Histone H1 Genes and Histone Gene Clusters in the Genus Drosophila

Whereas the genomes of many organisms contain several nonallelic types of linker histone genes, one single histone H1 type is known in Drosophila melanogaster that occurs in about 100 copies per genome. Amplification of H1 gene sequences from genomic DNA of wild type strains of D. melanogaster from Oregon, Australia, and central Africa yielded numerous clones that all exhibited restriction patterns identical to each other and to those of the known H1 gene sequence. Nucleotide sequences encoding the evolutionarily variable domains of H1 were determined in two gene copies of strain Niamey from central Africa and were found to be identical to the known H1 sequence. Most likely therefore, the translated sequences of D. melanogaster H1 genes do not exhibit intragenomic or intergenomic variations. In contrast, three different histone H1 genes were isolated from D. virilis and found to encode proteins that differ remarkably from each other and from the H1 of D. melanogaster and D. hydei. About 40 copies of H1 genes are organized in the D. virilis genome with copies of core histone genes in gene quintets that were found to be located in band 25F of chromosome 2. Another type of histone gene cluster is present in about 15 copies per genome and contains a variable intergenic sequence instead of an H1 gene. The H1 heterogeneity in D. virilis may have arisen from higher recombination rates than occur near the H1 locus in D. melanogaster and might provide a basis for formation of different chromatin subtypes. Received: 2 March 2000 / Accepted: 1 June 2000     

Evolution of Histone H4 and H3 Genes in Different Ciliate Lineages

The histones H4 are known as highly conserved proteins. However, in ciliates a high degree of variation was found compared both to other eukaryotes and between the ciliate species. To date, only H4 histones of species belonging to two distantly related classes have been investigated. In order to obtain more detailed information on histone H4 variation in ciliates we undertook a comprehensive sequence analysis of PCR-amplified internal H4 fragments from 12 species belonging to seven out of the nine currently recognized ciliate classes. In addition, we used PCR primers to amplify longer fragments of H3 and H4 genes including the intergenic region. The encoded amino acid sequences reveal a high number of differences when compared with those of other eukaryotes and the ciliate species investigated. Furthermore, in some species H4 gene variants were detected, which result in amino acid differences. The greatest number of substitutions and insertions found was in the amino terminal region of the H4 histones. However, all sequences possess a conserved region corresponding to those of all other eukaryotic H4 histones. The histone gene variations were used to reconstruct phylogenetic relationships. The tree from our data matches perfectly with the ribosomal RNA data: The heterotrichs, which were considered as a late branching lineage, diverge at the base of the ciliate tree and groups formerly thought to represent ancestral lineages now appear as highly derived ciliates. Received: 4 April 1997 / Accepted: 1 August 1997     

Mytilus edulis Histone Gene Clusters Containing Only H1 Genes

We isolated five different phage clones containing histone gene clusters with up to five H1 genes per phage clone from a Mytilus edulis genomic library. Among these H1 genes, nine gene types coding for five different H1 proteins have been identified. All H1 histone genes were located on repetitive restriction fragments with only slightly different sizes. The H1 coding regions show highly related sequences, suggesting that the multitude of H1 genes has evolved by gene duplication events. Core histone genes could not be found on these five Mytilus edulis genome fragments. Received: 28 July 1998 / Accepted: 17 May 1999     

Comparative Analysis of Evolution in a Rodent Histone H2a Pseudogene

Sequences were obtained from five species of rodents that are orthologous to an H2a histone pseudogene from Mus musculus. The pseudogene is part of the cluster of replication-dependent histone genes found on Mus musculus chromosome 13. Comparative analysis of these five sequences together with the previously published sequence from M. musculus shows that this gene has likely been a pseudogene throughout the evolution of the genus Mus, while the gene from Rattus norvegicus is likely functional. Three large (>20 bp) deletions were found among the Mus pseudogenes, a feature that is very unusual compared to surveys of processed pseudogenes. In addition, there are two single-base deletions and one 4-bp insertion among the Mus pseudogenes. The species distributions of one of the large deletions and the 4-bp insertion require either independent insertions of an identical sequence, independent deletions with identical boundaries, or a deletion followed by precise reintegration of the original sequence. The evidence favors the hypothesis of multiple deletions with identical boundaries. The ``coding' regions of the Mus pseudogenes show a much reduced level of among-species variability in the 3′ half of the pseudogene, compared both to the 5′ half and to flanking sequences. This supports a hypothesis that the 3′ end of the pseudogene is the target of frequent gene conversion by functional H2a genes. Received: 1 April 1997 / Accepted: 12 June 1997     

Common features of analogous replacement histone H3 genes in animals and plants

Phylogenetic analysis of histone H3 protein sequences demonstrates the independent origin of the replacement histone H3 genes in animals and in plants. Multiple introns in the replacement histone H3 genes of animals in a pattern distinct from that in plant replacement H3 genes supports this conclusion. It is suggested that replacement H3 genes arose at the same time that, independently, multicellular forms of animals and of plants evolved. Judged by the degree of invariant and functionally constrained amino acid positions, histones H3 and H4, which form together the tetramer kernel of the nucleosome, have co-evolved with equal rates of sequence divergence. Residues 31 and 87 in histone H3 are the only residues that consistently changed across each gene duplication event that created functional replacement histone H3 variant forms. Once changed, these residues have remained invariant across divergent speciation. This suggests that they are required to allow replacement histone H3 to participate in the assembly of nucleosomes in non–S-phase cells. The abundant occurrence of polypyrimidine sequences in the introns of all replacement H3 genes, and the replacement of an intron by a polypyrimidine motif upstream of the alfalfa replacement H3 gene, suggests a function. It is speculated that they may contribute to the characteristic cell-cycle-independent pattern of replacement histone H3 genes by binding nucleosome-excluding proteins.     

Histone H2A Ubiquitination Inhibits the Enzymatic Activity of H3 Lysine 36 Methyltransferases

Histone H3 lysine 27 (H3K27) methylation and H2A monoubiquitination (ubH2A) are two closely related histone modifications that regulate Polycomb silencing. Previous studies reported that H3K27 trimethylation (H3K27me3) rarely coexists with H3K36 di- or tri-methylation (H3K36me2/3) on the same histone H3 tails, which is partially controlled by the direct inhibition of the enzymatic activity of H3K27-specific methyltransferase PRC2. By contrast, H3K27 methylation does not affect the catalytic activity of H3K36-specific methyltransferases, suggesting other Polycomb mechanism(s) may negatively regulate the H3K36-specific methyltransferase(s). In this study, we established a simple protocol to purify milligram quantities of ubH2A from mammalian cells, which were used to reconstitute nucleosome substrates with fully ubiquitinated H2A. A number of histone methyltransferases were then tested on these nucleosome substrates. Notably, all of the H3K36-specific methyltransferases, including ASH1L, HYPB, NSD1, and NSD2 were inhibited by ubH2A, whereas the other histone methyltransferases, including PRC2, G9a, and Pr-Set7 were not affected by ubH2A. Together with previous reports, these findings collectively explain the mutual repulsion of H3K36me2/3 and Polycomb modifications.     

Molecular Evolution of the Aldo-keto Reductase Gene Superfamily

The aldo-keto reductase enzymes comprise a functionally diverse gene family which catalyze the NADPH-dependant reduction of a variety of carbonyl compounds. The protein sequences of 45 members of this family were aligned and phylogenetic trees were deduced from this alignment using the neighbor-joining and Fitch algorithms. The branching order of these trees indicates that the vertebrate enzymes cluster in three groups, which have a monophyletic origin distinct from the bacterial, plant, and invertebrate enzymes. A high level of conservation was observed between the vertebrate hydroxysteroid dehydrogenase enzymes, prostaglandin F synthase, and ρ-crystallin of Xenopus laevis. We infer from the phylogenetic analysis that prostaglandin F synthase may represent a recent recruit to the eicosanoid biosynthetic pathway from the hydroxysteroid dehydrogenase pathway and furthermore that, in the context of gene recruitment, Xenopus laevisρ-crystallin may represent a shared gene. Received: 26 August 1996 / Accepted: 5 June 1997     

Phosphorylation of Histone H2A.X by DNA-dependent Protein Kinase Is Not Affected by Core Histone Acetylation,but It Alters Nucleosome Stability and Histone H1 Binding

Phosphorylation of the C-terminal end of histone H2A.X is the most characterized histone post-translational modification in DNA double-stranded breaks (DSB). DNA-dependent protein kinase (DNA-PK) is one of the three phosphatidylinositol 3 kinase-like family of kinase members that is known to phosphorylate histone H2A.X during DNA DSB repair. There is a growing body of evidence supporting a role for histone acetylation in DNA DSB repair, but the mechanism or the causative relation remains largely unknown. Using bacterially expressed recombinant mutants and stably and transiently transfected cell lines, we find that DNA-PK can phosphorylate Thr-136 in addition to Ser-139 both in vitro and in vivo. Furthermore, the phosphorylation reaction is not inhibited by the presence of H1, which in itself is a substrate of the reaction. We also show that, in contrast to previous reports, the ability of the enzyme to phosphorylate these residues is not affected by the extent of acetylation of the core histones. In vitro assembled nucleosomes and HeLa S3 native oligonucleosomes consisting of non-acetylated and acetylated histones are equally phosphorylated by DNA-PK. We demonstrate that the apparent differences in the extent of phosphorylation previously observed can be accounted for by the differential chromatin solubility under the MgCl₂ concentrations required for the phosphorylation reaction in vitro. Finally, we show that although H2A.X does not affect nucleosome conformation, it has a de-stabilizing effect that is enhanced by the DNA-PK-mediated phosphorylation and results in an impaired histone H1 binding.     

Histone acetyltransferase-1 regulates integrity of cytosolic histone H3-H4 containing complex

Amounts of soluble histones in cells are tightly regulated to ensure supplying them for the newly synthesized DNA and preventing the toxic effect of excess histones. Prior to incorporation into chromatin, newly synthesized histones H3 and H4 are highly acetylated in pre-deposition complex, wherein H4 is di-acetylated at Lys-5 and Lys-12 residues by histone acetyltransferase-1 (Hat1), but their role in histone metabolism is still unclear. Here, using chicken DT 40 cytosolic extracts, we found that histones H3/H4 and their chaperone Asf1, including RbAp48, a regulatory subunit of Hat1 enzyme, were associated with Hat1. Interestingly, in HAT1-deficient cells, cytosolic histones H3/H4 fractions on sucrose gradient centrifugation, having a sedimentation coefficient of 5–6S in DT40 cells, were shifted to lower molecular mass fractions, with Asf1. Further, sucrose gradient fractionation of semi-purified tagged Asf1-complexes showed the presence of Hat1, RbAp48 and histones H3/H4 at 5–6S fractions in the complexes. These findings suggest the possible involvement of Hat1 in regulating cytosolic H3/H4 pool mediated by Asf1-containing cytosolic H3/H4 pre-deposition complex.     

Unexpected Histone H3 Tail-clipping Activity of Glutamate Dehydrogenase

Clipping of histone tails has been reported in several organisms. However, the significance and regulation of histone tail clipping largely remains unclear. According to recent discoveries H3 clipping has been found to be involved in regulation of gene expression and chromatin dynamics. Earlier we had provided evidence of tissue-specific proteolytic processing of histone H3 in White Leghorn chicken liver nuclei. In this study we identify a novel activity of glutamate dehydrogenase (GDH) as a histone H3-specific protease in chicken liver tissue. This protease activity is regulated by divalent ions and thiol-disulfide conversion in vitro. GDH specifically clips H3 in its free as well as chromatin-bound form. Furthermore, we have found an inhibitor that inhibits the H3-clipping activity of GDH. Like previously reported proteases, GDH too may have the potential to regulate/modulate post-translational modifications of histone H3 by removing the N-terminal residues of the histone. In short, our findings identify an unexpected proteolytic activity of GDH specific to histone H3 that is regulated by redox state, ionic concentrations, and a cellular inhibitor in vitro.     

Replication Orientation Affects the Rate and Direction of Bacterial Gene Evolution

In many bacterial genomes, the leading and lagging strands have different skews in base composition; for example, an excess of guanosine compared to cytosine on the leading strand. We find that Chlamydia genes that have switched their orientation relative to the direction of replication, for example by inversion, acquire the skew of their new ``host' strand. In contrast to most evolutionary processes, which have unpredictable effects on the sequence of a gene, replication-related skews reflect a directional evolutionary force that causes predictable changes in the base composition of switched genes, resulting in increased DNA and amino acid sequence divergence. Received: 27 April 2000 / Accepted: 1 August 2000