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.     

The human histone gene cluster at the D6S105 locus

The sequences and organization of the histone genes in the histone gene cluster at the chromosomal marker D6S105 have been determined by analyzing the Centre d’étude du Polymorphisme Humain yeast artificial chromosome (YAC) 964f1. The insert of the YAC was subcloned in cosmids. In the established contig of the histone-gene-containing cosmids, 16 histone genes and 2 pseudogenes were identified: one H1 gene (H1.5), five H2A genes, four H2B genes and one pseudogene of H2B, three H3 genes, and three H4 genes plus one H4 pseudogene. The cluster extends about 80 kb with a nonordered arrangement of the histone genes. The dinucleotide repeat polymorphic marker D6S105 was localized at the telomeric end of this histone gene cluster. Almost all human histone genes isolated until now have been localized within this histone gene cluster and within the previously described region of histone genes, about 2 Mb telomeric of the newly described cluster or in a small group of histone genes on chromosome 1. We therefore conclude that the data presented here complete the set of human histone genes. This now allows the general organization of the human histone gene complement to be outlined on the basis of a compilation of all known histone gene clusters and solitary histone genes. Received: 30 June 1997 / Accepted: 3 September 1997     

The human H2A and H2B histone gene complement

Sequences and expression patterns of newly isolated human histone H2A and H2B genes and the respective proteins were compared with previously isolated human H2A and H2B genes and proteins. Altogether, 15 human H2A genes and 17 human H2B genes have been identified. 14 of these are organized as H2A/H2B gene pairs, while one H2A gene and three H2B genes are solitary genes. Two H2A genes and two H2B genes turned outto be pseudogenes. The 13 H2A genes code for at least 6 different amino acid sequences, and the 15 H2B genes encode 11 different H2B isoforms. Each H2A/H2B gene pair is controlled by a divergent promoter spanning 300 to 330 nucleotides between the coding regions of the two genes. The highly conserved divergent H2A/H2B promoters can be classified in two groups based on the patterns of consensus sequence elements. Group I promoters contain a TATA box for each gene, two Oct-1 factor binding sites, and three CCAAT boxes. Group II promoters contain the same elements as group I promoters and an additional CCAAT box, a binding motif for E2F and adjacent a highly conserved octanucleotide (CACAGCTT) that has not been described so far. Five of the 6 gene pairs and 4 solitary genes with group I promoters are localized in the large histone gene cluster at 6p21.3-6p22, and one gene pair is located at 1q21. All group II promoter associated genes are contained within the histone gene subcluster at D6S105, which is located at a distance of about 2 Mb from the major subcluster at 6p21.3-6p22 containing histone genes with group I promoters. Almost all group II H2A genes encode identical amino acid sequences, whereas group I H2A gene products vary at several positions. Using human cell lines, we have analyzed the expression patterns of functional human H2A/H2B gene pairs organized within the two histone gene clusters on the short arm of chromosome 6. The genes show varying expression patterns in different tumor cell lines.     

A New Family of “H3L-Like” Histone Genes

The H3L histone variant gene in Paracentrotus lividus (sea urchin) shows almost all typical features of the replication-dependent histone genes, but it codes for the H3.3 histone protein with the S.//. A.IG amino acid motif, which is typical of the variants synthesized in a replication-independent manner. H3L-like histone genes have been found in several unrelated organisms. These genes are intronless and encode for the typical H3.3 histone proteins. The newly described family of H3L-like variants, nearly ubiquitous within the animal kingdom, could represent the common ancestor of H3 and H3.3 histone genes.     

The uni chromosome of Chlamydomonas: histone genes and nucleosome structure.

The uni linkage group (ULG) of Chlamydomonas reinhardtii contains many genes involved in the basal body-flagellar system. Recent evidence suggests that the corresponding uni chromosome is located in close proximity to the basal body complex. In the course of studies into its molecular organization, we have found a cluster of four histone genes on the ULG. The genes are arranged as divergently-transcribed pairs: H3-H4 and H2B-H2A. Genomic sequencing reveals that these genes lack introns and contain characteristic 3' palindromes similar to those of animals. The predicted amino acid sequences are highly conserved across species, with greatest similarities to the histone genes of Volvox. Southern analysis shows that each histone gene is present in 15-20 copies in Chlamydomonas and suggests a dispersed genomic organization. Northern analysis of mitotically-synchronized cells shows that, like the replication-dependent histones of higher eukaryotes, Chlamydomonas histone genes are expressed during S-phase. Using a gene-specific probe on Northern blots, we provide evidence that the ULG H4 gene is regulated in the same manner as other Chlamydomonas histone genes. Finally, micrococcal nuclease protection experiments show that the uni chromosome itself associates with histone proteins and displays a conventional nucleosomal banding pattern.     

Isolation of two murine H1 histone genes and chromosomal mapping of the H1 gene complement

The mammalian H1 histone gene complement consists of at least seven H1 protein isoforms. These include five S-phase-dependent H1 protein subtypes and two more distantly related proteins, which are expressed upon terminal differentiation (H1⁰) or during the pachytene stage of spermatogenesis (H1t). In the past, three replication-dependent murine H1 genes plus the H1 ⁰ and H1t genes have been isolated and characterized. In this report, we describe the sequences of two more H1 genes, and we show that all five murine replication-dependent H1 genes and the H1t gene map to the region A2-3 on Chromosome (Chr) 13. This is in agreement with our previous finding that the human H1 histone gene complement maps to 6p21.3, which corresponds to the A2-3 region on the murine Chr 13. Previous reports have shown that the replication-independent H1 ⁰ genes map to syntenic regions on Chrs 22 (human H1⁰) and 15 (murine H1 ⁰).     

Evolution of histone gene loci in chironomid midges.

In the present study we have localized the histone genes in the chromosomes of 16 different Chironomus species as well as in Prodiamesa olivacea, Glyptotendipes barbipes, and Acricotopus lucidus. In the genus of Chironomus we find four, five, or six different "major" chromosomal loci hybridizing with a histone gene cluster probe isolated from the genome of Chironomus thummi. These major histone gene loci probably contain clustered histone gene repeating units ("clustered" loci). They are located on one and the same chromosome arm in all but one of the species investigated. This shows that the histone gene clusters are rather conservative in their location over a long period of evolution. The comparison of the histone loci pattern from the chromosomes of the different chironomid species shows that there is good agreement with previously established chromosome maps and phylogenetic studies based on the chromosomal banding pattern. Stringent in situ hybridization with various histone gene containing clones suggest that the "clustered" histone gene loci are organized in a locus-specific way. In addition to the linked "clustered" histone gene loci, we found an isolated histone gene group ("orphon") present on chromosome IV in most Chironomus species. This gene group might be organized differently from the histone gene repeating unit described previously.     

Single-copy flanking sequences in human histone gene clusters map to chromosomes 1 and 6

Two single-copy sequences flanking two different human histone gene clusters were used as probes to map these clusters by in situ hybridization. pFF435B, a unique sequence subclone derived from a lambda genomic clone (lambda HHG55) containing H2A, H2B, H3, and H4 genes, mapped to chromosome 1q21 (chi 2 = 120.99, P less than 0.001). pST519E, a single-copy sequence derived from a lambda genomic clone (lambda HHG17) containing only H3 and H4 genes, mapped to chromosome 6p21 (chi 2 = 112.62, P less than 0.001). These findings agree with previous assignments of human histone genes to chromosomes 1 and 6 and demonstrate that the single-copy flanking sequences in different human histone gene clusters are unique for different chromosomes.     

Testis-Specific Expression of a Novel Human H3 Histone Gene

We have investigated the expression of a recently described, solitary human H3 histone gene. Using RNase protection assays, the corresponding mRNA could only be detected in RNA preparations from human testis, whereas several human cell lines and somatic tissues did not exhibit expression of this gene.In situhybridization of sections from human testis revealed expression to be confined to primary spermatocytes. In addition to H1t, this novel H3 gene, which is located on chromosome 1, is the second tissue-specific human histone gene that has been found to be expressed solely in the testis.     

Histone gene number and organisation in Xenopus: Xenopus borealis has a homogeneous major cluster

Using a Xenopus laevis H4 cDNA clone as a probe we have determined that the numbers of H4 histone genes in Xenopus laevis and Xenopus borealis are approximately the same. These numbers are dependent on the hybridization stringency and we measure about 90 H4 genes per haploid genome after a 60 degrees C wash in 3 X SSC. Using histone probes from both Xenopus and sea urchin we have studied the genomic organization of histone genes in these two species. In all of the X.borealis individuals analyzed about 70% of the histone genes were present in a very homogeneous major cluster. These genes are present in the order H1, H2B, H2A, H4 and H3, and the minimum length of the repeated unit is 16kb. In contrast, the histone gene clusters in X.laevis showed considerable sequence variation. However two major cluster types with different gene orders seem to be present in most individuals. The differences in histone gene organization seen in species of Xenopus suggest that even in closely related vertebrates the major histone gene clusters are quite fluid structures in evolutionary terms.     

Conserved organization of an avian histone gene cluster with inverted duplications of H3 and H4 genes

Summary The organization of histone gene clusters of the duckCairina moschata was studied in the DNA inserts of two recombinant phage that overlap and feature identical histone gene arrangements but differ in sequence details and in the extent of repetition of an AT-rich motif in one of the nontranscribed spacer regions. These few but substantial differences between otherwise nearly identical histone gene groups suggest that we have independently isolated alleles of the same site of the duck genome or that this gene arrangement occurs (with slight variations) more than once per haploid genome. Within the histone gene cluster described, H3 and H4 genes are duplicated (with inverted orientation), whereas one H1 gene is flanked by single H2A and H2B genes. The arrangement of duck histone genes described here is identical to a subsection of the chicken genome but differs from any other published histone gene cluster.     

<Emphasis Type="Italic">Mytilus edulis</Emphasis> Core Histone Genes Are Organized in Two Clusters Devoid of Linker Histone Genes

Abstract Comparison of histone gene cluster arrangements in several species has revealed a broad spectrum of histone gene patterns. To elucidate the core histone gene organization in a mollusk, we have analyzed a Mytilus edulis genomic library and have isolated eight phage clones containing core histone genes. Analysis of insert DNA revealed that the core histone genes are arranged as regular gene repeats of all four core histones. The repeats do not contain linker histone genes. The clones are distributed into two groups of dissimilar repeated units with a common size of about 5.6 kb. The genes of each core histone class in the distinct repeats encode identical histone proteins and have comparable gene arrangements in the two repeat units. However, the intergenic sequences differ significantly. The core histone genes are organized as large clusters of about 100 repeats each. Previously, we have shown that the linker histone genes in M. edulis are also organized in a cluster of repeats of solitary H1 genes. Hence, this is the first case of a separate, clustered organization of both core and linker histone genes, repectively.     

Increasing the complexity of chromatin: functionally distinct roles for replication-dependent histone H2A isoforms in cell proliferation and carcinogenesis

Replication-dependent histones are encoded by multigene families found in several large clusters in the human genome and are thought to be functionally redundant. However, the abundance of specific replication-dependent isoforms of histone H2A is altered in patients with chronic lymphocytic leukemia. Similar changes in the abundance of H2A isoforms are also associated with the proliferation and tumorigenicity of bladder cancer cells. To determine whether these H2A isoforms can perform distinct functions, expression of several H2A isoforms was reduced by siRNA knockdown. Reduced expression of the HIST1H2AC locus leads to increased rates of cell proliferation and tumorigenicity. We also observe that regulation of replication-dependent histone H2A expression can occur on a gene-specific level. Specific replication-dependent histone H2A genes are either up- or downregulated in chronic lymphocytic leukemia tumor tissue samples. In addition, discreet elements are identified in the 5′ untranslated region of the HIST1H2AC locus that confer translational repression. Taken together, these results indicate that replication-dependent histone isoforms can possess distinct cellular functions and that regulation of these isoforms may play a role in carcinogenesis.     

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.