Novel Interaction between the Transcription Factor CHOP (GADD153) and the Ribosomal Protein FTE/S3a Modulates Erythropoiesis

Abstract

The transcription factor CHOP (GADD153) heterodimerizes with other C/EBP family members, especially C/EBP␤, thus preventing their homodimerization and binding to DNA sequences specific for the homodimers. Some CHOP-C/EBP heterodimers apparently bind to alternative DNA sequence and thereby regulate the transcription of other genes. Recently, we demonstrated that CHOP is up-regulated during certain stages of erythroid differentiation and that ectopic overexpression of CHOP enhances this process (Coutts, M., Cui, K., Davis, K. L., Keutzer, J. C., and Sytkowski, A. J. (1999) Blood 93, 3369 -3378). In the present study, we report that CHOP also interacts with another non-C/EBP protein designated v-fos transformation effector (FTE) (Kho, C. J., and Zarbl, H. The growth of erythroid progenitor cells in the bone marrow and their differentiation into enucleate, hemoglobinized erythrocytes is regulated primarily by the glycoprotein hormone erythropoietin (Epo) 1 (1). This growth factor interacts with its cognate receptor on the surface of the erythroid cell and triggers a signal transduction cascade that results in cell proliferation (anti-apoptosis) and differentiation (2-13).A role in erythroid growth and development has been shown or suggested for several regulatory proteins including Myc, Myb, GATA-1, and NF-E2 (4, 7, 8, 14 -19). Recently, we found that Epo up-regulates the expression of CHOP (gadd153) (20 -24), a member of the C/EBP family of transcription factors (25). Gain-of-function studies indicated that increasing CHOP expression enhances hemoglobinization of erythroid cells, indicating a functional role for CHOP in erythroid differentiation. Additionally, we obtained evidence that CHOP protein can bind to several nuclear proteins from erythroid cells, potentially some that are not C/EBP family members.We have now used the yeast two-hybrid system (26) to screen an erythroid cell cDNA library for proteins that interact with CHOP. We report that CHOP interacts with a non-C/EBP protein designated v-fos transformation effector (FTE) (27) which is identical to ribosomal protein S3a (28). Our results indicate that this interaction inhibits the ability of CHOP to enhance erythroid differentiation. EXPERIMENTAL PROCEDURESRauscher Murine Erythroleukemia Cell cDNA Library Construction and Yeast Two-hybrid Screen-A library from Rauscher murine erythroleukemia cells (29, 30) was constructed into the pAD-Gal4 vector. Briefly, 5 g of poly(A) ϩ RNA was converted to double-stranded DNA using Stratascript RNase H Ϫ reverse transcriptase (Stratagene). First strand synthesis was primed with an oligonucleotide containing poly(dT) and an XhoI site (31). Second strand synthesis was carried out using Escherichia coli DNA polymerase I and RNase H (32). The cDNA ends were filled with T4 DNA polymerase, and the internal EcoRI sites of the cDNA were methylated with EcoRI methylase. Following EcoRI linker addition and size fractionation in a 1% agarose gel, doublestranded cDNA over 800 base pairs was cleaved with EcoRI and XhoI. The fragm...

Liposarcoma initiated by FUS/TLS-CHOP: the FUS/TLS domain plays a critical role in the pathogenesis of liposarcoma

“…These capacities contrast with the ability of CHOP to induce growth arrest (Barone et al, 1994) and enhance di erentiation of erythroid cells (Cui et al, 2000). Thus, our results demonstrate that some of the in vitro properties of CHOP do not correlate in vivo with the normal development of CHOP mice and indicate that the properties conferred The human CHOP and CHOP-FUS cDNAs containing a tag-sequence for the human c-Myc (amino acids 408 ± 439) at the 3' end were cloned into the pEF-BOS vector, which contains the EF-1 promoter sequences and polyadenylation and splice signals from the human G-CSF gene, to give pEF-CHOP and pEF-CHOP-FUS.…”

Crucial Role of Nrf3 in Smooth Muscle Cell Differentiation From Stem Cells

“…[27][28][29][30][31] In particular, during myogenesis ER stress enhances myoblast differentiation and myofiber formation. 32 Cho et al 31 have shown that ER stress increases during ESC differentiation toward neural cells and that this differentiation is further enhanced through chemical treatment with the ER stress inducers, thapsigargin and tunicamycin.…”

Autocrine Human Growth Hormone (hGH) Regulation of Human Mammary Carcinoma Cell Gene Expression

“…Furthermore, an altered form of CHOP, TLS-CHOP, formed by reciprocal translocation of the chop gene and a gene encoding an RNA-binding protein is associated with human myxoid liposarcoma (68,69). Erythropoietin has also been demonstrated to up-regulate CHOP to participate in the erythroid differentiation program (24,25). Here we have demonstrated that overexpression of CHOP in mammary carcinoma cells stimulated with hGH offers dramatic protection from apoptosis in a p38 MAP kinase-dependent manner.…”

“…Studies utilizing cells and animals with a targeted deletion of the chop gene have implicated CHOP in programmed cell death in response to endoplasmic reticulum (ER) stress (22), and overexpression of CHOP in growth factor-dependent 32D myeloid precursor cells (23) also results in apoptosis. CHOP protein has recently been demonstrated to be up-regulated by erythropoietin (24), resulting in erythroid differentiation (25).…”