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...

Studies on the active sites of the homodimer proteins by construction of hybrids of wild-type and site-directed mutant proteins have been reported (13-15). Wente and Schachman (13) used an approach which entails the formation of such hybrids in vitro, but this approach is only applicable to proteins which can sustain the dissociation and reassociation conditions. Larimer et al. (14) reported the production of a hybrid heterodimer ofwild type and a site-directed mutant by plasmid cotransformation. Nevertheless, the study was limited by the fact that the three species of dimeric proteins (wild-type homodimer, mutant homodimer, heterodimer) could not be separated from one another.In the present study, we have developed an approach to express a heterodimer (H194A/HNQOR) containing a wildtype subunit with a polyhistidine tag at the amino terminus of NQOR and a H194A mutant subunit by constructing two cDNAs under one promoter in Escherichia coli. This approach enabled us to separate heterodimer from the two homodimers by stepwise elution with imidazole from a nickel nitrilotriacetate (Ni-NTA) column under nondenaturing conditions. The functions of the heterodimer subunits were studied by characterizing the kinetics of the reduction of 2,6-dichloroindophenol (DCIP), menadione, and methyl red.

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The hematopoietic growth factor erythropoietin (Epo) triggers changes in the expression of genes that encode important regulators of erythroid cell growth and differentiation. We now report that Epo markedly upregulates chop (gadd153) expression and that this transcription factor plays a role in erythropoiesis. Using a differential hybridization assay, we isolated a full-length cDNA ofchop as an Epo upregulated gene in Rauscher murine erythroleukemia cells. RNase protection assays demonstrated that Epo or dimethyl sulfoxide induction increased steady-state mRNA levels 10- to 20-fold after 24 to 48 hours. Western blot analysis confirmed a marked increase in CHOP protein. Among the other c/ebp family members, only c/ebp β was also upregulated during erythroid differentiation. Among normal hematopoietic cells examined, steady-state mRNA levels were highest in erythroid cells, with levels peaking during terminal differentiation. Transient overexpression ofchop in Rauscher cells resulted in a significant increase in Epo- or dimethyl sulfoxide (DMSO)-induced hemoglobinization, further linking chop upregulation to erythroid differentiation. Artificial downregulation of chop in normal murine bone marrow cells with antisense oligodeoxynucleotides inhibited colony-forming unit-erythroid (CFU-E)–derived colony growth in a concentration-dependent manner. Burst-forming unit-erythroid (BFU-E)–derived colony growth was not affected. Using a Far Western type of analysis, we detected several potential CHOP binding partners among the nuclear proteins of Rauscher cells. Importantly, the number and relative abundance of these proteins changed with differentiation. The results strongly suggest that CHOP plays a role in erythropoiesis, possibly through interactions with both C/EBP and non-C/EBP family members.