Biochemical characterization of chromatin fractions isolated from induced and uninduced friend erythroleukemia cells

Summary Chromatin fractions from Friend erythroleukemia cells after induction of differentiation by dimethylsulfoxide (DMSO) were compared in their biochemical characteristics to fractions from uninduced cells. Fractions were prepared by extracting chromatin from nuclei after mild micrococcal nuclease treatment with increasing concentrations of NaCl according to Sanders [1]. This procedure has been found to release chromatin containing hyperacetylated histones preferentially [2]. The fractions obtained by this procedure were analysed in respect to the amount of chromatin released, the amount of histone H1, the degree of acetylation of histone H4, the presence of non-histone proteins and the concentration of transcribed and non-transcribed sequences. It was found that the fractions differ in the amount of histone H1 present, in several non-histone proteins and in the acetylation of histonie H4, regardless whether induced or uninduced cells were analysed. The distribution of transcribed sequences versus non-transcribed sequences among the fractions was the same, demonstrating that this fractionation procedure, although leading to fractions with biochemical differences, is not able to discriminate functional states of chromatin and that the biochemical characteristics of the fractions may be common to both, active as well as inactive states of chromatin.     

New gene expression in dimethylsulfoxide-treated friend erythroleukemia cells

Our previous studies had shown that a small amount of single-stranded DNA (ssDNA) separated from the bulk nuclear DNA of different animal cells by an improved method of hydroxylapatite chromatography (HAC) contains two distinct molecular fractions. The major fraction consists of non self-reassociating sequences that are reassociable to the unique component of bulk DNA and in great part hybridizable to homologous RNA. The minor fraction consists of self-reassociable sequences also reassociable to moderately repetitious bulk DNA. In the present work ssDNA from Friend leukemia cells induced to differentiate (ind FLC) by DMSO was compared with ssDNA from untreated control Friend cells (cont FLC). It was shown that the relative amount of ssDNA is greater in ind FLC than in cont FLC (1.5 – 1.6% and 1.2 – 1.3% of the total cell DNA respectively after a second step of HAC purification). The ind FLC-ssDNA contained a greater proportion of self-reassociable sequences (33–35%) as compared with cont FLC-ssDNA (18–20%). Also the relative amounts of ssDNA hybridizable to cytoplasmic RNA from homologous cells was slightly but constantly higher in ind FLC-ssDNA (33–34%) than in cont FLC-ssDNA (29–30%). Cross hybridizations were carried out between highly radioactive ssDNA and cellular RNAs in great excess, whether total cytoplasmic RNAs or polyadenylated mRNAs. At saturation levels, the hybridized ssDNA fraction was separated from the non-hybridized fraction, and both fractions were rehybridized to RNA from ind FLC or cont FLC. The results indicated that about 10% of ind FLC-ssDNA appeared to be specific for DMSO-treated cells. This may correspond to the expression of 1000–2000 different cytoplasmic mRNAs mostly belonging to the low abundance class.     

Affinity isolation of active murine erythroleukemia cell chromatin: uniform distribution of ubiquitinated histone H2A between active and inactive fractions.

This laboratory recently reported the development of a biotin-cellulose/streptavidin affinity chromatography method based on the DNase I sensitivity of active chromatin to isolate a DNA fraction from murine erythroleukemia (MEL) cells that is more than 15-fold enriched in active genes (Dawson et al.: Journal of Biological Chemistry 264:12830-12837, 1989). We now report the extension of this technique to isolate and characterize chromatin that is enriched in active genes. In this approach, DNA in nuclei isolated from MEL cells was nicked with DNase I at a concentration that does not digest the active beta-globin gene, followed by repair of the nicks with a cleavable biotinylated nucleotide analog, 5-[(N-biotin-amido)hexanoamido-ethyl-1,3'-dithiopropionyl-3- aminoallyl]-2'- deoxyuridine 5'-triphosphate (Bio-19-SS-dUTP), during a nick-translation reaction. After shearing and sonication of the nuclei to solubilize chromatin, chromatin fragments containing biotin were separated from non-biotinylated fragments by sequential binding to streptavidin and biotin cellulose. The bound complex contained approximately 10% of the bulk DNA. Reduction of the disulfide bond in the biotinylated nucleotide eluted approximately one-half of the affinity isolated chromatin. Hybridization analysis of DNA revealed that whereas inactive albumin sequences were equally distributed among the chromatin fractions, virtually all of the active beta-globin sequences were associated with chromatin fragments which had bound to the affinity complex. Western blot assessment for ubiquitinate histones revealed that ubiquitinated histone H2A (uH2A) was uniformly distributed among active (bound) and inactive (unbound) chromatin fractions.     

Action of hydrocortisone on the lipid composition of active and inactive chromatin fractions in the rat liver

A composition of phospholipids and neutral lipids of rat liver chromatin and its active and inactive fractions has been investigated. It is shown that the hydrocortisone action results in marked increase in phospholipid/neutral lipid ratio of both chromatin and its active fraction. The changes in lipid content is clearly expressed in active chromatin fraction, the lipid content of inactive fraction is not changed. It is concluded that the increase of content in certain phospholipids and simultaneous decrease of neutral lipids in chromatin promotes the hormonal activation of genome.     

Quantitation of DNase I sensitivity in Xenopus chromatin containing active and inactive globin, albumin and vitellogenin genes.

The disappearance of defined restriction fragments of the beta 1-globin, an albumin and the A1 vitellogenin gene was quantitated after DNase I digestion and expressed by a sensitivity factor defined by a mathematical model. Analysis of naked DNA showed that the gene fragments have similar but not identical sensitivity factors. DNase I digestion of chromatin revealed for the same gene fragments sensitivity factors differing over a much wilder range. This is correlated to the activity of the genes analyzed: the beta 1-globin gene fragment is more sensitive to DNase I in chromatin of erythrocytes compared to hepatocytes whereas the albumin gene fragment is more sensitive to DNase I in chromatin of hepatocytes. The A1 vitellogenin gene has the same DNase I sensitivity in both cell types. Comparing the DNase I sensitivity of the three genes in their inactive state we suggest that different chromatin conformations may exist for inactive genes.     

Presence of nucleosomal repeat in the transcribed alpha globin gene of induced murine erythroleukemia cells

The sensitivity of the mouse alpha-globin gene to micrococcal nuclease and its nucleosomal repeat were studied in three different functional states of the gene: inactive in EAT cells, potentially active in uninduced MEL cells and active in induced MEL cells. The results show that: 1. The nuclease sensitivity of the gene differs in the three different functional states. 2. Both the coding and the 5'-flanking regions of the induced actively transcribed gene show a typical nucleosomal repeat pattern. 3. Hypersensitive sites for micrococcal nuclease and for an endogenous nuclease appear upstream of the gene after induction of differentiation.     

Dependence of globin gene expression in mouse erythroleukemia cells on the NF-E2 heterodimer.

High-level, tissue-specific expression of the beta-globin genes requires the presence of an upstream locus control region (LCR). The overall enhancer activity of the beta-globin complex LCR (beta-LCR) is dependent on the integrity of the tandem NF-E2 sites of HS-2. The NF-E2 protein which binds these sites is a heterodimeric basic leucine zipper protein composed of a tissue-specific subunit, p45 NF-E2, and a smaller subunit, p18 NF-E2, that is widely expressed. In these studies, we sought to investigate the role of NF-E2 in globin expression. We show that expression of a dominant-negative mutant p18 greatly reduces the amount of functional NF-E2 complex in the cell. Reduced levels of both alpha- and beta-globin were associated with the lower levels of NF-E2 activity in this cell line. Globin expression was fully restored upon the introduction of a tethered p45-p18 heterodimer. We also examined CB3 cells, a mouse erythroleukemia (MEL) cell line that does not express endogenous p45 NF-E2, and demonstrated that the restoration of globin gene expression was dependent upon the levels of expressed tethered NF-E2 heterodimer. Results of DNase I hypersensitivity mapping and in vivo footprinting assays showed no detectable chromatin alterations in beta-LCR HS-2 due to loss of NF-E2. Finally, we examined the specificity of NF-E2 for globin gene expression in MEL cells. These experiments indicate a critical role for the amino-terminal domain of p45 NF-E2 and show that a related protein, LCRF1, is unable to restore globin gene expression in p45 NF-E2-deficient cells. From these results, we conclude that NF-E2 is specifically required for high level goblin gene expression in MEL cells.     

Increase in globin chains and globin mRNA in erythroleukemia cells in response to hemin.

Addition of 50 μm hemin to mouse erythroleukemia cells cultured in 0.5% dimethyl-sulfoxide (DMSO) resulted in >10-fold stimulation of globin chain synthesis as a percentage of acid precipitable protein. In cultures fully induced with 1.5% DMSO, addition of 15 mm 3-amino-1,2,4-triazole (AT), an inhibitor of heme synthesis, reduced globin chain synthesis to uninduced levels and reduced globin mRNA levels to less than 20% of induced values. The inhibition of AT was prevented by simultaneous addition of 25 μm hemin to the cultures. Using RNA-DNA hybridization analysis, the amount of globin mRNA sequences as a fraction of total cytoplasmic RNA was also increased by addition of 50 μm hemin to cultures with 0.5% DMSO. The results suggest that exogenous hemin can promote globin chain synthesis, that endogenously synthesized heme can be required for globin chain synthesis, and that hemin directly or indirectly also alters the appearance or degradation of globin mRNA sequences in the cytoplasm.