. Both HNF3 and the estrogen receptor activate transcription synergistically and are able to interact with chromatin reconstituted in vitro with three positioned nucleosomes. We propose that HNF3 is the cellular determinant which establishes a promoter environment favorable to a rapid transcriptional activation by the estrogen receptor.

Sequence-specific interaction of the Ets1 protein with the long terminal repeat of the human T-lymphotropic virus type I.

We recently demonstrated that members of the c-ets proto-oncogene family, Ets1 and Ets2, are sequence-specific transcriptional activators of the human T-lymphotropic virus type I (HTLV-I) long terminal repeat (LTR). We now report that the HTLV-I LTR contains two distinct Ets1-responsive regions, ERR-1 and ERR-2. Expression of Ets1 with reporter plasmids containing ERR-1 or ERR-2 upstream of a basal promoter resulted in an increase in transcriptional activity. By gel mobility shift assay, the interaction of Ets1 with the downstream ERR-1-binding region was found to be more stable than its interaction with the upstream ERR-2 region. By DNase I footprint, gel mobility shift, and methylation interference analyses, ERR-1 was found to contain two Ets1 binding sites, ERE-A and ERE-B. A recombinant Ets1 protein was found to bind with higher affinity to ERE-A than to ERE-B. Binding of Ets1 to these sites appears to result in a specific and sequential protection of a 37-nucleotide sequence of the HTLV-I LTR from -154 to -118. In view of the high-level expression of Ets1 in lymphoid cells, the c-ets proto-oncogenes encode transcription factors which could play an important role in both basal and Tax1-mediated HTLV-I transcription.

Regulation of parathyroid hormone-related protein (PTHrP) gene expression. Sp1 binds through an inverted CACCC motif and regulates promoter activity in cooperation with Ets1.

We have previously shown that mutations in the GGAA core motif of the Ets1 binding site, EBSI, or deletion of EBSI, reduced basal and Tax1 transactivation of the PTHrP P2 promoter. Here we demonstrate that, in addition to EBSI, a CACCC-like motif located between -53 and -58 is required for full basal activity of this promoter in Jurkat T-cells. Site-specific mutations in the CACCC motif decreased promoter activity approximately 5-fold. In an effort to identify transcription factors that bind to the CACCC element, we found that purified human Sp1, as well as Sp1 in HeLa nuclear extract, can specifically bind to a DNA probe that corresponds to the PTHrP-specific sequence between -94 and -34. Gel shift competition studies and DNase I footprinting analyses revealed that Sp1 specifically interacts with the CACCC motif. In the presence of Ets1, the mobility of the Sp1-specific gel shift complex with the PTHrP DNA decreased. DNase I footprint analysis of this gel shift complex showed an extended footprint over both the Sp1 and the Ets1 binding site, demonstrating that Sp1 and Ets1 form a ternary complex with the PTHrP DNA. Cotransfection of an Ets1 and Sp1 expression vector into Drosophila Schneider cells demonstrated that Sp1 can functionally cooperate with Ets1 to transactivate the PTHrP promoter. We conclude from these data that Ets1 and Sp1 can cooperatively regulate PTHrP P2 promoter activity.

[Preparation and characterization of antisera specific to various polypeptide domains corresponding to the v-ets oncogene of the avian leukemia virus E26]. / Préparation et caractérisation d'antisera specifiques de différents domaines polypeptidiques correspondant à l'oncogène v-ets du virus aviaire de leucémies aiguës E26.

We prepared antisera to three distinct portions of the v-ets oncogene of the avian leukemia virus E26. An antiserum directed against the middle v-ets-encoded domain identifies in different chicken cell lines and normal tissues a c-ets-encoded protein of Mr 54,000 (P54c-ets) and three proteins of Mr 60,000 62,000 and 64,000 partially related to P54c-ets. Antisera directed against the aminoterminal v-ets-encoded domain failed to precipitate P54c-ets or P60/P64. Thus, the E26 specific v-ets oncogene displays a complex structure that includes several distinct portions, the genetic origin of which could be different.

Identification and preferential expression in thymic and bursal lymphocytes of a c-ets oncogene-encoded Mr 54,000 cytoplasmic protein.

The avian retrovirus E26 is unique among acute leukemia viruses in its ability to induce transformation of cells belonging to either the myeloid or erythroid lineage. The genome of E26 carries two oncogenes, v-myb and v-ets, that are derived from distinct cellular loci, c-myb and c-ets. We have constructed a plasmid vector that allows expression of part of the coding region of v-ets in a bacterial host. Antisera to the bacterially synthesized ets protein specifically precipitated the E26-encoded P135gag-myb-ets transforming protein. These antisera permitted us to identify a chicken c-ets-encoded protein of Mr 54,000 (P54c-ets) that shares 7 out of 10 of its major [35S]methionine-containing tryptic peptides with the v-ets-encoded domain of P135gag-myb-ets. Unlike P135gag-myb-ets and the Mr 75,000 translation product of c-myb (P75c-myb), which are nuclear proteins, P54c-ets was found to be predominantly cytoplasmic. P54c-ets is expressed at low levels in most cell lines and tissues tested, including bone marrow cells and circulating lymphocytes. P54c-ets, together with a minor but closely related Mr 56,000 protein, was found to be expressed at high levels in chicken thymocytes and bursal lymphocytes.

The bovine leukemia virus p34 is a transactivator protein.

Recombinant Moloney murine retroviruses containing the BLV post-envelope long open reading frame were constructed and transfected into the psi 2 packaging cell line. They were shown to encode and to express a 34-kd protein able to transactivate the BLV long terminal repeat-directed gene expression in the respective transfected cells. These data demonstrate that the BLV X-LOR gene encodes a p34 transactivator product. Furthermore, the different cell lines produced infectious recombinant retroviruses capable of transferring X-LOR genes into recipient cells. The availability of the BLV transactivator protein should allow us to understand the role of the transactivator protein in BLV-induced leukemogenesis.

Analysis of the DNA binding and transcriptional activation properties of the Ets1 oncoprotein.

The c-ets1 gene product (Ets1) is the prototype of a family of sequence-specific transcriptional activators which have been implicated in various developmental processes and in the response of cells to a variety of extracellular stimuli. We report here a structure-function analysis of the DNA binding and transcriptional activation properties of Ets1. The minimal region required for specific DNA binding is located at the carboxy-terminus of Ets1, a domain highly conserved in all known members of the Ets family. Transcriptional activation by Ets1 in mammalian cells requires an additional domain of 110 amino acids characterized by a high content of acidic residues and localized in the amino-terminal half of the protein. This domain also functions as a transcriptional activation domain in yeast cells when linked to the heterologous DNA binding domain of Gal4. In contrast to its conservation in Ets1 proteins across vertebrate species, this activation domain is not conserved in other members of the Ets family. These results indicate that an important level of specificity between different members of the Ets family may reside in the differential interactions of their respective activation domains with distinct general transcription factors or different associated coactivators.

Chromosomal localization of the human proto-oncogene c-ets.

E26 is an acute leukaemia avian retrovirus which induces myeloblastosis and erythroblastosis in vivo and transforms erythroblasts and myeloblasts in vitro. It contains the oncogene v-myb (ref. 4), first described for avian myeloblastosis virus (AMV), as well as a second specific nucleotide sequence, v-ets located 3' to v-myb (refs 5,6). We have reported that v-ets has a cellular counterpart (c-ets) in chicken and human DNA. Now, using two independent methods--hybridization with human c-ets probe of sorted chromosomes and in situ hybridization--we report the localization of the ets locus on human chromosome 11 at bands q23-q24. This finding may be important, as specific breakpoints around this position have been reported for human malignancies such as acute monocytic leukaemia and Ewing's sarcoma.

Increased transcription of the c-myc oncogene in two methylcholanthrene-induced quail fibroblastic cell lines.

The expression of three c-onc genes (c-erb, c-myc, c-myb) was investigated in five cell lines established from fibrosarcomas induced with 20-methylcholanthrene (MCA) of Japanese quails. These cell lines showed low levels of the three c-onc genes, with the exception of two cell lines that accumulated moderate (MCAQ 1-4) and large amounts (MCAQ3-5) of c-myc RNA. Molecular cloning and restriction endonuclease analyses indicated that expression of c-myc in these two cell lines were not associated with detectable rearrangements in the c-myc locus, that the size of the c-myc transcript (2.7 kb) in MCAQ 3-5 was similar to that of the normal c-myc messenger RNAs (mRNA) and that the transcriptional activation observed in MCAQ 3-5 was not mediated by the LTR (long terminal repeat) of a proximate ALV (avian leukosis virus) provirus. Finally, when analysed with the restriction enzymes Msp I and Hpa II, the c-myc locus of MCAQ 3-5 and MCAQ 1-4 was found hypomethylated as compared with that of the other cell lines tested that show low levels of c-myc transcripts. Our results suggest that one of the ways methylcholantrene could mediate transformation is by inducing an abnormal regulation of the c-myc gene.

Molecular cloning of the avian acute transforming retrovirus MH2 reveals a novel cell-derived sequence (v-mil) in addition to the myc oncogene.

Mill-Hill-2 virus (MH2) proviral DNA was cloned from a transformed non-producer cell culture (MH2QB2) through insertion of randomly cut high mol. wt. cellular DNA in the lambdoid vector L47.1. Restriction analysis of a suitable recombinant phage by Southern DNA blotting and hybridization with different probes allowed us to characterize the genetic organization of the provirus and to identify a novel MH2-specific sequence of at least 1.1kbp. Such a sequence, for which we propose the name v-mil, from MilI-Hill-2 virus, is not homologous to v-myc, the previously described oncogene of MH2, nor to avian leukaemia virus-related sequences. Evidence is presented here that v-mil has a cellular counterpart (c-mil) phylogenetically conserved in birds and mammals, including man, and expressed as a single RNA species at least in some tissues. MH2 virus might thus be regarded, like avian erythroblastosis virus or E26, as another example of retroviruses having recombined with more than one cellular gene.

Characterization of a hepatoma mRNA transcribed from a third promoter of a 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-encoding gene and controlled by ets oncogene-related products.

6-Phosphofructo-2-kinase (EC 2.7.1.105)/fructose-2,6-bis-phosphatase (EC 3.1.3.46) catalyzes the synthesis and degradation of fructose 2,6-bisphosphate, a ubiquitous stimulator of glycolysis. The liver (L-type) and muscle (M-type) mRNAs for this bifunctional enzyme arise from distinct promoters of the same gene. We have now characterized in rat hepatoma FTO2B cells another mRNA, which is transcribed from a third promoter of that gene. This F-type mRNA is present in fetal rat liver and muscle, in rat placenta, and in several established rat cell lines. The F promoter contains no TATA box but contains several binding sites for Sp1 and for members of the ets oncogene family. Transfection of FTO2B cells with constructs containing the intact or mutagenized F promoter showed that its activity depends mainly on one of these sites. This site bound a heteromeric FTO2B cell protein indistinguishable from the ets-related GA binding protein alpha/ankyrin-repeats GA binding protein beta transcription factor.

The human DNA locus related to the oncogene myb of avian myeloblastosis virus (AMV): molecular cloning and structural characterization.

Chicken and human cell DNA contains sequences homologous to the avian myeloblastosis virus oncogene, v-myb. These cellular sequences, c-myb (human) and c-myb (chicken), were isolated from libraries of human or chicken cell DNA fragments, generated by partial digestion with the restriction enzymes AluI and HaeIII, and compared. The chicken c-myb locus isolated from two distinct overlapping recombinant phages, contained five contiguous EcoRI fragments of 5.4, 1.1, 2.1, 2.2 and 9 kbp, accounting for all the bands seen with a v-myb probe in a complete EcoRI digest of chicken cellular DNA. Likewise, the screening of the human library yielded a recombinant phage hybridizing with the v-myb specific probe, that contained five EcoRI fragments of 2.8, 2.6, 2.0, 1.2 and 5.0 kbp (the last ending with an artificial EcoRI site, due to the construction of the library) belonging to the c-myb (human) locus. Probes using the EcoRI chicken DNA cloned fragments revealed corresponding contiguous EcoRI fragments in the human clone. Subsequent analyses of cellular polyadenylated mRNA extracted from human and chicken cells allowed the identification of single RNA species of 3.8 and 4.0 kb, respectively, as the representative transcripts of the c-myb locus in the two species. Thus, c-myb appears as a single locus in man and chicken, conserved with a similar structure in the two distantly related species. Our preparation of a specific human c-myb probe with an increased sensitivity on DNA/RNA blots should facilitate analyses concerning this gene in human normal or tumour cells or tissues.

Identification in chicken macrophages of a set of proteins related to, but distinct from, the chicken cellular c-ets-encoded protein p54c-ets.

Using an antiserum to a bacterially expressed polypeptide corresponding to 56 amino acids of v-ets, we previously identified in chicken tissues a protein of 54 kd (p54c-ets) which shares extensive sequence homology to the v-ets-encoded domain of the E26-transforming protein p135gag-myb-ets and is thus apparently encoded by the c-ets proto-oncogene. We report here that the anti-ets serum specifically identifies in chicken cells a second set of proteins of 60 kd (p60), 62 kd (p62) and 64 kd (p64) which appear to be highly related to each other but display only a limited domain of homology with p54c-ets and p135gag-myb-ets and are thus probably encoded by a gene(s) partially related to, but different from c-ets. In contrast to p54c-ets which is expressed at high levels in chicken lymphoid tissues, prominent syntheses of p62 and p64 were found in both normal and transformed chicken macrophages but not in avian cells corresponding to immature stages of the myeloid differentiation pathway. These observations together with the fact that differentiation of avian myeloblastosis virus-transformed myeloblasts into macrophage-like cells after treatment with 12-O-tetradecanoylphorbol-13-acetate is accompanied by the synthesis of p62 and p64 suggest a role for these proteins in chicken macrophage differentiation or function. Induction of differentiation of human leukemia cell lines HL60 and U937 into macrophages is also accompanied by the increased synthesis of c-ets-encoded 68 kd, 62 kd and 58 kd proteins.