Multiple histone acetyltransferases are associated with a chicken erythrocyte chromatin fraction enriched in active genes.

We have examined salt-soluble chromatin released by micrococcal nuclease from a 15-day-old chicken embryo erythrocyte nuclei for histone acetyltransferase (HAT) activities. This chromatin is enriched in transcriptionally active sequences from within the active beta-globin locus and contains elevated levels of acetylated core histones. HAT activities present in this fraction target histones H4, H3, and H2A when the chromatin itself is used as the substrate. In gel HAT activity assay demonstrates that the salt-soluble chromatin fraction contains four acetyltransferase molecules distinguished by their different molecular masses (47, 33, 32, and 28 kDa). Further separation of the chromatin by centrifugation through sucrose gradients shows that the acetyltransferases segregate into chromatin-bound and chromatin-free populations. The 32- and 28-kDa HATs are associated with chromatin, whereas the 47- and 33-kDa HAT molecules are not. The chromatin-bound HAT activities predominantly target H4 to give the diacetyl and triacetyl species; some acetylation of H2A can also be seen. Our results suggest that the chromatin-associated acetyltransferases have a role in gene regulation.

The immunoglobulin heavy chain locus control region increases histone acetylation along linked c-myc genes.

In chromosome translocations characteristic of Burkitt lymphomas (BL) and murine plasmacytomas, c-myc genes become juxtaposed to immunoglobulin heavy-chain (IgH) sequences, resulting in aberrant c-myc transcription. Translocated c-myc alleles that retain the first exon exhibit increased transcription from the normally minor c-myc promoter, P1, and increased transcriptional elongation through inherent pause sites proximal to the major c-myc promoter, P2. We recently demonstrated that a cassette derived from four DNase I-hypersensitive sites (HS1234) in the 3'Calpha region of the IgH locus functions as an enhancer-locus control region (LCR) and directs a similar pattern of deregulated expression of linked c-myc genes in BL and plasmacytoma cell lines. Here, we report that the HS1234 enhancer-LCR mediates a widespread increase in histone acetylation along linked c-myc genes in Raji BL cells. Significantly, the increase in acetylation was not restricted to nucleosomes within the promoter region but also was apparent upstream and downstream of the transcription start sites as well as along vector sequences. Histone hyperacetylation of control c-myc genes, which was induced by the deacetylase inhibitor trichostatin A, mimics the effect of the HS1234 enhancer on expression from the c-myc P2 promoter, but not that from the P1 promoter. These results suggest that the HS1234 enhancer stimulates transcription of c-myc by a combination of mechanisms. Whereas HS1234 activates expression from the P2 promoter through a mechanism that includes increased histone acetylation, a general increase in histone acetylation is not sufficient to explain the HS1234-mediated activation of transcription from P1.

Chromosomal mapping of core histone acetylation by immunoselection.

Acetylation of specific lysine residues in the N-terminal domains of core histones is a biochemical marker of active genes. To determine the spatial and temporal distribution of this reversible posttranslational modification, affinity-purified polyclonal antibodies recognizing the epitope epsilon-acetyllysine have been used in immunoselection procedures with mononucleosomes and salt-soluble chromatin fragments generated by micrococcal nuclease. The DNA of the antibody-selected chromatin was slot-blotted and probed with a variety of gene sequences: an enhanced hybridization signal, with respect to that from the DNA of the input chromatin, demonstrated elevated acetylation levels on the histones associated with the probing sequences. Using chicken embryonic erythrocytes as chromatin source and probes from the beta globin locus, it was shown that both the embryonic epsilon and adult beta genes are acetylated at 5 and 15 days, and the acetylation uniformly covers the whole of the locus, precisely comapping with the 33 kb of open chromatin structure. Studies with proliferating human K562 cells show that the inactive but poised PDGF-beta gene is already hyperacetylated and that its acetylation status is not enhanced on induction. These results indicate that acetylation is not a consequence of transcription but a prerequisite and that it may be responsible for either generating or maintaining the open structure of poised and active genes.

Acetylation of histone H4 plays a primary role in enhancing transcription factor binding to nucleosomal DNA in vitro.

Core histones isolated from normal and butyrate-treated HeLa cells have been reconstituted into nucleosome cores in order to analyze the role of histone acetylation in enhancing transcription factor binding to recognition sites in nucleosomal DNA. Moderate stimulation of nucleosome binding was observed for the basic helix-loop-helix factor USF and the Zn cluster DNA binding domain factor GAL4-AH using heterogeneously acetylated histones. However, by coupling novel immunoblotting techniques to a gel retardation assay, we observed that nucleosome cores containing the most highly acetylated forms of histone H4 have the highest affinity for these two transcription factors. Western analysis of gel-purified USF-nucleosome and GAL4-AH-nucleosome complexes demonstrated the predominant presence of acetylated histone H4 relative to acetylated histone H3. Immunoprecipitation of USF-nucleosome complexes with anti-USF antibodies also demonstrated that these complexes were enriched preferentially in acetylated histone H4. These data show that USF and GAL4-AH preferentially interact with nucleosome cores containing highly acetylated histone H4. Acetylation of histone H4 thus appears to play a primary role in the structural changes that mediate enhanced binding of transcription factors to their recognition sites within nucleosomes.

Core histone hyperacetylation co-maps with generalized DNase I sensitivity in the chicken beta-globin chromosomal domain.

The distribution of core histone acetylation across the chicken beta-globin locus has been mapped in 15 day chicken embryo erythrocytes by immunoprecipitation of mononucleosomes with an antibody recognizing acetylated histones, followed by hybridization probing at several points in the locus. A continuum of acetylation was observed, covering both genes and intergenic regions. Using the same probes, the generalized sensitivity to DNase I was mapped by monitoring the disappearance of intact genomic restriction fragments from Southern transfers. Close correspondence between the 33 kb of sensitive chromatin and the extent of acetylation indicates that one role of the modification could be the generation and/or maintenance of the open conformation. The precision of acetylation mapping makes it a possible approach to the definition of chromosomal domain boundaries.

Histone acetylation and gene induction in human cells.

An antibody recognising acetylated core histones was used to immunoprecipitate chromatin fragments from proliferating human K562 cells and from cells induced to differentiate with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). The DNA of the acetylated chromatin was probed with sequences of platelet derived growth factor B chain (PDGF-B), a gene which is induced to strong expression upon differentiation. A high level of acetylation was observed before gene induction and no change seen following induction. This implies that core histone acetylation is an essential precondition for transcription.

Nucleosomal structure at hyperacetylated loci probed in nuclei by DNA-histone crosslinking.

Chemically induced histone-DNA crosslinking in nuclei is used to monitor structural changes in chromosomal domains containing hyperacetylated histones. Core particles harbouring the crosslinks are immunofractionated with antibodies specific for acetylated histones. Crosslinking is revealed by gel separation of tryptic peptides from core histones that carry 32P-labelled residual nucleotide. The large number of DNA-histone crosslinks retained indicates that acetylated core histone tails are not totally displaced from the DNA. Changes in the patterns of crosslinked peptides imply a restructuring of hyperacetylated histone-DNA interactions at several points within the nucleosome. This demonstrates that a distinct conformational state is adopted in acetylated nucleosomes, known to be concentrated at transcriptionally active loci.

Histone acetylation and globin gene switching.

An affinity-purified antibody that recognises the epitope epsilon-acetyl lysine has been used to fractionate chicken erythrocyte mononucleosomes obtained from 5 and 15 day embryos. The antibody bound chromatin was enriched in multiply acetylated forms of the core histones H3, H4 and H2B, but not in ubiquitinated H2A. The DNA of these modified nucleosomes was probed with genomic sequences from the embryonic beta rho gene (active at 5 days) and from the adult beta A gene (active at 15 days). Both genes were found to be highly enriched in the acetylated nucleosomes fractionated from both 5 day and from 15 day erythrocytes. We conclude that globin switching is not linked to a change in acetylation status of the genes and that a 'poised' gene carries histones acetylated to a similar level as a transcriptionally active gene. Core histone acetylation is not therefore a direct consequence of the transcriptional process and might operate at the level of the globin locus as a general enabling step for transcription.

A "minimal epitope" anti-protein antibody that recognises a single modified amino acid.

Antibodies that recognise proteins bind to epitopes of varying size, but a grouping of the order of six amino acids, contiguous or not, is regarded as a typical number. By using as immunogen a highly abundant and universal eukaryotic nuclear protein (histone H4) modified in a manner not typical of secreted proteins (acetylation of lysine side chains), antiserum has been raised in rabbits having the single amino acid epsilon-N-acetyl lysine as the recognition epitope. The affinity-purified antibody should be useful for studying the functional role of this modification. The methodology has potential for raising antibodies to other types of post-translationally modified proteins.

A direct link between core histone acetylation and transcriptionally active chromatin.

An antiserum raised against chemically acetylated histone H4 was found to recognize the epitope epsilon-N-acetyl lysine. Affinity-purified antibodies were used to fractionate oligo- and mononucleosomal chromatin fragments from the nuclei of 15-day chicken embryo erythrocytes. Antibody-bound chromatin was found to contain elevated levels of acetylated core histones. On probing with sequences of alpha D globin, an actively transcribed gene, the antibody-bound chromatin was 15- to 30-fold enriched relative to the input chromatin. Using ovalbumin sequences as a probe, no enrichment was observed. The results demonstrate directly that transcriptionally active genes carry acetylated core histones.