Expression of transfected vimentin genes in differentiating murine erythroleukemia cells reveals divergent cis-acting regulation of avian and mammalian vimentin sequences.

Abstract

We studied the expression of transfected chicken and hamster vimentin genes in murine erythroleukemia (MEL) cells. MEL cells normally repress the levels of endogenous mouse vimentin mRNA during inducermediated differentiation, resulting in a subsequent loss of vimentin filaments. (8,29,36,77).The generality and diversity of vimentin expression suggest that its regulation is necessarily complex. This complexity is particularly evident from a comparison of avian and mammalian erythropoiesis. The mature avian erythrocyte is a nucleated biconvex ellipsoidally shaped cell. Ultrastructural analyses of avian erythrocytes have revealed a network of intermediate filaments that spans the cytoplasm and appears to anchor the centrally located nucleus (34,89,92). The major subunit protein of these filaments is vimentin (36). * Corresponding author.In contrast, the anucleate, biconcave disk-shaped mammalian erythrocyte contains no intermediate filaments. Studies of human hematopoiesis in vivo have demonstrated that vimentin is expressed early in erythroid differentiation but is lost in the erythroblastic stages (22).In chicken erythroid cells, vimentin filaments are assembled rapidly and stably from a soluble pool of newly synthesized vimentin (5, 62). The efficiency and rapidity of vimentin assembly in vivo suggest that the extent of vimentin filament formation is determined primarily by the amount of vimentin synthesized. We have shown that the expression of vimentin protein during erythropoiesis is determined primarily by mRNA abundance (10, 69). In chicken embryonic erythropoiesis, vimentin mRNA is found at low levels in immature, mitotic primitive cells and accumulates to increasingly higher levels during terminal differentiation of the definitive erythroid lineage, apparently underlying similar changes at the protein level (10). During differentiation of murine erythroleukemia (MEL) cells in vitro, vimentin mRNA levels rapidly and extensively decline (-25-fold reduction), rendering a concomitant decrease in vimentin synthesis and a subsequent loss of vimentin filaments (69). The striking difference in the changes of vimentin mRNA abundances (and, ultimately, vimentin filaments) during mammalian and avian erythropoiesis presents an opportunity to study not only the factors responsible for the dynamic positive and negative regulation of the vimentin gene but also the basis for the evolutionary divergence of the two erythropoietic programs with regard to vimentin expression.In the present study, we addressed these issues by examining the behavior of transfected chicken and hamster vimentin genes in differentiating MEL cells. The utility of studying both resident and transfected genes during in vitro differentiation of MEL cells is well established (reviewed in references 13 and 55). We demonstrate that, in MEL cell lines harboring and expressing chicken vimentin genes,

Concentration dependence of transcriptional transactivation in inducible E1A-containing human cells.

“…Labeled RNA was isolated, and equivalent amounts of radioactivity (typically 1 x 10' to 2 x 107 cpm) were hybridized to a slot matrix of M13-adenovirus recombinant DNAs immobilized on nitrocellulose. Hybridization and washing conditions were as described previously (31).…”

“…RNA were isolated exactly as described previously (31). Transcription reactions were performed as described previously (31) except that the volume was increased to 200 ,ul containing 250 to 300 ,uCi of [a-32P]UTP (3,00OCi/mmol).…”

“…32P-labeled cRNA was synthesized from pSP6y-actin with 100 puM CTP at 100 Ci/mmol. The hybridization and digestion conditions were those previously described (31). Protected fragments were resolved on a 5% polyacrylamide-7 M urea gel and quantitated by scintillation counting of gel slices.…”

Expression of the chicken vimentin gene in transgenic mice: efficient assembly of the avian protein into the cytoskeleton.

“…2). However, the chicken gene expression was induced during in vitro differentiation of transfected MEL cells (35).…”

Intermediate filaments: regulation of gene expression and assembly

“…This element seems not to be conserved in the chicken vimentin promoter and the cis-acting sequences responsible for TPA induction still need to be identified (Stover and Zehner, 1992). Possibly there is some divergence, also considering the differences in expression of chicken and hamster vimentin gene constructs upon transfection and subsequent differentiation of erytroleukemia cells (Ngai et al, 1987). The overall pattern of vimentin expression regulation correlates well with the behavior of a growth-regulated gene, or a primary response gene (Connell and Rheinwald, 1983;Rittling and Baserga, 1987).…”