Structure of chromatin containing extensively acetylated H3 and H4

I have grown HeLa cells in 5 mM sodium n-butyrate leading to extensive in vivo histone acetylation, and have characterized the structure of chromatin containing the modified histones. Nuclear DNA in butyrate-treated cells is digested 5-10 fold more rapidly by DNAase I than the DNA of control cells. Staphylococcal nuclease degrades the two nuclear samples to acid-soluble material with identical rates; this nuclease, however, does excise nucleosomes with extensively acetylated histones from the nucleoprotein chain preferentially. The physical properties of unsheared chromatin and isolated core particles from control and butyrate-treated cells are closely similar, as are the rates of digestion of core particles from the two cell preparations by DNAase I. Determination of the relative susceptibilities of cleavage sites for DNAase I demonstrates that the site 60 bases from the ends of the DNA resistant in control cells, becomes susceptible to the nuclease in core particles containing acetylated histones. Similarly, the 5' terminal phosphate at the end of DNA in core prticles is removed by staphylococcal nuclease 2-3 fold faster in particles containing acetylated histones than in particles from control cells.     

Differentiation of eosinophilic leukemia EoL-1 cells into eosinophils induced by histone deacetylase inhibitors

EoL-1 cells differentiate into eosinophils in the presence of n-butyrate, but the mechanism has remained to be elucidated. Because n-butyrate can inhibit histone deacetylases, we hypothesized that the inhibition of histone deacetylases induces the differentiation of EoL-1 cells into eosinophils. In this study, using n-butyrate and two other histone deacetylase inhibitors, apicidin and trichostatin A, we have analyzed the relationship between the inhibition of histone deacetylases and the differentiation into eosinophils in EoL-1 cells. It was demonstrated that apicidin and n-butyrate induced a continuous acetylation of histones H4 and H3, inhibited the proliferation of EoL-1 cells without attenuating the level of FIP1L1-PDGFRA mRNA, and induced the expression of markers for mature eosinophils such as integrin beta7, CCR1, and CCR3 on EoL-1 cells, while trichostatin A evoked a transient acetylation of histones and induced no differentiation into eosinophils. These findings suggest that the continuous inhibition of histone deacetylases in EoL-1 cells induces the differentiation into mature eosinophils.     

n-Butyrate effects thyroid hormone stimulation of prolactin production and mRNA levels in GH1 cells

Using cultured GH1 cells, a growth hormone and prolactin-producing rat pituitary cell line, we have shown that n-butyrate and other short chain carboxylic acids stimulate histone acetylation and elicit a reduction of thyroid hormone nuclear receptor which is inversely related to the extent of acetylation (Samuels, H. H., Stanley, F., Casanova, J., and Shao, T. C. (1980) J. Biol. Chem. 255, 2499-2508). In this study, we compared the n-butyrate and propionate modulation of receptor levels to regulation of the growth hormone and prolactin response by 3,5,3'-triiodo-L-thyronine (L-T3). n-Butyrate (0.1-10 mM) did not stimulate growth hormone production. L-T3 stimulated the growth hormone response 4- to 5-fold and n-butyrate (0.5-1 mM) increased L-T3 stimulation of growth hormone production 1.5- to 2-fold compared to L-T3 alone. L-T3 stimulation of growth hormone production at higher n-butyrate concentrations decreased in parallel with the n-butyrate-mediated reduction of receptor levels. In contrast with the growth hormone response, n-butyrate (0.5 mM) increased basal prolactin production about 5-fold. Prolactin production, which is inhibited 25 to 50% by L-T3, was stimulated between 20- and 70-fold by L-T3 + n-butyrate (0.5-1 mM) and this decreased at higher n-butyrate levels. Prolactin mRNA and growth hormone mRNA levels paralleled the changes in prolactin and growth hormone production rates. These effects of L-T3, n-butyrate, or L-T3 + n-butyrate appeared unrelated to changes in cAMP levels or global changes in DNA methylation of the growth hormone or prolactin genes. Propionate elicited the same effects as n-butyrate but at a 5- to 10-fold higher concentration consistent with their relative effect on stimulating acetylation of chromatin proteins. These results suggest that prolactin gene expression is under partial regulatory repression which is reversed by a carboxylic acid-mediated postsynthetic modification event which allows for stimulation of the prolactin gene by thyroid hormone.     

In vivo acetylation of HMG1 protein enhances its binding affinity to distorted DNA structures.

The postsynthetic acetylation of HMG1 protein has been known for more than 20 years, but the effect of this modification on the properties of the protein has not been studied so far. Acetylated HMG1 was isolated from cells grown in the presence of sodium n-butyrate and identified as a monoacetylated protein, modified at lysine 2. Acetylated and parental forms of HMG1 were compared relative to their binding affinity to distorted DNA structures. By using mobility shift assay to determine the dissociation constants, we show that acetylation enhanced the ability of HMG1 to recognize UV light- or cisplatin-damaged DNA and four-way junctions. Since the modified lysine lies adjacent to the HMG1 DNA-binding domain, the results obtained were attributed to acetylation-induced conformational change in HMG1. The potential role of acetylation in modulating the interactions of HMG1 with both damaged DNA and other proteins is discussed.     

Hyperacetylation of core histones does not cause unfolding of nucleosomes. Neutron scatter data accords with disc shape of the nucleosome

Recent studies report that the frictional resistance of partially acetylated core particles increases when the number of acetyl groups/particle exceeds 10 (Bode, J., Gomez-Lira, M. M. & Schr?ter, H. (1983) Eur. J. Biochem. 130, 437-445). This was attributed to an opening of the core particle though other explanations, e.g. unwinding of the DNA ends were also suggested. Another possible explanation is that release of the core histone N-terminal domains by acetylation increased the frictional resistance of the particle. Neutron scatter studies have been performed on core particles acetylated to different levels up to 2.4 acetates/H4 molecule. Up to this level of acetylation the neutron scatter data show no evidence for unfolding of the core particle. The fundamental scatter functions for the envelope shape and internal structure are identical to those obtained previously for bulk core particles. The structure that gave the best fit to these fundamental scatter functions was a flat disc of diameter 11-11.5 nm and of thickness 5.5-6 nm with 1.7 +/- 0.2 turns of DNA coiled with a pitch of 3.0 nm around a core of the histone octamer. The data analysis emphasizes the changes in pair distance distribution functions at relatively low contrasts, particularly when the protein is contrast matched and DNA dominates the scatter. Under these conditions there is no evidence for the unwinding of long DNA ends in the hyperacetylated core particles. The distance distribution functions go to zero between 11.5 and 12 nm which gives the maximum chord length in a particle of dimension, 11 nm X 5.5 nm. The distance distribution function for the histone octamer contains 85% of the vectors within the 7.0-nm diameter of the histone core. 15% of the histone vectors lie between 7.0 and 12.0 nm, and these are attributed to the N-terminal domains of the core histones which extend out from the central histone core. Histone vectors extending beyond 7.0 nm are necessary to account for the measured radius of gyration of the histone core of 3.3 nm. A similar value of 3.2 nm is calculated for the recent ellipsoidal shape of 11.0 X 6.5 X 6.5 nm from the crystal structure of the octamer. However, the nucleosome model based on this structure is globular, roughly 11 nm in diameter, which does not accord with the flat disc shape core particle obtained from detailed neutron scatter data nor with the cross-section radii of gyration of the histone and DNA found previously for extended chromatin in solution.     

Structure-activity relationship between carboxylic acids and T cell cycle blockade

This study was designed to examine the potential structure-activity relationship between carboxylic acids, histone acetylation and T cell cycle blockade. Toward this goal a series of structural homologues of the short-chain carboxylic acid n-butyrate were studied for their ability to block the IL-2-stimulated proliferation of cloned CD4+ T cells. The carboxylic acids were also tested for their ability to inhibit histone deacetylation. In addition, Western blotting was used to examine the relative capacity of the carboxlic acids to upregulate the cyclin kinase-dependent inhibitor p21cip1 in T cells. As shown earlier n-butyrate effectively inhibited histone deacetylation. The increased acetylation induced by n-butyrate was associated with the upregulation of the cyclin-dependent kinase inhibitor p21cip1 and the cell cycle blockade of CD4+ T cells. Of the other carboxylic acids studied, the short chain acids, C3-C5, without branching were the best inhibitors of histone deacetylase. This inhibition correlated with increased expression of the cell cycle blocker p21cip1, and the associated suppression of CD4+ T cell proliferation. The branched-chain carboxylic acids tested were ineffective in all the assays. These results underline the relationship between the ability of a carboxylic acid to inhibit histone deacetylation, and their ability to block T cell proliferation, and suggests that branching inhibits these effects.     

Immunochemical detection of changes in chromatin subunits induced by histone H4 acetylation.

Native, reassociated, and reconstituted core particles from chicken erythrocytes were compared by both biophysical and immunochemical methods. No significant difference between the three types of core particles could be demonstrated by electron microscopy, circular dichroism, or immunochemical analysis with antisera to histone H2B, H2A, and H3. Core particles were also reconstituted with calf thymus non-acetylated H3, H2A, and H2B with either mono-, di-, or tri-acetylated H4 isolated from cuttle -fish testes. The hyperacetylation of H4 did not significantly alter the biophysical characteristics of core particles but it induced several changes in their immunochemical reactivity. Binding to core particles of antibodies specific for H2A, H3, and for the IRGERA (synthetic C-terminal) peptide of H3 was considerably decreased when di- or tri-acetylated H4 was used for reconstitution, whereas binding of H2B antibodies remained the same. Our results suggest that the presence of hyperacetylated H4 within core particles leads to conformational changes that alter the antigenic determinants of several of the histones present at the surface of chromatin subunits. Since histone acetylation is correlated with the open structure of active chromatin, it may become possible to monitor the activity of chromatin by immunochemical methods.     

Nucleosome linking number change controlled by acetylation of histones H3 and H4

High levels of acetylation of lysines in the amino-terminal domains of all four core histones, H2A, H2B, H3, and H4, have been shown to reduce the linking number change per nucleosome core particle in reconstituted minichromosomes (Norton, V. G., Imai, B. S., Yau, P., and Bradbury, E. M. (1989) Cell 57, 449-457). Because there is evidence to suggest that the acetylations of H3 and H4 have functions that are distinct from those of H2A and H2B, we have determined the nucleosome core particle linking number change in minichromosomes containing fully acetylated H3 and H4 and very low levels of acetylation in H2A and H2B. This linking number change was -0.81 +/- 0.05, in close agreement with the linking number change for hyperacetylated nucleosome core particles which contain high levels of acetylation in all four core histones (approximately 70% of full acetylation in H3 and H4). Therefore, high levels of acetylation of H3 and H4 alone are responsible for the reduction in the linking number change per nucleosome core particle.