Abstract
We report here the cloning and initial characterization of a novel growth-related gene (EEG-1) that is located on the short arm of chromosome 12. Two spliced transcripts were cloned from human bone marrow and human erythroid progenitor cells: EEG-1L containing a 4350-nucleotide open reading frame encoding a putative protein of 1077 amino acids including a C1q-like globular domain, and an alternatively spliced transcript lacking exon 5 (EEG-1S) encodes a significantly smaller coding region and no C1q-like domain. Quantitative PCR revealed expression of both EEG-1 transcripts in all analyzed tissues. Plasmids encoding green fluorescent protein-tagged genes (GFP-EEG-1) were transfected into Chinese hamster ovary cells for localization and functional assays. In contrast to the diffuse cellular localization of the GFP control, GFP-EEG-1L was detected throughout the cytoplasm and excluded from the nucleus, and GFP-EEG-1S co-localized with aggregated mitochondria. Transfection of both isoforms was associated with significantly increased levels of apoptosis. Stable transfection assays additionally demonstrated decreased growth in those cells expressing EEG-1 at higher levels. Quantitative PCR analyses of mRNA obtained from differentiating erythroid cells from blood donors were performed to determine the transcriptional pattern of EEG-1 during erythropoiesis. EEG-1 expression was highly regulated with increased expression at the stage of differentiation associated with the onset of global nuclear condensation and reduced cell proliferation. We propose that the regulated expression of EEG-1 is involved in the orchestrated regulation of growth that occurs as erythroblasts shift from a highly proliferative state toward their terminal phase of differentiation.
Highlights
Erythropoiesis is primarily regulated by the hormone erythropoietin released from the kidneys in response to hypoxia
Full-length cDNA was amplified by reverse transcription (RT)1-PCR with cDNA prepared from human erythroid progenitor cells, using the forward primer derived from 5Ј-end cDNA sequence and reverse primer derived from 3Ј-end sequence
Identification and Cloning of Human EEG-1 cDNA—As part of a systematic effort to identify and characterize novel genes expressed during erythropoiesis, we identified two erythroid expressed sequence tags (EST)
Summary
Erythropoiesis is primarily regulated by the hormone erythropoietin released from the kidneys in response to hypoxia. Stem cell factor may be involved in a proliferation response to acute erythroid stress (4). Erythroid proliferation and differentiation are controlled globally by expression of erythroid-specific genes as well as more generic factors. In addition to the expression of several genes already known to be involved in the control of erythroid proliferation, differentiation, and apoptosis, we hypothesized that novel growthregulating genes may be identified by gene profiling of developing erythroid cells. We described previously (9) a large number of growth-related genes that are up-regulated in response to the hormone erythropoietin. Those gene profiles include numerous genes known to be generally involved in tissue differentiation and apoptosis. We report the identification, cloning, and initial characterization of a novel growth-regulating gene called EEG-1 expressed during terminal erythroid maturation
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