The histone H3 and H4 mRNAs are polyadenylated in maize

Northern blot analysis revealed that the histone H3 and H4 mRNAs are of unusual large size in germinating maize embryos. S1-mapping experiments show that the 3'-untranslated regions of the mRNAs transcribed from 3 H3 and 2 H4 maize genes previously described are much longer than in the non-polyadenylated histone mRNAs which represent a major class in animals. Moreover, oligo d(T) cellulose fractionation of RNAs isolated at different developmental stages indicates that more than 99% of the maize H3 and H4 mRNAs are polyadenylated. A putative polyadenylation signal is present in all five genes 17 to 27 nucleotides before the 3'-ends of the mRNAs.     

Cross-talk between histone modifications in response to histone deacetylase inhibitors: MLL4 links histone H3 acetylation and histone H3K4 methylation

Histones are subject to a wide variety of post-translational modifications that play a central role in gene activation and silencing. We have used histone modification-specific antibodies to demonstrate that two histone modifications involved in gene activation, histone H3 acetylation and H3 lysine 4 methylation, are functionally linked. This interaction, in which the extent of histone H3 acetylation determines both the abundance and the "degree" of H3K4 methylation, plays a major role in the epigenetic response to histone deacetylase inhibitors. A combination of in vivo knockdown experiments and in vitro methyltransferase assays shows that the abundance of H3K4 methylation is regulated by the activities of two opposing enzyme activities, the methyltransferase MLL4, which is stimulated by acetylated substrates, and a novel and as yet unidentified H3K4me3 demethylase.     

Trimethylation of histone H3 lysine 4 impairs methylation of histone H3 lysine 9

Chromatin is broadly compartmentalized in two defined states: euchromatin and heterochromatin. Generally, euchromatin is trimethylated on histone H3 lysine 4 (H3K4^me3) while heterochromatin contains the H3K9^me3 marks. The H3K9^me3 modification is added by lysine methyltransferases (KMTs) such as SETDB1. Herein, we show that SETDB1 interacts with its substrate H3, but only in the absence of the euchromatic mark H3K4^me3. In addition, we show that SETDB1 fails to methylate substrates containing the H3K4^me3 mark. Likewise, the functionally related H3K9 KMTs G9A, GLP, and SUV39H1 also fail to bind and to methylate H3K4^me3 substrates. Accordingly, we provide in vivo evidence that H3K9^me2-enriched histones are devoid of H3K4^me2/3 and that histones depleted of H3K4^me2/3 have elevated H3K9^me2/3. The correlation between the loss of interaction of these KMTs with H3K4^me3 and concomitant methylation impairment leads to the postulate that, at least these four KMTs, require stable interaction with their respective substrates for optimal activity. Thus, novel substrates could be discovered via the identification of KMT interacting proteins. Indeed, we find that SETDB1 binds to and methylates a novel substrate, the inhibitor of growth protein ING2, while SUV39H1 binds to and methylates the heterochromatin protein HP1α. Thus, our observations suggest a mechanism of post-translational regulation of lysine methylation and propose a potential mechanism for the segregation of the biologically opposing marks, H3K4^me3 and H3K9^me3. Furthermore, the correlation between H3-KMTs interaction and substrate methylation highlights that the identification of novel KMT substrates may be facilitated by the identification of interaction partners.     

Lanosterol synthase in dicotyledonous plants

Sterols are important as structural components of plasma membranes and precursors of steroidal hormones in both animals and plants. Plant sterols show a wide structural variety and significant structural differences from those of animals. To elucidate the origin of structural diversity in plant sterols, their biosynthesis has been extensively studied [Benveniste (2004) Annu. Rev. Plant. Biol. 55: 429, Schaller (2004) Plant Physiol. Biochem. 42: 465]. The differences in the biosynthesis of sterols between plants and animals begin at the step of cyclization of 2,3-oxidosqualene, which is cyclized to lanosterol in animals and to cycloartenol in plants. However, here we show that plants also have the ability to synthesize lanosterol directly from 2,3-oxidosqualene, which may lead to a new pathway to plant sterols. The Arabidopsis gene At3g45130, designated LAS1, encodes a functional lanosterol synthase in plants. A phylogenetic tree showed that LAS1 belongs to the previously uncharacterized branch of oxidosqualene cyclases, which differs from the cycloartenol synthase branch. Panax PNZ on the same branch was also shown to be a lanosterol synthase in a yeast heterologous expression system. The higher diversity of plant sterols may require two biosynthetic routes in steroidal backbone formation.     

Polyadenylated H3 histone transcripts and H3 histone variants in alfalfa

Histone H3 mRNAs were found in polyA(+) fractions of total RNA prepared from alfalfa plants, calli and somatic embryos. The sequence analysis of cDNAs revealed the presence of a polyA tail on independent alfalfa H3 mRNAs. A highly conserved sequence motif AAUGAAA identified about 20bp upstream from the 3' ends of the alfalfa H3 cDNAs was suggested to be one of the possible regulatory elements in the 3' end formation and polyadenylation. Three out of the four analysed H3 cDNAs have more than 97% homology with a genomic clone and encode the same protein. While the fourth represents a minor species with only 78.8% homology to the coding region of the genomic clone and encodes a H3 histone with four amino acid replacements. On the basis of compilation analysis we suggest a consensus sequence for plant H3 histones which differs from that of animal's by four amino acid changes.     

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.     

Variability and inheritance of histone genes H3 and H4 in Vicia faba

Summary We have compared copy numbers and blothybridization patterns of histone genes (H3 plus H4) between and within individuals of broad bean (Vicia faba). Copy number differences among individuals in the population of 200 individuals were as great as 27 fold, and as much as 3.2 fold among separate leaves of the same plant. Among F₂ progeny from genetic crosses, up to a 5.4-fold range was seen (mean=3.5 fold), and among F₁ progeny of self-pollinated plants, up to a 5.9-fold range was observed (mean=2.3 fold). Histone gene blot-hybridization patterns for EcoRI and HindIII were also variable among individuals and indicated that the genes are probably clustered in only a few chromosomal loci. The degree of variation in histone gene copy number per haploid genome (2–55 copies, or 27 fold) was similar to that found previously for ribosomal RNA genes (230–22000, or 95 fold) of V. faba. However, the two gene families change independently, since individuals with a high or low copy number for one gene can have either a high or low copy number for the other. The mechanisms(s) for rapid gene copy number change may be similar for these gene families.     

Stomatal characteristics of some dicotyledonous plants in relation to the C4 and C3 pathways of photosynthesis

A comparative study of some stomatal characteristics and transpirationalrates of several C₄ and C₃ dicotyledonous plants was made. Amongthe plants studied, C₄ species had higher stomatal frequencyin upper leaf surfaces (relative to their lower leaf surfaces)than the C₃ ones. The maximal stomatal opening was at 12 noonin C₄ plants but at 10 AM in C₃ plants. C₄ plants had lowerstomatal conductance and and higher water-use efficiency thanC₃ plants. (Received November 13, 1976; )     

Nucleotide sequences of two corn histone H3 genes. Genomic organization of the corn histone H3 and H4 genes

Summary Two histone H3 genes have been cloned from a gtWES.B corn genomic library. The nucleotide sequences show 96% homology and both encode the same protein, which differs from its counterpart in wheat and pea by one amino acid substitution. The 5-flanking regions of the two corn H3 genes contain the classical histone-gene-specific consensus sequences and possess several regions of extensive nucleotide homology. A conserved octanucleotide 5-CGCGGATC-3 occurs at approximately 200 nucleotides upstream from the initiation ATG codon. This octanucleotide was found to exist in all of the 7 plant histone genes sequenced so far. Codon usage is characterized by a very high frequency of C (67%) and G (28%) at the third position of the codons, those ending by A (1%) and T (4%) being practically excluded.Comparison of Southern blots of EcoRI, EcoRV and BamHI digested genomic DNA suggests that the corn H3 and H4 genes are not closely associated. The H3 genes exist as 60 to 80 copies and the H4 genes as 100 to 120 copies per diploid genome. re]19851002 rv]19851212 ac]19851216     

An auxin-induced polypeptide in dicotyledonous plants

Antisera were raised to a 70-kD (kilodalton) soybean (Glycine max) protein encoded by a 2,4-dichlorophenoxyacetic acid (2,4-D) inducible mRNA, GH3. These antisera have been used to probe protein blots to study the kinetics and specificity of the GH3 induction response as well as the species specificity and intracellular location of the protein. Detectable levels of the GH3 protein are induced by 2,4-D within 2 h in elongating hypocotyl sections, root sections, and etiolated plumules, and within 30–60 min in soybean suspension cells. Synthesis of the GH3 protein is induced by a variety of auxins. Other plant hormones such as gibberellic acid, cytokinin and ethylene added with or whithout 2,4-D do not alter the level of GH3 protein induction. The GH3 protein is found only in the S100 fraction and is not associated with the nucleus or cell wall. This antiserum also reacts with a 2,4-D-inducible 70-kD protein in other dicots.     

Simultaneous induction of postabscission and germination mRNAs in cultured dicotyledonous embryos

Cloned mRNAs identify three programs of gene expression in cotton (Gossypium hirsutum L.) embryos that are associated with the maturation (reserve accumulation) stage, the postabscission stage, which is marked by expression of Late-embryogenesis-abundant (Lea) mRNAs, and germination (broadly defined as including all events through early postgerminative growth). In order to test if the regulation of these programs is the same in other dicotyledonous species, their expression was studied in normal and cultured maturation-stage, postabscission-stage, and mature embryo-stage embryos or seed of oilseed rape (Brassica napus L.), soybean (Glycine max [L.] Merr.), and tobacco (Nicotiana tabacum L.) using cotton and other cDNA probes. During postabscission, Lea mRNAs accumulated in all test species and were induced in earlier maturation-stage embryos by excision and culture on basal medium. Abscisic acid often enhanced this induction in the test species. Germinationspecific mRNAs were induced in cultured maturationstage and postabscission-stage embryos of all test species. These results indicate that the regulation of embryonic and germination programs is similar in all dicotyledons tested. Because excised embryos simultaneously induced postabscission and germination programs, the effects of exogenous growth regulators and other factors on such embryos probably reflect stress responses of germinating mature embryos rather than the identity of endogenous regulators of embryogenesis.Abbreviations ABA abscisic acid - GA₃ gibberellic acid - DPA days postanthesis - Lea late embryogenesis abundant - MAT maturation stage - PA postabscission stage - ME mature embryo stage We thank J.J. Harada (Department of Botany, University of California, Davis, USA) and S.L. Berry-Lowe (Department of Biology, University of Colorado, Colorado Springs, USA) for plasmids. John E. Stacy is acknowledged for help with the Figures. This work was supported by grant GM29495 from the National Institute of Health to G.A.G and by individual research/travel grants from the Norwegian Agricultural Research Council (NLVF) to each of the authors.     

Cyclic hydroxamic acids in dicotyledonous plants

DIBOA (2a) was identified in Scoparia dulcis (Scrophulariaceae) and in six Acanthaceae species: Acanthus mollis, A. spinosus, Aphelandra aurantiaca, A. squarrosa, Crossandra infundibuliformis and C. pungens. Both Crossandra species contained 7-hydroxyDIBOA (2c), and both Aphelandra species contained its methyl ether, DIMBOA (2b). No hydroxamates were found in 28 species from 19 other genera of Acanthaceae.     

Methods for the analysis of histone H3 and H4 acetylation in blood

LBH589 is one of the many histone deacetylase inhibitors (HDACi) that are currently in clinical trial. Despite their wide-spread use, there is little literature available describing the typical levels of histone acetylation in untreated peripheral blood, the treatment and storage of samples to retain optimal measurement of histone acetylation nor methods by which histone acetylation analysis may be monitored and measured during the course of a patient’s treatment. In this study, we have used cord or peripheral blood as a source of human leukocytes, performed a comparative analysis of sample processing methods and developed a flow cytometric method suitable for monitoring histone acetylation in isolated lymphocytes and liquid tumors. Western blotting and immunohistochemistry techniques have also been addressed. We have tested these methods on blood samples collected from four patients treated with LBH589 as part of an Australian Children’s Cancer Clinical Trial (CLBH589AAU03T) and show comparable results when comparing in vitro and in vivo data. This paper does not seek to correlate histone acetylation levels in peripheral blood with clinical outcome but describes methods of analysis that will be of interest to clinicians and scientists monitoring the effects of HDACi on histone acetylation in blood samples in clinical trials or in related research studies.     

Polyadenylation state of abundant mRNAs during Drosophila development

We have used a two-dimensional gel analysis of cell-free translation products to determine whether individual mRNAs present in Drosophila melanogaster embryos, larvae, pupae, and adults are predominantly polyadenylated or nonadenylated. While the majority of the embryonic mRNAs we detected exist mainly in the polyadenylated form, these mRNAs become more evenly distributed between the poly(A)+ and poly(A)- RNA fractions during postembryonic development. Although DNA:RNA hybridization experiments have indicated that Drosophila RNA populations contain a large group of rare class mRNAs restricted to the poly(A)- RNA compartment, this is not true for the 150 more abundant mRNA species analyzed by our methods. The histone mRNAs are the only abundant mRNA species which appear to be exclusively in the poly(A)- RNA class.     

Methods for the analysis of histone H3 and H4 acetylation in blood

LBH589 is one of the many histone deacetylase inhibitors (HDACi) that are currently in clinical trial. Despite their wide-spread use, there is little literature available describing the typical levels of histone acetylation in untreated peripheral blood, the treatment and storage of samples to retain optimal measurement of histone acetylation nor methods by which histone acetylation analysis may be monitored and measured during the course of a patient’s treatment. In this study, we have used cord or peripheral blood as a source of human leukocytes, performed a comparative analysis of sample processing methods and developed a flow cytometric method suitable for monitoring histone acetylation in isolated lymphocytes and liquid tumors. Western blotting and immunohistochemistry techniques have also been addressed. We have tested these methods on blood samples collected from four patients treated with LBH589 as part of an Australian Children’s Cancer Clinical Trial (CLBH589AAU03T) and show comparable results when comparing in vitro and in vivo data. This paper does not seek to correlate histone acetylation levels in peripheral blood with clinical outcome but describes methods of analysis that will be of interest to clinicians and scientists monitoring the effects of HDACi on histone acetylation in blood samples in clinical trials or in related research studies.