miRNA let-7c promotes granulocytic differentiation in acute myeloid leukemia.

MicroRNAs (miRNAs), small non-coding RNAs that regulate gene expression post-transcriptionally, are involved in many complex cellular processes. Several miRNAs are differentially expressed in hematopoietic tissues and play important roles in normal differentiation, but, when aberrantly regulated, contribute to the abnormal proliferation and differentiation of leukemic cells. Recently, we reported that a small subset of miRNAs is differentially expressed in acute promyelocytic leukemia (APL) blasts and is modulated by treatment with all-trans-retinoic acid (ATRA). In particular, PML/RARα-positive blasts from APL patients display lower levels of miRNA let-7c, a member of the let-7 family, than normal promyelocytes and its expression increases after ATRA treatment. In this study, we investigated the effects of let-7c in acute myeloid leukemia (AML) cells. We found that ectopic expression of let-7c promotes granulocytic differentiation of AML cell lines and primary blasts. Moreover, we identified PBX2, a well-known homeodomain protein whose aberrant expression enhances HoxA9-dependent leukemogenesis, as a novel let-7c target that may contribute to the AML phenotype. Together, these studies raise the possibility that perturbation of the let-7c-PBX2 pathway may have a therapeutic value in AML.

Overexpression of Ets-1 in human hematopoietic progenitor cells blocks erythroid and promotes megakaryocytic differentiation.

Ets-1 is a widely expressed transcription factor implicated in development, tumorigenesis and hematopoiesis. We analyzed Ets-1 gene expression during human erythroid and megakaryocytic (MK) differentiation in unilineage cultures of CD34+ progenitor cells. During erythroid maturation, Ets-1 is downmodulated and exported from the nucleus into the cytoplasm through an active mechanism mediated by a leucine-rich nuclear export signal. In contrast, during megakaryocytopoiesis Ets-1 increases and remains localized in the nucleus up to terminal maturation. Overexpression of Ets-1 in erythroid cells blocks maturation at the polychromatophilic stage, increases GATA-2 and decreases both GATA-1 and erythropoietin receptor expression. Conversely, Ets-1 overexpressing megakaryocytes are characterized by enhanced differentiation and maturation, coupled with upmodulation of GATA-2 and megakaryocyte-specific genes. We show that Ets-1 binds to and activates the GATA-2 promoter, in vitro and in vivo, indicating that one of the pathways through which Ets-1 blocks erythroid and promotes MK differentiation is via upmodulation of GATA-2 expression.

A pentamer transcriptional complex including tal-1 and retinoblastoma protein downmodulates c-kit expression in normal erythroblasts.

Human proerythroblasts and early erythroblasts, generated in vitro by normal adult progenitors, contain a pentamer protein complex comprising the tal-1 transcription factor heterodimerized with the ubiquitous E2A protein and linked to Lmo2, Ldb1, and retinoblastoma protein (pRb). The pentamer can assemble on a consensus tal-1 binding site. In the pRb(-) SAOS-2 cell line transiently transfected with a reporter plasmid containing six tal-1 binding site, pRb enhances the transcriptional activity of tal-1-E12-Lmo2 and tal-1-E12-Lmo2-Ldb1 complexes but not that of a tal-1-E12 heterodimer. We explored the functional significance of the pentamer in erythropoiesis, specifically, its transcriptional effect on the c-kit receptor, a tal-1 target gene stimulating early hematopoietic proliferation downmodulated in erythroblasts. In TF1 cells, the pentamer decreased the activity of the reporter plasmid containing the c-kit proximal promoter with two inverted E box-2 type motifs. In SAOS-2 cells the pentamer negatively regulates (i) the activity of the reporter plasmid containing the proximal human c-kit promoter and (ii) endogenous c-kit expression. In both cases pRb significantly potentiates the inhibitory effect of the tal-1-E12-Lmo2-Ldb1 tetramer. These data indicate that this pentameric complex assembled in maturing erythroblasts plays an important regulatory role in c-kit downmodulation; hypothetically, the complex may regulate the expression of other critical erythroid genes.

T-cell-directed TAL-1 expression induces T-cell malignancies in transgenic mice.

The TAL-1 gene specifies for a basic domain-helix-loop-helix protein, which is involved in the control of normal hematopoiesis. In human pathology, the TAL-1 gene product is expressed in a high percentage of T-cell acute lymphoblastic leukemias in the pediatric age range; however, it has not been established whether the expression has a causal role in oncogenesis. In this report, we describe the phenotype of mouse transgenic lines obtained by inducing tal-1 protein expression in lymphoid tissues using the LCK promoter. The survival rate of tal-1 transgenic animals was much lower as compared with control mice. Histopathological analysis revealed lymphomas of T-cell type, often comprising a minor B-cell component. Some mice showed marked splenic lymphocyte depletion. Primary lymphocyte cultures showed partial independence from exogenous growth stimuli and increased resistance to low-serum apoptosis. To further unravel the tal-1 oncogenic potential, a strain of tal-1 transgenic mice was crossbred with p53-/- mice; the survival rate in these animals was reduced by more than one-half when compared with that of tal-1 mice, and histopathological analysis revealed exclusively T-cell lymphomas. These data indicate that TAL-1, expressed in T cells, is per se a potent oncogene, which may exert a key leukemogenetic role in the majority of T-cell acute lymphoblastic leukemias.

Coordinate expression and developmental role of Id2 protein and TAL1/E2A heterodimer in erythroid progenitor differentiation.

The Id proteins and basic helix-loop-helix (bHLH) proteins play major roles in specifying cell fate decisions in diverse biologic settings. A potential role for Id and TAL1/E2A bHLH genes in hematopoiesis has been suggested by studies on immortalized cell lines. However, it is uncertain whether these observations reflect normal hematopoiesis. We have investigated the expression pattern of Id2 and TAL1/E2A genes in liquid suspension culture of purified hematopoietic progenitor cell (HPCs) undergoing erythroid or granulopoietic differentiation in the first culture week and maturation to terminal cells in the second week. In quiescent, freshly purified HPCs, Id2 mRNA is detected by reverse transcriptase-polymerase chain reaction (RT-PCR), whereas TAL1 and E2A mRNAs are not. At the onset of erythroid differentiation, Id2 mRNA is downregulated, while E2A and TAL1 mRNAs are concomitantly upregulated: their expression is further increased at erythroblast level. Conversely, Id2 is not downmodulated in granulopoietic culture, except for a late decline at day 10 to 12, while TAL1 and E2A are only transiently induced in the first week of granulopoietic differentiation. The expression pattern of the TAL1/E2A heterodimer, as evaluated by mobility shift assay, is consistent with RT-PCR results (except for lower levels of the heterodimer in late erythroid maturation). TAL1 protein level, analyzed by Western blot, shows a pattern consistent with gelshift results. Functional experiments were performed on purified HPCs treated with phosphorothioate antisense oligodeoxynucleotides to Id2 or TAL1 mRNA. The results are strictly consistent with the expression studies: anti-Id2 oligomer (alpha-Id2) causes a significant dose-dependent increase of erythroid colony formation, whereas alpha-TAL1 induces a selective dose-related inhibitory effect on erythroid colonies, as compared with untreated or scrambled oligomer-treated control HPCs. Finally, murine and human glutathione-S-transferase (GST)-Id2 polypeptides compete the TAL1/E2A-specific DNA binding activity when added to the nuclear extracts derived from erythroid culture cells, thus indicating biochemical and suggesting functional interaction of Id2 with the TAL1/E2A complex. These novel observations indicate a coordinate expression and function of an inhibitory Id protein (Id2) and a stimulatory bHLH/bHLH heterodimer (TAL1/E2A) in normal erythroid differentiation.

The mechanism of binding of exogenous DNA to sperm cells: factors controlling the DNA uptake.

Mature sperm cells have the spontaneous capability of taking up exogenous DNA. Potential substrates for the interaction of the DNA with the sperm heads are specific classes of DNA-binding proteins. In the present work three major classes of DNA-binding proteins were identified by Southwestern analysis of sperm head protein extracts: a first class of about 50 kDa in molecular weight, a second one of 30-35 kDa, and finally a third one below 20 kDa. The latter group most probably contains sperm protamines. Our attention was particularly focused on the 30- to 35-kDa proteins as a substrate for DNA binding, as they represented the only group whose electrophoretic mobility was conserved among mammalian species. In addition they were the only class of DNA-binding proteins accessible to exogenous DNA in intact sperm cells. The purified 30- to 35-kDa proteins interacted in vitro with exogenous DNA and generated discrete protein/DNA complexes as determined by band shift assay. A factor blocking the binding of exogenous DNA to sperm cells was also identified in the seminal fluid of mammals and in echinoid spermatoza. The factor also exerted a powerful inhibitory effect on DNA uptake in sperm cells of heterologous species. The 30- to 35-kDa DNA-binding proteins appeared to be the specific target through which the inhibition was mediated. In the presence of the inhibitory factor, the 30- to 35-kDa lost the ability to bind exogenous DNA. Thus, the interaction of exogenous DNA with sperm cells does not appear to be a casual event but, on the contrary, relies on a molecular mechanism based on the cooperation of specific protein factors.