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. 2013 Sep 26;6(1):31.
doi: 10.1186/1756-8935-6-31.

Transcription through enhancers suppresses their activity in Drosophila

Maksim Erokhin, Anna Davydova, Alexander Parshikov, Vasily M Studitsky, Pavel Georgiev, Darya Chetverina

Transcription through enhancers suppresses their activity in Drosophila

Maksim Erokhin et al. Epigenetics Chromatin. .

Abstract

Background: Enhancer elements determine the level of target gene transcription in a tissue-specific manner, providing for individual patterns of gene expression in different cells. Knowledge of the mechanisms controlling enhancer action is crucial for understanding global regulation of transcription. In particular, enhancers are often localized within transcribed regions of the genome. A number of experiments suggest that transcription can have both positive and negative effects on regulatory elements. In this study, we performed direct tests for the effect of transcription on enhancer activity.

Results: Using a transgenic reporter system, we investigated the relationship between the presence of pass-through transcription and the activity of Drosophila enhancers controlling the expression of the white and yellow genes. The results show that transcription from different promoters affects the activity of enhancers, counteracting their ability to activate the target genes. As expected, the presence of a transcriptional terminator between the inhibiting promoter and the affected enhancer strongly reduces the suppression. Moreover, transcription leads to dislodging of the Zeste protein that is responsible for the enhancer-dependent regulation of the white gene, suggesting a 'transcription interference' mechanism for this regulation.

Conclusions: Our findings suggest a role for pass-through transcription in negative regulation of enhancer activity.

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Figures

Figure 1
Figure 1
The upstream UAS promoter suppresses activity of the white enhancer. (A) UAS∆WY transgenic lines. The UAS promoter drives expression of the white gene; yellow gene was used as marker to select transgenes. “T” on the 3’-side of genes indicates terminators of transcription. Below the maps, phenotypes of parental lines and those after induction of GAL4 expression (+GAL4) are shown. The color scale for white is indicated above the horizontal line. Only the range of grades that were actually recorded in the flies is shown. Each entry in the frame is the number of transgenic lines with the corresponding pigmentation grade; the shaded region in each frame indicates the “mean level.”T is the total number of lines examined; for derivative constructs, N is the number of lines where the phenotype changed as compared with the parental construct. (B) (UAS)Ey(e)YW lines. The UAS promoter drives transcription through the eye enhancer (E) of the white gene, placed between wing (W) and body (B) enhancers of the yellow gene. Downward arrows indicate lox and frt sites. Below the maps are the expression data for the parental construct and for those derived after in vivo excision of the elements. (C) Quantification of (UAS)Ey(e)YW transcripts by RT-qPCR. Positions of primer pairs (1-2, 3-4, 5-6, 7-8) are indicated. Individual transcript levels were normalized relative to ras64B for the amount of input cDNA. The transgenic material of pupae was obtained from crosses between (P) homozygous parental line and yw1118 line, (P + GAL4) homozygous parental line and GAL4-expressing line, or (P∆UAS) homozygous derivative line with deleted UAS promoter and yw1118 line. Error bars indicate standard deviations. Statistical significance was analyzed using the Student’s t-test and expressed as a P-value. **P< 0.01; ***P< 0.005. Photographs show eye pigmentation in the heterozygous parental line and its derivatives used in RT-qPCRs.
Figure 2
Figure 2
Pass-through transcription is responsible for suppression of the eye enhancer. (A) (UAS)EyeRYW transgenic lines; the eye enhancer is inserted in the opposite orientation. (B) (UASR)EyeYW transgenic lines; the UAS promoter drives transcription in the direction from the enhancers. (C) (UAS)Ey(e)∆YtsW transgenic lines with deletion of the yellow gene promoter (indicated by the absence of an upstream arrow and by parentheses in front of the gene on the scheme); “ts” is the core 222-bp SV40 terminator. (D) Quantification of (UAS)Ey(e)∆YtsW transcripts by RT-qPCR. Positions of primer pairs (1-2, 3-4, 5-6, 9-10) are indicated. RT-qPCR was conducted on mRNAs isolated from transgenic lines at the mid-late pupae stage. Error bars indicate standard deviations. For other designations, see Figure  1.
Figure 3
Figure 3
Transcription through the yellow enhancers leads to their inactivation. (A) UAS∆YW transgenic lines. The UAS promoter drives expression of the yellow gene. The downstream white gene was used as a marker to select transgenes. The color scale for yellow (grades 5 to 2) is indicated above the horizontal line. Grade 5 corresponds to wild-type pigmentation; grades 4 and 3 correspond to partial stimulation of the yellow gene by enhancers; grade 2, to the basal level of yellow expression in the absence of enhancers. Grade 1, corresponding to complete loss of yellow expression, is not shown, because no lines with such a phenotype were obtained in this study. (B) (UAS)(Ey)∆W∆Y; the UAS promoter drives transcription through the yellow enhancers. The white gene with deleted promoter was used as a spacer. (C) Quantification of (UAS)(Ey)∆W∆Y transcripts by RT-PCR. Positions of primer pairs (11-12, 5-6, 13-14) are indicated. RT-qPCR was conducted on mRNAs isolated from transgenic lines at the mid-late pupae stage. Error bars indicate standard deviations. *P< 0.05; ***P< 0.005. For other designations, see Figure  1. (D) Summarized results of eye phenotype analysis in (UAS)(Ey)∆W∆Y transgenic lines. (E) (UASR)(Ey)∆W∆Y; the UAS promoter drives transcription in the direction from the enhancers.
Figure 4
Figure 4
Transcription initiated on the Ef1 promoter suppresses the enhancer activity, while the SV40 transcriptional terminator protects the enhancers from the repressive effect of transcription. (A) (EF1)Ey(e)YW, the Ef1 promoter drives transcription through eye enhancer. (B) (EF1)(Ey)∆W∆Y, the Ef1 promoter drives transcription through yellow enhancers. (C) UAS(Ts)Ey(e)∆YtsW, the 702-bp SV40 terminator (Ts) is inserted between the UAS promoter and the eye enhancer. (D) (UAS)Ts(Ey)∆W∆Y, the 702-bp SV40 terminator is inserted between the UAS promoter and yellow enhancers. For other designations, see Figures  1 and 2.
Figure 5
Figure 5
Transcription through eye enhancer leads to dislodging of Zeste from the enhancer. Results of ChIP with antibodies to the Zeste protein from (A) (UAS)Ey(e)YW, (B) UAS(Ts)Ey(e)∆YtsW and (C) (EF1)Ey(e)YW transgenic lines. Diagrams summarize the results of ChIP with specific antibodies followed by real-time PCR. The ordinate shows the percentage of target sequences in the immunoprecipitated material relative to the input (10% of total crosslinked chromatin), with the genome regions for which DNA enrichment was tested being indicated on the abscissa: pUbx, promoter of the Ubx gene, positive control; ras64B, negative control; E, eye enhancer of the white gene; pW, promoter of the white gene; codW, coding part of the white gene. “P” indicates that ChIP experiments were performed with a parental transgenic line indicated above diagram; “P∆UAS”deletion of the UAS promoter; “P∆Ts”deletion of the 702-bp SV40 terminator; “P∆EF1”deletion of the EF1 promoter. Vertical lines indicate standard deviations. All ChIP experiments were performed with chromatin isolated from heads of 2-to 5-day-old males from transgenic lines homozygous for the test construct. Background immunoprecipitation (the average normalized level after chromatin treatment with a nonspecific antibody) was subtracted from normalized specific ChIP signals (obtained with specific antibodies) at each position.
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