Probing p300/CBP associated Factor (PCAF)-dependent pathways with a small molecule inhibitor

PCAF (KAT2B) belongs to the GNAT family of lysine acetyltransferases (KAT) and specifically acetylates the histone H3K9 residue and several nonhistone proteins. PCAF is also a transcriptional coactivator. Due to the lack of a PCAF KAT-specific small molecule inhibitor, the exclusive role of the acetyltransferase activity of PCAF is not well understood. Here, we report that a natural compound of the hydroxybenzoquinone class, embelin, specifically inhibits H3Lys9 acetylation in mice and inhibits recombinant PCAF-mediated acetylation with near complete specificity in vitro. Furthermore, using embelin, we have identified the gene networks that are regulated by PCAF during muscle differentiation, further highlighting the broader regulatory functions of PCAF in muscle differentiation in addition to the regulation via MyoD acetylation.

Genome-wide methylation profiling identifies an essential role of reactive oxygen species in pediatric glioblastoma multiforme and validates a methylome specific for H3 histone family 3A with absence of G-CIMP/isocitrate dehydrogenase 1 mutation

Background. Pediatric glioblastoma multiforme (GBM) is rare, and there is a single study, a seminal discovery showing association of histone H3.3 and isocitrate dehydrogenase (IDH) 1 mutation with a DNA methylation signature. The present study aims to validate these findings in an independent cohort of pediatric GBM, compare it with adult GBM, and evaluate the involvement of important functionally altered pathways. Methods. Genome-wide methylation profiling of 21 pediatric GBM cases was done and compared with adult GBM data (GSE22867). We performed gene mutation analysis of IDH1 and H3 histone family 3A (H3F3A), status evaluation of glioma cytosine-phosphate-guanine island methylator phenotype (G-CIMP), and Gene Ontology analysis. Experimental evaluation of reactive oxygen species (ROS) association was also done. Results. Distinct differences were noted between methylomes of pediatric and adult GBM. Pediatric GBM was characterized by 94 hypermethylated and 1206 hypomethylated cytosine-phosphate-guanine (CpG) islands, with 3 distinct clusters, having a trend to prognostic correlation. Interestingly, none of the pediatric GBM cases showed G-CIMP/IDH1 mutation. Gene Ontology analysis identified ROS association in pediatric GBM, which was experimentally validated. H3F3A mutants (36.4%; all K27M) harbored distinct methylomes and showed enrichment of processes related to neuronal development, differentiation, and cell-fate commitment. Conclusions. Our study confirms that pediatric GBM has a distinct methylome compared with that of adults. Presence of distinct clusters and an H3F3A mutation-specific methylome indicate existence of epigenetic subgroups within pediatric GBM. Absence of IDH1/G-CIMP status further indicates that findings in adult GBM cannot be simply extrapolated to pediatric GBM and that there is a strong need for identification of separate prognostic markers. A possible role of ROS in pediatric GBM pathogenesis is demonstrated for the first time and needs further evaluation.

Hypothetical protein Rv3423.1 of Mycobacterium tuberculosis is a histone acetyltransferase

We isolated an 8 kDa mycobacterial hypothetical protein, Rv3423.1, from the chromatin of human macrophages infected with Mycobacterium tuberculosis H37Rv. Bioinformatics predictions followed by in vitro biochemical assays with purified recombinant protein showed that Rv3423.1 is a novel histone acetyltransferase that acetylates histone H3 at the K9/K14 positions. Transient transfection of macrophages containing GFP-tagged histone H1 with RFP-tagged Rv3423.1 revealed that the protein co-localizes with the chromatin in the nucleus. Co-immunoprecipitation assays confirmed that the Rv3423.1-histone interaction is specific. Rv3423.1 protein was detected in the culture filtrate of virulent but not avirulent M. tuberculosis. Infection of macrophages with recombinant Mycobacterium smegmatis constitutively expressing Rv3423.1 resulted in a significant increase in the number of intracellular bacteria. However, the protein did not seem to offer any growth advantage to free-living recombinant M. smegmatis. It is highly likely that, by binding to the host chromatin, this histone acetyltransferase from M. tuberculosis may manipulate the expression of host genes involved in anti-inflammatory responses to evade clearance and to survive in the intracellular environment.

Novel derivatives of spirohydantoin induce growth inhibition followed by apoptosis in leukemia cells

Hydantoin derivatives possess a variety of biochemical and pharmacological properties and consequently are used to treat many human diseases. However, there are only few studies focusing on their potential as cancer therapeutic agents. In the present study, we have examined anticancer properties of two novel spirohydantoin compounds, 8-(3,4-difluorobenzyl)-1'-(pent-4-enyl)-8-azaspiro[bicyclo[3.2.1] octane-3,4'-imidazolidine]-2',5'-dione (DFH) and 8-(3,4-dichlorobenzyl)-1'-(pent-4-enyl)-8-azaspiro[bicyclo[3.2.1]octane-3,4'-imidazolidine]-2',5'-dione (DCH). Both the compounds exhibited dose- and time-dependent cytotoxic effect on human leukemic cell lines, K562, Reh, CEM and 8ES. Incorporation of tritiated thymidine ([H-3) thymidine) in conjunction with cell cycle analysis suggested that DFH and DCH inhibited the growth of leukemic cells. Downregulation of PCNA and p-histone H3 further confirm that the growth inhibition could be at the level of DNA replication. Flow cytometric analysis indicated the accumulation of cells at subG1 phase suggesting induction of apoptosis, which was further confirmed and quantified both by fluorescence-activated cell sorting (FACS) and confocal microscopy following annexin V-FITC/propidium iodide (PI) staining. Mechanistically, our data support the induction of apoptosis by activation of the mitochondrial pathway. Results supporting such a model include, elevated levels of p53, and BAD, decreased level of BCL2, activation and cleavage of caspase 9, activation of procaspase 3, poly (ADP-ribosyl) polymerase (PARP) cleavage, downregulation of Ku70, Ku80 and DNA fragmentation. Based on these results we discuss the mechanism of apoptosis induced by DFH and its implications in leukemia therapy. (C) 2008 Elsevier Inc. All rights reserved.

Studies in terpenoids. Part 42. Synthesis of 3-(2-hydroxyisopropyl)5,8-dimethyl-1,2-naphthoquinone (emmotin-H)

Emmotin-H, a naturally occurring sesquiterpenoid 1,2-naphthoquinone pigment (1) has been synthesised in a four step sequence starting from the known 5,8-dimethyl-4-oxotetralin-2-carboxylic acid (3a). Selenium dioxide oxidation of its methyl ester (3b) gives 3-methoxycarbonyl-5,8-dimethyl-1,2-naphthoquinone (4) which on reductive acetylation affords the corresponding diacetoxynaphthalene ester (5). Its reaction with excess of methylmagnesium iodide is accompanied by aerial oxidation during work-up and furnishes emmotin-H (1).

Cloning Of Rice Dna And Identification Of Transfer-Rna Gene Clones

THE rapid development of recombinant DNA technology has brought forth a revolution in biology'>", it aids us to have a closer look at the 'way genes are organized, eS11 ecially in the complex eucaryotic genornes'<", Although many animal and yeast genes have been studied in detail using recombinant DNA technology, plant genes have seldom been targets for such studie., Germination is an ideal process to study gene expression .because it effects a . shift in the metabolic status of seeds from a state of 'dormancy to an active one. AJ;l understanding of gene organization and regulation darin.g germination can be accomplblted by molecular cloning of DNA from seeds lik.e rice. To study the status of histone, rRNA tRNA and other genes in the rice genome, a general method was developed to clone eucarvotic DNA in a' plasmid vector pBR 322. This essentially ~ involves the following steps. The rice embryo and plasmid pBR 322 DNAs were cut witll restriction endonuclease Bam Hi to generate stick.Y ends, The plasmid DNA was puosphatased, the DNA~ ware a~·tnealed and joined 'by T4 phage DNA ligase. The recombinant DNA molecules thus produced were transjerred into E. coli and colonies containing them Were selected by their sensitivity to tetracycline and resistance to ampicillin, Two clones were identified . 2S haVing tRNA genes by hybridization of the DNA in the clones \vitl1 32P-la.belled rice tRNAs.

Structural Polymorphism of the Nucleosomal DNA and Implications for Protein Binding

A nucleosome forms a basic unit of the chromosome structure. A biologically relevant question is how much of the nucleosomal conformational space is accessible to protein-free DNA, and what proportion of the nucleosomal conformations are induced by bound histones. To investigate this, we have analysed high resolution xray crystal structure datasets of DNA in protein-free as well as protein-bound forms, and compared the dinucleotide step parameters for the two datasets with those for high resolution nucleosome structures. Our analysis shows that most of the dinucleotide step parameter values for the nucleosome structures lie within the range accessible to protein-free DNA, indirectly indicating that the histone core plays more of a stabilizing role. The nucleosome structures are observed to assume smooth and nearly planar curvature, implying that ‘normal’ B-DNA like parameters can give rise to a curved geometry at the gross structural level. Different nucleosome

DNase I Site Mapping and Micrococcal Nuclease Digestion of Pachytene Chromatin Reveal Novel Structural Features

A comparison of the DNase I digestion products of the 32P-5’-end-labeled pachytene nucleosome core particles (containing histones H2A, TH2A, X2, H2B, THPB, H3a, nd H4) and liver nucleosome core particles (containing somatic histones H2A, H2B, H3, and H4) revealed that the cleavage sites that are 30, 40, and 110 nucleotidesa way from the 5’-enda re significantly more accessiblei n the pachytene core particles than in the liver core particles. These cleavage sites correspond to the region wherein H2B interacts with the nucleosome core DNA. These results, therefore, suggest that the histone-DNA interactiona t these sites in the pachytene core particles is weaker, possibly because of the presence of the histone variant THBB interacting at similar topological positions in the nucleosome core as that of its somatic counterpart H2B. Such a loosened structumrea y also be maintainede ven in the native pachytene chromatin since micrococcal nuclease digestion of pachytene nuclei resulted in a higher ratio of subnucleosomes (SN4 + SN?) to mononucleosomes than that observed liinv er chromatin

Nucleosome core protein: Asymmetric dissociation of the octamer

The octameric nucleosomal core-histone complex, (H2A)2-(H2B)2-(H3)2-(H4)2, isolated from rat liver, undergoes dissociation during gel exclusion chromatography as a result of dilution occurring in the columns. The elution pattern at pH 7.0 and 4°C showed a sharp leading peak containing all four histones but predominantly H3 and H4, and a trailing peak containing equal amounts of histones H2A and H2B. As column length was increased the area under the leading peak decreased and that under the trailing peak increased. In addition the relative positions of the two peaks varied with column length. From an analysis of the data on increase in elution volume of the leading peak in relation to column length an apparent molecular weight of 86 000 was calculated for the undissociated molecule. Its apparent molecular weight, histone composition and pattern of further dissociation in relation to column length suggest that this species is the hexamer, (H2A-H2B)-(H3)2-(H4)2. At pH 7.0 and 4°C the dissociation of the core complex appears to be as follows: (H2A)2-(H2B)2-(H3)2-(H4)2 → (H2A-H2B) + (H2A-H2B)-(H3)2-(H4)2 → 2(H2A-H2B) + (H3)2-(H4)2 This dissociation was accelerated by an increase in temperature or decrease in pH and was accompanied by marked conformational changes as judged by circular dichroism measurements.

Chemical Modification Studies On Ricinus-Communis (Castor Bean) Agglutinin

Ricinus communis agglutinin was subjected to various chemical treatments and the effect on its hemagglutinating and saccharide-binding properties was studied. Acetylation, succinylation and citraconylation led to a complete loss in the activity of the agglutinin, whereas reductive methylation had no effect on the activity, showing that charged amino groups were involved in the hemagglutinating and saccharide-binding activity of Ricinus agglutinin. Modification of tryptophyl, arginyl and carboxyl-group-containing residues did not lead to any loss in the activity of the agglutinin. Acetylation of tyrosyl groups with N-acetylimidazole strongly reduced the hemagglutinating and saccharide-binding property of Ricinus agglutinin. The loss in activity was restored on deacetylation of the tyrosyl groups. Modification of tyrosyl residues also led to a change in the immunological properties of the agglutinin. The initial rate of modification of tyrosyl and amino groups and the concomitant loss of activity was reduced in the presence of lactose.

Embryogenesis in flowering plants: Recent approaches and prospects

The overall architectural pattern of the mature plant is established during embryogenesis. Very little is known about the molecular processes that underlie embryo morphogenesis. Last decade has, nevertheless, seen a burst of information on the subject. The synchronous somatic embryogenesis system of carrot is largely being used as the experimental system. Information on the molecular regulation of embryogenesis obtained with carrot somatic embryos as well as observations on sandalwood embryogenic system developed in our laboratory are summarized in this review. The basic experimental strategy of molecular analysis mostly relied on a comparison between genes and proteins being expressed in embryogenic and non-embryogenic cells as well as in the different stages of embryogenesis. Events such as expression of totipotency of cells and establishment of polarity which are so critical for embryo development have been characterized using the strategy, Several genes have been identified and cloned from the carrot system, These include sequences that encode certain extracellular proteins (EPs) that influence cell proliferation and embryogenesis in specific ways and sequences of the abscisic acid (ABA) inducible late embryogenesis abundant (LEA) proteins which are most abundant and differentially expressed mRNAs in somatic embryos. That LEAs are expressed in the somatic embryos of a tree flora also is evidenced from studies on sandalwood Several undescribed or novel sequences that are enhanced in embryos were identified. A sequence of this nature exists in sandalwood embryos was demonstrated using a Cuscuta haustorial (organ-specific) cDNA probe. Somatic embryogenesis systems have been used to assess the expression of genes isolated from non-embryogenic tissues. Particular attention has been focused on both cell cycle and histone genes.

Positional Preferences of Polypurine/Polypyrimidine Tracts in Saccharomyces cerevisiae Genome: Implications for cis Regulation of Gene Expression

The complete genome of the baker's yeast S. cerevisiae was analyzed for the presence of polypurine/polypyrimidine (poly[pu/py]) repeats and their occurrences were classified on the basis of their location within and outside open reading frames (ORFs). The analysis reveals that such sequence motifs are present abundantly both in coding as well as noncoding regions. Clear positional preferences are seen when these tracts occur in noncoding regions. These motifs appear to occur predominantly at a unit nucleosomal length both upstream and downstream of ORFs. Moreover, there is a biased distribution of polypurines in the coding strands when these motifs occur within open reading frames. The significance of the biased distribution is discussed with reference to the occurrence of these motifs in other known mRNA sequences and expressed sequence tags. A model for cis regulation of gene expression is proposed based on the ability of these motifs to form an intermolecular triple helix structure when present within the coding region and/or to modulate nucleosome positioning via enhanced histone affinity when present outside coding regions.

DNA- and chromatin-condensing properties of rat testes H1a and H1t compared to those of rat liver H1bdec: H1t is a poor condenser of chromatin

Histones H1a and H1t are two major linker histone variants present at the pachytene interval of mammalian spermatogenesis. The DNA- and chromatin-condensing properties of these two variants isolated from rat testes were studied and compared with those from rat liver. For this purpose, the histone H1 subtypes were purified from the respective tissues using bath acid and salt extraction procedures, Circular dichroism studies revealed that acid exposure during isolation affects the alpha-helical structure of both the globular domain (in the presence of 1 M NaCl) and the C-terminal lambda-tail (in the presence of 60% trifluoroethanol). The condensation of rat oligonucleosomal DNA, as measured by circular dichroism spectroscopy, by the salt-extracted histone H1 was at least 10 times more efficient than condensation by the acid-extracted histone H1. A site size of 16-20 base pairs was calculated for the salt-extracted histone H1. Among the different histone H1 subtypes, somatic histone H1bdec had the highest DNA-condensing property, followed by histone H1a and histone H1t. All the salt-extracted histones condensed rat oligonucleosomal DNA more efficiently than linear pBR-322 DNA, Histones H1bdec and H1a condensed histone H1-depleted chromatin, prepared from rat liver nuclei, with relatively equal efficiency. On the other hand, there was no condensation of histone H1-depleted chromatin with the testes specific histone H1t. A comparison of the amino acid sequences of histone H1d (rat) and histone H1t (rat) revealed several interesting differences in the occurrence of DNA-binding motifs at the C-terminus. A striking observation is the presence of a direct repeat of an octapeptide motif K(A)T(S)PKKA(S)K(T)K(A) in histone H1d that is absent in histone H1t.

DNA-condensing and chromatin-condensing properties of rat testes hla and hit compared to those of rat-liver hlbdec - hlt is a poor condenser of chromatin

Histones H1a and H1t are two major linker histone variants present at the pachytene interval of mammalian spermatogenesis. The DNA- and chromatin-condensing properties of these two variants isolated from rat testes were studied and compared with those from rat liver. For this purpose, the histone H1 subtypes were purified from the respective tissues using bath acid and salt extraction procedures, Circular dichroism studies revealed that acid exposure during isolation affects the alpha-helical structure of both the globular domain (in the presence of 1 M NaCl) and the C-terminal lambda-tail (in the presence of 60% trifluoroethanol). The condensation of rat oligonucleosomal DNA, as measured by circular dichroism spectroscopy, by the salt-extracted histone H1 was at least 10 times more efficient than condensation by the acid-extracted histone H1. A site size of 16-20 base pairs was calculated for the salt-extracted histone H1. Among the different histone H1 subtypes, somatic histone H1bdec had the highest DNA-condensing property, followed by histone H1a and histone H1t. All the salt-extracted histones condensed rat oligonucleosomal DNA more efficiently than linear pBR-322 DNA, Histones H1bdec and H1a condensed histone H1-depleted chromatin, prepared from rat liver nuclei, with relatively equal efficiency. On the other hand, there was no condensation of histone H1-depleted chromatin with the testes specific histone H1t. A comparison of the amino acid sequences of histone H1d (rat) and histone H1t (rat) revealed several interesting differences in the occurrence of DNA-binding motifs at the C-terminus. A striking observation is the presence of a direct repeat of an octapeptide motif K(A)T(S)PKKA(S)K(T)K(A) in histone H1d that is absent in histone H1t.

A novel DNA intercalator, butylamino-pyrimido[4',5':4,5]selenolo(2,3-b)quinoline, induces cell cycle arrest and apoptosis in leukemic cells

DNA intercalators are one of the most commonly used chemotherapeutic agents. Novel intercalating compounds of pyrimido[4',5':4,5]selenolo(2,3-b)quinoline series having a butylamino or piperazino group at fourth position (BPSQ and PPSQ, respectively) are studied. Our results showed that BPSQ induced cytotoxicity whereas PPSQ was cytostatic. The cytotoxicity induced by BPSQ was concentration- and time-dependent. Cell cycle analysis and tritiated thymidine assay revealed that BPSQ affects the cell cycle progression by arresting at S phase. The absence of p-histone H3 and reduction in the levels of PCNA in the cells treated with BPSQ further confirmed the cell cycle arrest. Further, annexin V staining, DNA fragmentation, nuclear condensation and changes in the expression levels of BCL2/BAD confirmed the activation of apoptosis. Activation of caspase 8 and lack of cleavage of caspase 9, caspase 3 and PARP suggest the possibility of BPSQ triggering extrinsic pathway for induction of apoptosis, which is discussed. Hence, we have identified a novel compound which would have clinical relevance in cancer chemotherapeutics.