Transcriptional repression by the retinoblastoma protein through the recruitment of a histone methyltransferase.

The E2F transcription factor controls the cell cycle-dependent expression of many S-phase-specific genes. Transcriptional repression of these genes in G(0) and at the beginning of G(1) by the retinoblasma protein Rb is crucial for the proper control of cell proliferation. Rb has been proposed to function, at least in part, through the recruitment of histone deacetylases. However, recent results indicate that other chromatin-modifying enzymes are likely to be involved. Here, we show that Rb also interacts with a histone methyltransferase, which specifically methylates K9 of histone H3. The results of coimmunoprecipitation experiments of endogenous or transfected proteins indicate that this histone methyltransferase is the recently described heterochromatin-associated protein Suv39H1. Interestingly, phosphorylation of Rb in vitro as well as in vivo abolished the Rb-Suv39H1 interaction. We also found that Suv39H1 and Rb cooperate to repress E2F activity and that Suv39H1 could be recruited to E2F1 through its interaction with Rb. Taken together, these data indicate that Suv39H1 is involved in transcriptional repression by Rb and suggest an unexpected link between E2F regulation and heterochromatin.

Physical association between the histone acetyl transferase CBP and a histone methyl transferase.

CBP (CREB-binding protein) is involved in transcriptional activation by a great variety of sequence-specific transcription factors. CBP has been shown to activate transcription through its histone acetyl transferase activity. Acetylation is a common post-translational modification of nucleosomal histone N-terminal tails, which generally correlates with transcriptional activation. Histone N-terminal tails are also modified by methylation but its functional consequences are largely unknown. Here we found that immunoprecipitation of CBP, or of the highly related p300, led to the co-immunoprecipitation of a robust histone methyl transferase (HMT) activity, indicating that CBP physically interacts with an HMT in living cells. The CBP-associated HMT is specific for lysines 4 and 9 of histone H3, which are known to be methylated in living cells. These results suggest that histone methylation could be involved in transcriptional activation. Furthermore, they raise the question of the link between histone methylation and acetylation.

Teashirt is required for transcriptional repression mediated by high Wingless levels.

During development, extracellular signals often act at multiple thresholds to specify distinct transcriptional and cellular responses. For example, in the embryonic midgut of Drosophila, low Wingless levels stimulate the transcription of homeotic genes whereas high Wingless levels repress these genes. Wingless- mediated transcriptional activation is conferred by Drosophila: T-cell factor (dTCF) and its co-activator Armadillo, but the nuclear factors mediating transcriptional repression are unknown. Here we show that teashirt is required for Wingless-mediated repression of Ultrabithorax: in the midgut. Teashirt is also a repressor of the homeotic gene labial in this tissue. Furthermore, the target sequence for Tsh within the Ultrabithorax: midgut enhancer coincides with the response sequence for Wingless-mediated repression. Finally, we demonstrate that the zinc finger protein Teashirt behaves as a transcriptional repressor in transfected mammalian cells. It thus appears that the response to high Wingless levels in the Drosophila: midgut is indirect and based on transcriptional activation of the Teashirt repressor.

Residues phosphorylated by TFIIH are required for E2F-1 degradation during S-phase.

The transcription factor E2F-1 plays a key role in regulating cell cycle progression. Accordingly, E2F-1 activity is itself tightly controlled by a series of transcriptional and post-transcriptional events. Here we show that the E2F-1 activation domain interacts with a kinase activity which phosphorylates two sites, Ser403 and Thr433, within the activation domain. We demonstrate that TFIIH is responsible for the E2F-1 phosphorylation observed in cell extracts and that endogenous E2F-1 interacts in vivo with p62, a component of TFIIH, during S phase. When the two phosphorylation sites in E2F-1 are mutated to alanine, the stability of the E2F-1 activation domain is greatly increased. These results suggest that TFIIH-mediated phosphorylation of E2F-1 plays a role in triggering E2F-1 degradation during S phase.

The HMG-box transcription factor HBP1 is targeted by the pocket proteins and E1A.

A yeast two-hybrid screen has identified HBP1 as a transcription factor capable of interacting with the pocket protein family. We show that HBP1 can interact with one of these, RB, both in vitro and in mammalian cells. Two distinct RB binding sites are present within HBP1--a high affinity binding site, mediated by an LXCXE motif and a separate low affinity binding site present within an activation domain. GAL4-fusion experiments indicate that HBP1 contains a masked activation domain. Deletion of two independent N- and C-terminal inhibitor domains unmasks an activation domain which is 100-fold more active than the full length protein. The released activation capacity is repressed by RB, p130 and p107. In addition, E1A can repress the activity of HBP1 via conserved region 1 sequences in a manner independent of the CBP co-activator. We show by stable expression in NIH3T3 cells that HBP1 has the capacity to induce morphological transformation of cells in culture.

Stepwise transformation of rat embryo fibroblasts: c-Jun, JunB, or JunD can cooperate with Ras for focus formation, but a c-Jun-containing heterodimer is required for immortalization.

Among the Jun family of transcription factors, only c-Jun displays full transforming potential in cooperation with activated c-Ha-Ras in primary rat embryo fibroblasts. c-Jun in combination with Ras can both induce foci of transformed cells from rat embryo fibroblast monolayers and promote the establishment of these foci as tumoral cell lines. JunB can also cooperate with Ras to induce foci but is unable to promote immortalization. We report here that JunD, in cooperation with Ras, induces foci with an efficiency similar to that of JunB. Artificial Jun/eb1 derivatives from each of the three Jun proteins were also analyzed. These constructs carry a heterologous homodimerization domain from the viral EB1 transcription factor and are thought to form only homodimers in the cell. We show here that these Jun/eb1 chimeras are potent transactivators of AP1 sites and that they can cooperate with c-Ha-Ras to induce foci. However, among all the Ras-Jun and Ras-Jun/eb1 combinations tested, only foci from Ras-c-Jun can be efficiently expanded and maintained as long-term growing cultures. Therefore, we suggest that a heterodimer containing c-Jun might be required for in vitro establishment of these primary mammalian cells.

Tumor induction by v-Jun homodimers in chickens.

To study the contribution of v-Jun homodimers to oncogenesis, we constructed artificial v-Jun derivatives in which the natural dimerization domain of v-Jun was replaced by an heterologous homodimerization domain from either the viral EB1 or the yeast GCN4 transcription factor. The resulting v-Jun chimeric proteins, called v-Juneb1 and v-Jungcn4, which can no longer dimerize with Jun or Fos, should only form homodimers in the cell. Helper-independent retroviruses expressing v-Jun, v-Juneb1 and v-Jungcn4 were generated. All three viruses transformed primary cultures of chick embryo cells with the same high efficiency and promoted local tumor growth after subcutaneous injection of infected cells in young animals. In contrast, after intravenous injection of viral suspensions into chick embryos, only the chimeric proteins produced internal tumors that were lethal. These tumors were leiomyosarcomas located within the liver and along the digestive tract. Thus, in vivo, v-Juneb1 and v-Jungcn4 are more potent oncoproteins than v-Jun. These data demonstrate that when forced to accumulate, v-Jun homodimers can induce tumors efficiently. They also show that the oncogenic potential of v-Jun can be regulated through the properties of its dimerization domain.

Increased transforming activity of JunB and JunD by introduction of an heterologous homodimerization domain.

The closely-related proteins c-Jun, JunB and JunD form a family of transcription factors which require dimerization for DNA-binding and transcriptional activity. Dimerization is mediated by a conserved amphipathic alpha-helix located adjacent to a highly charged DNA-binding domain. The Jun proteins can form both homo- and heterodimers within the Jun family and can also cross-dimerize with the Fos proteins. When expressed at high levels in primary chicken cells, each mouse Jun displays distinct transforming capacities: c-Jun transforms efficiently, JunB transforms poorly, and JunD does not transform at all. The composition of the transforming dimers, however, is unknown. To study the activity of Jun-Jun homodimers we constructed artificial derivatives, denoted Juneb1, in which the naturally occurring dimerization domain has been replaced by an heterologous homodimerization domain from the Epstein-Barr virus transcription factor EB1. These derivatives were introduced into chicken cells and assayed for their ability to affect growth. Unexpectedly, all three Juneb1 proteins conferred a transformed phenotype to primary cultures, promoting sustained growth in low-serum medium and colony formation from single cells in agar. These data demonstrate that when forced to accumulate as homodimers, both JunB and JunD can transform cells. They also suggest that the poor transforming activity of JunB and the absence of transforming activity of JunD may be due to their inability to accumulate to high levels as homodimers.

Cloning and sequence analysis of the gene for a carbapenem-hydrolyzing class A beta-lactamase, Sme-1, from Serratia marcescens S6.

Serratia marcescens S6 produces a pI 9.7 carbapenem-hydrolyzing beta-lactamase that is probably encoded by the chromosome (Y. Yang, P. Wu, and D. M. Livermore, Antimicrob. Agents Chemother. 34:755-758, 1990). A total of 11.3 kb of genomic DNA from this strain was cloned into plasmid pACYC184 in Escherichia coli. After further subclonings, the carbapenem-hydrolyzing beta-lactamase gene (blaSme-1) was sequenced (EMBL accession number Z28968). The gene corresponded to an 882-bp open reading frame which encoded a 294-amino-acid polypeptide. This open reading frame was preceded by a -10 and a -35 region consistent with a putative promoter sequence of members of the family Enterobacteriaceae. This promoter was active in E. coli and S. marcescens, as demonstrated by primer extension analysis. N-terminal sequencing showed that the Sme-1 enzyme had a 27-amino-acid leader peptide and enabled calculation of the molecular mass of the mature protein (29.3 kDa). Sequence alignment revealed that Sme-1 is a class A serine beta-lactamase and not a class B metalloenzyme. The earlier view that the enzyme was zinc dependent was discounted. Among class A beta-lactamases, Sme-1 had the greatest amino acid identity (70%) with the pI 6.9 carbapenem-hydrolyzing beta-lactamase, NMC-A, from Enterobacter cloacae NOR-1. Comparison of these two protein sequences suggested a role for specific residues in carbapenem hydrolysis. The relatedness of Sme-1 to other class A beta-lactamases such as the TEM and SHV types was remote. This work details the sequence of the second carbapenem-hydrolyzing class A beta-lactamase from an enterobacterial species and the first in the genus Serratia.

HLA polymorphism and T cell recognition of a conserved region of p190, a malaria vaccine candidate.

We have examined T cell recognition of a recombinant polypeptide (190L), corresponding to a 175-amino-acid-long conserved region of the major surface antigen (p190) of Plasmodium falciparum merozoites. We show that 190L contains a variety of T cell epitopes, and can be recognized in association with many different MHC class II molecules, including HLA-DR, DP, and DQ antigens. Most of the epitope-containing peptides are able to bind to more than one DR, and a single DR molecule can bind to different peptides. These findings, together with the fact that humans are generally heterozygous at the DR, DQ, and DP beta chain loci, suggest that MHC restriction should not be a major constraint in the development of malaria subunit vaccines.