Analysis of model replication origins in Drosophila reveals new aspects of the chromatin landscape and its relationship to origin activity and the prereplicative complex.
ABSTRACT Epigenetic regulation exerts a major influence on origins of DNA replication during development. The mechanisms for this regulation, however, are poorly defined. We showed previously that acetylation of nucleosomes regulates the origins that mediate developmental gene amplification during Drosophila oogenesis. Here we show that developmental activation of these origins is associated with acetylation of multiple histone lysines. Although these modifications are not unique to origin loci, we find that the level of acetylation is higher at the active origins and quantitatively correlated with the number of times these origins initiate replication. All of these acetylation marks were developmentally dynamic, rapidly increasing with origin activation and rapidly declining when the origins shut off and neighboring promoters turn on. Fine-scale analysis of the origins revealed that both hyperacetylation of nucleosomes and binding of the origin recognition complex (ORC) occur in a broad domain and that acetylation is highest on nucleosomes adjacent to one side of the major site of replication initiation. It was surprising to find that acetylation of some lysines depends on binding of ORC to the origin, suggesting that multiple histone acetyltransferases may be recruited during origin licensing. Our results reveal new insights into the origin epigenetic landscape and lead us to propose a chromatin switch model to explain the coordination of origin and promoter activity during development.
Article: Drosophila double parked: a conserved, essential replication protein that colocalizes with the origin recognition complex and links DNA replication with mitosis and the down-regulation of S phase transcripts.[show abstract] [hide abstract]
ABSTRACT: We identified a Drosophila gene, double parked (dup), that is essential for DNA replication and belongs to a new family of replication proteins conserved from Schizosaccharomyces pombe to humans. Strong mutations in dup cause embryonic lethality, preceded by a failure to undergo S phase during the postblastoderm divisions. dup is required also for DNA replication in the adult ovary, establishing that dup is needed for DNA replication at multiple stages of development. Strikingly, DUP protein colocalizes with the origin recognition complex to specific sites in the ovarian follicle cells. This suggests that DUP plays a direct role in DNA replication. The dup transcript is cell cycle regulated and is under the control of E2F and Cyclin E. Interestingly, dup mutant embryos fail both to downregulate S phase genes and to engage a checkpoint preventing mitosis until completion of S phase. This could be either because these events depend on progression of S phase beyond the point blocked in the dup mutants or because DUP is needed directly for these feedback mechanisms.Genes & Development 08/2000; 14(14):1765-76. · 11.66 Impact Factor
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ABSTRACT: We show that during Caenorhabditis elegans male spicule development, the specification of a glial versus neuronal cell fate in a canonical neurogenic sublineage is dependent on Wnt signaling. Inactivation of a Wnt signaling pathway mediated by the Wnt receptor LIN-17 transforms the SPD sheath cell into its sister, the SPD neuron. We discovered a new mutant, son-1, that displays this same cell fate transformation. The son-1 mutation enhances the phenotypes of reduction-of-function lin-17 mutants in several developmental processes, including vulva development, somatic gonad development, and male tail patterning. son-1 encodes an HMG1/2-like DNA-binding protein and is localized in all cell nuclei through development as revealed by a GFP reporter construct. Disruption of son-1 function by RNA-mediated interference results in the same spicule defect as caused by overexpression of POP-1, a TCF/LEF class HMG protein known to act downstream of the Wnt signaling pathway. Our results provide in vivo evidence for the functional involvement of an HMG1/2-like protein, SON-1, in Wnt signaling. The sequence nonspecific HMG protein SON-1 and the sequence specific HMG protein POP-1 might both act in the Wnt responding cells to regulate gene transcription in opposite directions.Genes & Development 05/1999; 13(7):877-89. · 11.66 Impact Factor
Article: Radioimmunoassay of apolipoprotein A-i. application of a non-ionic detergent (Tween-20) and solid-phase staphylococcus.[show abstract] [hide abstract]
ABSTRACT: We describe two techniques for radioimmunoassay of apolipoprotein A-I (apoA-I) in human plasma, each involving use of a non-ionic detergent, Tween-20, to expose antigenic sites, and one involving "IgG SORB" (a suspension of killed staphylococci) as a solid-phase separator. Tween-20 (3.75 g/L) decreased nonspecific binding and unmasked the antigenic sites on the apoA-I molecule in plasma to the same extent as did a tedious delipidation procedure, without altering the binding affinity between apoA-I and apoA-I antibodies as determined by Scatchard analysis (Ka congruent to 2.83 X 10(8) L/mol). The widely accepted double-antibody immunoprecipitation technique for separating bound and unbound 125I-labeled apoA-I is time-consuming, owing to extended periods of incubation and centrifugation IgG SORB effectively separates bound from unbound 125I-labeled apoA-I and the reaction is complete within 10 min. On comparing concentrations of apoA-I in human plasma by the conventional second-antibody (y) and solid-phase IgG SORB methods (x), we found results by the two techniques to be reasonably identical (r = 0.98, y = 1.2x -- 0.17). The mean concentrations of apoA-I in plasma from 65 normal and five hyperlipidemic patients were 1.33 (SD 0.32) and 0.78 (SD 0.35) g/L, respectively, and apoA-I and high-density lipoprotein cholesterol were significantly correlated (r = 0.72, p less than 0.001).Clinical Chemistry 02/1982; 28(1):199-204. · 7.91 Impact Factor