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First published online 23 June 2004
doi: 10.1242/dev.01223

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Mechanisms of HP1-mediated gene silencing in Drosophila

Department of Biochemistry, University of Iowa, Iowa City, IA 52242, USA

View larger version (44K): [in a new window]   Fig. 1. lacI-HP1 association silences heat shock inducible expression of hsp26 and hsp70 reporter genes. (A) The lac-hsp26-hsp70 reporter contains 256 lac operator sites positioned 1.9 and 3.7 kb upstream from the transcriptional start site of the heat shock inducible promoters hsp26 and hsp70, respectively. The hsp26 gene is fused to a barley cDNA fragment as a unique sequence tag and hsp70 is fused to the white reporter gene as a transformation marker. (B) RNA was isolated from adult flies carrying either the lac-hsp26-hsp70 reporter transposon alone, the GFP-lacI expressor and the reporter transposon, or the lacI-HP1 expressor and the reporter transposon. The flies were raised with daily heat shock treatments or non-heat shock conditions. RNA was analyzed for hsp26-tag expression (upper autoradiograph) and rp49 expression (lower autoradiograph) as a loading control. (C) Northern analysis of hsp70-white expression (upper autoradiograph) and rp49 expression (lower radiograph).

View larger version (34K): [in a new window]   Fig. 2. lacI-HP1 association renders the hsp26 promoter less accessible. (A) The hsp26 reporter gene (not to scale) contains a TATA box, HSEs and (GA)n elements within two DNaseI hypersensitive regions. XbaI sites within the HSEs were used for restriction enzyme accessibility. (B) Accessibility of the hsp26 promoter region was determined under non-tethering, GFP-tethering and HP1-tethering conditions. Nuclei were isolated and treated with an excess of XbaI. The DNA was purified, digested to completion with SalI, and analyzed by Southern analysis using the unique hsp26-tag sequences for hybridization. The percent accessibility is shown below each lane. (C) MNase accessibility of the hsp26 promoter was determined in homozygous larvae containing either the lac-hsp26-hsp70 reporter and the lacI-HP1 expressor transgene, or the reporter gene alone. Nuclei isolated from homozygous third instar larvae were treated with increasing amounts of MNase. The DNA was purified and assayed by Southern analysis using the unique hsp26 tag sequences for hybridization. The left pair of membranes was re-hybridized with heterochromatic sequences upstream of the rolled locus (shown on right). A densitometry trace through the fourth lane (top to bottom) of each membrane is plotted below.

View larger version (36K): [in a new window]   Fig. 3. HP1 associates with the silenced hsp26 and hsp70 reporter genes. Chromatin immunoprecipitation experiments were performed to determine whether the lac-hsp26-hsp70 transposon in stocks carrying the lacI-HP1 expressor and the transposon (+ lacI-HP1) or the reporter transposon alone (- lacI-HP1) was associated with HP1. Polyclonal HP1 antibodies were used and polyclonal GFP antibodies served as a negative control. (A) Primer sets corresponding to unique sequences within the reporter genes were used to PCR amplify the immunoprecipitated material. The amount of immunoprecipitated material (designated % input) was quantitated by dividing its signal intensity by the signal intensity generated from a 1:100 dilution of the input material. (B) The limits of HP1 spreading were determined using primer sets corresponding to sequences over the reporter transposon in stock hsp26-4D5. Primers corresponding to sequences located 1.9 (hsp26), 3.7 (hsp70) and 10.1 kb from the 3' end of the lac repeat array and 0.5 and 3.1 kb from the 5' end of the lac repeat array were assayed for HP1 association. (C) The amount of HP1 associated material (% input) determined from immunoprecipitation with anti-HP1 antibodies, with or without lacI-HP1 expression, was compared at each primer site (n=3). A probability (P value) of less than 0.05 was considered to be a significant change.

View larger version (33K): [in a new window]   Fig. 4. Effects of Su(var)3-906 on silencing. The expression levels of the hsp26 and hsp70 reporters in adult flies carrying the lac-hsp26-hsp70 transposon and the lacI-HP1 expressor were compared to adults carrying the lac-hsp26-hsp70 transposon, the lacI-HP1 expressor, and Su(var)3-906. (A) Northern analysis of RNA isolated from adults showing hsp26 expression (upper autoradiograph) and rp49 expression (lower autoradiograph). (B) Northern analysis of RNA isolated from adults showing hsp70 expression (upper autoradiograph) and rp49 expression (lower autoradiograph). (C) Summary of the expression data (n=3).