Histone H2A Gene Research Articles

Potentials and limitations of histone repeat sequences for phylogenetic reconstruction of Sophophora.

Simplified DNA sequence acquisition has provided many new data sets that are useful for phylogenetic reconstruction, including single- and multiple-copy nuclear and organellar genes. Although transcribed regions receive much attention, nontranscribed regions have recently been added to the repertoire of sequences suitable for phylogenetic studies, especially for closely related taxa. We evaluated the efficacy of a small portion of the histone repeat for phylogenetic reconstruction among Drosophila species. Histone repeats in invertebrates offer distinct advantages similar to those of widely used ribosomal repeats. First, the units are tandemly repeated and undergo concerted evolution. Second, histone repeats include both highly conserved coding and variable intergenic regions. This composition facilitates application of "universal" primers spanning potentially informative sites. We examined a small region of the histone repeat, including the intergenic spacer segments of coding regions from the divergently transcribed H2A and H2B histone genes. The spacer (about 230 bp) exists as a mosaic with highly conserved functional motifs interspersed with rapidly diverging regions; the former aid in alignment of the spacer. There are no ambiguities in alignment of coding regions. Coding and noncoding regions were analyzed together and separately for phylogenetic information. Parsimony, distance, and maximum-likelihood methods successfully retrieve the corroborated phylogeny for the taxa examined. This study demonstrates the resolving power of a small histone region which may now be added to the growing collection of phylogenetically useful DNA sequences.

The expression of human H2A–H2B histone gene pairs is regulated by multiple sequence elements in their joint promoters

The majority of human H2A and H2B histone genes are organized as gene pairs: 14 H2A–H2B gene pairs, one solitary H2A gene and three solitary H2B genes have been described. Two of the H2A genes and two of the H2B genes arranged within gene pairs are pseudogenes. The gene pairs are organized with divergent transcriptional orientation, and the coding regions of the respective H2A and H2B genes are separated by about 320 nucleotide pairs that form overlapping promoter regions. Comparison of promoters of H2A–H2B gene pairs has previously shown that these belong to two different groups (groups I and II) which are characterized by specific patterns of conserved sequence elements. We have constructed a reporter gene vector that allows the simultaneous analysis of both genes regulated by the divergent promoters belonging to group I or II, respectively. Firefly-luciferase and β-galactosidase genes were taken as reporter genes. Site directed mutagenesis performed at individual promoter elements revealed that individual sequence elements within both groups of promoters functionally depend on each other and may contribute to a coordinate expression of paired H2A and H2B genes through assembly of their joint promoter into a mutually dependent promoter complex. Group II promoters are characterized by the presence of an E2F binding site upstream of the H2A gene-proximal TATA box. Immediately upstream of the E2F element, we have identified a highly conserved octanucleotide CACAGCTT (RT-1) that exists in all human group II H2A–H2B gene promoters. Protein binding studies at the RT-1 element indicate factor binding to this sequence. Site directed mutagenesis indicates that both the E2F element and the RT-1 motif are essential for full promoter activity.

Regulation of the sea urchin early H2A histone gene expression depends on the modulator element and on sequences located near the 3' end.

Transcription of the sea urchin early histone genes occurs transiently during early cleavage, reaching the maximum at the morula stage and declining to an undetectable level at the gastrula stage. To identify the regulatory elements responsible for the timing and the levels of transcription of the H2A gene, we used promoter binding studies in nuclear extracts and microinjection of a CAT transgene driven by the early H2A promoter. We found that morula and gastrula nuclear proteins produced indistinguishable DNase I footprint patterns on the H2A promoter. Two sites of interactions, centred on the modulator/enhancer and on the CCAAT box respectively, were detected. Deletion of the modulator or coinjection of an excess of modulator sequences severely affected the expression of two transgenes driven by the enhancer-less and modulator-containing H2A promoter. Finally, a DNA fragment containing 3' coding and post-H2A spacer sequences, where upon silencing three micrococcal nuclease hypersensitive sites were previously mapped, specifically repressed at the gastrula stage the expression of the transgene driven by the H2A promoter. These results indicate that the modulator is essential for the expression of early H2A gene and that sequences for downregulation are localized near the 3' end of the H2A gene.

Male gametic cell-specific expression of H2A and H3 histone genes.

Formation of the generative cell in flowering plants initiates the male spermatogenesis pathway which eventually culminates in the process of double fertilization. Little is known about the molecular mechanisms operative in the generative cell. Here, we report the isolation and characterization of cDNA clones encoding generative cell-specific histones, gcH2A and gcH3. In situ hybridization analysis revealed that both genes are activated after formation of the generative cell and accumulation of transcripts increases progressively during generative cell maturation. These results suggest that gcH2A and gcH3 mRNAs are products of transcriptional activation of the generative cell nucleus and thus provide the first evidence for male germ line cell-specific gene expression in flowering plants.

Molecular analysis and chromosome mapping of the H2A, H3 and H4 histone genes from the malaria vector Anopheles gambiae

In this article we report the cloning and analysis of PCR generated fragments that encode H2A, H3 and H4 histone genes from the malaria vector An. gambiae. Sequence analysis indicated that some conservative changes are present in the An. gambiae H2A and H4 genes as compared with histone genes from other organisms. Divisional mapping showed that these genes map in division 20 on the left arm of the second chromosome. Southern blot experiments and the molecular characterization of the genomic fragment containing the H2A, H2B, H3 and H4 genes showed that they are organized in a cluster with an orientation different from the one found in other dipterans.

Control mechanisms of the H2A genes expression in Trypanosoma cruzi

In a previous report we have described that the T. cruzi histone H2A gene is encoded in two independent gene clusters located in a single chromosome. In the present paper we show that both gene cluster are actively transcribed as two sized classes of polyadenylated mRNAs demonstrating, moreover, the existence of alternative splicing sites and microheterogeneities at the polyadenylation site. We also describe that while the expression of the H2A genes in the non replicative trypomastigote forms is only residual, in the replicative forms there is constitutive transcription of these genes and that the transcription is not associated to DNA replication. The data show, moreover, that in the replicative forms the steady state levels of the H2A mRNAs are controlled at a post-transcriptional level which is associated to DNA replication.

The mouse histone H2a gene contains a small element that facilitates cytoplasmic accumulation of intronless gene transcripts and of unspliced HIV-1-related mRNAs.

Histone mRNAs are naturally intronless and accumulate efficiently in the cytoplasm. To learn whether there are cis-acting sequences within histone genes that allow efficient cytoplasmic accumulation of RNAs, we made recombinant constructs in which sequences from the mouse H2a gene were cloned into a human beta-globin cDNA. By using transient transfection and RNase protection analysis, we demonstrate here that a 100-bp sequence within the H2a coding region permits efficient cytoplasmic accumulation of the globin cDNA transcripts. We also show that this sequence appears to suppress splicing and can functionally replace Rev and the Rev-responsive element in the cytoplasmic accumulation of unspliced HIV-1-related mRNAs. Like the Rev-responsive element, this sequence acts in an orientation-dependent manner. We thus propose that the sequence identified here may be a member of the cis-acting elements that facilitate the cytoplasmic accumulation of naturally intronless gene transcripts.

Centrifugation does not alter spatial distribution of `BEP4' mRNA in paracentrotus lividus EGG

Paracentrotus lividus unfertilized eggs were centrifuged in a sucrose gradient, so to split each into two parts: a nucleated light fragment and an anucleated heavy fragment. Northern blot analyses utilizing a bep4 probe as animal marker and H2A histone gene and 12S-mit RNA as controls indicate that the eggs are elongated along the animal-vegetal axis during centrifugation and thereafter split into an animal and a vegetal half. Treatment of the eggs with colchicine before centrifugation abolishes the animal localization of bep4 mRNA.

Enhancer blocking activity located near the 3' end of the sea urchin early H2A histone gene.

The sea urchin early histone repeating unit contains one copy of each of the five histone genes whose coordinate expression during development is regulated by gene-specific elements. To learn how within the histone repeating unit a gene-specific activator can be prevented to communicate with the heterologous promoters, we searched for domain boundaries by using the enhancer blocking assay. We focused on the region near the 3' end of the H2A gene where stage-specific nuclease cleavage sites appear upon silencing of the early histone genes. We demonstrated that a DNA fragment of 265 bp in length, defined as sns (for silencing nucleoprotein structure), blocked the enhancer activity of the H2A modulator in microinjected sea urchin embryos only when placed between the enhancer elements and the promoter. We also found that sns silenced the modulator elements even when placed at 2.7 kb from the promoter. By contrast, the enhancer activity of the modulator sequences, located downstream to the coding region, was not affected when sns was positioned in close proximity to the promoter. Finally, the H2A sns fragment placed between the simian virus 40 regulative region and the tk promoter repressed chloramphenicol acetyltransferase expression in transfected human cell lines. We conclude that 3' end of the H2A gene contains sequence elements that behave as functional barriers of enhancer function in the enhancer blocking assay. Furthermore, our results also indicate that the enhancer blocking function of sns lacks enhancer and species specificity and that it can act in transient assays.

Gene expression during early embryogenesis of sea urchin: the histone and homeobox genes

Summary Transcriptional regulators are thought to play a key role in cell fate determination and territorial specification in sea urchin. Our goals are to clone transcription factors for studying embryonic development. One approach has been to use promoter binding and gene transfer technology to investigate the mechanisms of transcriptional activation and repression of the early H2A histone gene. By this analysis we identified a transcriptional activator, the MBF-1, that binds to the modulator element of the H2A gene and enhances the activity of the H2A promoter. However, the enhancer activity of the modulator and its interaction with MBF-1 also occurs at the gastrula stage when the early histone genes are shut off. Therefore, the silencing of the early H2A histone gene at late stages of development requires the inactivation of the modulator function. To search for antimodulator sequence elements, we took advantage of our previous work showing the presence of phased nucleosomes specifically positioned on the 3′-spacer and in the modulator of the repressed H2A gene. Evidence is described indicating that a 3′-spacer DNA fragment cloned between the modulator and the basal promoter behaves as an antimodulator element. However, this element does not confer temporal capability to the modulator in its function, suggesting that other elements have to be involved in the regulation of the early H2A expression. The second approach relied on the cloning of genes controlling development using probes of regulators known to be involved in regional specification, such as the homeobox genes, with the aim to understand their possible role through the study of their temporal and territorial expression and through the analysis of the mechanism of regulation of their expression. We present evidence that PIHbox 12, a homeodomain encoding gene, is transiently expressed during the early/mid cleavage stages. The abundance of the transcripts reach their maximum in embryos at the 64/128-cell stage concomitantly with the segregation of the specified embryonic territories. Expression of PIHbox l2 is drastically reduced in the absence of cell contacts and/or Ca ions, suggesting that this gene is transcriptionally activated by signal transduction mechanisms. Whole mount in situ hybridization showed that PIHbox 12 transcripts are asymmetrically distributed along the A-V axis, being spatially localized in the blastomeres of the ectodermal lineage. We suggest that PIHbox l2 might be involved in the initial events that lead to the specification of the ectodermal territories.

An H3 coding region regulatory element is common to all four nucleosomal classes of mouse histone-encoding genes

We have previously identified the α element within the mouse H2A and H3 histone gene coding region activating sequences (CRAS). This common element is required for normal in vivo expression of these two replication-dependent genes and interacts with nuclear factor(s). Here we report that the CRAS α element is present in the coding region sequences of two other replication-dependent mouse H genes, H2B and H4. The DNA-protein interactions were examined by DNase I footprinting and methylation-interference assays, and are very similar, if not identical, for these replication-dependent genes, confirming that the α element is the binding site for common nuclear protein(s) in H genes of all four nucleosomal classes. Moreover, we show that the same nuclear factor is involved in these DNA-protein interactions. Our findings, together with the fact that a replication-independent H gene, H3.3, has a mutated α element that fails to interact with nuclear proteins, suggest that this regulatory element is involved in the coordinate expression of the replication-dependent core H genes in the eukaryotic cell cycle.

Characterization of the 55-kb mouse histone gene cluster on chromosome 3.

The histone gene cluster on mouse chromosome 3 has been isolated as a series of overlapping P1 clones, covering 110-120 kb, by probing with the histone H3-614 gene that had been mapped previously to mouse chromosome 3. There are genes for 10 core histone proteins present in a 55-kb cluster within this contig. There are three histone H3 genes, two of which are identical; four histone H2a genes, two of which are identical, one histone H4 gene; and two histone H2b genes. These histone H3 and H2a genes encode approximately 40% of the total H3 and H2a mRNA, whereas the histone H4 and histone H2b genes encode < 10% of the total H4 and H2b mRNA. There are no histone H1 genes present in this cluster. All of the histone H2a genes encode histone H2a.2 proteins (or variants of H2a.2), and account for all the H2a.2 genes in the mouse genome. All three histone H3 genes encode the histone H3.2 protein. A 21-kb region containing the adjacent H3-614 and H2a-614 genes has been duplicated and is present in an inverted repeat separated by 4.5 kb. The other two H2a genes are adjacent, with the 3' ends of their mRNAs separated by only 49 nucleotides in the DNA and the U7 snRNP binding sites separated by only 20 nucleotides. One of the histone H2b genes has lost the stem-loop sequence characteristic of the replication-dependent histone mRNAs and encodes only polyadenylated mRNAs.

Cloning and characterization of the major histone H2A genes completes the cloning and sequencing of known histone genes of Tetrahymena thermophila.

A truncated cDNA clone encoding Tetrahymena thermophila histone H2A2 was isolated using synthetic degenerate oligonucleotide probes derived from H2A protein sequences of Tetrahymena pyriformis. The cDNA clone was used as a homologous probe to isolate a truncated genomic clone encoding H2A1. The remaining regions of the genes for H2A1 (HTA1) and H2A2 (HTA2) were then isolated using inverse PCR on circularized genomic DNA fragments. These partial clones were assembled into intact HTA1 and HTA2 clones. Nucleotide sequences of the two genes were highly homologous within the coding region but not in the noncoding regions. Comparison of the deduced amino acid sequences with protein sequences of T. pyriformis H2As showed only two and three differences respectively, in a total of 137 amino acids for H2A1, and 132 amino acids for H2A2, indicating the two genes arose before the divergence of these two species. The HTA2 gene contains a TAA triplet within the coding region, encoding a glutamine residue. In contrast with the T. thermophila HHO and HTA3 genes, no introns were identified within the two genes. The 5'- and 3'-ends of the histone H2A mRNAs; were determined by RNase protection and by PCR mapping using RACE and RLM-RACE methods. Both genes encode polyadenylated mRNAs and are highly expressed in vegetatively growing cells but only weakly expressed in starved cultures. With the inclusion of these two genes, T. thermophila is the first organism whose entire complement of known core and linker histones, including replication-dependent and basal variants, has been cloned and sequenced.

Gene activation by UV light, fungal elicitor or fungal infection in Petroselinum crispum is correlated with repression of cell cycle‐related genes

The effects of UV light or fungal elicitors on plant cells have so far been studied mostly with respect to defense-related gene activation. Here, an inverse correlation of these stimulatory effects with the activities of several cell cycle-related genes is demonstrated. Concomitant with the induction of flavonoid biosynthetic enzymes in UV-irradiated cell suspension cultures of parsley (Petroselinum crispum), total histone synthesis declined to about half the initial rate. A subclass of the histone H3 gene family was selected to demonstrate the close correlation of its expression with cell division, both in intact plants and cultured cells. Using RNA-blot and run-on transcription assays, it was shown that one arbitrarily selected subclass of each of the histone H2A, H2B, H3 and H4 gene families and of the genes encoding a p34cdc2 protein kinase and a mitotic cyclin were transcriptionally repressed in UV-irradiated as well as fungal elicitor-treated parsley cells. The timing and extent of repression differed between the two stimuli; the response to light was more transient and smaller in magnitude. These differential responses to light and elicitor were inversely correlated with the induction of phenylalanine ammonia-lyase, a key enzyme of phenylpropanoid metabolism. Essentially the same result was obtained with a defined oligopeptide elicitor, indicating that the same signaling pathway is responsible for defense-related gene activation and cell cycle-related gene repression. A temporary (UV light) or long-lasting (fungal elicitor) cessation of cell culture growth is most likely due to an arrest of cell division which may be a prerequisite for full commitment of the cells to transcriptional activation of full commitment of the cells to transcriptional activation of pathways involved in UV protection or pathogen defense. This conclusion is corroborated by the observation that the histone H3 mRNA level greatly declined around fungal infection sites in young parsley leaves.

The organization structure and regulatory elements of Chlamydomonas histone genes reveal features linking plant and animal genes.

The genome of the green alga Chlamydomonas reinhardtii contains approximately 15 gene clusters of the nucleosomal (or core) histone H2A, H2B, H3 and H4 genes and at least one histone H1 gene. Seven non-allelic histone gene loci were isolated from a genomic library, physically mapped, and the nucleotide sequences of three isotypes of each core histone gene species and one linked H1 gene determined. The core histone genes are organized in clusters of H2A-H2B and H3-H4 pairs, in which each gene pair shows outwardly divergent transcription from a short (< 300 bp) intercistronic region. These intercistronic regions contain typically conserved promoter elements, namely a TATA-box and the three motifs TGGCCAG-G(G/C)-CGAG, CGTTGACC and CGGTTG. Different from the genes of higher plants, but like those of animals and the related alga Volvox, the 3' untranslated regions contain no poly A signal, but a palindromic sequence (3' palindrome) essential for mRNA processing is present. One single H1 gene was found in close linkage to a H2A-H2B pair. The H1 upstream region contains the octameric promoter element GGTTGACC (also found upstream of the core histone genes) and two specific sequence motifs that are shared only with the Volvox H1 promoters. This suggests differential transcription of the H1 and the core histone genes. The H1 gene is interrupted by two introns. Unlike Volvox H3 genes, the three sequenced H3 isoforms are intron-free. Primer-directed PCR of genomic DNA demonstrated, however, that at least 8 of the about 15 H3 genes do contain one intron at a conserved position. In synchronized C. reinhardtii cells, H4 mRNA levels (representative of all core histone mRNAs) peak during cell division, suggesting strict replication-dependent gene control. The derived peptide sequences place C. reinhardtii core histones closer to plants than to animals, except that the H2A histones are more animal-like. The peptide sequence of histone H1 is closely related to the V. carteri VH1-II (66% identity). Organization of the core histone gene in pairs, and non-polyadenylation of mRNAs are features shared with animals, whereas peptide sequences and enhancer elements are shared with higher plants, assigning the volvocalean histone genes a position intermediate between animals and plants.

High level histone H2A gene expression during early stages of adventitious bud formation in Norway spruce (Picea abies)

We have demonstrated the correlation between cell division and the expression of a histone H2A‐encoding gene, His2A, in Norway spruce. Picea abies (L.) Karst and used a cDNA clone in in situ hybridization experiments to monitor the cytokinin‐induced cell division during early stages of adventitious bud development. A general stimulation of division of epidermal and cortical cells followed upon the cytokinin treatment. After two weeks in culture a high mitotic activity was detected only in single cells or small groups of cells in the epidermis and subepidermal cell layers. These cells presumably constitute the early stages of meristem primordia. The small clusters of dividing cells enlarge and subsequently form adventitious buds. Cells of the meristem and needle primordia of adventitious buds divide frequently as do the corresponding cells in vegetative buds. A quiescent center is distinguished within the apical meristem of vegetative buds. These cells, in the summit of the domed meristem, divide with a considerably lower frequency than cells in the flanking region. Differences in the temporal expression pattern of the histone H2A gene in cells of the vascular tissue, detected between embryos germinating in vitro and bud‐induced embryos, suggest that the cytokinin treatment affects the timing of cell divisions in the differentiating procambium.

High level histone H2A gene expression during early stages of adventitious bud formation in Norway spruce (Picea abies)

High level histone H2A gene expression during early stages of adventitious bud formation in Norway spruce (Picea abies)

Differential expression of the two types of histone H2A genes in wheat

Five histone H2A cDNA clones have been isolated from a wheat cDNA library. They were divided into two groups, termed type 1 and type 2, based on their deduced amino acid sequences and their gene expression patterns. Three type 1 clones had ORFs encoding proteins similar to angiosperm histone H2As known so far, whereas two type 2 clones encoded an identical protein, which was more similar to Norway spruce (gymnosperm) H2A than to the angiosperm H2As. The C-terminus of the type 2 H2A was shorter than that of the type 1 H2As and lacked the characteristic SPKK motif that is conserved in angiosperm H2As. Northern analysis revealed that the mRNA levels of the type 1 H2A genes were high in proliferating cells during germination and in various tissues of young seedlings, while the mRNA levels of the type 2 genes were high in non-proliferating cells in which the type 1 gene was poorly expressed. This result suggests that the expression of these two groups of H2A genes is differently regulated during development in wheat.

The coding sequences of mouse H2A and H3 histone genes contains a conserved seven nucleotide element that interacts with nuclear factors and is necessary for normal expression.

Expression of replication-dependent histone genes of all classes is up-regulated coordinately at the onset of DNA synthesis. The cellular signals involved in coordinate regulation of these genes are not known. Here we report identification of an alpha element, present within the mouse histone coding region activating sequence (CRAS). We show evidence that this element is present in histone genes from two classes, H2a and H3, in the mouse. This element has two biological functions in histone gene expression, i.e. the element interacts with nuclear proteins in regulation of gene expression, as well as encoding the amino acids of the histone proteins. We present both in vivo and in vitro evidence that interaction of nuclear proteins with this element is required for normal expression. The binding site for nuclear protein(s) has been precisely defined by means of synthetic oligonucleotides, as well as DNase I protection and methylation interference. It is interesting to note that the histone CRAS alpha element is mutated in a replication-independent H3.3 gene; 5 of 7 nt in the CRAS alpha box are changed in this gene.

RFLP analysis of preferential transmission in interspecific hybrids of Phaseolus vulgaris and P. coccineus.

The inheritance of RFLP markers in interspecific hybrids of Phaseolus vulgaris and P. coccineus was analyzed. Of 280 cDNA probes used, 70%-85% revealed polymorphism between species while intraspecific RFLP ranged from 8% to 37%. Segregation of 63 clearly scorable markers was examined in 177 P. vulgaris x P. coccineus F2's maintained as callus. Preferential transmission of the P. vulgaris alleles was observed for 24 of the 28 loci exhibiting non-Mendelian ratios. Although the segregation ratios at 17 loci fit gametic selection, also other factors such as nuclear-cytoplasmic or embryo-endosperm interactions may be involved. In the reciprocal F2, a relatively high frequency of maternal alleles was recovered for several loci, while the paternal allele was favored at others. The cDNA clone detecting the most extreme segregation, with no P. coccineus type detected among 165 P. vulgaris x P. coccineus F2 progeny, showed high homology to histone H2A genes. The markers were mapped to nine linkage groups. Aggregation of markers with preferential maternal transmission was observed, which could be due to selection of individual chromosomes, although false linkage detection cannot be excluded. The results obtained with RFLPs may explain the skewed distribution of phenotypic traits following interspecific hybridization.