Epigenetic inheritance of active chromatin after removal of the main transactivator

Science

Volumn 286, Issue 5441, 1999, Pages 955-958

  Cavalli, Giacomo ^a,b   Paro, Renato ^a  

^a UNIVERSITY OF HEIDELBERG   (Germany)

^b INSTITUTE OF HUMAN GENETICS   (France)

Author keywords

[No Author keywords available]

Indexed keywords

TRANSACTIVATOR PROTEIN;

ACETYLATION; ARTICLE; DROSOPHILA; EMBRYO DEVELOPMENT; GENE EXPRESSION REGULATION; GENETIC TRANSCRIPTION; HERITABILITY; NONHUMAN; PRIORITY JOURNAL; TRANSACTIVATION;

ACETYLATION; ANIMALS; ANIMALS, GENETICALLY MODIFIED; CHROMATIN; DNA-BINDING PROTEINS; DROSOPHILA; DROSOPHILA PROTEINS; FUNGAL PROTEINS; GENE EXPRESSION REGULATION, DEVELOPMENTAL; HISTONES; INSECT PROTEINS; LAC OPERON; REGULATORY SEQUENCES, NUCLEIC ACID; SACCHAROMYCES CEREVISIAE PROTEINS; TRANS-ACTIVATORS; TRANSCRIPTION FACTORS;

EID: 0033615616     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.286.5441.955     Document Type: Article

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25. Immunostaining of tetra-acetylated histones on polytene chromosomes used antibodies from Up-state Biotechnology. For heat shock induction of GAL4, embryos were collected at 18°C in standard apple juice-agar plates and heat shocked at 37°C by immersion in a water bath for 45 min. After heat shock, embryos were transferred to 18°C in fresh tubes containing standard Drosophila medium, allowed to develop into third instar larvae, and used for polytene chromosome studies. Late wandering third instar larvae were collected and heat shocked at 37°C for 45 min in a 1.5-ml Eppendorf tube. In all studies with GAL4 induction, memory of activated chromatin states was monitored by inspection of adult eye pigmentation as described in (17). In the FLFW-1 line, derepressed flies have red eyes, as opposed to controls, which have yellow eyes at hatching. Male salivary glands were used for experiments on polytene chromosomes, because activation is stronger in males than in females (a strong heat shock pulse of GAL4 results in up to 80% fully red-eyed males). Activation of lacZ expression upon GAL4 pulses was monitored by β-Gal staining of salivary glands. At 18°C, most salivary glands show a variegated staining pattern. Upon administration of a GAL4 pulse to third instar larvae, all glands stained strongly and homogeneously. When the GAL4 pulse was given to embryos, the large majority of salivary glands of third instar larvae stained homogeneously, although severalfold less strongly than upon a pulse at third instar larvae. Only in a few glands was a variegated pattern retained. All analyses of polytene chromosomes were performed in two to five independent experiments, and 10 to 20 chromosomes were inspected in each experiment to ensure detection of many activated chromosomes in each case.
26. 7-1 balanced over the CyO chromosome. Larvae raised from control embryos grown at 18°C or from embryos submitted to an embryonic GAL4 pulse showed the same pattern. In contrast, a transient hyperacetylation of histone H4 could be observed at 93B upon a 45-min 37°C heat shock at third instar larvae. The signal was lost 100 min after the end of the GAL4 pulse (30). Therefore, a line that does not show trxG-mediated inheritance of active chromatin states does not show stable H4 hyperacetylation.
27. Association of RNA polymerase II during transcriptional activation of the UAS-lacZ reporter in the Fab-7 template of the FLFW-1 line was studied by immunostaining polytene chromosomes in a time course experiment of heat shock GAL4 induction. Strong recruitment of RNA polymerase II was observed 40 and 80 min after the end of a 45-min 37°C heat shock (30). This suggests that hyperacetylation at the same template could have been observed (note the absence of hyperacetylated H4 signal at Fab-7 in Fig. 3, K and L) if it did occur to a substantial degree.
28. 2 embryos from activated FLW-1 flies was now independent of a heat shock treatment. Furthermore, the absence of the GAL4 transgene was directly demonstrated by Southern (DNA) analysis of genomic DNA of activated FLW-1 flies (30).
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30. C. Cavalli and R. Paro, data not shown.
31. We thank F. Sauer, M. Méchali, L. Ringrose, and I. Chen for comments and critical reading of the manuscript; C. Grimaud for support in genetic analyses of PcG and trxG mutant strains; H. Ehret for technical support; and Y. Cully for photographic work. G.C. was supported by a European Molecular Biology Organization long-term fellowship. The work of G.C. is funded by grants from the Association pour la Recherche sur le Cancer and by the Fondation pour la Recherche Médicale. The work of R.P. is funded by grants from the Deutsche Forschungsgemeinschaft and the Human Frontier Science Program and by the Fond der Chemischen Industrie.