Proteomic changes of malignant cells induced by short-term treatment with valproic acid.

Cancers are clonal disorders of multicellular organisms that arise by accumulation of genetic mutations and step-wise induction of epigenetic events that silence transcription of tumor suppressing genes and facilitate expression of tumor promoting genes. This allows cancer cells in combination with altered response of host organism to overcome the regulatory cascades that orchestrate proper cooperation of cells within the multicellular organism. Epigenetic regulation is executed on the level of DNA by methylation of CpG islands and on the level of post-translational modification of chromatin proteins, histones in the first place. Post-translational modifications of histones include histone phosphorylation, acetylation, methylation, biotinylation, poly(ADP ribosylation), ubiquitination and sumoylation. Histone acetylation is connected with transcription activation or "openness" of chromatin to regulation by transcriptional factors. Histones are acetylated by histone acetyltranferases (HATs). Histone acetylation is a dynamic process that is reversed by histone deacetylases (HDACs), enzymes that are able to remove the acetyl residue from the acetylated histones. A decrease in gene expression brought about by low acetylation of histones is part of the cancer specific transcription profile that is characterized..

Trichoplax adhaerens reveals a network of nuclear receptors sensitive to 9-cis-retinoic acid at the base of metazoan evolution

Trichoplax adhaerens, the only known species of Placozoa is likely to be closely related to an early metazoan that preceded branching of Cnidaria and Bilateria. This animal species is surprisingly well adapted to free life in the World Ocean inhabiting tidal costal zones of oceans and seas with warm to moderate temperatures and shallow waters. The genome of T. adhaerens (sp. Grell) includes four nuclear receptors, namely orthologue of RXR (NR2B), HNF4 (NR2A), COUP-TF (NR2F) and ERR (NR3B) that show a high degree of similarity with human orthologues. In the case of RXR, the sequence identity to human RXR alpha reaches 81% in the DNA binding domain and 70% in the ligand binding domain. We show that T. adhaerens RXR (TaRXR) binds 9-cis retinoic acid (9-cis-RA) with high affinity, as well as high specificity and that exposure of T. adhaerens to 9-cis-RA regulates the expression of the putative T. adhaerens orthologue of vertebrate L-malate-NADP+ oxidoreductase (EC 1.1.1.40) which in vertebrates is regulated by a heterodimer of RXR and thyroid hormone receptor. Treatment by 9-cis-RA alters the relative expression profile of T. adhaerens nuclear receptors, suggesting the existence of natural ligands. Keeping with this, algal food composition has a profound effect on T. adhaerens growth and appearance. We show that nanomolar concentrations of 9-cis-RA interfere with T. adhaerens growth response to specific algal food and causes growth arrest. Our results uncover an endocrine-like network of nuclear receptors sensitive to 9-cis-RA in T. adhaerens and support the existence of a ligand-sensitive network of nuclear receptors at the base of metazoan evolution

Perilipin-related protein regulates lipid metabolism in C. elegans

Perilipins are lipid droplet surface proteins that contribute to fat metabolism by controlling the access of lipids to lipolytic enzymes. Perilipins have been identified in organisms as diverse as metazoa, fungi, and amoebas but strikingly not in nematodes. Here we identify the protein encoded by the W01A8.1 gene in Caenorhabditis elegans as the closest homologue and likely orthologue of metazoan perilipin. We demonstrate that nematode W01A8.1 is a cytoplasmic protein residing on lipid droplets similarly as human perilipins 1 and 2. Downregulation or elimination of W01A8.1 affects the appearance of lipid droplets resulting in the formation of large lipid droplets localized around the dividing nucleus during the early zygotic divisions. Visualization of lipid containing structures by CARS microscopy in vivo showed that lipid-containing structures become gradually enlarged during oogenesis and relocate during the first zygotic division around the dividing nucleus. In mutant embryos, the lipid containing structures show defective intracellular distribution in subsequent embryonic divisions and become gradually smaller during further development. In contrast to embryos, lipid-containing structures in enterocytes and in epidermal cells of adult animals are smaller in mutants than in wild type animals. Our results demonstrate the existence of a perilipin-related regulation of fat metabolism in nematodes and provide new possibilities for functional studies of lipid metabolism

Rescue experiment of <i>gei-8(ok1671)</i> with overlapping amplified regions of genomic DNA injected into the gonads of parents.

<p>Rescue experiment of <i>gei-8(ok1671)</i> with overlapping amplified regions of genomic DNA injected into the gonads of parents.</p

Comparison of N-terminal regions of GEI-8-related proteins to NCoR/SMRT.

<p>Sequence alignment of GEI-8 nematode orthologues with their nearest Metazoa/Fungi homologues, both human orthologues NCoR1 and SMRT (NCoR2) are shown. Green bars indicate the position of the alpha-helices in the structure of the upstream DAD domain of human SMRT and homology predicted positions in the second SANT domain. Residues indispensable for regulating HDAC interactions and function are highlighted in blue (needed for the structural integrity), magenta (interaction with HDAC) and red (activation of HDAC). Only the N-terminal part of the sequences is shown. The identical and similar residues are highlighted by different intensity of shading. Sequence identifiers: <i>C. elegans</i>: GEI8_CAEEL, <i>C. brenneri</i>: CN15693, <i>C. briggsae</i>: A8X8F0_CAEBR, <i>C. remanei</i>: RP40355, <i>C. japonica</i>: JA23925 ABLE03010463.1 ABLE03032768.1 ABLE03032771.1 ABLE03032769.1 ABLE03032772.1, <i>Loa loa</i>: E1FVE0_LOALO, <i>Brugia malayi</i>: A8NSC3_BRUMA, <i>Ixodes scapularis</i>: B7PZ26_IXOSC, <i>Saccharomyces cerevisiae</i>: SNT1_YEAST, <i>Drosophila melanogaster</i>: Q9VYK0_DROME, <i>Trichoplax adhaerens</i>: B3SAN1_TRIAD, <i>Branchiostoma floridae</i>: C3XV35_BRAFL, <i>Danio rerio</i>: A8B6H7_DANRE, <i>Xenopus tropicalis</i>: NCOR1_XENTR AAMC01044136.1, <i>Anolis carolinensis</i>: ANOCA15679 2 ENSACAP00000014806; ENSACAT00000015107, <i>Gallus gallus</i>: UPI0000E813A6, <i>Homo sapiens</i>: NCOR2_HUMAN (NCoR2), <i>Homo sapiens</i>: NCOR1_HUMAN (NCoR1). PDB structure 1XC5 was used to determine the position of the helices.</p

GEI-8, a Homologue of Vertebrate Nuclear Receptor Corepressor NCoR/SMRT, Regulates Gonad Development and Neuronal Functions in <em>Caenorhabditis elegans</em>

<div><p>NCoR and SMRT are two paralogous vertebrate proteins that function as corepressors with unliganded nuclear receptors. Although <i>C. elegans</i> has a large number of nuclear receptors, orthologues of the corepressors NCoR and SMRT have not unambiguously been identified in <i>Drosophila</i> or <i>C. elegans</i>. Here, we identify GEI-8 as the closest homologue of NCoR and SMRT in <i>C. elegans</i> and demonstrate that GEI-8 is expressed as at least two isoforms throughout development in multiple tissues, including neurons, muscle and intestinal cells. We demonstrate that a homozygous deletion within the <i>gei-8</i> coding region, which is predicted to encode a truncated protein lacking the predicted NR domain, results in severe mutant phenotypes with developmental defects, slow movement and growth, arrested gonadogenesis and defects in cholinergic neurotransmission. Whole genome expression analysis by microarrays identified sets of de-regulated genes consistent with both the observed mutant phenotypes and a role of GEI-8 in regulating transcription. Interestingly, the upregulated transcripts included a predicted mitochondrial sulfide:quinine reductase encoded by Y9C9A.16. This locus also contains non-coding, 21-U RNAs of the piRNA class. Inhibition of the expression of the region coding for 21-U RNAs leads to irregular gonadogenesis in the homozygous <i>gei-8</i> mutants, but not in an otherwise wild-type background, suggesting that GEI-8 may function in concert with the 21-U RNAs to regulate gonadogenesis. Our results confirm that GEI-8 is the orthologue of the vertebrate NCoR/SMRT corepressors and demonstrate important roles for this putative transcriptional corepressor in development and neuronal function.</p> </div

Analysis of neuromuscular function of <i>gei-8(ok1671)</i> mutant (VC1213).

<p>Aldicarb and levamisole sensitivity assays revealed increased sensitivity of <i>gei-8</i> mutants towards the acetylcholinesterase inhibitor aldicarb (<b>A</b>) and levamisole (<b>B</b>) suggesting a synaptic defect in cholinergic transmission.</p

Development of the germline in <i>gei-8(ok1671)</i> mutants and additional phenotypic changes induced by RNAi targeted against Y9C9A.16 (<i>sqrd-2</i>) in homozygous <i>gei-8(ok1671)</i> mutants.

<p>(<b>A</b>) The reproductive structures of a wild-type larva at the L4 stage is shown. The vulva is indicated by an arrowhead and formation of the uterus is visible next to vulval structures. The position of the lead migrating cell for the gonad (distal tip cell) during the larval L4 stage is indicated by arrow. (<b>B</b>) Development of the gonad in a young adult N2 animal. The distal gonad arm continues in growth beyond the position of the vulva (marked by arrowhead) and makes contact with the proximal gonad arm (arrow). (<b>C</b>) <i>gei-8(ok1671)</i> mutant gonadogenesis by Nomarski optics. The arrested gonad arm in a position similar to wild type L4 larva is indicated by arrow. The vulva is marked by an arrowhead. (<b>D</b>) A <i>gei-8(ok1671)</i> mutant with arrested growth of the gonad as visualized by DAPI staining. The distal tip of arrested gonad is marked by an arrow and the vulva by an arrowhead. (<b>E</b>, <b>F</b>, <b>G</b>, <b>H</b>, <b>I</b> and <b>J</b>) Additional phenotypic changes induced by RNAi targeted against Y9C9A.16 (<i>sqrd-2</i>) region including three 21U-RNAs: 21ur-2020, 21ur-11733 and 21ur-9201 in <i>gei-8(ok1671)</i> homozygous mutant animals. (<b>E</b>) A <i>gei-8(ok1671)</i> mutant treated with <i>sqrd-2</i> RNAi shows growth of the gonad beyond the usual arrest point, reaching the position of the vulva (marked by arrow and arrowhead, respectively). (<b>F</b>) Additional phenotypes of <i>gei-8(ok1671)</i> animals treated with <i>sqrd-2</i> RNAi. Nomarski optics view of homozygous <i>gei-8(ok1671)</i> larva treated with <i>sqrd-2</i> RNAi revealing frequent growth defects, including irregular body shapes, (distention of proximal part of the body and thin elongation of the distal part of the body) and extended growth of the distal part of the gonad. The gonad is visualized by DAPI staining in panel <b>G</b> (distal arm of the gonad is marked by right arrow, proximal arm of the gonad is marked by left arrow). Arrowhead indicates the position of vulva in panels E, F and G. (<b>H</b>) Additional growth defects induced by <i>sqrd-2</i> RNAi in homozygous <i>gei-8(ok1671)</i> worms including a Pvul phenotype (arrowhead), accumulation of gonadal cells with a possible incomplete second vulva formation (left arrow) and a distal arm of germline that fails to turn and instead continues to grow in the direction of the thin and elongated tail (right arrow). (<b>I</b>) A mutant animal with germline growth directional changes of both gonad arms induced by <i>sqrd-2</i> RNAi: anterior gonad arm makes an incomplete turn dorsally and continues to grow in the anterior direction (left arrow) while the posterior gonad arm fails to turn and continues in additional growth towards the tail (right arrow). The position of vulva is indicated by arrowhead. (<b>J</b>) A homozygous <i>gei-8(ok1671)</i> mutant developing a convoluted irregular accumulation of cells of distal gonad arm in the position of gonad turn (marked by arrows). The position of vulva is indicated by arrowhead. Scale <b>A</b>, <b>B</b>, <b>D</b>, <b>E</b> and <b>J</b> 50 µm, <b>C</b>, <b>F</b>, <b>G</b>, <b>H</b> an <b>I</b> 100 µm.</p

Induction of additional gonad and body shape phenotypes in homozygous <i>gei-8</i>(<i>ok1671</i>) mutant worms by RNAi directed against <i>sqrd-2</i> or <i>sqrd-1</i>.

<p>Induction of additional gonad and body shape phenotypes in homozygous <i>gei-8</i>(<i>ok1671</i>) mutant worms by RNAi directed against <i>sqrd-2</i> or <i>sqrd-1</i>.</p

Analysis of the pharyngeal pumping rate of <i>gei-8(ok1671)</i> mutant animals and controls.

<p>Pharyngeal pumping rate is regulated by cholinergic transmission. In <i>gei-8</i> mutants the pumping rate is low compared to wild-type animals and decreases with age (n = 10 for each category).</p