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. 2006 Dec 15;127(6):1209-21.
doi: 10.1016/j.cell.2006.10.039.

Transcription of bxd noncoding RNAs promoted by trithorax represses Ubx in cis by transcriptional interference

Svetlana Petruk  1 , Yurii Sedkov, Kristen M Riley, Jacob Hodgson, Francois Schweisguth, Susumu Hirose, James B Jaynes, Hugh W Brock, Alexander Mazo
Affiliations

"VSports app下载" Transcription of bxd noncoding RNAs promoted by trithorax represses Ubx in cis by transcriptional interference

Svetlana Petruk et al. Cell. .

"V体育官网" Abstract

Much of the genome is transcribed into long noncoding RNAs (ncRNAs). Previous data suggested that bithoraxoid (bxd) ncRNAs of the Drosophila bithorax complex (BX-C) prevent silencing of Ultrabithorax (Ubx) and recruit activating proteins of the trithorax group (trxG) to their maintenance elements (MEs) VSports手机版. We found that, surprisingly, Ubx and several bxd ncRNAs are expressed in nonoverlapping patterns in both embryos and imaginal discs, suggesting that transcription of these ncRNAs is associated with repression, not activation, of Ubx. Our data rule out siRNA or miRNA-based mechanisms for repression by bxd ncRNAs. Rather, ncRNA transcription itself, acting in cis, represses Ubx. The Trithorax complex TAC1 binds the Ubx coding region in nuclei expressing Ubx, and the bxd region in nuclei not expressing Ubx. We propose that TAC1 promotes the mosaic pattern of Ubx expression by facilitating transcriptional elongation of bxd ncRNAs, which represses Ubx transcription. .

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Figures

Figure 1
Figure 1. Ubx and ncRNAs are expressed in complementary embryonic domains
(A) Map of Ubx and intergenic transcripts. The exons of Ubx and intergenic transcripts (Lipshitz et al., 1987) are shown in black. bxd ME (Tillib et al., 1999) is shown by the black bar below DNA line. Described locations of three bxd transcripts (Sanchez-Elsner et al., 2006) are shown by grey bars. Probes used for in situ hybridization are diagrammed below the cDNAs as black bars. The bottom map shows the extent of deleted regions in pbx1 and pbx2 mutants. Promoters of ncRNAs are indicated by arrows below the probes. (B) Time-course of nascent Ubx and ncRNA transcription. Nascent ncRNA was detected with probes indicated in A. At syncytial blastoderm, RNA 1–7 is expressed earlier than Ubx (left panels). The initial expression domain of Ubx is anterior to that of RNA 1–7 (middle panels, including blow-up of merge). During germband elongation, expression domains of RNA 1–7 in each parasegment are anterior to those of Ubx (right panels). Ubx-expressing cells do not express RNA 1–7, or any ncRNAs detected by probes shown in Figure 1A at any stage (some data not shown). (C) RNA containing exons 1–7, 4, 5, and bxd RNA are expressed within the same intersegmental domains, although their expression differs in different germ layers. (D) Effects of pbx1 and pbx2 deletions on expression of the indicated ncRNAs and endogenous Ubx.
Figure 2
Figure 2. Intergenic transcription represses Ubx in cis
(A) Products of ncRNAs do not directly repress Ubx transcription. A mixture of dsRNAs specific to exons 1, 3, 7, and part of the bxd region, and control dsRNA specific to GFP were injected either into adult females (top panels) or into preblastoderm embryos. Embryos were analyzed by in situ hybridization with probes to Ubx, exons 1–7 and the mixture of probes to exons 1–7 and bxd, as indicated. (B) Immunostaining of embryo carrying the Ubx-GFP transgenic construct N (see map in C) with GFP antibody. (C) Map of the Ubx-GFP transgenic construct N. The transgene contains ncRNA promoters P2 and P5 but lacks promoters P1 and P4. (D) The GFP reporter gene is ectopically expressed (arrows) at blastoderm and germband extension relative to endogenous Ubx. (E) A diagram of predicted expression patterns of Ubx and GFP, assuming repression occurs only in cis. In the absence of the P1 and P4 promoters, the GFP transgene is expected to be expressed in cells where the corresponding endogenous RNAs are expressed. A, P, anterior and posterior of the embryo. (F) Expression of the GFP reporter gene overlaps with expression of ncRNAs that are produced from the P1 (probe ex 1–7 left panels) and P4 (probe ex 4, right panels) promoters. (G) Comparison of the expression of endogenous Ubx and the GFP reporter gene with RNA 1–7 at the blastoderm stage. Expression of GFP is seen in both cytoplasm and nuclei, and RNA 1–7 is detected in the nuclei of the same cells (left).
Figure 3
Figure 3. Ubx is repressed by read-through transcription from intergenic transcription units
(A) Map of the intergenic region between the 3’-exons 7 and 8 of ncRNAs and the Ubx start site. cDNA was synthesized from the primer close to the Ubx start site (RT). Primer sets for PCR amplification are indicated below the map. Probe P5 for in situ hybridization is shown at the bottom. For coordinates of primers and in situ probes see Supplementary material. (B) RT-PCR detection of transcripts in the region proximal to the Ubx promoter. Primer sets are those shown in (A). (C) Transcripts from the region proximal to the Ubx start site are expressed in the same cells as transcripts of the ncRNAs containing exon 7. (D) Transcripts from the region proximal to the Ubx start site are expressed earlier than Ubx in blastoderm embryos (left panels), and they are expressed in cells that do not express Ubx (right panels).
Figure 4
Figure 4. NcRNAs are not required for activation of Ubx in larval imaginal discs
(A). RT-PCR analysis of the amounts of ex1, ex5, bxd, and Ubx RNAs in wing, haltere and 3rd leg imaginal discs in wildtype (wt) and transgenic (N) larvae. Controls without RT are shown in the third row. rp49 was used to normalize the amount of RNA in discs between the two strains (bottom). Primer sets are the same as in Figures 5B,E. (B) In situ hybridization of Ubx and bxd probes to wing (W), haltere (H) and 3rd leg (L) larval imaginal discs in wildtype (wt) and transgenic (N) larvae. Probes are the same as in Figure 1B,C. (C) High magnification detection of Ubx (red) and bxd (green) nascent RNAs by fluorescent in situ hybridization in 3rd leg disc of transgenic larvae. Probes are the same as in (B).
Figure 5
Figure 5. Association of TAC1 complex with active and silenced Ubx in embryos
(A) Expression of GFP reporter (top) and endogenous Ubx (bottom) in mid-stage Drosophila embryos. Non-specific fluorescence is indicated. (B) RT-PCR analysis of the amounts of GFP, Ubx, intergenic and rp49 transcripts in RNA from Ubx/GFP-positive (+) and Ubx/GFP-negative (-) nuclei from 7–13 hr embryos. Primers for exons 4 and 5 and the bxd region were designed according to (Lipshitz et al., 1987; Rank et al., 2002). rp49 was used to normalize the amount of RNA. Controls without RT are shown on the right. (C) Top: map showing the regions of endogenous Ubx and the GFP-Ubx transgene that were tested for association of TAC1 components. Trx binding sites/response elements (TREs) in the bxd region (Tillib et al., 1999) are shown as grey bars, primer sets used for ChIP analysis are indicated by arrows, with distances between them. Bottom: ChIP analyses were performed with antibodies specific to Trx and Sbf1 (as shown on the left) from chromatin isolated from either Ubx/GFP-positive (+) or Ubx/GFP-negative (–) nuclei. Data shown is for the transcribed region 2 kb downstream of the endogenous Ubx start site; indistinguishable results were obtained with the corresponding primers for the GFP transgene. Control, no antibody. Input is shown in the middle. (D) Graph of the relative levels of Trx and Sbf1 in the Ubx transcription unit. Background levels of cross-linking were determined by omitting the precipitating antibody and were then subtracted from the signals. (E) RT-PCR analysis of the amounts of Ubx and ncRNAs in wildtype (wt) and trxB11 (B11) mutant embryos. rp49 was used to normalize the amount of RNA. For coordinates of primer sets for Ubx and exons 1, 7 and 8 see Supplementary material and Figure 1A. Controls without RT are shown on the right. (F) ChIP analysis of histone modifications in the coding region of Ubx. Immunoprecipitations were performed with antibodies against acetylated H3 (AcH3) and H3 dimethylated at K4 (H3-meK4) from the chromatin isolated from Ubx/GFP+ and Ubx/GFP– nuclei. Immunoprecipitated material was PCR amplified using the primers for the region 2 kb downstream of the Ubx start site. Input is the same as in Figure 5C. (G) Chromatin prepared from wildtype (wt) and trxB11 homozygous mutant embryos (B11) was immunoprecipitated with antibodies against AcH3 and H3-meK4. Primers were those for the sequence 2 kb downstream of the Ubx promoter. Control, without antibody.
Figure 6
Figure 6. Elongation factors are required for TAC1 recruitment to Ubx
Chromatin was prepared from wildtype (wt) and the homozygous mutant embryos indicated above each set, and immunoprecipitated with antibodies against Trx, Sbf1 and histone H3-meK4 as indicated on the right. Immunoprecipitated material was PCR amplified with primers for the region 2 kb downstream of the start site (A) and to the promoter (B). Primer sets are the same as in Figure 5C. Control, no antibody.
Figure 7
Figure 7. TAC1 is essential for association of FACT
(A) Chromatin prepared from wildtype (wt) and trxB11 (B11) homozygous mutant embryos was immunoprecipitated with antibodies against Trx or Spt16. Immunoprecipitated material was PCR amplified in with primers for the region 2 kb downstream of the start site (top), for the Ubx promoter (middle), and for the central C bxd region (bottom) (Figure 5C). C, no antibody control. Input is shown at the bottom. (B) Salivary gland polytene chromosomes were prepared from wildtype 3rd instar larvae and a line expressing trx RNAi. The overall structure of chromosomes is indistinguishable (first row). Binding by Trx (row 2) and Spt16 (right row 3) is almost completely abolished in the trx RNAi line, while Ecdysone Receptor (EcR) binding is unaffected (row 3 left). Merge is shown at the bottom. (C) A model for the role of TAC1 in transcription. TAC1 recruitment along with elongation factors and histone modification is necessary for efficient transcriptional elongation of Ubx and ncRNAs in complementary cells in the posterior and anterior regions of each parasegment, respectively. Efficient elongation of Pol II from promoters of ncRNAs proceeds through the 5’-regulatory elements of Ubx, thus preventing its expression. This generates a mosaic pattern of Ubx expression within embryonic parasegments.
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References

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