Globin RNA synthesis in vitro by isolated erythroleukemic cell nuclei: direct evidence for increased transcription during erythroid differentiation.

Abstract

Murine erythroleukemic cells accumulate cytoplasmic globin mRNA during differentiation induced in tissue culture by dimethyl sulfoxide. Cellular accumulation of globin RNA may reflect transcriptional activation of the globin genes and/or posttranscriptional stabilization of globin RNA during differentiation. To evaluate possible transcriptional controls directly, globin RNA synthesis by isolated erythroleukemic cell nuclei was studied.Conditions were established for optimal nuclear RNA synthesis in vitro in the presence of a mercurinucleotide (Hg-CTP Isolation of Nuclei. Washed cell pellets were suspended in 0.3 M sucrose (Schwarz/Mann ultrapure) containing 2 mM Mg(OAc)2, 3 mM CaCI2, 10 mM Tris-HCl at pH 8.0, and 2 mM 2-mercaptoethanol. After addition of Triton X-100 to 0.25%, the cell suspension was stirred on a vortex mixer for 30 sec. The crude nuclei were gently sedimented at 1000 X g for 5 min at 40, and then resuspended in the same buffer minus Triton X-100. An equal volume of 2 M sucrose containing 5 mM Mg(OAc)2, 10 mM Tris at pH 8.0, and 2 mM mercaptoethanol was immediately added, and the mixture was homogenized with five strokes of a tight-fitting Teflon-glass homogenizer. Purified nuclei were sedimented through a cushion of 2 M sucrose containing 2 mM Mg(OAc)2, 3 mM CaCl2, and 10 mM Tris at pH 8.0 by centrifugation at 25,000 rpm in a Beckman SW-40 rotor for 45 min at 40. The nuclear pellet was resuspended in 25% (vol/vol) glycerol/5 mM Mg(OAc)2/50 mM Tris at pH 8.0/2 mM mercaptoethanol at 1 to 2 X 108 nuclei per ml, and frozen in liquid nitrogen.RNA Synthesis by Isolated Nuclei. Conditions for RNA synthesis were modified slightly from those described for other systems (8, 9). Each reaction mixture contained 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) at pH 8.0, 5 mM Mg(OAc)2, 1 mM MnCl2, 150 mM KCI, 12.5% glycerol, 4 mM phosphoenolpyruvate, pyruvate kinase (3 units/ml), 0.185 mM of three ribonucleoside triphosphates, 0.037 mM of the fourth ribonucleoside triphosphate labeled with 3H, and approximately 5 X 107 rapidly thawed nuclei per ml. Mercurinucleotide, either Hg-CTP or Hg-UTP (P.L. Biochemicals), replaced the corresponding unmodified nucleotide at the same concentration in the reaction mixture when RNA containing mercury (Hg-RNA) was prepared. Mercaptans, as noted below, were present at 10 mM final concentration in the reaction mixture. The reaction mixture was incubated at 230 with intermittent, gentle shaking and aliquots were withdrawn at appropriate times, spotted on Whatman 3MM paper, and processed as described by Ernest et al. (9) to monitor RNA synthesis. No adjustment was made for background cpm at time zero.Preparative Isolation of RNA after Nuclear Synthesis. After incubation, nuclei were diluted 10-fold with 20 mM Tris at pH 7.5/50 mM NaCl/1 mM EDTA/1% sodium dodecyl sulfate and extracted vigorously in a vortex mixer with an equal volume of 600 phenol previously equilibrated with Tris buffer. The aqueous phase was reextracted three times with chloroform/is...

Specific pattern of gene expression during induction of mouse erythroleukemia cells.

“…Previous studies have shown and our results confirm that induction of MEL cells with DMSO is accompanied by a decrease in transcriptional activity (Orkin and Swerdlow 1977;Patel and Lodish 1984). Transcriptional output, measured in terms of total yield of TCA-precipi- table counts, dropped greater than 50% from day 0 to day 3 ( Fig.…”

“…Induction is most notably characterized by a marked increase in globin gene transcription and accumulation of globin mRNA as has been reported by a number of independent investigators (Aviv et al 1976;Curtis and Weissmann 1976;Nudel et al 1977;Orkin and Swerdlow 1977;Lowenhaupt et al 1978;Hofer et al 1982). Our results show that even though the total transcriptional output of the cell is drastically reduced during induction, globin gene transcription increases greater than 40-fold.…”

“…Among the observed changes are progressively smaller cells and increasingly condensed nuclei, leading to a reduction in RNA content and a decreased rate of synthesis of total RNA (Sherton and Kabat 1976). The most dramatic and oft noted change is the marked increase in the rate of transcription of a-and/3-globin genes resulting in accumulation of globin mRNA and hemoglobin (Aviv et al 1976;Curtis and Weissmann 1976;Nudel et al 1977;Orkin and Swerdlow 1977;Lowenhaupt et al 1978;Hofer et al 1982). Also observed is an increase of newly synthesized carbonic anhydrase II mRNA (Curtis 1983) as well as increased synthesis of oL-and [3-spectrin (Eisen et al 1977;Pfeffer et al 1986), band 3 (Sabban et al 1980), band 4 (Pfeffer andRedman 1981), and other erythroid-specific polypeptides.…”

Linker scanning mutagenesis of the 5'-flanking region of the mouse beta-major-globin gene: sequence requirements for transcription in erythroid and nonerythroid cells.

“…In these studies, cloned globin genes are stably introduced into mouse erythroleukemia (MEL) cells which are derived from adult hematopoietic precursor cells transformed with the Friend virus complex (19; see reference 36 for review). Treatment of these cells with dimethyl sulfoxide (DMSO) or a variety of other chemical inducers leads to a series of morphological and biochemical modifications that mimic events in normal erythroid cell differentiation (19,36), including a dramatic increase in the rate of transcription of adult globin genes (8,29,49). When cloned mouse or human ,-globin genes are introduced into MEL cells by DNA-mediated gene transfer, they are coregulated with the endogenous mouse globin genes (8,60).…”

Transcription factor EKLF (KLF1) recruitment of the histone chaperone HIRA is essential for β-globin gene expression

“…Treatment of MEL cells with hexamethylene bis-acetamide (HMBA) or DMSO results in high-level induction of erythroid genes, including β-globin, and eventual terminal differentiation (50,51). The expression of EKLF, GATA-1, and importantly, HIRA proteins, are stable during the differentiation of MEL cells (Fig.…”