Bettina Sarg

Postsynthetic trimethylation of histone H4 at lysine 20 in mammalian tissues is associated with aging.

Methylation of the N-terminal region of histones was first described more than 35 years ago, but its biological significance has remained unclear. Proposed functions range from transcriptional regulation to the higher order packing of chromatin in progress of mitotic condensation. Primarily because of the recent discovery of the SET domain-depending H3-specific histone methyltransferases SUV39H1 and Suv39h1, which selectively methylate lysine 9 of the H3 N terminus, this posttranslational modification has regained scientific interest. In the past, investigations concerning the biological significance of histone methylation were largely limited because of a lack of simple and sensitive analytical procedures for detecting this modification. The present work investigated the methylation pattern of histone H4 both in different mammalian organs of various ages and in cell lines by applying mass spectrometric analysis and a newly developed hydrophilic-interaction liquid chromatographic method enabling the simultaneous separation of methylated and acetylated forms, which obviates the need to work with radioactive materials. In rat kidney and liver the dimethylated lysine 20 was found to be the main methylation product, whereas the monomethyl derivative was present in much smaller amounts. In addition, for the first time a trimethylated form of lysine 20 of H4 was found in mammalian tissue. A significant increase in this trimethylated histone H4 was detected in organs of animals older than 30 days, whereas the amounts of mono- and dimethylated forms did not essentially change in organs from young (10 days old) or old animals (30 and 450 days old). Trimethylated H4 was also detected in transformed cells; although it was present in only trace amounts in logarithmically growing cells, we found an increase in trimethylated lysine 20 in cells in the stationary phase.

The Microheterogeneity of the Mammalian H10Histone EVIDENCE FOR AN AGE-DEPENDENT DEAMIDATION

Histone H10 is known to consist of two subfractions named H10a and H10b. The present work was performed with the aim of elucidating the nature of these two subfractions. By using reversed-phase high performance liquid chromatography in combination with hydrophilic interaction liquid chromatography, we fractionated human histone H10 into even four subfractions. Hydrophilic interaction liquid chromatographic analysis of the peptide fragments obtained after cleavage with cyanogen bromide and digestion with chymotrypsin suggested that the four H10 subfractions differ only in their small N-terminal end of the H10 molecule (30 residues). Edman degradation of the N-terminal H10 peptide fragments and mass spectra analysis have indicated that human histone H10 consists of intact histones H10 (named H10 Asn-3) and deamidated H10 forms (H10 Asp-3) having an aspartic acid residue at position 3 instead of asparagine. Moreover, both H10 Asn-3 and H10 Asp-3 are blocked (H10a Asn-3, H10a Asp-3) and unblocked (H10b Asn-3, H10b Asp-3) on their N terminus. Acid-urea gel electrophoretic analysis has shown that the histone subfraction, in the literature originally named H10a, actually consists of a mixture of H10a Asn-3 and H10a Asp-3, whereas H10b consists of H10b Asn-3 and H10b Asp-3. Furthermore, we found that hydrophilic interaction liquid chromatography separates rat and mouse histone H10 just like human H10 into four subfractions. Hydrophilic interaction liquid chromatographic analysis of brain and liver histone H10 from rats of different ages revealed an age-dependent increase of both the N-terminally acetylated and the deamidated forms of H10. In addition, we found that the relative proportions of the four forms of H10 histones differ from tissue to tissue.

Hyperphosphorylation of histone H2A.X and dephosphorylation of histone H1 subtypes in the course of apoptosis.

Chromatin condensation paralleled by DNA fragmentation is one of the most important nuclear events occurring during apoptosis. Histone modifications, and in particular phosphorylation, have been suggested to affect chromatin function and structure during both cell cycle and cell death. We report here that phosphate incorporation into all H1 subtypes decreased rapidly after induction of apoptosis, evidently causing a strong reduction in phosphorylated forms of main H1 histone subtypes. H1 dephosphorylation is accompanied by chromatin condensation preceding the onset of typical chromatin oligonucleosomal fragmentation, whereas H2A.X hyperphosphorylation is strongly correlated to apoptotic chromatin fragmentation. Using various kinase inhibitors we were able to exclude some of the possible kinases which can be involved directly or indirectly in phosphorylation of histone H2A.X. Neither DNA-dependent protein kinase, protein kinase A, protein kinase G, nor the kinases driven by the mitogen-activated protein kinase (MAP) pathway appear to be responsible for H2A.X phosphorylation. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA), however, markedly reduced the induction of apoptosis in TNFα-treated cells with a simultaneous change in the phosphorylation pattern of histone H2A.X. Hyperphosphorylation of H2A.X in apoptotic cells depends indirectly on activation of caspases and nuclear scaffold proteases as shown in zVAD-(OMe)-fmk- or zAPF-cmk-treated cells, whereas the dephosphorylation of H1 subtypes seems to be influenced solely by caspase inhibitors. Together, these results illustrate that H1 dephosphorylation and H2A.X hyperphosphorylation are necessary steps on the apoptotic pathway.

Substrate and sequential site specificity of cytoplasmic histone acetyltransferases of maize and rat liver

The cytoplasmic B‐type histone acetyltransferase was purified to apparent homogeneity from maize embryos. We established a novel protocol for easy large‐scale preparation of acetylated core histone species, using preparative acetic acid‐urea‐Triton PAGE. The pure maize histone acetyltransferase B was highly specific for histone H4 under various assay conditions, modifying H4 up to the di‐acetylated isoform. Only non‐acetylated H4 isoform was accepted as substrate, whereas mono‐acetylated H4 could not be further acetylated. The enzyme selectively acetylated lysines 12 and 5 in a sequential manner. The same results were obtained with a partially purified cytoplasmic histone acetyltransferase of rat liver. Protein sequencing results were supported by immunological characterization of acetylated H4 subspecies with site‐specific H4‐acetyllysine antibodies.

Regulation and Processing of Maize Histone Deacetylase Hda1 by Limited Proteolysis

A maize histone deacetylase gene was identified as a homolog of yeast Hda1. The predicted protein corresponds to a previously purified maize deacetylase that is active as a protein monomer with a molecular weight of 48,000 and is expressed in all tissues of germinating embryos. Hda1 is synthesized as an enzymatically inactive protein with an apparent molecular weight of 84,000 that is processed to the active 48-kD form by proteolytic removal of the C-terminal part, presumably via a 65-kD intermediate. The enzymatically inactive 84-kD protein also is part of a 300-kD protein complex of unknown function. The proteolytic cleavage of ZmHda1 is regulated during maize embryo germination in vivo. Expression of the recombinant full-length protein and the 48-kD form confirmed that only the smaller enzyme form is active as a histone deacetylase. In line with this finding, we show that the 48-kD protein is able to repress transcription efficiently in a reporter gene assay, whereas the full-length protein, including the C-terminal part, lacks full repression activity. This report on the processing of Hda1-p84 to enzymatically active Hda1-p48 demonstrates that proteolytic cleavage is a mechanism to regulate the function of Rpd3/Hda1-type histone deacetylases.