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Comparative Study
. 2006 Nov 21;16(22):2222-7.
doi: 10.1016/j.cub.2006.09.035.

Element 1360 and RNAi components contribute to HP1-dependent silencing of a pericentric reporter

Karmella A Haynes  1 , Amy A Caudy, Lynne Collins, Sarah C R Elgin
Affiliations
Comparative Study

Element 1360 and RNAi components contribute to HP1-dependent silencing of a pericentric reporter

Karmella A Haynes et al. Curr Biol. .

Abstract

In eukaryotes, distinct regions of the genome are packaged as euchromatin (less condensed, more active) or heterochromatin (condensed, silenced). Studies in yeast, plants, and flies suggest that RNA interference (RNAi) is linked to heterochromatin formation and transcriptional silencing of transposable element (TE) sequences. We previously reported that insertion of a mobile hsp70-white reporter within 10 kb of a 1360 element on chromosome four of Drosophila melanogaster correlates with variegation (silencing). Here, we report small RNAs (approximately 23 nt) corresponding to 1360, indicating processing by the RNAi machinery. To directly test the ability of 1360 to silence a nearby gene in vivo, we introduced a P element construct carrying a single copy of 1360 upstream of the hsp70-white reporter into flies. This 1360 element contributes to HP1-dependent variegation at a pericentric insertion site, as demonstrated by a decrease in silencing after FLP-mediated removal of 1360. In euchromatin, 1360 is not sufficient to induce silencing, suggesting that proximity to pericentric heterochromatin and/or a high local TE density contributes to heterochromatin formation. Silencing of the 1360, hsp70-white reporter is sensitive to mutations in RNAi components. Our results implicate 1360 as a target for sequence-specific heterochromatic silencing through an RNAi-dependent mechanism.

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Figures

Figure 1
Figure 1
Short RNAs Correspond to Sense and Antisense Strands of 1360 (A) A 1360 fragment from the Su(Ste) repeat (pSY15.1) [18] and copies of 1360 annotated within coordinates 300000..500000 in region 102B of chromosome four (Flybase Release 4.2) compared to the ProtoP consensus [8]. ProtoP (white horizontal bar) contains an ORF that encodes transposase (1194-3784) [8] and a sequence identical (except for a single mismatch at 1897) to the predicted 1360 promoter (1869-1919) [9]; the predicted transcription start site is indicated by a bent arrow (1909) [9]. The Flybase identifier of each 1360 fragment is followed by + or -indicating the orientation of the homologous sequence (black and grey bars, total length shown at right) relative to ProtoP. Arrow heads (far left) indicate cases where a line has been recovered with a P reporter within 10 kb of the 1360; in all such cases, the reporter is variegating (described in [3]). The Su(Ste) sequence contains three putative transcription start sites within < 450 bp of the TIR [18]. The 1360 fragments have large internal deletions, but maintain highly conserved sequences near the ends; many show conservation of Su(Ste) transcription start sites. (B) The siRNA profile of TEs from Drosophila Kc cells as shown by Northern blot. RNA of ca. 23 nt is observed using either the plus strand or minus strand of 1360{}1503 as a probe.
Figure 2
Figure 2
PInsertion Lines Generated by Microinjection and T1-90E Mobilization The silencing capacity of 1360 is assessed by white variegation in the transformant lines. Construct maps (upper left) show P[W], a reference for maximum hsp70-white expression, and P[T1] which contains a FRT-flanked 1360{}1503 placed upstream of hsp70-white. Slashed box = P element ends 3’P (left) and 5’P (right), white arrowheads = FLP recognition sequences (FRT), blue arrows = primers. Primers 1, 2 and 3 (see Supplemental Experimental Procedures for primer sequences) were used for PCR amplification of adult genomic DNA to determine the state of the transgenic 1360. The annotated euchromatic D. melanogaster genome (FlyBase Genome Browser Release 4.1, 2005) is shown (white rectangles = chromosome arms, numbers = polytene divisions, grey circles = centromeres). The P insertion site in each fly line (described in detail in Table S1) is marked by a triangle (red = full red eye, orange = orange eye, speckled = variegated eye, black dot = 5’P end). A horizontal line under T190-177 indicates its two putative map positions. Representative photos of variegating, orange and full red eye phenotypes are shown (top right). In most instances, the single copy of 1360 present in the reporter construct is not sufficient to induce silencing, but in one case (T190-177), a variegating phenotype is observed.
Figure 3
Figure 3
FLP-mediated Removal of 1360 Causes Loss of Silencing at T190-177 (A) Comparison of eye pigment levels in hemizygotes from +1360 vs. -1360 lines. FLP-mediated excision of 1360 is described in Supplemental Experimental Procedures. Total pigment levels are higher when two copies of the transgene are present. (B) Quantitative measurement of eye pigmentation was carried out as described [3]. Bars represent the mean of triplicate samples (10 flies each) with standard error indicated (thin black line). white+ males typically show increased levels of red eye pigment compared to females. Lines lacking the 1360 element show increased expression. (C) Northern blot analysis of hsp70-white transcripts. Numbers represent the means of triplicate white:RpL32 (signal:loading control) ratios normalized to W-43E (control line carrying P[W], set at 100 %). FLP-mediated excision of the 1360 element restores inducible expression from the silenced locus.
Figure 4
Figure 4
HP1-dependent Silencing at T190-177 is Sensitive to Mutations in the RNAi MachineryPhotos (A) and eye pigment levels (B) of male progeny from a cross between males carrying either variegating insert T190-177 (+1360) or FLP1 (indentical except for the excision of 1360) and the following females: yw67c23, wild type (“w.t.”); Su(var)2-502, HP1 loss of function; Dp(2;2)P90, HP1 duplication; Su(var)3-906, HMTase null; piwi1, aubQC42, hlsD125, mutations in RNAi components; dcr-1Q1147X, Dicer-1 loss of function; dcr-2R416X, Dicer-2 loss of function. (C) Each bar represents the mean of triplicate values normalized to the mean wild type eye pigment value for T190-177 (red and pink) or FLP1 (blue and light blue). Silencing at this locus is dependent upon HP1, SU(VAR)3-9 and the RNAi machinery.
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