Dalal Asker

Comparative analysis of metazoan chromatin organization

Genome function is dynamically regulated in part by chromatin, which consists of the histones, non-histone proteins and RNA molecules that package DNA. Studies in Caenorhabditis elegans and Drosophila melanogaster have contributed substantially to our understanding of molecular mechanisms of genome function in humans, and have revealed conservation of chromatin components and mechanisms. Nevertheless, the three organisms have markedly different genome sizes, chromosome architecture and gene organization. On human and fly chromosomes, for example, pericentric heterochromatin flanks single centromeres, whereas worm chromosomes have dispersed heterochromatin-like regions enriched in the distal chromosomal ‘arms’, and centromeres distributed along their lengths. To systematically investigate chromatin organization and associated gene regulation across species, we generated and analysed a large collection of genome-wide chromatin data sets from cell lines and developmental stages in worm, fly and human. Here we present over 800 new data sets from our ENCODE and modENCODE consortia, bringing the total to over 1,400. Comparison of combinatorial patterns of histone modifications, nuclear lamina-associated domains, organization of large-scale topological domains, chromatin environment at promoters and enhancers, nucleosome positioning, and DNA replication patterns reveals many conserved features of chromatin organization among the three organisms. We also find notable differences in the composition and locations of repressive chromatin. These data sets and analyses provide a rich resource for comparative and species-specific investigations of chromatin composition, organization and function.

Haloferax alexandrinus sp. nov., an extremely halophilic canthaxanthin-producing archaeon from a solar saltern in Alexandria (Egypt)

An extremely halophilic red micro-organism designated strain TM(T) was isolated from a solar saltern in Alexandria, Egypt. The micro-organism stains gram-negative, is very pleomorphic, non-motile and strictly aerobic and requires at least 10 g NaCl l(-1) for growth. The growth optimum is 250 g NaCl l(-1). Growth is also observed over a wide range of MgSO4 concentrations (10-40 g l(-1)). Aerobic reduction of nitrate without gas production was detected. Cells grew aerobically in a minimal salts medium containing ammonium chloride and glucose. Strain TM(T) produced acid from fructose, glucose, rhamnose, maltose and glycerol. The G+C content of the DNA was 59.5+/-0.3 mol %. On the basis of polar lipid analysis, the isolate belonged to the genus Haloferax. Analysis of the 16S rDNA sequence showed the highest similarity (>99%) to be to the type strain Haloferax volcanii. Although the spectrum of antibiotic susceptibility was similar to that of validly described species of the genus Haloferax, the strain could be distinguished from them by its different response to josamycin and rifampicin. Strain TM(T) is unique within the genus Haloferax in producing canthaxanthin. Comparative analysis of phenotypic properties and DNA-DNA hybridization between strain TM(T) and Haloferax species supported the conclusion that TM(T) is a novel species within this genus, for which the name Haloferax alexandrinus sp. nov. is proposed. The type strain is TM(T) (= JCM 10717T = IFO 16590T).

Unique diversity of carotenoid-producing bacteria isolated from Misasa, a radioactive site in Japan

We obtained carotenoid-producing microorganisms at a high frequency from water samples collected at Misasa (Tottori, Japan), a region known for its high natural radioactivity content. A comprehensive 16S rRNA gene-based phylogenetic analysis revealed that the 104 potential carotenoid producers isolated from Misasa could be classified into 38 different species belonging to seven bacterial classes (Flavobacteria, Sphingobacteria, α-Proteobacteria, γ-Proteobacteria, Deinococci, Actinobacteria, and Bacilli). Of these 38 species, 14 showed sequence similarities less than 97% to their closest identified relatives, and 9 were related to genera that have not been described earlier in terms of carotenoid production. The red-pigmented isolates belonging to Deinococci showed marked resistance to γ rays and UV irradiation, while those related to Sphingomonas showed weak resistance. The carotenoids produced by the isolates were zeaxanthin (6 strains), dihydroxyastaxanthin (24 strains), astaxanthin (27 strains), canthaxanthin (10 strains), and unidentified molecular species that were produced by the isolates related to Deinococcus, Exiguobacterium, and Flectobacillus. UV irradiation would be useful for the selective isolation of carotenoid-producing microorganisms, and that new microbial producers and other molecular species of carotenoids may potentially be identified from radioactive environments.

Enrichment of HP1a on Drosophila Chromosome 4 Genes Creates an Alternate Chromatin Structure Critical for Regulation in this Heterochromatic Domain

Chromatin environments differ greatly within a eukaryotic genome, depending on expression state, chromosomal location, and nuclear position. In genomic regions characterized by high repeat content and high gene density, chromatin structure must silence transposable elements but permit expression of embedded genes. We have investigated one such region, chromosome 4 of Drosophila melanogaster. Using chromatin-immunoprecipitation followed by microarray (ChIP–chip) analysis, we examined enrichment patterns of 20 histone modifications and 25 chromosomal proteins in S2 and BG3 cells, as well as the changes in several marks resulting from mutations in key proteins. Active genes on chromosome 4 are distinct from those in euchromatin or pericentric heterochromatin: while there is a depletion of silencing marks at the transcription start sites (TSSs), HP1a and H3K9me3, but not H3K9me2, are enriched strongly over gene bodies. Intriguingly, genes on chromosome 4 are less frequently associated with paused polymerase. However, when the chromatin is altered by depleting HP1a or POF, the RNA pol II enrichment patterns of many chromosome 4 genes shift, showing a significant decrease over gene bodies but not at TSSs, accompanied by lower expression of those genes. Chromosome 4 genes have a low incidence of TRL/GAGA factor binding sites and a low Tm downstream of the TSS, characteristics that could contribute to a low incidence of RNA polymerase pausing. Our data also indicate that EGG and POF jointly regulate H3K9 methylation and promote HP1a binding over gene bodies, while HP1a targeting and H3K9 methylation are maintained at the repeats by an independent mechanism. The HP1a-enriched, POF-associated chromatin structure over the gene bodies may represent one type of adaptation for genes embedded in repetitive DNA.

Nubsella zeaxanthinifaciens gen. nov., sp. nov., a zeaxanthin-producing bacterium of the family Sphingobacteriaceae isolated from freshwater

A yellow-pigmented, Gram-negative, strictly aerobic, rod-shaped bacterium (TDMA-5(T)) was isolated from a freshwater sample collected at Misasa (Tottori, Japan). The DNA G+C content was 38.6 mol%. Major fatty acids were iso-C(15 : 0), iso-C(17 : 0) 3-OH and summed feature 4 (iso-C(15 : 0) 2-OH and/or C(16 : 1)omega7c). MK-7 was the predominant respiratory quinone. Zeaxanthin was the major carotenoid pigment produced; flexirubin-type pigments were not produced. TDMA-5(T) was sensitive to gamma-irradiation. The strain degraded gelatin, casein, starch, Tween 80 and DNA. Phylogenetic analysis based on the 16S rRNA gene sequence placed TDMA-5(T) in a distinct lineage in the family Sphingobacteriaceae, sharing 89.4-93.4 % sequence similarity with members of the nearest genus Pedobacter. Strain TDMA-5(T) could be distinguished from the other members of the family Sphingobacteriaceae by a number of chemotaxonomic and phenotypic characteristics. Based on its unique phenotypic, genotypic and phylogenetic features, strain TDMA-5(T) represents a novel genus and species, for which the name Nubsella zeaxanthinifaciens gen. nov., sp. nov. is proposed. The type strain of Nubsella zeaxanthinifaciens is TDMA-5(T) (=NBRC 102579(T) =CCUG 54348(T)).

Genetic control for light-induced carotenoid production in non-phototrophic bacteria

Carotenoids are naturally occurring yellow or orange pigments that serve as a protectant against photo-oxidative damages. Among the wide variety of producers, the prokaryotes generate a broad spectrum of carotenoids with diverse chemical structures that are expected to have a high potential in biotechnological applications. Bacterial carotenogenesis occurs in a constitutive or light-induced manner, which suggests the diversity of the regulatory mechanism. The mechanism for light-induced carotenoid production in non-phototrophic bacteria has been studied in detail in Myxococcus xanthus, a Gram-negative gliding bacterium. The complicated mechanism involves the activation of an extracytoplasmic function (ECF) sigma factor (CarQ), which leads to the sequestration of a MerR family transcriptional regulator (CarA) that represses the expression of the carotenoid biosynthesis genes in the dark. Recently, we identified another regulatory mechanism for light-induced carotenogenesis in Streptomyces coelicolor A3(2), a Gram-positive soil bacterium. In this organism, the transcription of the carotenoid biosynthesis gene cluster is specified by LitS, a photo-inducible ECF sigma factor. The evidence indicates that the photo-dependent transcription of litS is mediated by LitR, a MerR family transcriptional regulator. In addition, it is suggested that the conformational alteration of LitR upon receiving the illumination signal determines its binding to DNA. The carboxy-terminal domain of LitR contains a possible binding site for Vitamin B12, which may serve as a capturing apparatus for the illumination signal.

Deinococcus aquiradiocola sp. nov., isolated from a radioactive site in Japan

A gamma- and UV-radiation-tolerant, pale-pink strain (TDMA-uv53T) was isolated from a freshwater sample collected at Misasa (Tottori, Japan), after exposure of the water sample to UV radiation. The cells stained Gram-positive and were non-motile, rod-shaped and non-spore-forming. The DNA G+C content of the strain was 69.1 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain TDMA-uv53T belongs to the genus Deinococcus, the highest sequence similarities being found with Deinococcus claudionis PO-04-19-125T (96 %), D. altitudinis ME-04-01-32T (96 %), D. radiomollis PO-04-20-132T (95 %), D. deserti VCD115T (91.5 %), D. hopiensis KR-140T (91.0 %) and D. sonorensis KR-87T (91.0 %). Major fatty acids were iso-15 : 0, 15 : 1omega6c, 15 : 0, 16 : 0 and summed feature 3 (iso-15 : 0 2-OH and/or 16 : 1omega7c). MK-8 was the predominant respiratory quinone. Phylogenetic distinctiveness and unique phenotypic characteristics differentiated strain TDMA-uv53T from closely related Deinococcus species. The results of our polyphasic taxonomic analyses suggested that TDMA-uv53T represents a novel Deinococcus species, for which the name Deinococcus aquiradiocola sp. nov. is proposed. The type strain is TDMA-uv53T (=JCM 14371(T) [corrected] =NBRC 102118T =CCUG 53612T).

Deinococcus depolymerans sp. nov., a gamma- and UV-radiation-resistant bacterium, isolated from a naturally radioactive site

Four gamma- and UV-radiation-resistant bacterial strains, designated TDMA-24(T), TDMA-24-2, TDMA-24-3 and TDMA-24-4, were isolated from a fresh-water sample collected at Misasa, Tottori, Japan. Cells of these strains were Gram-reaction-positive, non-motile, non-spore-forming, rod-shaped and formed red colonies. The genomic DNA G+C contents ranged from 70.5 to 70.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the novel isolates belong to the genus Deinococcus, the highest sequence similarities being with Deinococcus aquaticus PB314(T) (98%) and Deinococcus caeni Ho-08(T) (97 %). The polar lipid profile of strain TDMA-24(T) comprised three unidentified phosphoglycolipids, five unidentified glycolipids and seven unidentified polar lipids. MK-8 was the predominant respiratory quinone. Major fatty acids were iso-C(15 : 0), C(15 : 1)ω6c, C(15 : 0), C(16 : 0) and summed feature 3 (iso-C(15 : 0) 2-OH and/or C(16 : 1)ω7c). On the basis of their phylogenetic positions and chemotaxonomic and phenotypic characteristics, the novel isolates represent a novel species of the genus Deinococcus, for which the name Deinococcus depolymerans sp. nov. is proposed. The type strain is TDMA-24(T) ( = JCM 14369(T)  = NBRC 102115(T)  = CCUG 53609(T)).

Lipids of Haloferax alexandrinus strain TMT: an extremely halophilic canthaxanthin-producing archaeon

The core lipids, polar and non polar lipids, of the novel canthaxanthin-producing archaeon, Haloferax alexandrinus strain TM(T), were investigated using thin-layer chromatography (TLC), spectrophotometry, electron ionization-mass spectroscopy (EI-MS), fast atom bombardment mass spectrometry (FAB-MS), nuclear magnetic response spectrometry (13C-NMR) and high performance liquid chromatography (HPLC). This archaeon appeared to contain diphytanyl diether lipids (C20-C20) as core lipids. The major phospholipids were found to be phosphatidylglycero-phosphate-methyl ester (PGP-Me) and phosphatidylglycerol (PG), but no phosphatidylglycero-sulfate was detected. The strain contained two glycolipids, sulfated diglycosyl diether (S-DGD-1) and unsulfated diglycosyl diether (DGD). Analysis of the non polar lipids revealed the presence of beta-carotene, 3-hydroxyechinenone, gamma-carotene, cis-astaxanthin, lycopene, trisanhydro-bacterioruberin,monanhydro-bacterioruberin, bacterioruberin isomer, bacterioruberin and canthaxanthin. Although the polar lipids profile of Hfx. alexandrinus strain TM(T) was similar to those of other species of the genus Haloferax, the non polar lipid (carotenoids) profile was markedly different. Further experiments on the influence of NaCl concentration on the lipids composition of Hfx. alexandrinus strain TM(T) demonstrated the dependence of the proportion of each of bacterioruberin, beta-carotene and canthaxanthin on the NaCl concentration in the growth media.

Regulation of Polycomb group genes Psc and Su(z)2 in Drosophila melanogaster.

Certain Polycomb group (PcG) genes are themselves targets of PcG complexes. Two of these constitute the Drosophila Psc-Su(z)2 locus, a region whose chromatin is enriched for H3K27me3 and contains several putative Polycomb response elements (PREs) that bind PcG proteins. To understand how PcG mechanisms regulate this region, the repressive function of the PcG protein binding sites was analyzed using reporter gene constructs. We find that at least two of these are functional PREs that can silence a reporter gene in a PcG-dependent manner. One of these two can also display anti-silencing activity, dependent on the context. A PcG protein binding site near the Psc promoter behaves not as a silencer but as a down-regulation module that is actually stimulated by the Pc gene product but not by other PcG products. Deletion of one of the PREs increases the expression level of Psc and Su(z)2 by twofold at late embryonic stages. We present evidence suggesting that the Psc-Su(z)2 locus is flanked by insulator elements that may protect neighboring genes from inappropriate silencing. Deletion of one of these regions results in extension of the domain of H3K27me3 into a region containing other genes, whose expression becomes silenced in the early embryo.