Oligomerization of ETO is obligatory for corepressor interaction.

Nearly 40% of cases of acute myelogenous leukemia (AML) of the M2 subtype are due to a chromosomal translocation that combines a sequence-specific DNA binding protein, AML1, with a potent transcriptional repressor, ETO. ETO interacts with nuclear receptor corepressors SMRT and N-CoR, which recruit histone deacetylase to the AML1-ETO oncoprotein. SMRT-N-CoR interaction requires each of two zinc fingers contained in C-terminal Nervy homology region 4 (NHR4) of ETO. However, here we show that polypeptides containing NHR4 are insufficient for interaction with SMRT. NHR2 is also required for SMRT interaction and repression by ETO, as well as for inhibition of hematopoietic differentiation by AML1-ETO. NHR2 mediates oligomerization of ETO as well as AML1-ETO. Fusion of NHR4 polypeptide to a heterologous dimerization domain allows strong interaction with SMRT in vitro. These data support a model in which NHR2 and NHR4 have complementary functions in repression by ETO. NHR2 functions as an oligomerization domain bringing together NHR4 polypeptides that together form the surface required for high-affinity interaction with corepressors. As nuclear receptors also interact with corepressors as dimers, oligomerization may be a common mechanism regulating corepressor interactions.

Terminal megakaryocytic differentiation of TF-1 cells is induced by phorbol esters and thrombopoietin and is blocked by expression of PML/RARalpha fusion protein.

We have analyzed the differentiation program of growth factor-dependent TF-1 erythroleukemia cells as well as clones with inducible expression of the APL-specific PML/RARalpha protein. We have shown that TF-1 cells may be induced to megakaryocytic differentiation by phorbol ester (phorbol dibutyrate, PDB) addition, particularly when combined with thrombopoietin (Tpo). RT-PCR studies showed that Tpo induces Tpo receptor (TpoR or c-mpl), whose expression was further potentiated by PDB addition. When the cells are induced with both PDB and Tpo erythropoietin receptor (EpoR) expression was inhibited. In the absence of Zn2+-induced PML/RARalpha expression, PDB and Tpo induced megakaryocytic differentiation of TF-1 MTPR clones as observed in 'wild-type' TF-1 cells. Conversely, when PML/RARalpha expression was induced by Zn2+, PDB and Tpo treatment of these clones caused only a reduced level of megakaryocytic differentiation. These observations indicate that: (1) TF-1 cells as well as other erythroleukemic cells, possess the capacity to differentiate to megakaryocytic cells when grown in the presence of protein kinase (PKC) activators and more efficiently when combined with Tpo; (2) the PML/RARalpha gene has a wide capacity to interfere with the program of hematopoietic differentiation, including megakaryocytic differentiation. Finally, we also observed that PML/RARalpha expression in TF-1 cells induces an up-modulation of interleukin-3 receptor, c-kit and c-mpl, a phenomenon which may offer these cells a growth advantage.

Binding specificity and in vivo targets of the EH domain, a novel protein-protein interaction module.

EH is a recently identified protein-protein interaction domain found in the signal transducers Eps15 and Eps15R and several other proteins of yeast nematode. We show that EH domains from Eps15 and Eps15R bind in vitro to peptides containing an asparagine-proline-phenylalanine (NPF) motif. Direct screening of expression libraries with EH domains yielded a number of putative EH interactors, all of which possessed NPF motifs that were shown to be responsible for the interaction. Among these interactors were the human homolog of NUMB, a developmentally reguated gene of Drosophila, and RAB, the cellular cofactor of the HIV REV protein. We demonstrated coimmunoprecipitation of Eps15 with NUMB and RAB. Finally, in vitro binding of NPF-containing peptides to cellular proteins and EST database screening established the existence of a family of EH-containing proteins in mammals. Based on the characteristics of EH-containing and EH-binding proteins, we propose that EH domains are involved in processes connected with the transport and sorting of molecules within the cell.

Opposite effects of the p52shc/p46shc and p66shc splicing isoforms on the EGF receptor-MAP kinase-fos signalling pathway.

Shc proteins are targets of activated tyrosine kinases and are implicated in the transmission of activation signals to Ras. The p46shc and p52shc isoforms share a C-terminal SH2 domain, a proline- and glycine-rich region (collagen homologous region 1; CH1) and a N-terminal PTB domain. We have isolated cDNAs encoding for a third Shc isoform, p66shc. The predicted amino acid sequence of p66shc overlaps that of p52shc and contains a unique N-terminal region which is also rich in glycines and prolines (CH2). p52shc/p46shc is found in every cell type with invariant reciprocal relationship, whereas p66shc expression varies from cell type to cell type. p66shc differs from p52shc/p46shc in its inability to transform mouse fibroblasts in vitro. Like p52shc/p46shc, p66shc is tyrosine-phosphorylated upon epidermal growth factor (EGF) stimulation, binds to activated EGF receptors (EGFRs) and forms stable complexes with Grb2. However, unlike p52shc/p46shc it does not increase EGF activation of MAP kinases, but inhibits fos promoter activation. The isolated CH2 domain retains the inhibitory effect of p66shc on the fos promoter. p52shc/p46shc and p66shc, therefore, appear to exert different effects on the EGFR-MAP kinase and other signalling pathways that control fos promoter activity. Regulation of p66shc expression might, therefore, influence the cellular response to growth factors.

Altered myeloid development and acute leukemia in transgenic mice expressing PML-RAR alpha under control of cathepsin G regulatory sequences.

Acute promyelocytic leukemia (APML) is characterized by abnormal myeloid development, resulting an accumulation of leukemic promyelocytes that are often highly sensitive to retinoic acid. A balanced t(15;17) (q22;q21) reciprocal chromosomal translocation is found in approximately 90% of APML patients; this translocation fuses the PML gene on chromosome 15 to the retinoic acid receptor alpha (RAR alpha) gene on chromosome 17, creating two novel fusion genes, PML-RAR alpha and RAR alpha-PML. The PML-RAR alpha fusion gene product, which is expressed in virtually all patients with t(15;17), is thought to play a direct role in the pathogenesis of APML. To determine whether PML-RAR alpha is sufficient to cause APML in an animal model, we used the promyelocyte-specific targeting sequences of the human cathepsin G (hCG) gene to direct the expression of a PML-RAR alpha cDNA to the early myeloid cells of transgenic mice. Mice expressing the hCG-PML-RAR alpha transgene were found to have altered myeloid development that was characterized by increased percentages of immature and mature myeloid cells in the peripheral blood, bone marrow, and spleen. In addition, approximately 30% of transgene-expressing mice eventually developed acute myeloid leukemia after a long latent period. The splenic promyelocytes of mice with both the nonleukemic and leukemic phenotypes responded to all-trans retinoic acid (ATRA) treatment, which caused apoptosis of myeloid precursors. Although low-level expression of the hCG-PML-RAR alpha transgene is not sufficient to directly cause acute myeloid leukemia in mice, its expression alters myeloid development, resulting in an accumulation of myeloid precursors that may be susceptible to cooperative transforming events.

Developmental analysis of murine Promyelocyte Leukemia Zinc Finger (PLZF) gene expression: implications for the neuromeric model of the forebrain organization.

Promyelocyte Leukemia Zinc Finger (PLZF) is a Kruppel-like zinc finger gene previously identified in a unique case of acute promyelocytic leukemia (APL) as the counterpart of a reciprocal chromosomal translocation involving the retinoic acid receptor alpha gene (RAR alpha). PLZF is highly conserved throughout evolution from yeast to mammals. To elucidate its role, we isolated the murine PLZF gene and studied its expression during embryogenesis. PLZF is expressed in an extremely dynamic pattern with transcripts appearing at E 7.5 in the anterior neuroepithelium and quickly spreading to the entire neuroectoderm until E 10. At E 8.5, PLZF is transcribed in most of the endoderm. During mid to late gestation PLZF is expressed in restricted domains of the developing CNS as well as in specific organs and body structures. We have focused our attention on the developing forebrain where PLZF is transcribed in a transverse, segment-like domain corresponding to the anterior pretectum, in the alarmost part of the dorsal thalamus, in the epithalamus, and in the hypothalamus along a defined longitudinal subdomain. Furthermore, PLZF is expressed in several segmentary boundaries, among them, the zona limitans intrathalamica. Combined analysis with other regionally restricted genes, such as Orthopedia and Dlx1, indicates that in the hypothalamus the PLZF domain is contained within that of Orthopedia and both are complementary to that of Dlx1. Our data suggest a role for PLZF in the establishment and maintenance of transverse identities, longitudinal subdomains, and interneuromeric boundaries, providing additional evidences in favor of the neuromeric organization of the forebrain.

Inhibition of CD4/p56lck signaling by a dominant negative mutant of the Shc adaptor protein.

T-cell antigen receptor stimulation results in phosphorylation of the SH2 containing Shc proteins and recruitment of the Grb2/mSos complex suggesting that Shc proteins are involved in transducing T-cell activating signals to Ras. We have measured the effects of the isolated Shc-SH2 domain and the dominant negative RasN17 protein on activation of the T-cell specific transcription factor NF-AT. The isolated Shc-SH2 domain was designed to compete with endogenous Shc binding to upstream tyrosine phosphorylated proteins and to interfere with coupling to regulators of Ras activation. We have demonstrated that both the Shc-SH2 domain and the RasN17 protein significantly inhibited NF-AT activation by the CD4 coreceptor and the CD4 associated tyrosine kinase p56lck. In contrast, only the RasN17 protein reduced NF-AT activation by the TCR/CD3 complex. Furthermore, tyrosine kinase activity and p56lck protein were found in complexes immunoprecipitated with Shc specific antisera after CD4 triggering but not after CD3 triggering. These results indicate that both CD4 and CD3 signal to Ras and that this signaling is mediated by independent pathways of activation of the Shc adaptor protein.

Clinical relevance of the PML/RAR-a gene rearrangement in acute promyelocytic leukaemia.

The molecular mechanisms underlying the t(15;17) cytogenetic translocation of acute promyelocytic leukaemia (APL) have been recently elucidated. Together with new insights into the understanding of APL pathogenesis, such investigations also provided the availability of a novel leukemia-specific marker to be used for both diagnostic and monitoring studies. The chromosome breakpoints of the t(15;17) have been shown to involve the retinoic acid receptor alpha (RAR-a) gene and the newly described PML gene on chromosomes 17 and 15, respectively. Rearrangements of these loci are found in virtually 100% of APLs, including the microgranular variant subtype and cases displaying apparently normal karyotypes. In cases with ambiguous morphology, molecular diagnosis may therefore enable the prompt administration of APL-specific therapies, such as all-trans retinoic acid. Significantly, clinical response to this differentiating agent is predictable based on the presence of the specific PML/RAR-a rearrangement. Appropriate oligoprimers complementary to PML and RAR-a sequences nearby the DNA breakpoints may be successfully used in PCR experiments to amplify the PML/RAR-a hybrid gene and sensitively detect minimal residual disease. Preliminary PCR studies indicate that this approach might indeed prove a very important and reliable prognostic indicator in the follow up monitoring of APL patients. Early identification of impending relapse by PCR may suggest the use of additional treatment in patients at risk and significantly increase the probability of cure of the disease.