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Transcription through enhancers suppresses their activity in Drosophila.

Erokhin M, Davydova A, Parshikov A, Studitsky VM, Georgiev P, Chetverina D - Epigenetics Chromatin (2013)

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

View Article: PubMed Central - HTML - PubMed

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.

No MeSH data available.


Related in: MedlinePlus

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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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.

Mentions: We compared binding of Zeste to the eye enhancer by chromatin immunoprecipitation (X-ChIP) assay in transgenic lines homozygous for either the (UAS)Ey(e)YW construct or its (∆)Ey(e)YW derivative obtained by deletion of the UAS promoter (Figure 5A). In all experiments, the Ubx promoter region known to be bound by Zeste[29] and the ras64B coding region were used as the positive and negative control sequences, respectively. As a result, we detected an enrichment of Zeste on the eye enhancer only in derivative transgenic line carrying the transgene lacking the UAS promoter (∆UAS) (Figure 5A), which indicated that even low level of transcription interfered with Zeste binding to the eye enhancer.


Transcription through enhancers suppresses their activity in Drosophila.

Erokhin M, Davydova A, Parshikov A, Studitsky VM, Georgiev P, Chetverina D - Epigenetics Chromatin (2013)

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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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.
Mentions: We compared binding of Zeste to the eye enhancer by chromatin immunoprecipitation (X-ChIP) assay in transgenic lines homozygous for either the (UAS)Ey(e)YW construct or its (∆)Ey(e)YW derivative obtained by deletion of the UAS promoter (Figure 5A). In all experiments, the Ubx promoter region known to be bound by Zeste[29] and the ras64B coding region were used as the positive and negative control sequences, respectively. As a result, we detected an enrichment of Zeste on the eye enhancer only in derivative transgenic line carrying the transgene lacking the UAS promoter (∆UAS) (Figure 5A), which indicated that even low level of transcription interfered with Zeste binding to the eye enhancer.

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

View Article: PubMed Central - HTML - PubMed

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.

No MeSH data available.


Related in: MedlinePlus