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Intergenic transcription through a polycomb group response element counteracts silencing - PubMed

  • ️Sat Jan 01 2005

Comparative Study

. 2005 Mar 15;19(6):697-708.

doi: 10.1101/gad.326205. Epub 2005 Mar 1.

Affiliations

Comparative Study

Intergenic transcription through a polycomb group response element counteracts silencing

Sabine Schmitt et al. Genes Dev. 2005.

Abstract

Polycomb group response elements (PREs) mediate the mitotic inheritance of gene expression programs and thus maintain determined cell fates. By default, PREs silence associated genes via the targeting of Polycomb group (PcG) complexes. Upon an activating signal, however, PREs recruit counteracting trithorax group (trxG) proteins, which in turn maintain target genes in a transcriptionally active state. Using a transgenic reporter system, we show that the switch from the silenced to the activated state of a PRE requires noncoding transcription. Continuous transcription through the PRE induced by an actin promoter prevents the establishment of PcG-mediated silencing. The maintenance of epigenetic activation requires transcription through the PRE to proceed at least until embryogenesis is completed. At the homeotic bithorax complex of Drosophila, intergenic PRE transcripts can be detected not only during embryogenesis, but also at late larval stages, suggesting that transcription through endogenous PREs is required continuously as an anti-silencing mechanism to prevent the access of repressive PcG complexes to the chromatin. Furthermore, all other PREs outside the homeotic complex we tested were found to be transcribed in the same tissue as the mRNA of the corresponding target gene, suggesting that anti-silencing by transcription is a fundamental aspect of the cellular memory system.

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Figures

Figure 1.
Figure 1.

Transgenic reporter constructs to study the function of noncoding transcription through PREs. The Fab-7 PRE is located downstream of either the constitutively active actin5c promoter (pFAs and pFAas) or the transiently active zygotic hunchback (hb) promoter (pFHs and pFHas). The orientation of the Fab-7 element is either proximal-distal (p-d) or distal-proximal (d-p) in relation to its orientation in the BX-C (Martin et al. 1995; see also Fig. 5). As such, either sense (d-p, pFAs and pFHs) or antisense (p-d, pFAas and pFHas) strand with respect to the Abdominal B (AbdB) mRNA direction is produced. The Fab-7 PRE is flanked by FRT sites (triangle), and the promoters are cloned between loxP sites (oval), allowing the separate excision of these sequences from the transgene by Flp/FRT and Cre/loxP recombination, respectively.

Figure 2.
Figure 2.

Fab-7 is activated upon constitutive transcription through the PRE. Shown are pictures of adult fly eyes of the pFAs and pFAas transgenic lines before and after all possible recombination steps. (A,E) Expression of the miniwhite marker, when the Fab-7 PRE is transcribed from the actin5c promoter. (B,F) Eye pigmentation in the presence of the Fab-7 PRE but absence of the actin5c promoter on the transgene. (C,G) miniwhite expression in the absence of Fab-7 when the actin5c promoter is in the vicinity of the marker gene. (D,H) Level of eye pigmentation when neither Fab-7 nor the actin5c promoter are on the transgene. (I) miniwhite expression level determined by photometric pigment measurements before and after Cre/loxP and/or Flp/FRT recombination in four independent transgenic lines. Shown are the mean values of three independent measurements.

Figure 3.
Figure 3.

Epigenetic activation requires transcription to pass through the Fab-7 PRE. In the pFTA construct, the hsp70 3′UTR transcription termination signal (txterm) is inserted between the Fab-7 element and the actin5c promoter. In the pFLA transgene, the Fab-7 PRE is separated from the actin5c promoter by λ DNA of the same length as the hsp70 3′UTR lacking termination sequences. The termination signal and λ DNA are flanked by loxP sites, the Fab-7 PRE by FRT sequences. (A,B) miniwhite expression in the pFTA line before and after excision of the hsp70 3′UTR from the transgene, respectively. (A′,B′) In situ hybridizations detecting Fab-7 RNA to show that transcription from the actin5c promoter is efficiently terminated before the PRE is reached. (C,D) The level of eye pigmentation remains constant independent of the presence or absence of λ DNA on the transgene. C′,D′) RNA in situ hybridization to confirm that increasing the distance between the Fab-7 element and the actin5c promoter does not influence transcription through the PRE.

Figure 4.
Figure 4.

The maintenance of epigenetic activation requires continuous transcription. (A-H) miniwhite expression in the pFHs and pFHas transgenic lines before and after all possible recombination steps. (A,E) Eye pigmentation when the Fab-7 PRE is transcribed from the zygotic hb promoter. (B,F) Level of miniwhite expression after removal of the zygotic hb promoter. (C,G) Excision of the Fab-7 element leads to derepression of the miniwhite marker. (D,H) Further excision of the zygotic hb promoter from the transgene does not change the activity of the miniwhite gene. (I-N) Change in the level of eye pigmentation by expression of the conditionally active Cre-EBD fusion protein. (I) In the absence of estrogen, the expression of the miniwhite gene was not changed. (J) Induction of Cre-EBD activity by transferring first-instar larvae to food containing 0.06 mg/mL estrogen resulted in clones with low eye pigmentation (red circle) in one out of 20 adult flies. (K) Expected level of eye pigmentation when the Cre-EBD and eyGAL4 transgenes are combined with the pFAas transgene. (L-N) Similar conditions as in I-K for a line lacking the Fab-7 PRE (no clones were observed in these cases.

Figure 5.
Figure 5.

Noncoding transcripts of the BX-C were also transcribed at later stages of development. (A) In situ hybridizations using RNA probes which detect the Ubx and AbdB mRNAs as well as the noncoding bxd, Mcp, and Fab-7 PRE sense RNAs in the brain of third-instar larvae. Below the in situ hybridizations is a drawing showing the organization of the BX-C and the location of the sequences used for the preparation of RNA probes. (B) Fluorescent in situ hybridization showing the Fab-7 sense transcript in the posterior tip of the brain costained with DAPI to visualize the DNA.

Figure 6.
Figure 6.

Noncoding transcription through predicted PREs. Shown are in situ hybridizations using strand-specific probes to detect mRNAs as well as transcripts spanning predicted PRE sequences. (A) The en PRE was transcribed in sense and antisense orientation in a pattern that reflects the en mRNA distribution. (B) Both sense and antisense transcription through the predicted slou PRE were detected in the same cells that express the slou mRNA. (C) The predicted salm PRE was transcribed in both orientations in the same pattern as the respective mRNA. (D) Transcription through the predicted tll PRE was found only in the antisense direction, in a pattern that reflects the tll mRNA expression domain. For this PRE, transcripts were also detected in the optic lobes of third-instar larvae.

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