Heterologous expression of NodZ and NolL proteins in Rhizobium leguminosarum bv. viciae led to the production of acetyl fucosylated lipo-chitin oligosaccharides (LCOs), indicating that the NolL ...protein obtained from Mesorhizo-bium loti functions as an acetyl transferase. We show that the NolL-dependent acetylation is specific for the fucosyl penta-N-acetylglucosamine species. In addition, the NolL protein caused elevated production of LCOs. Efficient nodulation of Lotus japonicus by the NodZ/NolL-producing strain was demonstrated. Nodulation efficiency was further improved by the addition of the ethylene inhibitor L-α-(2-aminoethoxyvinyl) glycine (AVG).
Abstract
Transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) controls cell proliferation and myeloid differentiation. In ∼10% of patients with acute myeloid leukemia (AML) C/EBPA is ...either mutated or epigenetically silenced. C/EBPA mutated leukemias differ from C/EBPA silenced leukemias in prognosis and phenotype, yet both leukemias cluster together based on genome wide gene expression signatures, indicating a unifying mechanism of disease. So far, the key downstream events required for C/EBPA loss to trigger leukemogenesis are still unclear.
Based on microarray gene expression analysis, we used a shRNA screening platform to search for mediators of leukemic outgrow of C/EBPα-deficient stem/progenitor cells (C/EBPα-/- HSC/HPCs, lineage-c-kit+ScaI+). In our screen, oncogene Sox4 was identified as a gene that was up-regulated in C/EBPα-/- HSC/HPCs and whose down-regulation abrogated aberrant self-renewal ability and restored myeloid differentiation of C/EBPα-/- HSC/HPCs, as demonstrated by in vitro serial-replating and differentiation assays.
Chromatin immunoprecipitation confirmed the endogenous binding of C/EBPα at the proximal promoter of Sox4 in the HSC/HPCs enriched population (lineage-c-kit+) and the mature myeloid population (Mac1+Gr1+). In vitro promoter reporter assay demonstrated that wild-type human C/EBPA, but none of the C/EBPA mutants identified from AML patients, repressed Sox4 transcription through its binding to a highly conserved C/EBPα binding site.
C/EBPα and Sox4 showed reciprocal expression patterns in both HSCs and various hematopoietic compartments during myeloid maturation of wild type mice. Furthermore, expression of Sox4 was up-regulated in HSCs of C/EBPα-deficient mice as well as in leukemia-initiating cells (LICs) of a murine C/EBPα mutant AML model. To further genetically dissect the role of Sox4 in driving leukemia in the absence of functional CEBPα, we generated Sox4, C/EBPα double deficient mice and observed that loss of Sox4 alleviated the abnormal HSC/HPCs expansion and defective myeloid programming caused by C/EBPα deficiency. In addition, comparisons of the murine C/EBPα mutant AML model with a Sox4-induced AML model revealed that LICs of both leukemia models were enriched in immunophenotypically similar populations and exhibited comparable gene expression signatures. Importantly, down-regulation of Sox4 by shRNA in LICs from murine C/EBPα mutant AML was sufficient to abolish their augmented serial-replating ability. Intriguingly, enhanced expression of SOX4 in AML patients with either mutated or epigenetically silenced C/EBPA compared to other AML subtypes confirmed the findings in our mouse model systems.
Our data demonstrate that failure to suppress Sox4 expression is the underlying mechanism of leukemias with mutated or silenced C/EBPα. These data also uncover a promising rationale for a therapeutic target in these leukemias.
Citation Format: Hong Zhang, Min Ye, Meritxell Alberich-Jordà, Giovanni Amabile, Henry Yang, Rob Welner, Philipp Staber, Véronique Lefebvre, Peter J.M. Valk, Ruud Delwel, Constanze Bonifer, Daniel G. Tenen. Identification of Sox4 as a key oncogene in leukemias with mutated or silenced C/EBPα. abstract. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2317. doi:10.1158/1538-7445.AM2013-2317
In blood, transcription factor C/EBPa is essential for myeloid differentiation and has been implicated in regulating self-renewal of fetal liver (FL) hematopoietic stem cells (HSCs). However, its ...function in adult HSCs has remained unknown. Here, using an inducible knockout model we found that C/EBPa deficient adult HSCs underwent a pronounced expansion with enhanced proliferation, characteristics resembling FL HSCs. Consistently, transcription profiling of C/EBPa deficient HSCs revealed a gene expression programme similar to FL HSCs. Moreover we observed that age-specific C/EBPa expression correlated with its inhibitory effect on HSC cell cycle. Mechanistically we identified N-Myc as C/EBPa downstream target, and loss of C/EBPa resulted in de-repression of N-Myc. Our data establish C/EBPa as a central determinant in the switch from fetal to adult HSCs.
: By means of retroviral insertional mutagenesis, we identified a novel common virus integration site (cVIS) (Evi11) in murine leukemias, and demonstrated that Cb2, encoding the peripheral ...cannabinoid receptor, is the potential proto‐oncogene located in that region. Cb2 is a 7‐transmembrane G protein‐coupled receptor (GPCR), which is normally expressed on B lymphocytes. Using transwell assays we observed strong migration of Cb2‐expressing cells upon stimulation with 2‐arachidonoylglycerol (2‐AG), a potent endocannabinoid. Overexpression of Cb2 receptor on murine myeloid precursor cells causes a block of neutrophilic differentiation, a major characteristic of myeloid leukemia. Intriguingly, we could not detect functional Cb2 receptors on normal murine bone marrow precursor cells. Furthermore, analysis of human acute myeloid leukemia (AML) samples revealed the presence of CB2 mRNA transcripts in several cases. Furthermore, migration could be induced by 2‐AG when analyzed in one of the patient samples. Our data suggest that the initially identified cVIS, Evi11, encodes for a murine onco‐protein and that human CB2 may be involved in certain cases of human AML as well.
The gene encoding the peripheral cannabinoid receptor
Cb2 is located in the common virus integration site
Evi11 and is associated with hematopoietic malignancies in mice. To determine the effect of ...Cb2 overexpression on hematopoietic development in vivo,
Cb2 transgenic mice were generated.
A
Cb2 expression vector was constructed containing a
Cb2 cDNA fragment cloned into the 14kb
Sca-1 (Ly-6E.1) gene. Two transgenic lines in which Cb2 expression is controlled by the
Sca-1 promoter were generated, and the effect on hematopoietic development was studied. Expression of
Cb2 mRNA or protein was studied by RNase protection analysis and ligand binding assays, respectively. Leukemic predisposition was investigated by injecting newborn transgenic as well as control animals with Cas-Br-M murine leukemia virus (Cas-Br-M MuLV).
Although increased expression of the
Cb2 gene was observed in hematopoietic tissues, follow-up of more than 1 year did not reveal any hematologic defect. Interestingly, infection of newborn
pSca-1/Cb2 transgenic mice with Cas-Br-M MuLV revealed that significantly more transgenic mice developed leukemia than virus-treated control littermates. Because these studies provide evidence for the cooperative potential of Cb2 in leukemia progression, we wished to identify genes that may collaborate with
Cb2 in leukemic transformation. Our study suggests that
Evi1, another common target for proviral integration in mouse leukemias, may be overexpressed in virus-induced leukemias in
pSca-1/Cb2 transgenic mice.
The data indicate that hematopoietic precursor cells that express high levels of Cb2 possess increased susceptibility for leukemia development and that
Cb2 and
Evi1 might collaborate in leukemogenesis.
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