The yan gene is highly conserved in Drosophila and its expression suggests a complex role throughout development

 Competence for cell fate determination and cellular differentiation is under tight control of regulatory genes. Yan, a nuclear target of receptor tyrosine kinase (RTK) signaling, is an E twenty six (ETS) DNA-binding protein that functions as a negative regulator of cell differentiation and proliferation in Drosophila. Most members of RTK signaling pathways are highly conserved through evolution, yet no yan orthologues have been identified to date in vertebrates. To investigate the degree of yan conservation during evolution, we have characterized a yan homologue from a sibling species of D. melanogaster, D. virilis. Our results show that the organization, primary structure and expression pattern of yan are highly conserved. Both genes span over 20 kb and contain four exons with introns at identical positions. The areas with highest amino acid similarity include the Pointed and ETS domain but there are other discrete regions with a high degree of similarity. Phylogenetic analysis reveals that yan’s closest relative is the human tel gene, a negative regulator of differentiation in hematopoetic precursors. In both species, Yan is dynamically expressed beginning as early as stage 4/5 and persisting throughout embryogenesis. In third instar larvae, Yan is expressed in and behind the morphogenetic furrow of the eye imaginal disc as well as in the laminar precursor cells of the brain. Ovarian follicle cells also contain Yan protein. Conservation of the structure and expression patterns of yan genes strongly suggests that regulatory mechanisms for their expression are also conserved in these two species. Received: 3 November 1998 / Accepted: 9 December 1998     

Localized activation of RTK/MAPK pathways during Drosophila development

Receptor tyrosine kinase (RTK) signaling is mediated by a signaling cascade culminating in activation of mitogen-activated protein kinase (MAPK) by double phosphorylation on threonine and tyrosine residues. The pattern of MAPK activation can now be directly visualized in situ during embryonic and adult development using an antiserum is specific for the double phosphorylated form of MAPK (db-P MAPK).^1,2 The pattern of MAPK activation detected by this antiserum in developing embryos and larval imaginal discs conforms remarkably well to the inferred pattern of known RTK function. In addition, db-P MAPK staining directly reveals features of signaling such as the range of signal spreading and the kinetics of RTK activation, which would be difficult to measure by other methods. The ability to visualize the output of RTK signaling also permits detailed establishment of epistatic relationships between signaling components of RTK cascades. BioEssays 20 :189–194, 1998.© 1998 John Wiley & Sons, Inc.     

Bile Acid Signal-induced Phosphorylation of Small Heterodimer Partner by Protein Kinase Cζ Is Critical for Epigenomic Regulation of Liver Metabolic Genes

Bile acids (BAs) are recently recognized key signaling molecules that control integrative metabolism and energy expenditure. BAs activate multiple signaling pathways, including those of nuclear receptors, primarily farnesoid X receptor (FXR), membrane BA receptors, and FXR-induced FGF19 to regulate the fed-state metabolism. Small heterodimer partner (SHP) has been implicated as a key mediator of these BA signaling pathways by recruitment of chromatin modifying proteins, but the key question of how SHP transduces BA signaling into repressive histone modifications at liver metabolic genes remains unknown. Here we show that protein kinase Cζ (PKCζ) is activated by BA or FGF19 and phosphorylates SHP at Thr-55 and that Thr-55 phosphorylation is critical for the epigenomic coordinator functions of SHP. PKCζ is coimmunopreciptitated with SHP and both are recruited to SHP target genes after bile acid or FGF19 treatment. Activated phosphorylated PKCζ and phosphorylated SHP are predominantly located in the nucleus after FGF19 treatment. Phosphorylation at Thr-55 is required for subsequent methylation at Arg-57, a naturally occurring mutation site in metabolic syndrome patients. Thr-55 phosphorylation increases interaction of SHP with chromatin modifiers and their occupancy at selective BA-responsive genes. This molecular cascade leads to repressive modifications of histones at metabolic target genes, and consequently, decreased BA pools and hepatic triglyceride levels. Remarkably, mutation of Thr-55 attenuates these SHP-mediated epigenomic and metabolic effects. This study identifies PKCζ as a novel key upstream regulator of BA-regulated SHP function, revealing the role of Thr-55 phosphorylation in epigenomic regulation of liver metabolism.     

Insulation of the chicken beta-globin chromosomal domain from a chromatin-condensing protein,MENT

Active genes are insulated from developmentally regulated chromatin condensation in terminally differentiated cells. We mapped the topography of a terminal stage-specific chromatin-condensing protein, MENT, across the active chicken beta-globin domain. We observed two sharp transitions of MENT concentration coinciding with the beta-globin boundary elements. The MENT distribution profile was opposite to that of acetylated core histones but correlated with that of histone H3 dimethylated at lysine 9 (H3me2K9). Ectopic MENT expression in NIH 3T3 cells caused a large-scale and specific remodeling of chromatin marked by H3me2K9. MENT colocalized with H3me2K9 both in chicken erythrocytes and NIH 3T3 cells. Mutational analysis of MENT and experiments with deacetylase inhibitors revealed the essential role of the reaction center loop domain and an inhibitory affect of histone hyperacetylation on the MENT-induced chromatin remodeling in vivo. In vitro, the elimination of the histone H3 N-terminal peptide containing lysine 9 by trypsin blocked chromatin self-association by MENT, while reconstitution with dimethylated but not acetylated N-terminal domain of histone H3 specifically restored chromatin self-association by MENT. We suggest that histone H3 modification at lysine 9 directly regulates chromatin condensation by recruiting MENT to chromatin in a fashion that is spatially constrained from active genes by gene boundary elements and histone hyperacetylation.     

Highly conserved ENY2/Sus1 protein binds to Drosophila CTCF and is required for barrier activity

Chromatin insulators affect interactions between promoters and enhancers/silencers and function as barriers for the spreading of repressive chromatin. Drosophila insulator protein dCTCF marks active promoters and boundaries of many histone H3K27 trimethylation domains associated with repressed chromatin. In particular, dCTCF binds to such boundaries between the parasegment-specific regulatory domains of the Bithorax complex. Here we demonstrate that the evolutionarily conserved protein ENY2 is recruited to the zinc-finger domain of dCTCF and is required for the barrier activity of dCTCF-dependent insulators in transgenic lines. Inactivation of ENY2 by RNAi in BG3 cells leads to the spreading of H3K27 trimethylation and Pc protein at several dCTCF boundaries. The results suggest that evolutionarily conserved ENY2 is responsible for barrier activity mediated by the dCTCF protein.     

Receptor tyrosine kinases as targets for anticancer drugs.

Receptor tyrosine kinases (RTKs) are the primary mediators of the signaling network that transmit extracellular signals into the cell. Gene amplification and/or overexpression of RTK proteins or functional alterations caused by mutations in the corresponding genes or abnormal autocrine-paracrine growth factor loops contribute to constitutive RTK signaling, ultimately resulting in the manifestation of dysregulated cell growth and cancer. The mechanism of uncontrolled RTK signaling that leads to cancer has provided the rationale for anti-RTK drug development. Strategies towards the prevention and interception of RTK signaling include monoclonal antibodies, small-molecule inhibitors, immunotoxins and antisense oligonucleotides.     

Keeping fingers crossed: heterochromatin spreading through interdigitation of nucleosome arrays

Interphase eukaryotic nuclei contain diffuse euchromatin and condensed heterochromatin. Current textbook models suggest that chromatin condensation occurs via accordion-type compaction of nucleosome zigzag chains. Recent studies have revealed several structural aspects that distinguish the highly compact state of condensed heterochromatin. These include an extensive lateral self-association of chromatin fibers, prominent nucleosome linker 'stems', and special protein factors that promote chromatin self-association. Here I review the molecular and structural determinants of chromatin compaction and discuss how heterochromatin spreading may be mediated by lateral self-association of zigzag nucleosome arrays.