Isolation and characterization of a mouse fully replication-dependent H1 gene within a genomic cluster of core histone genes

We have used an oligonucleotide complementary to a sequence coding for the conserved central globular domain of H1s to screen a mouse genomic library for H1 genes. We then used a series of universal histone oligonucleotides to identify five different H1 genes which were linked to core histone genes. We characterized one of the H1 genes which was linked to an H2a, an H2b, an H3, and an H4 histone gene. This characterization involved: 1) sequencing of the coding region of the gene and several hundred base pairs of flanking region. 2) Comparison of this sequence to other H1 sequences from other organisms. This sequence analysis clearly showed that the gene coded for an H1 and identified H1 consensus sequences in the 5'- and 3'-flanking region. 3) Mapping of the 5'- and 3'-ends of the mRNA complementary to this gene by S1 nuclease analysis. 4) Identifying this gene and an adjacent H3 gene as being of the fully replication-dependent expression class, by measuring changes in the steady state levels of their mRNAs in the presence of hydroxyurea and during differentiation of murine erythroleukemia cells.     

Structure and organization of the chicken H2B histone gene family.

The results of Southern blotting experiments confirm that the chicken H2B histone gene family contains eight highly homologous members. One or two more sequences which are considerably divergent from the others appear to exist in the chicken genome. Seven of the eight H2B genes have been cloned and sequenced. All seven genes fall in two histone gene clusters, but no common arrangement exists for the clusters themselves. Three different H2B protein variants are encoded by these seven genes. The nucleotide sequence homology among the genes within their coding sequences appears to exceed that required for the corresponding protein sequences, suggesting that histone H2B mRNA sequence and structure are both selected during evolution. An analysis of the 5' flanking sequence data reveals that these genes possess CCAAT and TATA boxes, elements commonly associated with genes transcribed by RNA polymerase II. In addition, these genes all share an H2B-specific element of the form: ATTTGCATA. The 3' sequences of these genes contain the hyphenated symmetrical dyad homology and downstream purine-rich sequence shared by histone genes in general.     

Nucleotide sequences of Caenorhabditis elegans core histone genes. Genes for different histone classes share common flanking sequence elements

We have determined the nucleotide sequence of core histone genes and flanking regions from two of approximately 11 different genomic histone clusters of the nematode Caenorhabditis elegans. Four histone genes from one cluster (H3, H4, H2B, H2A) and two histone genes from another (H4 and H2A) were analyzed. The predicted amino acid sequences of the two H4 and H2A proteins from the two clusters are identical, whereas the nucleotide sequences of the genes have diverged 9% (H2A) and 12% (H4). Flanking sequences, which are mostly not similar, were compared to identify putative regulatory elements. A conserved sequence of 34 base-pairs is present 19 to 42 nucleotides 3' of the termination codon of all the genes. Within the conserved sequence is a 16-base dyad sequence homologous to the one typically found at the 3' end of histone genes from higher eukaryotes. The C. elegans core histone genes are organized as divergently transcribed pairs of H3-H4 and H2A-H2B and contain 5' conserved sequence elements in the shared spacer regions. One of the sequence elements, 5' CTCCNCCTNCCCACCNCANA 3', is located immediately upstream from the canonical TATA homology of each gene. Another sequence element, 5' CTGCGGGGACACATNT 3', is present in the spacer of each heterotypic pair. These two 5' conserved sequences are not present in the promoter region of histone genes from other organisms, where 5' conserved sequences are usually different for each histone class. They are also not found in non-histone genes of C. elegans. These putative regulatory sequences of C. elegans core histone genes are similar to the regulatory elements of both higher and lower eukaryotes. The coding regions of the genes and the 3' regulatory sequences are similar to those of higher eukaryotes, whereas the presence of common 5' sequence elements upstream from genes of different histone classes is similar to histone promoter elements in yeast.     

The human and mouse replication-dependent histone genes

The multigene family encoding the five classes of replication-dependent histones has been identified from the human and mouse genome sequence. The large cluster of histone genes, HIST1, on human chromosome 6 (6p21-p22) contains 55 histone genes, and Hist1 on mouse chromosome 13 contains 51 histone genes. There are two smaller clusters on human chromosome 1: HIST2 (at 1q21), which contains six genes, and HIST3 (at 1q42), which contains three histone genes. Orthologous Hist2 and Hist3 clusters are present on mouse chromosomes 3 and 11, respectively. The organization of the human and mouse histone genes in the HIST1 cluster is essentially identical. All of the histone H1 genes are in HIST1, which is spread over about 2 Mb. There are two large gaps (>250 kb each) within this cluster where there are no histone genes, but many other genes. Each of the histone genes encodes an mRNA that ends in a stemloop followed by a purine-rich region that is complementary to the 5' end of U7 snRNA. In addition to the histone genes on these clusters, only two other genes containing the stem-loop sequence were identified, a histone H4 gene on human chromosome 12 (mouse chromosome 6) and the previously described H2a.X gene located on human chromosome 11. Each of the 14 histone H4 genes encodes the same protein, and there are only three histone H3 proteins encoded by the 12 histone H3 genes in each species. In contrast, both the mouse and human H2a and H2b proteins consist of at least 10 non-allelic variants, making the complexity of the histone protein complement significantly greater than previously thought.     

Mouse histone H2A and H2B genes: four functional genes and a pseudogene undergoing gene conversion with a closely linked functional gene.

The sequence of five mouse histone genes, two H2a and three H2b genes on chromosome 13 has been determined. The three H2b genes all code for different proteins, each differing in two amino acids from the others. The H2b specific elements present 5' to H2b genes from other species are present in all three mouse H2b genes. All three H2b genes are expressed in the same relative amounts in three different mouse cell lines and fetal mice. The H2b gene with the H2b specific sequence closest to the TATAA sequence is expressed in the highest amount. One of the H2a genes lacks the first 9 amino acids, the promoter region, the last 3 amino acids and contains an altered 3' end sequence. Despite these multiple defects, there is only one nucleotide change between the two H2a genes from codon 9 to 126. This indicates that a recent gene conversion has occurred between these two genes. The similarity of the nucleotide sequences in the coding regions of mouse histone genes is probably due to gene conversion events targeted precisely at the coding region.     

Vertebrate histone genes: nucleotide sequence of a chicken H2A gene and regulatory flanking sequences.

The DNA sequence of a chicken genomal fragment containing a histone H2A gene has been determined. It contains extensive 5' and 3' flanking regions and encodes a protein identical in sequence to the histone H2A protein isolated from chicken erythrocytes. In the 5' flanking region, a possible "TATA box" and three possible "cap sites" can be recognised upstream from the initiation codon. To the 5' side of the "TATA box" is found an unusual sequence of 21 A's interrupted by a central G residue. It occupies the same relative position as the P. miliaris H2A gene-specific 5' dyad symmetry sequence and the "CCAAT box" seen in other eukaryotic polymerase II genes but is clearly different from both. A significant feature of the 3' non-coding region is the presence of a 23 base-pair sequence that is nearly identical to a conserved region found in sea urchin histone genes. The coding region is extremely GC rich, with strong selection for these bases in the third position of codons. Not a single coding triplet ends in U. No intervening sequences were found in this gene.     

The sea urchin histone gene complement

The only eukaryotic mRNAs that are not polyadenylated are the replication-dependent histone mRNAs in metazoans. The sea urchin genome contains two sets of histone genes that encode non-polyadenylated mRNAs. One of these sets is a tandemly repeated gene cluster with a 5.6-kb repeat unit containing one copy of each of the five alpha-histone genes and is present as a single large cluster which spans over 1 Mb. There is a second set of genes, consisting of 39 genes, containing two histone H1 genes, 34 genes encoding core histone proteins (H2a, H2b, H3 and H4) and three genes expressed only in the testis. Unlike vertebrates where these genes are clustered, the sea urchin late histone genes, expressed in embryos, larvae and adults, are dispersed throughout the genome. There are also genes encoding polyadenylated histone mRNAs, which encode histone variants, including all variants found in other metazoans, as well as a unique set of five cleavage stage histone proteins expressed in oocytes. The cleavage stage histone H1 is the orthologue of an oocyte-specific histone H1 protein found in vertebrates.     

Structural characteristics of two wheat histone H2A genes encoding distinct types of variants and functional differences in their promoter activity

To investigate the regulation of plant histone H2A gene expression, we isolated two H2A genes (TH254 and TH274) from wheat, which encode two variants of H2A. Both genes had an intron in the coding region. In the promoters, some characteristic sequences, such as Oct and Nona motifs, which are conserved among plant histone genes, were located in a short region (about 120 bp) upstream from the putative TATA box. Transient expression analyses of promoter activity with H2A–GUS fusion genes using tobacco protoplasts revealed novel types of positive cis/-acting sequences in the TH254 promoter: a direct repeat of a 13 bp sequence (AGTTACATTATTG) and a stretch composed of an AT-rich sequence (ATATAGAAAATTAAAA) and a G-box (CACGTG). Quantitative S1 assay of the mRNA amounts from the TH254/GUS and TH274/GUS chimeric genes in stably transformed and cell cycle-synchronized tobacco cell lines showed that the promoters of both genes contained at least one cis/-acting element responsible for S phase-specific expression. Histochemical analysis of transgenic tobacco plants carrying the chimeric genes showed that the promoters of the two H2A genes were active in developing seedlings and flower organs but were regulated in a different manner.     

The human histone H3 complement anno 2011

Histones are highly basic, relatively small proteins that complex with DNA to form higher order structures that underlie chromosome topology. Of the four core histones H2A, H2B, H3 and H4, it is H3 that is most heavily modified at the post-translational level. The human genome harbours 16 annotated bona fide histone H3 genes which code for four H3 protein variants. In 2010, two novel histone H3.3 protein variants were reported, carrying over twenty amino acid substitutions. Nevertheless, they appear to be incorporated into chromatin. Interestingly, these new H3 genes are located on human chromosome 5 in a repetitive region that harbours an additional five H3 pseudogenes, but no other core histone ORFs. In addition, a human-specific novel putative histone H3.3 variant located at 12p11.21 was reported in 2011. These developments raised the question as to how many more human histone H3 ORFs there may be. Using homology searches, we detected 41 histone H3 pseudogenes in the current human genome assembly. The large majority are derived from the H3.3 gene H3F3A, and three of those may code for yet more histone H3.3 protein variants. We also identified one extra intact H3.2-type variant ORF in the vicinity of the canonical HIST2 gene cluster at chromosome 1p21.2. RNA polymerase II occupancy data revealed heterogeneity in H3 gene expression in human cell lines. None of the novel H3 genes were significantly occupied by RNA polymerase II in the data sets at hand, however. We discuss the implications of these recent developments.     

Isolation and characterization of two replication-dependent mouse H1 histone genes.

Mice contain at least seven nonallelic forms of the H1 histones, including the somatic variants H1a-e and less closely related variants H1 degrees and H1t. The mouse H1 degrees and H1c (H1var.1) genes were isolated and characterized previously. We have now isolated, sequenced and studied the expression properties of two additional mouse H1 genes, termed H1var.2 and H1var.3. Extensive amino acid and nucleotide sequence comparisons were made between the two genes and other mammalian H1 histone genes. A high degree of nucleotide sequence identity was seen between the H1var.2, rat H1d and human H1b genes, even well beyond the coding region, indicating that these genes are likely homologues. Unlike the previously characterized mouse H1var.1 gene which produces both nonpolyadenylated and polyadenylated mRNAs, the H1var.2 and H1var.3 genes produce only typical, replication dependent, nonpolyadenylated mRNAs.     

Isolation of a genomal clone containing chicken histone genes.

We have used enriched chicken histone cDNA to select genomal clones from a chicken library. Because the cDNA probe also contained other sequences, a further screening of positive plagues with negative probes eliminated most non-histone gene clones. One 'positively-selected' genomal clone, lambda CH-01, hybridised with cloned sea-urchin histone genes and also detected histone genes in EcoRI-digested genomal sea-urchin DNA. Limited DNA sequencing of HaeIII fragments identified two sequences within the coding region of chicken histone H2A. A third fragment predicted an amino acid sequence with strong homology to an H1 histone sequence.     

An H3-H4 histone gene pair in the marine copepod Tigriopus californicus, contains an intergenic dyad symmetry element.

Histone genes are one of the most widely studied multigene families in eucaryotes. Over 200 histone genes have been sequenced, primarily in vertebrates, echinoderms, fungi and plants. We present here the structure and genomic orientation of an H3-H4 histone gene pair from the marine copepod, Tigriopus californicus. These histone gene sequences are the first to be determined for the class Crustacea and among the first to be determined for protostomes. The H4 and H3 genes in Tigriopus are shown to be adjacent, to have opposite polarity, and to contain a 26 bp region of dyad symmetry centrally located within the spacer region between the two genes. A similarly located dyad element has been found in yeast which contributes to the coordinated cell cycle control of the adjacent histone genes. The Tigriopus H3-H4 histone gene pair is clustered with one H2A and two H2B histone genes on a 15 kb genomic Bam H1 fragment. The H4 gene sequence predicts an H4 protein with an unusual serine to threonine substitution at the amino terminal residue. The H3 gene sequence predicts an H3 protein which is identical to the vertebrate H3.2 histone.     

Organization of the histone H3 genes in soybean, barley and wheat

Several variants of the replacement histone H3 genes from soybean, barley and wheat have been cloned and sequenced. Analysis of segregating populations in barley and soybean, as well as analysis of clones isolated from a soybean genomic library, suggested that these genes are dispersed throughout the genome. Several genes contain introns located in similar positions, but of different lengths and sequence. Comparison of mRNA levels in different tissues revealed that the intron-containing and intronless genes have different expression patterns. The distribution of the introns in the histone H3 genes across several plant species suggests that some of the introns might have been lost during the evolution of the gene family. Sequence divergence among introns and gene-flanking sequences in cloned gene variants allowed us to use them as specific probes for localizing individual gene copies and analyzing the genomic distribution of these variants across a range of genotypes.Journal paper No. J-16127 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IowaMention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the United States Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable     

Nucleotide sequence and expression of two cDNA coding for two histone H2B variants of maize

The complete amino acid sequences of two variants of histone H2B of maize were deduced from the cDNAs isolated from a maize cDNA library. The two encoded proteins are 150 (H2B(1)) and 149 (H2B(2)) amino acids long and shows the classical organization of H2B histones. The hydrophobic C-terminal region is highly conserved as compared to that of the animal counterparts with only 21 changes (13 conservative) among the 90 residues. Between the N-terminal part and the C-terminal region we note the presence of a basic cluster (9 residues) characteristic of histones H2B. The N-terminal third is extended as compared to the animal consensus H2B and has the same size as the H2B histone of wheat. Up to 9 acidic residues and a five time repeated pentapeptide PA/KXE/KK are present in this region. Southern-blot hybrization showed that the H2B histones are encoded by a multigenic family like the other core histones (H3 and H4) of plants. The general expression pattern of these genes was not significantly different from that of the H3 and H4 genes neither in germinating seeds nor in different tissues of adult maize.