Capture of cytokine-responsive genes (NACA and RBM3) using a gene trap approach

Abstract

We have developed a gene trap approach to select specific cytokine receptor/ligand responsive genes in the cell line TF-1. This cell line exhibits a dependency on granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) and responds to interleukin-5 (IL-5). In an attempt to detect genes modulated by one of these factors, cells were infected with the Rosaβgeo retrovirus in the presence of GM-CSF, IL-3, or IL-5 and clones were selected for retroviral integration on the basis of G418 resistance. Housekeeping and cytokine-regulated trapped genes were then differentiated on the basis of G418 resistance versus sensitivity in the presence of the different cytokines. To determine the reliability of this screen, DNA sequences upstream of the proviral integration site were identified by 5′ rapid amplification of DNA ends polymerase chain reaction (RACE PCR) from selected GM-CSF–treated and –infected clones. Comparison of the sequences with those in the Genbank database revealed that 2 sequences correspond to known genes: NACA and RBM3. NACAwas recently defined as a coactivator of c-jun–mediated transcription factors in osteoblasts, and RBM3 as a protein from the heterogeneous nuclear ribonucleoprotein family. Data from transcriptional analysis of these 2 genes in TF-1 cells showed a specific up-regulation by GM-CSF. Both transcripts were also found to be up-regulated in purified CD34+ cells, suggesting their involvement in proliferative processes during hematopoiesis. Interestingly, down-regulation was observed during monocytic differentiation of TF-1 cells, suggesting their extinction could contribute to monocytic lineage development. This study demonstrates that this gene trap approach is a useful method for identifying novel, specific cytokine-responsive genes that are involved in the regulation of hematopoiesis.

Cardiac transcriptional response to acute and chronic angiotensin II treatments

“…For instance, RNA binding motif protein 3 (RBM3) was ϩ2.4-fold upregulated in response to acute ANG II treatment; RBM3 is a cold-stress-induced RNA binding protein that affects posttranscriptional regulation of gene expression, possibly facilitating translation by binding 18S ribosomal RNA (17). RBM3 is upregulated in response to cytokines and may be associated with tissue growth and differentiation as it is also upregulated in proliferative processes during hematopoesis (6). Also strongly upregulated acutely (ϩ1.7-fold) was angiomotin, a cell-surface protein that transduces angiostatin-induced inhibition of cell motility and induction of apoptosis (65).…”

“…As mentioned above, Rho GTPase is an important second messenger system in transducing ANG II effects on heart and other tissues. Integrins play a crucial role in cardiac hypertrophy, linking the ECM to the cytoskeleton and transducing extracellular mechanical stress and chemical signals (6). The significant cellular component GO terms were primarily cytosolic terms for ribosome and microtubules, although there were also significant terms for lysosome and cell-substrate adhesion junctions.…”

RBM3 upregulates ARPC2 by binding the 3'UTR and contributes to breast cancer progression

“…RBM3 contributes to the response of cellular stressors, such as degenerative and hypoxic conditions (7,14). Moreover, RBM3 has been documented to promote cell proliferation and erythropoietic differentiation (15,16). Recent studies have demonstrated that RBM3 plays a promoting role in human colorectal cancer (17,18) and prostate cancer (15).…”

The interferon-inducible gene, Ifi204, is transcriptionally activated in response to M-CSF, and its expression favors macrophage differentiation in myeloid progenitor cells

“…The retroviral vector ROSA␤EGFP, a gift of Dr. S. Gomez (INSERM U119, Marseille, France), was derived from the ROSA␤geo vector [11,25], and its construction will be described elsewhere (Sophie Gomez and Patrice Dubreuil, unpublished data). Briefly, this vector has a deletion of viral enhancers and a splice acceptor (SA) sequence in front of a promoterless EGFP gene, allowing the production of a chimeric transcript containing at least the ATG of EGFP, from which the EGFP protein expression can be obtained and monitored by fluorescence analysis.…”

“…In an attempt to isolate M-CSF-responsive genes, we used an in vitro gene trap strategy [11], taking advantage of the retroviral vector ROSA␤EGFP, which places the enhanced green fluorescent protein (EGFP) reporter gene under the control of the regulatory elements of genes disrupted by retroviral insertion. As a model, we used FDC-P1 cells expressing the murine M-CSF-R (FD-Fms cells).…”