Key Points• Runx1 is necessary for survival and development of B cell-specified progenitors and also the transition through the pre-B-cell stage.• Genomewide expression and Runx1 occupancy analyses identified critical target genes and collaborating transcription partners.The t(12;21) chromosomal translocation, targeting the gene encoding the RUNX1 transcription factor, is observed in 25% of pediatric acute lymphoblastic leukemia (ALL) and is an initiating event in the disease. To elucidate the mechanism by which RUNX1 disruption initiates leukemogenesis, we investigated its normal role in murine B-cell development. This study revealed 2 critical functions of Runx1: (1) to promote survival and development of progenitors specified to the B-cell lineage, a function that can be substituted by ectopic Bcl2 expression, and (2) to enable the developmental transition through the pre-B stage triggered by the pre-B-cell antigen receptor (pre-BCR). Gene expression analysis and genomewide Runx1 occupancy studies support the hypothesis that Runx1 reinforces the transcription factor network governing early B-cell survival and development and specifically regulates genes encoding members of the Lyn kinase subfamily (key integrators of interleukin-7 and pre-BCR signaling) and the stage-specific transcription factors SpiB and Aiolos (critical downstream effectors of pre-BCR signaling). Interrogation of expression databases of 257 ALL samples demonstrated the specific down-regulation of the SPIB and IKZF3 genes (the latter encoding AIOLOS) in t(12;21) ALL, providing novel insight into the mechanism by which the translocation blocks B-cell development and promotes leukemia. (Blood. 2013; 122(3):413-423)

Key Points• Mef2c and Mef2d are activated by the pre-B-cell receptor and are essential for pre-B-cell transition.• Mef2c complexes with B-cell transcription factors to shut down the immediate early response and to initiate a new transcriptional network.The sequential activation of distinct developmental gene networks governs the ultimate identity of a cell, but the mechanisms involved in initiating downstream programs are incompletely understood. The pre-B-cell receptor (pre-BCR) is an important checkpoint of B-cell development and is essential for a pre-B cell to traverse into an immature B cell. Here, we show that activation of myocyte enhancer factor 2 (Mef2) transcription factors (TFs) by the pre-BCR is necessary for initiating the subsequent genetic network. We demonstrate that B-cell development is blocked at the pre-B-cell stage in mice deficient for Mef2c and Mef2d TFs and that pre-BCR signaling enhances the transcriptional activity of Mef2c/d through phosphorylation by the Erk5 mitogen-activating kinase. This activation is instrumental in inducing Krüppel-like factor 2 and several immediate early genes of the AP1 and Egr family. Finally, we show that Mef2 proteins cooperate with the products of their target genes (Irf4 and Egr2) to induce secondary waves of transcriptional regulation. Our findings uncover a novel role for Mef2c/d in coordinating the transcriptional network that promotes early B-cell development. (Blood. 2016;127(5):572-581)

• Runx1 is a key determinant of megakaryocyte cell-fate decisions in multipotent progenitors.• Runx1 downregulates celladhesion factors that promote residency of stem cells and megakaryocytes in their bone marrow niche.Disrupting mutations of the RUNX1 gene are found in 10% of patients with myelodysplasia (MDS) and 30% of patients with acute myeloid leukemia (AML). Previous studies have revealed an increase in hematopoietic stem cells (HSCs) and multipotent progenitor (MPP) cells in conditional Runx1-knockout (KO) mice, but the molecular mechanism is unresolved. We investigated the myeloid progenitor (MP) compartment in KO mice, arguing that disruptions at the HSC/MPP level may be amplified in downstream cells. We demonstrate that the MP compartment is increased by more than fivefold in Runx1 KO mice, with a prominent skewing toward megakaryocyte (Meg) progenitors. Runx1-deficient granulocyte-macrophage progenitors are characterized by increased cloning capacity, impaired development into mature cells, and HSC and Meg transcription signatures. An HSC/MPP subpopulation expressing Meg markers was also increased in Runx1-deficient mice. Rescue experiments coupled with transcriptome analysis and Runx1 DNA-binding assays demonstrated that granulocytic/monocytic (G/M) commitment is marked by Runx1 suppression of genes encoding adherence and motility proteins (Tek, Jam3, Plxnc1, Pcdh7, and Selp) that support HSC-Meg interactions with the BM niche. In vitro assays confirmed that enforced Tek expression in HSCs/MPPs increases Meg output. Interestingly, besides this key repressor function of Runx1 to control lineage decisions and cell numbers in progenitors, our study also revealed a critical activating function in erythroblast differentiation, in addition to its known importance in Meg and G/M maturation. Thus both repressor and activator functions of Runx1 at multiple hematopoietic stages and lineages likely contribute to the tumor suppressor activity in MDS and AML. (Blood. 2016;127(26):3369-3381)

Significance Immunodeficient mice are important tools to define stem cells that drive malignancies (cancers). Primary myelofibrosis (PMF) is a chronic myeloproliferative neoplasm that can progress to malignant leukemia. In a study to define PMF stem cells in transplanted mice, we observed a high incidence of mouse leukemia. We show that endogenous retrovirus (ERV), whose replication is unrestricted in immunodeficient mice, are pathogenic in the PMF-xenograft microenvironment, likely because of increased numbers of proliferating mouse cells stimulated by PMF-derived cells. Proliferating cells are targets of retroviral transformation and spontaneous mutations, and thus susceptible to leukemia induction. These results substantiate the importance of paracrine mechanisms in PMF disease and expose the presence of replicating ERVs in mice commonly used to model human diseases.

The expression of Moloney murine leukemia virus is restricted in embryonal carcinoma (EC) cells. To characterize specific mutations necessary for expression of retroviruses in EC cells, we analyzed the expression of retrovirus mutants and recombinants thereof in EC cell lines F9 and PCC4. DNA sequence comparison and functional studies allowed us to define three point mutations in the enhancer region of the viral mutants at positions -345, -326, and -166 and two point mutations within the 5'-untranslated region of the viral genome at positions +164 and +165 that were essential for retrovirus expression in EC cells. DNA fragments derived from either the wild type or mutant viruses were used to search for sequence-specific DNA-binding factors in nuclear extracts from undifferentiated PCC4 cells. A cellular factor was found to bind strongly to sequences within the enhancer region (-354 to -306) of wild-type viruses but only weakly to sequences derived from mutant viruses. This factor was named ECF-I (for EC cell factor I). Retroviral expression in EC cells correlates with decreased binding affinity for ECF-I.

The expression of Moloney murine leukemia virus (Mo-MuLV) and Mo-MuLV-derived vectors is restricted in undifferentiated mouse embryonal carcinoma and embryonal stem (ES) cells. We have previously described the isolation of retroviral mutants with host range properties expanded to embryonal cell lines. One of these mutants, the murine embryonic stem cell virus (MESV), is expressed in ES cell lines. Expression of MESV in these cells relies on DNA sequence motifs within the enhancer region of the viral long terminal repeat (LTR). Here we show that replacement of the Mo-MuLV enhancer region by sequences derived from the MESV LTR results in the activation of the Mo-MuLV LTR in ES cells. The enhancer regions of MESV and Mo-MuLV differ by seven point mutations. Of these, a single point mutation at position-166 is sufficient to activate the Mo-MuLV LTR and to confer enhancer-dependent expression to Mo-MuLV-derived retroviral vectors in ES cells. This point mutation creates a recognition site for a sequence-specific DNA-binding factor present in nuclear extracts of ES cells. This factor was found by functional assays to be the murine equivalent to human Spl.

Murine leukemia virus (MuLV) M813 was originally isolated from the Southeast Asian rodent Mus cervicolor. As with the ecotropic MuLVs derived from Mus musculus, its host range is limited to rodent cells. Earlier studies have mapped its receptor to chromosome 2, but it has not been established whether M813 shares a common receptor with any other MuLVs. In this study, we have performed interference assays with M813 and viruses from four interference groups of MuLV. The infection efficiency of M813 was not compromised in cells expressing any one of the other MuLVs, demonstrating that M813 must use a distinct receptor for cell entry. The entire M813 env coding region was molecularly cloned. Sequence analysis revealed high similarity with other MuLVs but with a unique receptor-binding domain. Substitution of M813 env sequences in Moloney MuLV resulted in a replication-competent virus with a host range and interference profile similar to those of the biological clone M813. M813 thus defines a novel receptor interference group of type C MuLVs.Viral subgroups of the murine C-type viruses have been defined on the basis of host range, generally corresponding to distinct interference groups defined by receptor usage (40). Five distinct interference groups have been established for the well-studied isolates of Mus musculus, and the cellular receptors for these viruses have all been identified (Table 1). Three families of receptors have been characterized for these viruses, all of which share multiple transmembrane-spanning topology and are implicated in transporter activity: (i) cationic amino acid transport for the cationic amino acid transporter (CAT) family (20, 48), (ii) inorganic phosphate transport for the Pit family (18, 37), and (iii) a presumed phosphate transport activity for the newly identified Sgy1 family (44). Whereas some viruses, such as 10A1 murine leukemia virus (10A1 MuLV), recognize several members of the same transporter family (e.g., Pit1 and Pit2) as well as homologous receptors of different species (e.g., mouse, rat, and human) (34), other viruses recognize only one or at most two members of a receptor family and are unable to recognize homologous receptors of other species (e.g., ecotropic MuLV recognizes only the rodent CAT1 and, with less affinity, the CAT3 receptor, but not the CAT2 receptor or CAT homologues of other species) (19, 30). Interestingly, viruses of different interference groups may use the same receptor but show different affinities to homologues in other species (e.g., the xenotropic MuLV does not recognize all murine homologues of its human cellular receptor, all of which, however, are recognized by polytropic MuLVs [5,28,44,50]). Determining the domains in the viral Env protein complex and the receptor molecule which dictate this specificity has been a subject of intense research over the last few years (11).In addition to being isolated from M. musculus, type C retroviruses have also been isolated from Mus caroli (25), Mus cervicolor (7), and, more recently, Mus dunni (9). With ...

The Asian wild mouse species Mus caroli harbors an endogenous retrovirus (McERV) that is closely related to but distinct from the endogenous retrovirus family defined by the Mus dunni endogenous virus and the Mus musculus endogenous retrovirus. McERV could infect some cell types from humans, dogs, and rats, but not all, and did not infect any mouse cell line tested. Because of its interesting host range and proposed ancestral relationship to primate retroviruses and because none of the entry receptors for this family of retroviruses had been identified, we began a search for the McERV receptor. We determined the chromosomal location of the receptor gene in the human genome by phenotypic screening of the G3 human-hamster radiation hybrid cell line panel and confirmed the localization by assaying for receptor activity conferred by bacterial artificial chromosome (BAC) clones spanning the region. We next localized the gene more precisely in one positive BAC by assaying for receptor activity following BAC digestion with several restriction enzymes that cleaved different sets of genes, and we confirmed that the final candidate gene, plasmolipin (PLLP; TM4SF11), is the novel receptor by showing that the expression of the human PLLP cDNA renders hamster and mouse cells susceptible to McERV infection. PLLP functions as a voltage-dependent potassium ion channel and is expressed primarily in kidney and brain, helping to explain the limited range of cell types that McERV can infect. Interestingly, mouse PLLP also functioned well as a receptor for McERV but was simply not expressed in the mouse cell types that we originally tested.An endogenous gammaretrovirus can be induced from cells of the Asian wild mouse Mus caroli by treatment of the cells with bromodeoxyuridine (22). This virus is distinct from the murine leukemia virus (MuLV) family of active retroviruses found in the laboratory mouse Mus musculus and appeared to be similar to exogenous retroviruses from gibbon apes (gibbon ape leukemia virus [GALV]) and woolly monkeys (simian sarcoma-associated virus [SSAV]). These similarities included a xenotropic host range (the ability to infect cells from species other than mice but not cells from mice) and reported cross interference between the viruses, indicative of a common cell entry receptor. It was therefore hypothesized that mice were the source of the exogenous simian viruses. Recently, a retrovirus induced by bromodeoxyuridine treatment of Mus caroli (Mus caroli endogenous retrovirus [McERV]) that has properties similar to those of the original virus studied over 30 years ago (22) was cloned and sequenced (C. Stocking, M. Ziegler, U. Bergholz, K. Weber, M. Eiden, and V. Prassolov, unpublished results). However, further analysis indicated that McERV does not use the cell entry receptor used by GALV and SSAV (Pit1; Slc20a1), weakening the argument that McERV is the progenitor of GALV and/or SSAV. McERV also did not use the receptors for xenotropic MuLV (Xpr1) (Mus musculus) or those for the endogenous feline leukemia v...