The Chromatin Remodelers PKL and PIE1 Act in an Epigenetic Pathway That Determines H3K27me3 Homeostasis in Arabidopsis.

Selective, tissue-specific gene expression is facilitated by the epigenetic modification H3K27me3 (trimethylation of lysine 27 on histone H3) in plants and animals. Much remains to be learned about how H3K27me3-enriched chromatin states are constructed and maintained. Here, we identify a genetic interaction in Arabidopsis thaliana between the chromodomain helicase DNA binding chromatin remodeler PICKLE (PKL), which promotes H3K27me3 enrichment, and the SWR1-family remodeler PHOTOPERIOD INDEPENDENT EARLY FLOWERING1 (PIE1), which incorporates the histone variant H2A.Z. Chromatin immunoprecipitation-sequencing and RNA-sequencing reveal that PKL, PIE1, and the H3K27 methyltransferase CURLY LEAF act in a common gene expression pathway and are required for H3K27me3 levels genome-wide. Additionally, H3K27me3-enriched genes are largely a subset of H2A.Z-enriched genes, further supporting the functional linkage between these marks. We also found that recombinant PKL acts as a prenucleosome maturation factor, indicating that it promotes retention of H3K27me3. These data support the existence of an epigenetic pathway in which PIE1 promotes H2A.Z, which in turn promotes H3K27me3 deposition. After deposition, PKL promotes retention of H3K27me3 after DNA replication and/or transcription. Our analyses thus reveal roles for H2A.Z and ATP-dependent remodelers in construction and maintenance of H3K27me3-enriched chromatin in plants.

The chromatin remodeler chd5 is necessary for proper head development during embryogenesis of Danio rerio.

The chromatin remodeler CHD5 plays a critical role in tumor suppression and neurogenesis in mammals. CHD5 contributes to gene expression during neurogenesis, but there is still much to learn regarding how this class of remodelers contributes to differentiation and development. CHD5 remodelers are vertebrate-specific, raising the prospect that CHD5 plays one or more conserved roles in this phylum. Expression of chd5 in adult fish closely mirrors expression of CHD5 in adult mammals. Knockdown of Chd5 during embryogenesis suggests new roles for CHD5 remodelers based on resulting defects in craniofacial development including reduced head and eye size as well as reduced cartilage formation in the head. In addition, knockdown of Chd5 results in altered expression of neural markers in the developing brain and eye as well as a profound defect in differentiation of dopaminergic amacrine cells. Recombinant zebrafish Chd5 protein exhibits nucleosome remodeling activity in vitro, suggesting that it is the loss of this activity that contributes to the observed phenotypes. Our studies indicate that zebrafish is an appropriate model for functional characterization of CHD5 remodelers in vertebrates and highlight the potential of this model for generating novel insights into the role of this vital class of remodelers.

PICKLE is a CHD subfamily II ATP-dependent chromatin remodeling factor.

PICKLE plays a critical role in repression of genes that regulate development identity in Arabidopsis thaliana. PICKLE codes for a putative ATP-dependent chromatin remodeler that exhibits sequence similarity to members of subfamily II of animal CHD remodelers, which includes remodelers such as CHD3/Mi-2 that also restrict expression of developmental regulators. Whereas animal CHD3 remodelers are a component of the Mi-2/NuRD complex that promotes histone deacetylation, PICKLE promotes trimethylation of histone H3 lysine 27 suggesting that it acts via a distinct epigenetic pathway. Here, we examine whether PICKLE is also a member of a multisubunit complex and characterize the biochemical properties of recombinant PICKLE protein. Phylogenetic analysis indicates that PICKLE-related proteins in plants share a common ancestor with members of subfamily II of animal CHD remodelers. Biochemical characterization of PICKLE in planta, however, reveals that PICKLE primarily exists as a monomer. Recombinant PICKLE protein is an ATPase that is stimulated by ssDNA and mononucleosomes and binds to both naked DNA and mononucleosomes. Furthermore, recombinant PICKLE exhibits ATP-dependent chromatin remodeling activity. These studies demonstrate that subfamily II CHD proteins in plants, such as PICKLE, retain ATP-dependent chromatin remodeling activity but act through a mechanism that does not involve the ubiquitous Mi-2/NuRD complex.

Evolution of I-SceI homing endonucleases with increased DNA recognition site specificity.

Elucidating how homing endonucleases undergo changes in recognition site specificity will facilitate efforts to engineer proteins for gene therapy applications. I-SceI is a monomeric homing endonuclease that recognizes and cleaves within an 18-bp target. It tolerates limited degeneracy in its target sequence, including substitution of a C:G(+4) base pair for the wild-type A:T(+4) base pair. Libraries encoding randomized amino acids at I-SceI residue positions that contact or are proximal to A:T(+4) were used in conjunction with a bacterial one-hybrid system to select I-SceI derivatives that bind to recognition sites containing either the A:T(+4) or the C:G(+4) base pairs. As expected, isolates encoding wild-type residues at the randomized positions were selected using either target sequence. All I-SceI proteins isolated using the C:G(+4) recognition site included small side-chain substitutions at G100 and either contained (K86R/G100T, K86R/G100S and K86R/G100C) or lacked (G100A, G100T) a K86R substitution. Interestingly, the binding affinities of the selected variants for the wild-type A:T(+4) target are 4- to 11-fold lower than that of wild-type I-SceI, whereas those for the C:G(+4) target are similar. The increased specificity of the mutant proteins is also evident in binding experiments in vivo. These differences in binding affinities account for the observed ∼36-fold difference in target preference between the K86R/G100T and wild-type proteins in DNA cleavage assays. An X-ray crystal structure of the K86R/G100T mutant protein bound to a DNA duplex containing the C:G(+4) substitution suggests how sequence specificity of a homing enzyme can increase. This biochemical and structural analysis defines one pathway by which site specificity is augmented for a homing endonuclease.

Involvement of snapdragon benzaldehyde dehydrogenase in benzoic acid biosynthesis.

Benzoic acid (BA) is an important building block in a wide spectrum of compounds varying from primary metabolites to secondary products. Benzoic acid biosynthesis from L-phenylalanine requires shortening of the propyl side chain by two carbons, which can occur via a beta-oxidative pathway or a non-beta-oxidative pathway, with benzaldehyde as a key intermediate. The non-beta-oxidative route requires benzaldehyde dehydrogenase (BALDH) to convert benzaldehyde to BA. Using a functional genomic approach, we identified an Antirrhinum majus (snapdragon) BALDH, which exhibits 40% identity to bacterial BALDH. Transcript profiling, biochemical characterization of the purified recombinant protein, molecular homology modeling, in vivo stable isotope labeling, and transient expression in petunia flowers reveal that BALDH is capable of oxidizing benzaldehyde to BA in vivo. GFP localization and immunogold labeling studies show that this biochemical step occurs in the mitochondria, raising a question about the role of subcellular compartmentalization in BA biosynthesis.

A point mutation produced a class 3 aldehyde dehydrogenase with increased protective ability against the killing effect of cyclophosphamide.

Cyclophosphamides are pro-drugs whose killing agent is produced from an aldehyde that is formed by the action of a P450 oxidation step. The mustard from the aldehyde can destroy bone marrow cells as well as the tumor. Aldehyde dehydrogenase (EC 1.2.1.3) can oxidize the aldehyde and hence inactivate the cytotoxic intermediate but bone marrow has little, if any, of the enzyme. Others have shown that over-expression of the enzyme can afford protection of the marrow. A T186S mutant of the human stomach enzyme (ALDH3) that we developed has increased activity against the aldehyde compared to the native enzyme and HeLa cells transformed with the point mutant are better protected against the killing effect of the drug. It took threefold more drug to kill 90% of the cells transformed with the mutant compared to the native enzyme (15.8 compared to 5.1mM of a precursor of the toxic aldehyde). Analysis of molecular models makes it appear that removing the methyl group of threonine in the T186S mutant allows the bulky aldehyde to bind better. The mutant was found to be a poorer enzyme when small substrates such as benzaldehyde derivatives were investigated. Thus, the enzyme appears to be better only with large substrates such as the one produced by cyclophosphamide.

MORPHOLOGICAL AND PHYLOGENETIC STUDIES ON UNICELLULAR DIAZOTROPHIC CYANOBACTERIA (CYANOPHYTES) ISOLATED FROM THE COASTAL WATERS AROUND SINGAPORE(1).

Six unicellular diazotrophic cyanobacteria were isolated from the coast around Singapore. The isolates grew under both light:dark (L:D) cycles and continuous illumination (CL) in media without combined nitrogen and exhibited an ability to fix nitrogen (as measured by acetylene reduction) under aerobic conditions. The cells of all isolates were surrounded by a thick fibrous outer wall layer, and they divided by transverse binary fission. The arrangement of photosynthetic thylakoids was of the dispersed type. Three isolates were identified as form-genus Gloeothece as cells were divided in a single plane, and the other three isolates were identified as form-genus Gloeocapsa as cells were divided in multiple planes. Phylogenetic analyses based on the DNA sequences of the genes encoding 16S rRNA and dinitrogenase reductase (nifH) revealed the following: (i) Our six isolates formed a monophyletic cluster. (ii) The monophyletic cluster was subdivided into two phylogenetic groups, which taxonomically corresponded with the form-genera Gloeothece and Gloeocapsa. However, (iii) a diazotrophic strain of form-genus Gloeothece, Gloeothece membranacea (Rabenh.) Bornet PCC6501, was not closely related to our isolates, and (iv) some, but not all, diazotrophic unicellular strains of form-genus Cyanothece were observed to be in a close relationship with our isolates.

Oxidative inhibition of the mitochondrial aldehyde dehydrogenase promotes nitroglycerin tolerance in human blood vessels.

OBJECTIVES: We tested the hypothesis of whether an inhibition of the nitroglycerin (GTN) bioactivating enzyme mitochondrial aldehyde dehydrogenase (ALDH-2) contributes to GTN tolerance in human blood vessels. BACKGROUND: The hemodynamic effects of GTN are rapidly blunted by the development of tolerance, a phenomenon associated with increased formation of reactive oxygen species (ROS). Recent studies suggest that ROS-induced inhibition of ALDH-2 accounts for tolerance in animal models. METHODS: Segments of surgically removed arteria mammaria and vena saphena from patients undergoing coronary bypass surgery were used to examine the vascular responsiveness to GTN and the endothelium-dependent vasodilator acetylcholine. The ALDH-2 activity and expression in these segments were assessed by the conversion of a benzaldehyde or its derivative to the benzoic acid metabolite and by Western blotting technique. RESULTS: In contrast to patients not treated with nitrates (n = 36), patients treated with GTN for 48 h (n = 14) before surgery showed tolerance to GTN and endothelial dysfunction in arterial and venous vessels. In vivo GTN tolerance was mimicked in vitro by incubation of nontolerant vessels with the ALDH-2 inhibitor benomyl. In vivo GTN treatment decreased vascular aldehyde dehydrogenase activity compared with nontolerant vessels and decreased the expression of ALDH-2 in arterial tissue. Incubation of control venous vessels with GTN caused a significant attenuation of aldehyde dehydrogenase activity that was reversed by presence of the sulfhydryl group donor dithiothreitol. CONCLUSIONS: Long-term GTN treatment induces tolerance and endothelial dysfunction in human vessels, associated with an inhibition and down-regulation of vascular ALDH-2. Thus, these findings extend results of previous animal studies to humans.

Enantioselective oxidation of trans-4-hydroxy-2-nonenal is aldehyde dehydrogenase isozyme and Mg2+ dependent.

trans-4-Hydroxy-2-nonenal (HNE) is a cytotoxic alpha,beta-unsaturated aldehyde implicated in the pathology of multiple diseases involving oxidative damage. Oxidation of HNE by aldehyde dehydrogenases (ALDHs) to trans-4-hydroxy-2-nonenoic acid (HNEA) is a major route of metabolism in many organisms. HNE exists as two enantiomers, (R)-HNE and (S)-HNE, and in intact rat brain mitochondria, (R)-HNE is enantioselectively oxidized to HNEA. In this work, we further elucidated the basis of the enantioselective oxidation of HNE by brain mitochondria. Our results showed that (R)-HNE is oxidized enantioselectively by brain mitochondrial lysates with retention of stereoconfiguration of the C4 hydroxyl group. Purified rat ALDH5A enantioselectively oxidized (R)-HNE, whereas rat ALDH2 was not enantioselective. Kinetic data using (R)-HNE, (S)-HNE, and trans-2-nonenal in combination with computer-based modeling of ALDH5A suggest that the selectivity of (R)-HNE oxidation by ALDH5A is the result of the carbonyl carbon of (R)-HNE forming a more favorable Bürgi-Duntiz angle with the active site cysteine 293. The presence of Mg2+ ions altered the enantioselectivity of ALDH5A and ALDH2. Mg2+ ions suppressed (R)-HNE oxidation by ALDH5A to a greater extent than that of (S)-HNE. However, Mg2+ ions stimulated the enantioselective oxidation of (R)-HNE by ALDH2 while suppressing (S)-HNE oxidation. These results demonstrate that enantioselective utilization of substrates, including HNE, by ALDHs is dependent upon the ALDH isozyme and the presence of Mg 2+ ions.

Selective alteration of the rate-limiting step in cytosolic aldehyde dehydrogenase through random mutagenesis.

Random mutagenesis followed by a filter-based screening assay has been used to identify a mutant of human class 1 aldehyde dehydrogenase (ALDH1) that was no longer inhibited by Mg(2+) ions but was activated in their presence. Several mutants possessed double, triple, and quadruple amino acid substitutions with a total of seven different residues being altered, but each had a common T244S change. This point mutation proved to be responsible for the Mg(2+) ion activation. An ALDH1 T244S mutant was recombinantly expressed and was used for mechanistic studies. Mg(2+) ions have been shown to increase the rate of deacylation. Consistent with the rate-limiting step for ALDH1 being changed from coenzyme dissociation to deacylation was finding that chloroacetaldehyde was oxidized more rapidly than acetaldehyde. Furthermore, Mg(2+) ions only in the presence of NAD(H) increased the rate of hydrolysis of p-nitrophenyl acetate showing that the metal only affects the binary complex. Though the rate-limiting step for the T244S mutant was different from that of the native enzyme, the catalytic efficiency of the mutant was just 20% that of the native enzyme. The basis for the change in the rate-limiting step appears to be related to NAD(+) binding. Using the structure of a sheep class 1 ALDH, it was possible to deduce that the interaction between the side chain of T244 and its neighboring residues with the nicotinamide ring of NAD(+) were an essential determinant in the catalytic action of ALDH1.

Differential effects of Mg2+ ions on the individual kinetic steps of human cytosolic and mitochondrial aldehyde dehydrogenases.

Although the structures of mammalian cytosolic and mitochondrial ALDH have been determined, several differences, mainly functional, between these two 70% identical isozymes remain unexplained. A major difference is the differential effect of Mg(2+) ions that inhibits the cytosolic and activates the mitochondrial isozyme. Here, we have investigated the effect of Mg(2+) ions on each individual kinetic step of ALDH1 and ALDH2. The metal ions were found not to affect either acylation or hydride transfer for either isozyme. The lack of a Mg(2+) ion effect on hydride transfer was further demonstrated with an E399Q mutant of ALDH1 whose rate-limiting step had been changed from NADH dissociation to hydride transfer. The other steps, however, were affected by Mg(2+) ions for both isozymes. The metal ions inhibited NADH dissociation, the rate-limiting step for ALDH1, and enhanced deacylation, the rate-limiting step for ALDH2. Our results indicated that, with both isozymes, Mg(2+) ions tightened the binding of NADH, and by binding to the coenzyme, they increased the nucleophilicity of the nucleophile Cys302. The inhibition of ALDH1 and activation of ALDH2 at pH 7.4 are due to their different rate-limiting steps. Mg(2+) ions affected similarly the NADH activation of the esterase reaction for both isozymes. In contrast, the metal ions affected only the NAD(+) activation of ALDH1. This latter finding and other features described here can be rationalized on the basis of the known three-dimensional structures of the isozymes.

Isolation and characterization of an aldehyde dehydrogenase encoded by the aldB gene of Escherichia coli.

An aldehyde dehydrogenase was detected in crude cell extracts of Escherichia coli DH5alpha. Growth studies indicated that the aldehyde dehydrogenase activity was growth phase dependent and increased in cells grown with ethanol. The N-terminal amino acid sequence of the purified enzyme identified the latter as an aldehyde dehydrogenase encoded by aldB, which was thought to play a role in the removal of aldehydes and alcohols in cells that were under stress. The purified enzyme showed an estimated molecular mass of 220 +/- 8 kDa, consisting of four identical subunits, and preferred to use NADP and acetaldehyde. MgCl2 increased the activity of the NADP-dependent enzyme with various substrates. A comparison of the effect of Mg2+ ions on the bacterial enzyme with the effect of Mg2+ ions on human liver mitochondrial aldehyde dehydrogenase revealed that the bacterial enzyme shared kinetic properties with the mammalian enzyme. An R197E mutant of the bacterial enzyme appeared to retain very little NADP-dependent activity on acetaldehyde.

The crystal structure of a cyanobacterial water-soluble carotenoid binding protein.

Carotenoids undergo a wide range of photochemical reactions in animal, plant, and microbial systems. In photosynthetic organisms, in addition to light harvesting, they perform an essential role in protecting against light-induced damage by quenching singlet oxygen, superoxide anion radicals, or triplet-state chlorophyll. We have determined the crystal structure of a water-soluble orange carotenoid protein (OCP) isolated from the cyanobacterium Arthrospira maxima at a resolution of 2.1 A. OCP forms a homodimer with one carotenoid molecule per monomer. The carotenoid binding site is lined by a striking number of methionine residues. The structure reveals several possible ways in which the protein environment influences the spectral properties of the pigment and provides insight into how the OCP carries out its putative functions in photoprotection.