Abstract
The Drosophila Polycomb and trithorax group proteins act through chromosomal elements such as Fab-7 to maintain repressed or active gene expression, respectively. A Fab-7 element is switched from a silenced to a mitotically heritable active state by an embryonic pulse of transcription. Here, histone H4 hyperacetylation was found to be associated with Fab-7 after activation, suggesting that H4 hyperacetylation may be a heritable epigenetic tag of the activated element. Activated Fab-7 enables transcription of a gene even after withdrawal of the primary transcription factor. This feature may allow epigenetic maintenance of active states of developmental genes after decay of their early embryonic regulators.
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- Immunostaining of tetra-acetylated histones on polytene chromosomes used antibodies from Upstate 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.
- Immunostaining of polytene chromosomes with antibodies against tetra-acetylated histone H4 was carried out in the LW-1 line. This line contains the pU/l5 construct inserted at the 93B cytological location and hsGAL4 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.
- 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.
- The line FLFW-1 carrying a construct with two Fab-7 elements is very inefficient in the meiotic transmission of the activated state. The FLW-1 line with the Fab-7 transgene p5F24 at cytological position 13F showed effective meiotic transmission of active states. Flies of the FLW-1 line were activated by an embryonic pulse of GAL4. Twenty red-eyed F 0 females were crossed with thirty males of the 5F24 25 2 line (9) carrying the same Fab-7 transgene but no GAL4-expressing construct. GAL4-less F 1 flies were selected for strong eye pigmentation (25 to 30% of the total progeny) and recrossed further to obtain F 2 and F 3 . β-Gal staining of F 2 and F 3 embryos was performed as described by B. Zink Y. Engström W. J. Gehring and R. Paro [ EMBO J. 10 153 (1991)]. The staining pattern was compared with control FLW-1 embryos at 18°C or upon a 45-min heat shock followed by 2 hours of recovery or with embryos of the lines U/l5 1 1 and U/l5 2 1 grown at 18°C. Further proof of the absence of the hsp70-GAL4 transgene from activated FLW-1 flies was obtained by demonstrating that β-Gal staining of F 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).
10.1016/S0092-8674(00)80740-0
- G. Cavalli and R. Paro data not shown.
- 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.
Dates
Type | When |
---|---|
Created | 23 years, 1 month ago (July 27, 2002, 5:49 a.m.) |
Deposited | 1 year, 7 months ago (Jan. 13, 2024, 5:34 a.m.) |
Indexed | 3 weeks, 3 days ago (Aug. 6, 2025, 9:37 a.m.) |
Issued | 25 years, 10 months ago (Oct. 29, 1999) |
Published | 25 years, 10 months ago (Oct. 29, 1999) |
Published Print | 25 years, 10 months ago (Oct. 29, 1999) |
@article{Cavalli_1999, title={Epigenetic Inheritance of Active Chromatin After Removal of the Main Transactivator}, volume={286}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.286.5441.955}, DOI={10.1126/science.286.5441.955}, number={5441}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Cavalli, Giacomo and Paro, Renato}, year={1999}, month=oct, pages={955–958} }