Regulation of myofibroblast transdifferentiation by DNA methylation and MeCP2: implications for wound healing and fibrogenesis.

Myofibroblasts are critical cellular elements of wound healing generated at sites of injury by transdifferentiation of resident cells. A paradigm for this process is conversion of hepatic stellate cells (HSC) into hepatic myofibroblasts. Treatment of HSC with DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-azadC) blocked transdifferentiation. 5-azadC also prevented loss of IkappaBalpha and PPARgamma expression that occurs during transdifferentiation to allow acquisition of proinflammatory and profibrogenic characteristics. ChIP analysis revealed IkappaBalpha promoter is associated with transcriptionally repressed chromatin that converts to an active state with 5-azadC treatment. The methyl-CpG-binding protein MeCP2 which promotes repressed chromatin structure is selectively detected in myofibroblasts of diseased liver. siRNA knockdown of MeCP2 elevated IkappaBalpha promoter activity, mRNA and protein expression in myofibroblasts. MeCP2 interacts with IkappaBalpha promoter via a methyl-CpG-dependent mechanism and recruitment into a CBF1 corepression complex. We conclude that MeCP2 and DNA methylation exert epigenetic control over hepatic wound healing and fibrogenesis.

Targeted and extended acetylation of histones H4 and H3 at active and inactive genes in chicken embryo erythrocytes.

Affinity-purified polyclonal antibodies recognizing the most highly acetylated forms of histones H3 and H4 were used in immunoprecipitation assays with chromatin fragments derived from 15-day chicken embryo erythrocytes by micrococcal nuclease digestion. The distribution of hyperacetylated H4 and H3 was mapped at the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and the tissue-specific gene, carbonic anhydrase (CA). H3 and H4 acetylation was found targeted to the CpG island region at the 5' end of both these genes, falling off in the downstream direction. In contrast, at the beta(A)-globin gene, both H3 and H4 are highly acetylated throughout the gene and at the downstream enhancer, with a maximum at the promoter. Low level acetylation was observed at the 5' end of the inactive ovalbumin gene. Run-on assays to measure ongoing transcription showed that the GAPDH and CA genes are transcribed at a much lower rate than the adult beta(A)-globin gene. The extensive high level acetylation at the beta(A)-globin gene correlates most simply with its high rate of transcription. The targeted acetylation of histones H3 and H4 at the GAPDH and CA genes is consistent with a role in transcriptional initiation and implies that transcriptional elongation does not necessarily require hyperacetylation.

A human genomic library enriched in transcriptionally active sequences (aDNA library).

Core histone hyperacetylation, in particular of H4, is concentrated in the promoter-upstream regions of active genes and in certain cases is locuswide. Antibodies to hyperacetylated H4 were used to immunoprecipitate dinucleosomal chromatin derived from K562 human erythroleukemic cells by micrococcal nuclease digestion. The extracted DNA was made into a genomic library and was expected to contain sequences from genes active in K562 cells (an active, 'aDNA' library). Clones (180) were randomly selected from the library; 24 of 103 tested (23%) contained highly repeated sequences, as determined by their hybridization to total genomic DNA, and were not analyzed further. An additional 10 clones (6%) were shown to contain no insert DNA. The remaining 146 were sequenced and compared with the nucleic acid databases and in all six frames to the protein databases: Sixeen clones could be assigned to known genes, the majority of which (12) were tissue specific. All but 2 of these 16 corresponded to segments 5' of the coding sequences, as expected if H4 acetylation is concentrated at promoter regions. Thirty-three clones (23%) displayed high sequence identity to cDNAs in the expressed sequence tag database (dbEST). Northern blots and reverse transcription (RT)-PCR were used to determine the proportion of clones representing sequences expressed in K562 cells: Although only 1 of 34 tested clones showed a band in Northern hybridization, RT-PCR demonstrated that at least 12 of 40 tested clones (30%) were present in the mRNA population. Because a further 8 of these 40 clones were identified as gene fragments by database sequence comparisons, it follows that about half of this subset of 40 clones is derived from genes. The aDNA library is thus very gene rich and not skewed toward the most highly expressed sequences, as in mRNA libraries. The aDNA library is also rich in promoters and could be a valuable source of such sequences, particularly those that lack CpG islands or other features that allow their specific selection.

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.

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.

Patterns of histone acetylation.

The N-terminal domains of all four core histones are subject to reversible acetylation at certain lysine residues. This modification has been functionally linked to transcription, histone deposition at replication and to histone removal during spermatogenesis. To increase understanding of the significance of this modification we have studied the specificity of site utilisation in the monoacetyl, diacetyl and triacetyl forms of histones H3, H4 and H2B (histone H2A has only a single modification site), using pig thymus and HeLa cells as the source of histones. The HeLa histones were extracted from cells grown both with and without butyrate treatment. It is found that for histone H3 there is a fairly strict order of site occupancy: Lys14, followed by Lys23, followed by Lys18 in both pig and HeLa histones. Since the order and specificity is the same when butyrate is added to the HeLa cell cultures, we conclude that addition of the fatty acid does not scramble the specificity of site utilisation, but merely allows more of the natural forms of modified histone to accumulate. For histone H4, the monoacetyl form is exclusively modified at Lys16, but further addition of acetyl groups is less specific and progresses through sites 12, 8 and 5 in an N-terminal direction. Similar results were obtained for H4 from both pig thymus and butyrate-treated HeLa cells. Histone H2B could be studied in detail only from butyrate-treated HeLa cells and a much lower level of site specificity was found: sites 12 and 15 were preferred to the more N- and C-terminal sites at Lys5 and Lys20. The data reinforces the view that lysine acetylation in core histones is a very specific phenomenon that plays several functionally distinct roles. The high degree of site specificity makes it unlikely that the structural effects of acetylation are mediated merely by a generalised reduction of charge in the histone N-terminal domains.

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.

The structure of ubiquitinated histone H2B.

Ubiquitinated histone H2B (uH2B) has been purified from both calf and pig thymus by exclusion chromatography in 7 M urea. Digestion of uH2B with Staphylococcus aureus V8 protease yielded the peptide 114-125 containing the ubiquitin moiety. Further digestion of this peptide with trypsin removed the ubiquitin and three H2B residues from the N-terminus. Edman degradations of both peptides established that ubiquitin is attached to the epsilon-amino group of lysine 120 in both calf and pig uH2B by an iso-peptide bond to the C-terminal glycine 76 of ubiquitin.

Proteins from the sperm of the bivalve mollusc Ensis minor. Co-existence of histones and a protamine-like protein.

Analysis of the total protein of the mature sperm of the bivalve mollusc Ensis minor (razor shell) using gel electrophoresis, amino acid analysis, nuclear magnetic resonance, circular dichroism and trypsin digestion, show it to contain all five histones plus a protamine-like protein. The histones H3, H4 and probably H2A are similar to those from calf thymus or sea urchin sperm, but the putative H2B appears to have a very high molecular mass (about 20 kDa). The histone H1 molecule is unusual, having little or no proline and 8-10 residues of histidine. The protamine-like species is rich in both lysine as well as arginine and is of much higher molecular mass than fish sperm protamines. Nucleosomes containing the four core histones have been prepared and the nucleosomal repeat shown to be 200 +/- 5 base pairs. Checks for the absence of contaminating cells reinforce the conclusion that a histone-containing nucleosomal structure co-exists with a protamine-like protein in this sperm chromatin.

Effect of HMG protein 17 on the thermal stability of control and acetylated HeLa oligonucleosomes.

Many studies have implicated histone acetylation and HMG proteins 14 and 17 in the structure of active chromatin. Studies of the binding of HMG 14 and 17 to chromatin core particles have shown that there are two binding sites for HMG 14 or 17 located within 20-25 bp of the DNA ends of the core particles [13-15]. Such binding sites may result from the free DNA ends in the core particle being available for the binding of HMG 14 and 17. We have studied the effects of the binding of HMG 17 on the thermal denaturation of DNA in mono, di and trinucleosomes. In each case the binding of 1 HMG 17 molecule per nucleosome reduces the DNA premelt region by 50%, while the binding of 2 HMG 17 molecules per nucleosome abolishes the premelt region. From this it is concluded that there are two HMG 17 binding sites per nucleosome which are located between the entry and exit points to the nucleosome and the strongly complexed central DNA region. Highly acetylated mono, di and trinucleosomes have been isolated from butyrate treated HeLa S3 cells. For this series of acetylated oligonucleosomes, it has been found that there are also two HMG 17 binding sites per acetylated nucleosome.

Thermal denaturation studies of acetylated nucleosomes and oligonucleosomes.

The thermal melting behaviors of control and acetylated mononucleosomes, dinucleosomes and trinucleosomes have been studied. Along each series of oligonucleosomes, the melting profiles change in a manner consistent with the increasing number of nucleosomes. For the control mononucleosome, the melting profile exhibits a premelting region at about 61-64 degrees C and a major cooperative transition at 75-77 degrees C. The melting profiles of the control dinucleosomes and trinucleosomes show a premelt at 61-62 degrees C (similar to that of the nucleosome core); an intermediate transition at 73-74 degrees C for the dinucleosome and at 76-77 degrees C for the trinucleosome and a major cooperative transition at 79-80 degrees C for the dinucleosome and at 81-82 degrees C for the trinucleosome. The major cooperative transition at the highest melting temperatures in the melting profiles of the mononucleosome, dinucleosome and trinucleosome comes from the melting of the central region of DNA in the nucleosome which complexed with the core histones; the premelt region is attributed to two DNA segments per nucleosome which flank this central DNA region and are free or weakly complexed with histones. The origin of the intermediate transition found for the dinucleosomes and trinucleosomes is not fully understood but probably results from the melting of DNA at the entry to and exit from the nucleosome and the linker DNA which are complexed with histones. A very similar pattern of behavior is observed for the acetylated oligonucleosomes. Direct comparison of the melting profiles of acetylated and control mononucleosomes, dinucleosomes and trinucleosomes show that the premelt region is unaffected by histone acetylation whereas the intermediate and major cooperative transitions for the acetylated oligonucleosomes are broader and occur consistently at lower temperatures than for the controls. These differences support proposals that the N-terminal regions of core histones interact within the nucleosome and on linker DNA.