The transcription factor PU.1 is a hematopoietic-specific member of the ets family. Mice carrying a mutation in the PU.1 locus were generated by gene targeting. Homozygous mutant embryos died at a late gestational stage. Mutant embryos produced normal numbers of megakaryocytes and erythroid progenitors, but some showed an impairment of erythroblast maturation. An invariant consequence of the mutation was a multilineage defect in the generation of progenitors for B and T lymphocytes, monocytes, and granulocytes. Thus, the developmental programs of lymphoid and myeloid lineages require a common genetic function likely acting at the level of a multipotential progenitor.

https://science.sciencemag.org/content/sci/265/5178/1573.full.pdf

Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages

Compilation of vertebrate-encoded transcription factors.

Interest in the Ets proteins has grown enormously over the last decade. The v-ets oncogene was originally discovered as part of a fusion protein expressed by a transforming retrovirus (avian E26), and later shown to be transduced from a cellular gene. About 30 related proteins have now been found in species ranging from flies to humans, that resemble the vEts protein in the so-called 'ets domain'. The ets domain has been shown to be a DNA-binding domain, that specifically interacts with sequences containing the common core trinucleotide GGA. Furthermore, it is involved in protein-protein interactions with co-factors that help determine its biological activity. Many of the Ets-related proteins have been shown to be transcription activators, like other nuclear oncoproteins and anti-oncoproteins (Jun, Fos, Myb, Myc, Rel, p53, etc.). However, Ets-like proteins may have other functions, such as in DNA replication and a general role in transcription activation. Ets proteins have been implicated in regulation of gene expression during a variety of biological processes, including growth control, transformation, T-cell activation, and developmental programs in many organisms. Signals regulating cell growth are transmitted from outside the cell to the nucleus by growth factors and their receptors. G-proteins, kinases and transcription factors. We will discuss how several Ets-related proteins fit into this scheme, and how their activity is regulated both post- and pre-translationally. Loss of normal control is often associated with conversion to an oncoprotein. vEts has been shown to have different properties from its progenitor, which might explain how it has become oncogenic. Oncogene-related products have been implicated in the control of various developmental processes. Evidence is accumulating for a role for Ets family members in Drosophila development, Xenopus oocyte maturation, lymphocyte differentiation, and viral infectious cycles. An ultimate hope in studying transformation by oncoproteins is to understand how cells become cancerous in humans, which would lead to more effective treatments. vEts induces erythroblastosis in chicken. Cellular Ets-family proteins can be activated by proviral insertion in mice and, most interestingly, by chromosome translocation in humans. We are at the beginning of understanding the multiple facets of regulation of Ets activity. Future work on the Ets family promises to provide important insights into both normal control of growth and differentiation, and deregulation in illness.

https://febs.onlinelibrary.wiley.com/doi/epdf/10.1111/j.1432-1033.1993.tb19864.x

The Ets family of transcription factors

Transcription factors, normal myeloid development, and leukemia.

Molecular biology of the Ets family of transcription factors.

Previous studies identified a common site (Sfpi-1) for proviral integration in immortalized Friend erythroleukemias. cDNAs corresponding to a 1.5-kb Sfpi-1 mRNA were isolated and sequenced. These were larger than an independently isolated Sfpi-1 cDNA described by researchers from another laboratory, and they contained common differences from that sequence, including in the coding region four extra nucleotides that altered the reading frame. The properly translated protein is identical to Pu.1, a transcription activation factor that is related to the ets oncogene family. Genetic methods were used to map Sfpi-1 with respect to other loci on mouse chromosome 2. Our results suggest that Pu.1 blocks erythroblast differentiation and thereby causes immortalization.

The Sfpi-1 proviral integration site of Friend erythroleukemia encodes the ets-related transcription factor Pu.1.

In vivo studies of Friend virus erythroleukemia have implied that proviral integrations adjacent to the gene for the Ets-related transcription factor PU.1 may inhibit the commitment of erythroblasts to differentiate and cause their capability for indefinite transplantation (C. Spiro, B. Gliniak, and D. Kabat, J. Virol. 62:4129-4135, 1988; R. Paul, S. Schuetze, S. L. Kozak, C. Kozak, and D. Kabat, J. Virol. 65:464-467, 1991). To test this hypothesis, we ligated PU.1 cDNA into a retroviral vector and studied its effects on cultured cells. Infection of fibroblasts with PU.1-encoding retrovirus resulted in PU.1 synthesis followed by nuclear pyknosis, cell rounding, and degeneration. In contrast, in long-term bone marrow cultures, erythroblasts were efficiently and rapidly immortalized. The resulting cell lines were polyclonal populations that contained PU.1, were morphologically blast-like, required erythropoietin and bone marrow stromal cells for survival and proliferation, and spontaneously differentiated at low frequency to synthesize hemoglobin. After 9 months in culture, erythroblasts became stroma independent, and they then grew as clonal cell lines. We conclude that PU.1 perturbs the pathway(s) that controls potential for indefinite proliferation and that it can be used to generate permanent erythroblast cell lines.

The Ets-related transcription factor PU.1 immortalizes erythroblasts.

Both viral and cellular genes have been directly implicated in pathogenesis of Friend viral erythroleukemia. The virus-encoded gp55 glycoprotein binds to erythropoietin receptors to cause mitogenesis and differentiation of erythroblasts. However, if the provirus integrates adjacent to the gene for the PU.1 transcription factor, the cell loses its commitment to terminally differentiate and becomes immortal, as indicated by its transplantability and by its potential for indefinite growth in culture (C. Spiro, B. Gliniak, and D. Kabat, J. Virol. 63:4434-4437, 1989; R. Paul, S. Schuetze, S. L. Kozak, and D. Kabat, J. Virol. 65:464-467, 1991). To test the implications of these results, we produced polyclonal antiserum to bacterially synthesized PU.1, and we used it to analyze PU.1 expression throughout leukemic progression and during chemically induced differentiation of Friend erythroleukemia (F-MEL) cell lines. This antiserum identified three electrophoretically distinct PU.1 components in extracts of F-MEL cells and demonstrated their nuclear localization. Although PU.1 proteins are abundant in F-MEL cells, they are absent or present in only trace amounts in normal erythroblasts or in differentiating erythroblasts from the preleukemic stage of Friend disease. Furthermore, chemicals (dimethyl sulfoxide or N,N'-hexamethylenebisacetamide) that overcome the blocked differentiation of F-MEL cells induce rapid declines of PU.1 mRNA and PU.1 proteins. The elimination of PU.1 proteins coincides with recommitment to the program of erythroid differentiation and with loss of immortality. These results support the hypothesis that PU.1 interferes with the commitment of erythroblasts to differentiate and that chemicals that reduce PU.1 expression reinstate the erythropoietic program.

Role of the PU.1 transcription factor in controlling differentiation of Friend erythroleukemia cells.

By using a tagged derivative of Friend spleen focus-forming virus, we previously obtained evidence that proviral integration(s) in the host genome can cause erythroblast immortality by abrogating the commitment of the cell to differentiate (C. Spiro, B. Gliniak, and D. Kabat, J. Virol. 62:4129-4135, 1988). Exploiting the fact that each leukemia was a single clone that contained one tagged provirus, we have now molecularly cloned and characterized one common genomic site for immortalizing proviral integrations.

Scott Schuetze

Papers

The Sfpi-1 proviral integration site of Friend erythroleukemia encodes the ets-related transcription factor Pu.1.

The Ets-related transcription factor PU.1 immortalizes erythroblasts.

Role of the PU.1 transcription factor in controlling differentiation of Friend erythroleukemia cells.

A common site for immortalizing proviral integrations in Friend erythroleukemia: molecular cloning and characterization.

A CommonSite forImmortalizing Proviral Integrations inFriend Erythroleukemia: Molecular Cloning andCharacterization