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Transcription through enhancers suppresses their activity in Drosophila

Maksim Erokhin12, Anna Davydova13, Alexander Parshikov1, Vasily M Studitsky34, Pavel Georgiev12* and Darya Chetverina12*

Author Affiliations

1 Department of the Control of Genetic Processes, Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov St., Moscow 119334, Russia

2 LIA 1066, Laboratoire Franco-Russe de recherche en oncologie, Moscow 119334, Russia

3 Laboratory of Epigenetic Regulation of Transcription, Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov St., Moscow 119334, Russia

4 Department of Pharmacology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA

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Epigenetics & Chromatin 2013, 6:31  doi:10.1186/1756-8935-6-31


The electronic version of this article is the complete one and can be found online at: http://www.epigeneticsandchromatin.com/content/6/1/31


Received:21 June 2013
Accepted:27 August 2013
Published:26 September 2013

© 2013 Erokhin et al.; licensee BioMed Central Ltd.

This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

Keywords:
Chromatin enhancer; Transcription interference; Transcription regulation; Enhancer suppression; Pass-through transcription

Background

The development of multicellular organisms involves differentiation of various cell types, which is achieved by the establishment of requisite spatial and temporal patterns of gene expression. Regulation of transcription is a highly complex process involving different regulatory DNA elements, enhancers in particular. Enhancers are positive DNA sequences containing multiple binding sites for a variety of transcription factors. These regulatory elements can activate genes over long distances, up to several tens of thousands of base pairs, and act independently of the distance and orientation with respect to the promoters of target genes [1,2].

A number of experiments performed to date indicate that a major portion of the genome is being transcribed and that a large percentage of the transcripts are accounted for by long non-protein-coding sequences (lncRNAs), either in mammals or in Drosophila[3-6]. Recent data suggest that many of lncRNAs have important roles in the regulation of transcription [7]. However, it was found that the expression of lncRNA clusters did not correlate absolutely, either positively or negatively, with the expression of the nearest mRNAs [8]. For instance, transcripts detected in the Drosophila bithorax complex correlate with the repressed state of the locus [9]. In vertebrates, many clusters of imprinted genes contain lncRNAs, and some of them have been implicated in the transcriptional silencing [10]. Similarly, the X chromosome inactivation relies on the expression of a lncRNA named Xist [11]. There is also evidence that a lncRNA expressed from the HOXC locus may negatively affect the expression of genes in the HOXD locus, which is located on a different chromosome [12].

On the other hand, there are data indicative of a positive role of lncRNAs. For example, it has been shown that intergenic transcription through the PRE element counteracts silencing [13]. Some of non-coding RNAs proved to have a positive influence on expression of neighboring protein-coding genes [14]. Moreover, there is a large class of mammalian lncRNAs originating from and/or near the enhancers, named eRNAs. They are associated with active enhancers, and the resulting bidirectional eRNAs can be spliced and polyadenylated. However, regulatory functions of eRNAs remain unknown [15-17].

The detailed mechanism of the lncRNAs action is also not clear. One possibility is that these transcripts can recruit different enzymatic complexes and act as molecular scaffolds [18]. Another possibility involves the mechanism of 'transcription interference’ in which the moving RNA pol II complex can disturb protein complexes associated with DNA [19,20]. For example, transcription across the yeast SER3 promoter interferes with the binding of activators, resulting in gene repression [21]. Another illustration from the yeasts is the dislodging of Rap1 and Gcr1 factors from the ADH1 promoter by non-coding intergenic RNA ZRR1 [22].

In order to evaluate the possible role of intergenic transcription in modulation of enhancer action, we have examined the effect of transcription on the activity of yellow and white gene enhancers using transgenic reporter systems. Here, we present evidence that intergenic transcription counteracts the ability of enhancers to stimulate the promoter of the target gene. Moreover, transcription leads to displacement of the Zeste protein that is required for activity of the enhancer that stimulates white expression in the eyes.

Results

Transcription suppresses the activity of the enhancer that stimulates white gene expression in the eyes

To test the role of transcription in modulation of enhancer action, we used the yellow and white genes. The white gene is required for eye pigmentation, with the eye-specific enhancer being responsible for the high level of its transcription [23]. The yellow gene is responsible for dark pigmentation of the larval and adult cuticle and its derivatives. Two upstream enhancers stimulate its expression in the body cuticle and wing blades [24,25]. At first, we examined the effect of transcription on the activity of the eye enhancer of the white gene.

As a test system, we chose the P-element-based transgenic integration providing the possibility to obtain, in parallel, several independent transgene insertions in different genome locations. To control the potential position effect, the main elements in all constructs used in functional tests were flanked by frt or lox sites for Flp- and Cre-recombinase, respectively. The presence of the frt and lox sites allowed us to delete the flanked DNA fragments and to compare the expression of the reporter gene before and after the deletion of the regulatory elements in one genome position.

To induce transcription, we selected the UAS promoter consisting of the minimal hsp70 promoter (from –43 to +204 bp) and five binding sites for the GAL4 activator [26]. To confirm the ability of this promoter to drive transcription in the eyes, we constructed transgenic lines UAS∆WY in which the white gene was expressed under control of the UAS promoter (Figure  1A). In the absence of GAL4 stimulation, flies carrying the white transgene under control of the UAS promoter displayed eye color phenotypes ranging from pale yellow to dark yellow, which indicated that the UAS promoter only weakly drove white transcription in the eyes. Induction of this promoter by GAL4 resulted in flies with the red eye color corresponding to a high level of white expression.

thumbnailFigure 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.

Next, we tested whether the transcription process could influence the activity of eye enhancer ((UAS)Ey(e)YW construct) (Figure  1B). Hereafter, parentheses in construct designations enclose the elements flanked by the frt or lox sites. The yellow gene was used as a spacer sequence. As a result, the distance between the eye enhancer and the white promoter was 7.1 kb. The eye enhancer (“e”) flanked by frt sites was inserted in direct orientation relative to the white gene in the genomic position between the wing and body enhancers of the yellow gene. The UAS promoter flanked by lox sites was inserted upstream of the enhancers. In all transgenic lines tested, flies had moderate levels of eye pigmentation, suggesting partial suppression of the eye enhancer. In most of the lines, however, eye pigmentation increased significantly after deletion of the UAS promoter ((∆)Ey(e)YW). Thus, the eye enhancer was partially suppressed by the UAS promoter in the absence of GAL4. Induction of the UAS promoter by GAL4 led to complete suppression of the eye enhancer, resulting in eye phenotypes identical to those observed in the absence of enhancers ((UAS)Ey(∆)YW).

At the next step, we performed RT-qPCR analysis of RNA isolated from heterozygous mid-late pupae (the stage of high white expression) of the one transgenic line containing the (UAS)Ey(e)YW parental (P) construct and from its derivatives with GAL4 activator (P + GAL4) or with deletion of UAS promoter (P∆UAS). The results showed that the level of white transcription correlated well with the phenotypic data (Figure  1C). Transcription from the UAS promoter was relatively weak in the absence of GAL4 but increased to a high level (approximately 200-fold) upon induction by GAL4. The increased transcription was detected both upstream and downstream of the white enhancer but not downstream of the yellow terminator sequences. In agreement with phenotypic data, the level of white gene transcription (relative to parental lines) was reduced upon GAL4 induction but increased after deletion of the UAS promoter. A decrease in transcription level was observed downstream of yellow enhancers. This could be explained by the presence of AATAAA in the enhancer sequences, which could contribute to transcription termination.

Next, we tested the importance of eye enhancer orientation in the construct for its sensitivity to transcription from the UAS promoter ((UAS)EyeRYW) (Figure  2A). Eye pigmentation in transgenic flies increased after deletion of this promoter ((∆)EyeRYW); at the same time, induction of transcription resulted in complete inactivation of the eye enhancer. Thus, the enhancer orientation proved to be not important for the observed suppressive effect of transcription.

thumbnailFigure 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.

The suppression of the eye enhancer could be explained either by transcription through the enhancers or by competition for the enhancer between the UAS and white promoters. To determine the role of transcription in suppression of the eye enhancer, we inserted the UAS promoter in the opposite orientation ((UASR)EyeYW) (Figure  2B) and found that all the resulting transgenic lines had an almost wild-type level of eye pigmentation, which did not decrease after either deletion of the UAS promoter ((∆)EyeYW) or induction of transcription by GAL4. These results contradict the promoter competition model, since the opposite orientation of the white and UAS promoters should not affect their ability to compete for the eye enhancer. Thus, transcription leads to suppression of the eye enhancer.

In the transgenic lines described above, the eye enhancer should stimulate white across the yellow promoter, which could reduce the activity of the enhancer and affect the observed result of intergenic transcription. To test this possibility we made the construct with the deleted yellow promoter (Figure  2C). The core 222-bp SV40 terminator (ts) fragment was added downstream of the yellow terminator to stabilize it. In general, lines with deletion of the yellow promoter showed darker eye pigmentation (cf. Figures  1B and 2C), providing indirect evidence for the ability of the yellow promoter to partially insulate the eye enhancer. However, deletion of the UAS promoter increased eye pigmentation in most of the transgenic lines tested, suggesting that the low level of transcription produced by the UAS promoter was still sufficient for affecting the activity of the eye enhancer (Figure  2C). As expected, induction of strong transcription by GAL4 completely repressed the eye enhancer.

One of the transgenic lines with this construct and its derivatives was selected for RT-qPCR analysis (Figure  2D). As in the previously tested line with the (UAS)Ey(e)YW construct, weak transcription from the UAS promoter increased drastically (approximately 440-fold) upon induction by GAL4. Once again, we observed a significant decrease in transcription downstream of the enhancers, suggesting partial termination of transcription in this region. In agreement with phenotypic data, the level of white gene transcription was reduced upon GAL4 induction but increased after deletion of the UAS promoter (Figure  2D). Thus, promoter of the yellow gene in this system did not affect the suppressive effect of transcription from the UAS promoter.Taken together, these results suggest that the ability of the eye enhancer to stimulate the white promoter is sensitive to pass-through transcription.

The effect of transcription is not unique for the eye enhancer: yellow gene enhancers

In all transgenic lines described above, the eye enhancer was inserted between the wing and body enhancers of the yellow gene. We noticed that the UAS promoter weakly affected wing and body pigmentation only when it was located in direct orientation relative to the yellow enhancers (Additional file 1: Figure S1).

Additional file 1: Figure S1. Summarized results of wing and body phenotype analysis in (a) (UAS)Ey(e)YW and (b) (UASR)EyeYW transgenic lines.

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To further test the suppressive effect of transcription on the yellow enhancers, we primarily tested the ability of the UAS promoter to drive transcription in tissues where the yellow gene is transcribed. For this purpose, we constructed transgenic lines carrying the yellow gene under control of the UAS promoter (Figure  3A). In the absence of GAL4 stimulation, the wings and bodies of transgenic flies were pigmented only slightly (grade 2), indicating a low level of yellow transcription, while induction of GAL4 stimulated a high level of transcription, resulting in the wild-type phenotype (grade 5) of flies.

thumbnailFigure 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)∆WY; 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)∆WY 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)∆WY transgenic lines. (E) (UASR)(Ey)∆WY; the UAS promoter drives transcription in the direction from the enhancers.

Next, we tested whether the transcription process could influence the activity of yellow enhancers (Figure  3B). In the (UAS)(Ey)∆WY construct, the white gene with the deleted promoter and 3’-Wari insulator (∆W) was used as a spacer inserted between the enhancers flanked by frt sites and the yellow promoter, so that the distance between the enhancers and the promoter was 4.6 kb. To induce transcription through the enhancers, the UAS promoter flanked by lox sites was placed immediately upstream of the enhancers.

In all transgenic lines obtained, flies had weak pigmentation of the wing and body cuticle that corresponded to the basal level of yellow transcription in the absence of enhancers (grade 2) or to its weak stimulation (grade 3). Induction of GAL4 resulted in the basal level of wing and body pigmentation in all transgenic lines, suggesting complete inactivation of the enhancers (Figure  3B). Deletion of the UAS promoter ((∆)(Ey)∆WY) in the transgenic lines provided for a darker pigmentation of flies, indicating that enhancers recovered their ability to stimulate the target promoter. This result confirmed that the yellow enhancers were strongly suppressed in the presence of the UAS promoter. Deletion of the yellow enhancers ((UAS)(∆)∆WY) resulted in the basal level of wing and body pigmentation of flies in all transgenic lines, confirming that the enhancers accounted for weak yellow stimulation in parental lines. These results showed that the yellow enhancers are very sensitive even to uninduced UAS promoter and that a high level of transcription completely inhibited their activity.

An RT-qPCR analysis of RNA isolated from mid-late pupae of one transgenic (UAS)(Ey)∆WY line and its derivatives (the stage of high yellow expression) showed that transcription from the UAS promoter was relatively weak in the absence of GAL4 but increased approximately 250-fold upon induction by GAL4, with a higher transcription level being detected both upstream and downstream of the yellow enhancers but not downstream of white terminator sequences (Figure  3C). In agreement with phenotypic data, the level of yellow gene transcription (relative to that in the parental line) was reduced upon GAL4 induction but increased after deletion of the UAS promoter (Figure  3C). As in constructs tested previously, transcription was partially terminated on the yellow enhancers.

Termination of transcription by yellow enhancers was also observed by white phenotype (Figure  3D). The white gene contains an IRES-like element [27], which allows its expression to be used for measuring the level of upstream transcription from a distantly placed promoter. Deletion of the yellow enhancers from transgenic lines with the (UAS)(Ey)∆WY construct resulted in increasing eye pigmentation, suggesting that transcription from the UAS promoter was partially terminated on these enhancers. Induction of UAS promoter by GAL4 led to red eye phenotype in transgenic flies (Figure  3D), indicating that the level of transcription downstream of the yellow enhancers was relatively high.

To exclude the role of promoter competition in repression of the yellow enhancers, we reinserted the UAS promoter in the opposite orientation ((UASR)(Ey)∆WY) (Figure  3E). The deletion of the UAS promoter or its induction by GAL4 did not lead to decrease in wing and body pigmentation, indicating that transcription through the yellow enhancers was responsible for their inactivation. The deletion of the yellow enhancers resulted in the basal level of wing and body pigmentation, confirming the role of the enhancers in yellow stimulation.

Taken together, the results of these experiments confirm that transcription through the yellow enhancers leads to their inactivation. Moreover, as in case of eye enhancer, even the very low level of transcription produced by the UAS promoter in the absence of GAL4 is sufficient for strong suppression of the enhancer activity.

Transcription from the Ef1 promoter inhibits the activity of the enhancers

To verify that the observed effect was not unique to the UAS promoter, we tested the strong constitutive promoter of the Elongation factor 1α48D (Ef1).

The (EF1)Ey(e)YW construct was made for the eye enhancer (Figure  4A). In these transgenic lines, all flies had the weak eye pigmentation indicative of strong suppression of the enhancer. Deletion of enhancer ((EF1)Ey(∆)YW) did not change pigmentation, indicating that the enhancer is completely inactivated in the presence of Ef1 promoter, while deletion of the promoter ((∆)Ey(e)YW) resulted in darker pigmentation, restoring the ability of the enhancer to stimulate transcription. Thus, the Ef1 promoter was also found to effectively inhibit the activity of eye enhancer.

thumbnailFigure 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)∆WY, 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)∆WY, the 702-bp SV40 terminator is inserted between the UAS promoter and yellow enhancers. For other designations, see Figures  1 and 2.

We also confirmed that the Ef1 promoter could suppress the activity of the yellow enhancers (Figure  4B). In (EF1)(Ey)∆WY transgenic lines, all flies had the basal level of wing and body pigmentation, indicative of strong suppression of the yellow enhancers. Deletion of enhancers ((EF1)(∆)∆WY) confirmed that they were inactive in the presence of the Ef1 promoter. At the same time deletion of the promoter ((∆)(Ey)∆WY) restored the ability of these enhancers to stimulate transcription. Thus, the Ef1 promoter effectively inhibits the activity of yellow enhancers.

The SV40 transcription terminator strongly reduces the inhibiting effect of transcription on activity of the enhancers

To further confirm that transcription is responsible for repression of enhancer activity, we used the strong transcriptional terminator from SV40 to stop transcription from the UAS promoter. To test the UAS promoter-eye enhancer pair, we inserted the 702-bp SV40 terminator flanked by lox sites between the UAS promoter and the eye enhancers (UAS(Ts)Ey(e)∆YtsW) (Figure  4C). The UAS promoter was placed at 1 kb from the SV40 terminator. As a result, the distance between the UAS promoter and the eye enhancer was 3.1 kb. As expected, induction of the UAS promoter by GAL4 did not affect the activity of the eye enhancer, confirming that SV40 terminator protects enhancer from the negative effect of the transcription. At the same time, deletion of the terminator (UAS(∆)Ey(e)∆YtsW) resulted in reduction of eye pigmentation in only three out of nine transgenic lines, suggesting that suppressive effect of transcription produced by the UAS promoter was weaker then at 1.2 kb (cf. Figures  4C and 2C). However, induction of transcription by GAL4 considerably reduced white expression, indicating that the eye enhancer was still sensitive to high level of transcription.

In the next construct, the SV40 terminator was inserted between the UAS promoter and the yellow enhancers ((UAS)Ts(Ey)∆WY) (Figure  4D). The UAS promoter was flanked by lox sites and inserted immediately upstream of the SV40 terminator. All flies in the resulting transgenic lines had a wild-type level of wing and body pigmentation. This level decreased upon deletion of the enhancers ((UAS)Ts(∆)∆WY), which confirmed that they were active in the parental transgenic lines. On the other hand, no changes in pigmentation were observed upon deletion of the UAS promoter ((∆)Ts(Ey)∆WY) or its induction by GAL4, indicating that terminator effectively protected from suppressive effect of transcription. Thus, the SV40 transcription terminator protects the enhancers from repression mediated by transcription through the enhancer in transgenic lines.

Transcription through eye enhancer leads to dislodging of Zeste protein from the enhancer

Several mechanisms may be involved in suppression of the enhancer activity by pass-through transcription. In particular, transcription may disturb binding of proteins forming active complexes on the enhancers. To test such a possibility, we compared binding of the Zeste protein to the eye enhancer in absence or presence of pass-through transcription. The white gene enhancer contains five binding sites for Zeste, the protein that is known to be important for communication of the eye enhancer with the white promoter [23,28].

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.

thumbnailFigure 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.

To further confirm these results, we performed the same experiment with a pair of transgenic lines carrying either the transgene with the SV40 transcriptional terminator inserted between the UAS promoter and the eye enhancer or its derivative in which this transcriptional terminator was deleted (Figure  5B). In accordance with the previous observation, the Zeste protein was detected by X-ChIP on the eye enhancer only in the presence of the transcriptional terminator.

When we tested Zeste binding to the eye enhancer in the presence or absence of the Ef1 promoter, a positive result was also obtained in the transgenic line lacking the Ef1 promoter (Figure  5C). Taken together these results suggest that transcription through the enhancer leads to dislodging of Zeste from DNA.

Finally, we tested whether pass-through transcription could recruit repression complexes to the enhancer. As shown previously, the Zeste protein is involved in regulation of transcription by Trx/PcG proteins [30,31]. Therefore, we used X-ChIP to examine binding of PcG proteins, PH, the core subunit of PRC1 [32], and Sfmbt, the core subunit of the PhoRC complex [33], to the eye enhancer in transgenic lines used for the analyzes of the Zeste binding (Additional file 2: Figure S2). As a result, we observed no enrichment with these proteins on the eye enhancer in either of these lines (Additional file 2: Figure S2). Thus, our current results do not support the model that pass-through transcription leads to recruitment of the PcG complex to the eye enhancer.

Additional file 2: Figure S2. Results of ChIP with antibodies to (a) Ph and (b) Sfmbt. 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 cross-linked chromatin), with the genome regions for which DNA enrichment was tested being indicated on the abscissa: bxd, positive control; ras64B, negative control; E, eye enhancer of the white gene. “P” indicates that ChIP experiments were performed with a parental transgenic line indicated above the diagram; “PΔUAS,”deletion of the UAS promoter; “PΔTs,”deletion of the 702-bp SV40 terminator; “PΔEF1,”deletion of the EF1 promoter. Vertical lines indicate standard deviations. All ChIP experiments were performed with heads of homozygous transgenic lines in the w1118 background lacking the endogenous eye enhancer and white gene. 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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Discussion

In this study we have demonstrated that transcription suppresses the activity of enhancers. Several mechanisms may be involved in suppression of the enhancer activity by pass-through transcription. The first possibility is 'transcription interference’ by transcription complex that can disturb the association of enhancer-bound proteins with DNA [19,20]. There are several examples demonstrating that transcription leads to dissociation of transcription factors from the promoters in yeast [21,22]. In Drosophila, transcription initiated from the distal promoter of the Adh gene can repress activity of the proximal promoter at the late developmental stages [34]. Similarly bxd untranslated RNAs are involved in repression of Ubx expression [9]. In mammalian cells, it has been shown that pass-through transcription induces dissociation of a CTCF protein from an insulator [35]. Here we have found that transcription through the white enhancer prevents binding of Zeste. Since this protein is critical for communication between the white enhancer and promoter [28], reduction of Zeste binding may account for inactivation of the eye enhancer by pass-through transcription. Such an explanation may also hold for inactivation of the yellow enhancers by transcription.

Suppression of enhancers might be also explained by the ability of some transcripts to recruit chromatin remodeling complexes, known in vertebrates [36]. In particular, experiments with mammals provided evidence for the recruitment of PcG complexes via ncRNA [37,38]. Our results suggest that inactivation of the white enhancer by transcription is not accompanied by the recruitment of the PRC1 and PhoRC complexes. However, we cannot exclude the recruitment of other chromatin remodeling complexes capable of suppressing the activity of enhancers.

Suppression of enhancer activity by transcription may play a general role in the regulation of enhancer activity. It is well known that many functionally active enhancers are located in the introns and exons of transcribed genes [39,40], and the activity of these enhancers is likely to depend on the level of interfering transcription. These types of enhancers could be regulated by a negative feedback mechanism: an increase in transcription leads to a decrease in enhancer activity, thereby preventing excessive activation of the target gene.

It is known today that some enhancer regions are transcribed into non-coding RNAs [15,16]. Similarly to feedback regulation, transcription from one cell-type-specific active enhancer can suppress the activity of neighboring enhancers that would be negatively regulated in a given group of cells or a tissue.

Conclusions

We have analyzed the relationship between the presence of pass-through transcription and the activity of Drosophila enhancers using a transgenic reporter system. The results confirm that pass-through transcription suppresses the ability of enhancers to stimulate the target gene promoters. The effect of enhancer suppression has been observed in experiments with the enhancers of two different genes transcribed from two different promoters. Thus, the effect of transcription appears to be common to different Drosophila enhancers and not specific to the promoter driver. Even the low level of transcription induced by the UAS promoter in the absence of GAL4 activator is sufficient for noticeable inactivation of the enhancers. Accordingly, the presence of the uninduced UAS promoter leads to dislodgement of Zeste protein from the enhancer, which is important for enhancer-promoter communication.

Methods

Drosophila strains, germline transformation, and genetic crosses

All flies were maintained at 25°C on the standard yeast medium. The construct, together with a P element containing defective inverted repeats (P25.7wc) that was used as a transposase source [41], was injected into yacw1118 preblastoderm embryos as described [42,43]. The resulting flies were crossed with yacw1118 flies, and the transgenic progeny were identified by their eye or cuticle pigmentation. The transformed lines were tested for transposon integrity and copy number by Southern blot hybridization. Only single-copy transformants were included in the study.

The lines with DNA fragment excisions were obtained by crossing the transposon-bearing flies with the Flp (w1118; S2CyO, hsFLP, ISA/Sco; +) or Cre (y1w1; Cyo, P[w+,cre]/Sco; +) recombinase-expressing lines [44,45]. All excisions were confirmed by PCR analysis. To induce GAL4 expression, we used the modified yw1118; P[w¯, tubGAL4]117/TM3,Sb line (Bloomington Stock Center #5138), in which the marker mini-white gene was deleted as described [46].

To estimate the levels of yellow and white expression, we visually determined the degree of pigmentation in the abdominal cuticle and wing blades (yellow) and in the eyes (white) of 3-to 5-day-old males developing at 25°C, with reference to standard color scales. Pigmentation of all flies was analyzed in heterozygote. For white, the pigmentation scale ranges from red (R) in wild type, through brownish red (BrR), brown (Br), dark orange (dOr), orange (Or), dark yellow (dY), yellow (Y) and pale yellow (pY), to white (W) in the absence of expression. For yellow, 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, to complete loss of yellow expression.The pigmentation scores were independently determined by two investigators.

The details of crosses used for genetic analysis and for excision of functional elements are available upon request.

Plasmid construction

The constructs were made on the basis of the CaSpeR vector [47]. The 5-kb BamHI-BglII fragment containing the yellow coding region (yc) was inserted in direct orientation into the C∆ plasmid [48] cleaved with BamHI (C∆-yc). The 3-kb SalI - BamHI fragment containing the yellow gene regulatory region (yr) was cloned into the pGEM7 cleaved with XhoI and BamHI (yr-pGEM7). The XbaI-BamHI fragment containing the yellow regulatory region (yr) was then cloned from the yr-pGEM7 vector into C∆-yc cleaved with XbaI and BamHI (C∆-y). The 5-kb BamHI-BglII fragment of the yellow gene coding region (yc) was cloned into the pCaSpeR2 plasmid (yc-C2). The production of the pCaSpeR∆700 plasmid, containing deletion of the Wari insulator at the 3’-side of the mini-white gene was described previously [49]. The DNA sequences of the white gene corresponding to the promoter region (-328 to +169) were deleted from the pCaSpeR∆700 vector [50] [∆prw-pCaSpeR∆700]. The AorI-SmaI fragment of the yellow coding region (yc) with 893-bp upstream sequence lacking enhancers was then cloned from the C∆-y vector into ∆prw-pCaSpeR∆700 cleaved with EcoRI [∆prw-C2∆700-y(-893)]. The HindIII-EcoRI fragment containing the minimal hsp70 promoter with five GAL4 binding sites upstream of it was excised from the pUAST vector (26) and cloned into the pBluescript SK + vector between lox sites to produce the lox(UAS) plasmid.

UAS∆WY

The XbaI-XbaI fragment of the lox(UAS) plasmid was inserted into ∆prw-C2∆700-y(-893) cleaved with XbaI.

(UAS)Ey(e)YW

The eye enhancer (Ee) corresponding to the white gene regulatory sequences from position–1180 to -1849 bp relative to the transcription start site (23) was cloned into pBluescript SK + between frt sites to produce the frt(Ee) plasmid. The HincII-BamHI fragment (containing the eye enhancer) of the frt(Ee) plasmid was inserted in direct orientation into the yr-pGEM7 plasmid cleaved with BglII [yr-frt(Ee)]. The XbaI-BamHI fragment of the yr-frt(Ee) plasmid was cloned into the yc-C2 plasmid cleaved with XbaI and BamHI [yr-frt(Ee)-yc-C2]. The XbaI-XbaI fragment of the lox(UAS) plasmid was inserted into the yr-frt(Ee)-yc-C2 plasmid cleaved with XbaI.

(UAS)EyeRYW

The eye enhancer without flanking frt sites was cut out of the Ee-pBluescript SK + plasmid and cloned in reverse orientation into the yr plasmid cleaved with BglII (yr-EeR). The XbaI-BamHI fragment from the yr-EeR plasmid was cloned into the yc-C2 plasmid cleaved with XbaI and BamHI (yr-EeR-yc-C2). The XbaI-XbaI fragment of the lox(UAS) plasmid was inserted into the yr-EeR-yc-C2 plasmid cleaved with XbaI.

(UASR)EyeYW

The eye enhancer without flanking frt sites was cut out of the Ee- pBluescript SK + plasmid and cloned in direct orientation into the yr plasmid cleaved with BglII (yr-Ee). The XbaI-BamHI fragment from the yr-Ee plasmid containing enhancers was cloned into the yc-C2 plasmid cleaved with XbaI and BamHI (yr-Ee-yc-C2). The XbaI-XbaI fragment of the lox(UAS) plasmid was inserted into the yr-Ee-yc-C2 plasmid cleaved with XbaI.

(UAS)Ey(e)∆YtsW

The 222-bp SV40 terminator from the pGL3basic vector (Promega) was inserted into the pBluescript SK + plasmid cleaved with EcoRV [SV40(s)-pSK]. The XhoI-BamHI fragment of the SV40(s)-pSK was cloned into yc-C2 cleaved with BglII [yc-SV40(s)-C2]. The SpeI-KpnI fragment of the (UAS)Ey(e)YW construct (containing the minimal hsp70 promoter with GAL4-binding sites and the enhancers of yellow and white genes) was inserted into yc-SV40(s)-C2 cleaved with BamHI.

UAS∆YW

The yellow translation start containing AflII-AflII fragment was cut out of the C∆-y plasmid and inserted into the pBluescript SK + plasmid cleaved with EcoRV [y(ATG)-pSK]. The XbaI-XbaI fragment of the lox(UAS) plasmid was inserted into the y(ATG)-pSK plasmid cleaved with SmaI. The BamHI-BamHI fragment corresponding to the lox(UAS)-y(ATG) was cloned into the yc-C2 cleaved with BamHI.

(UAS)(Ey)∆WY

The XbaI-AorI fragment containing the yellow gene enhancers was cut out of the yr plasmid and inserted between frt sites in pGEM-7zf [frt(yr)]. The lox(UAS) sequence was inserted into the frt(yr) plasmid cleaved with XbaI [lox(UAS)-frt(yr)]. The KpnI–NotI fragment of the lox(UAS)-frt(yr) plasmid was cloned into ∆prw-C2∆700-y(-893) cleaved with XbaI.

(UASR)(Ey)∆WY

The SalI-BamHI fragment of the frt(yr) plasmid was inserted into the lox(UAS) plasmid cleaved with BamHI [lox(U)R-frt(yr)]. The sequence corresponding to the lox(UAS)R-frt(yr) was cloned into ∆prw-C2∆700-y(-893) cleaved with XbaI.

(EF1)Ey(e)YW

The promoter of Elongation factor 1α48D gene was PCR-amplified with primers 5’-attgttaactgatttcgcaagc-3’ and 5’-tggatgattacactatggctgtt-3’. The PCR product was inserted into pBluescript SK + between lox sites [lox(prEf1)]. The resulting lox(prEf1) plasmid was sequenced to confirm that no unwanted changes had been introduced into the promoter sequence. The XbaI-XbaI fragment of the lox(prEF1) plasmid was inserted into the yr-frt(Ee)-yc-C2 plasmid cleaved with XbaI.

(EF1)(Ey)∆WY

The SalI–SacII fragment of the frt(yr) plasmid was inserted into ∆prw-pCaSpeR∆700-y(-893) cleaved with XbaI [frt(yr)-∆prw-C2∆700-y(-893)]. The XbaI-XbaI fragment of the lox(prEf1) plasmid was inserted into frt(yr)-∆prw-C2∆700-y(-893) cleaved with XbaI.

UAS(Ts)Ey(e)∆YtsW

The HindIII-EcoRI fragment of the pUAST vector (containing the minimal hsp70 promoter with five GAL4 binding sites upstream of it) was cloned into pBluescript SK + [UAS-pSK]. The 717-bp fragment consisting of the GFP coding region (used as a spacer) was cloned into the UAS-pSK plasmid cleaved with HincII [UAS-gfp]. The XbaI-BamHI fragment of the pUAST vector containing the 702-bp SV40 terminator was inserted into pBluescript SK + between lox sites [lox(SV40b)-pSK]. The XbaI–XbaI fragment of lox(SV40b)-pSK was cloned into the UAS-gfp plasmid cleaved with XhoI [UAS-gfp-lox(SV40b)]. The XbaI–KpnI fragment containing the yellow and white enhancers was cut out of the yr-frt(Ee) plasmid and cloned into UAS-gfp-lox(SV40b) cleaved by BamHI [UAS-gfp-lox(SV40b)-yr-frt(Ee)]. The SpeI–SpeI fragment of UAS-gfp-lox(SV40b)-yr-frt(Ee) was inserted into yc-SV40(s)-C2 cleaved with BamHI.

(UAS)Ts(Ey)∆WY

The XbaI–BamHI fragment of the pUAST vector containing the 702-bp SV40 terminator was inserted into the lox(UAS) plasmid cleaved with ApaI [lox(UAS)-SV40b]. The XbaI-XbaI fragment of the lox(UAS)-SV40b plasmid was inserted into ∆prw-C2∆700-y(-893) cleaved with XbaI.

RT-PCR

RNA was isolated from 20 mid-late pupae with TRI reagent (Ambion) according to the manufacturer’s instructions. Purified RNA pools were digested by RNase-free DNase I (BioLabs) and re-purified using the RNeasy Mini kit (Quagen). For reverse transcription, 3 μg of the generated RNA was incubated with ArrayScript Reverse Transcriptase (Ambion) in the presence of dNTPs, Oligo(dT) (Fermentas) and RNase inhibitor (Ambion) in the supplied reaction buffer at 42°C for 1.5 h, according to the manufacturer’s instructions. The reverse transcriptase was inactivated by heating at 95°C for 5 min. To control DNA digestion by DNase I, additional negative control experiments were performed without reverse transcriptase in the reaction mixture. The generated cDNA pools were used as templates in real-time qPCR using a C1000™ Thermal Cycler with the CFX96 real-time PCR detection module (Bio-Rad). Each PCR was performed in triplicate; cDNA pools were obtained in technical duplicate. Relative levels of mRNA expression were calculated in the linear amplification range by calibration to a DNA fragment standard curve (for genomic DNA) to account for differences in primer efficiency. The results of RT-PCR detection of ras64B were used to standardize the overall amount of cDNA used in PCR assays. Primers used for Q-PCR are given in Additional file 3: Table S1.

Additional file 3: Table S1. Primers used for RT-qPCR analysis of transcripts from transgenic flies.

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X-ChIP

For each experiment, 200 heads from 2-to 5-day-old flies were collected. The material was homogenized in 5 ml of buffer A1 (15 mM HEPES, pH 7.6; 60 mM KCl, 15 mM NaCl, 4 mM MgCl2, 0.5% Triton X-100, 0.5 mM DTT) supplemented with the EDTA-free protease inhibitor cocktail (Roche, Switzerland) and formaldehyde as a crosslinking agent (final concentration 1.8%). The reaction was stopped by adding glycine (final concentration 225 mM). The homogenate was cleared by passing through 100-μm nylon cell strainer (BD Falcon) and pelleted by centrifugation at 4,000 g, 4°C for 5 min. After washing in three 3-ml portions of buffer A1 at 4°C (5 min each) and 3 ml of lysis buffer without SDS, the pellet was treated with 0.5 ml of complete lysis buffer (15 mM HEPES, pH 7.6; 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1%Triton X-100, 0.5 mM DTT, 0.1% sodium deoxycholate, 0.1% SDS, 0.5% N-lauroylsarcosine, EDTA-free protease inhibitor cocktail) and sonicated to break chromatin into fragments with an average length of 700 bp. The material was pelleted by centrifugation at 18,000 g for 5 min, and the supernatant fluid was transferred to a new tube. The pellet was treated with the second 0.5-ml portion of lysis buffer, and the preparation was centrifuged at 18,000 g for 5 min. The two portions of the supernatant fluid were pooled, cleared by centrifuging twice at 18,000 g for 10 min, and the resultant chromatin extract (1 ml) was used in four ChIP experiments after preincubation with A-Sepharose or G-Sepharose (see below). One aliquot (1/10 volume) of chromatin extract after preincubation with Sepharose was kept as a control sample (Input).

ChIP experiments involved incubation with rat antibody to Zeste, rabbit antibody to Sfmbt and rabbit antibody to PH. Corresponding nonimmune IgGs were used as nonspecific antibody controls. Antibody-chromatin complexes were collected with either protein A-Sepharose (Sfmbt and PH) or G-Sepharose (Zeste) beads (Thermo Scientific). The enrichment of specific DNA fragments was analyzed by real-time qPCR, using a C1000™ Thermal Cycler with CFX96 real-time PCR detection module (Bio-Rad).Primers used in ChIP/real-time PCR analyses are listed in Additional file 4: Table S2.

Additional file 4: Table S2. Primers used for PCR in X-ChIP experiments with DNA fragments from the genome or transgenic constructs.

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Antibodies

Antibodies against Zeste (C-end 105 aa of Zeste protein) were raised in rats. Antibodies against Sfmbt (1-348 aa of Sfmbt protein isoform B) and PH (87-521 aa of Ph-p protein isoform A) were raised in rabbits. In all cases, epitopes for antibody production were expressed as 6 × His-tagged fusion proteins in Escherichia coli, affinity purified on Ni Sepharose 6 Fast Flow (GE Healthcare) according to the manufacturer’s protocol and injected into rats/rabbits following the standard immunization procedure. Antibodies were affinity-purified on the same epitope as was used for immunization and tested by Western blotting from wild-type and null material or by IP to confirm their specificity (Additional file 5: Figure S3 and Additional file 6: Supplementary methods).

Additional file 5: Figure S3. (A) Testing of Zeste antibodies by Western blot. Protein extract was prepared from wild-type (WT) or zv77h larvae. Upper panel, antibodies against Zeste; lower panel, control anti-tubulin antibodies. (B) Western blot analysis of nuclear extracts (Input line), PH immunoprecipitates (PH line) and control IgG immunoprecipitates (IgG line) from Sg4 cells with antibodies against PH. (C) Western blot analysis of nuclear extracts (Input line), Sfmbt immunoprecipitates (Sfmbt line) and control IgG immunoprecipitates (IgG line) from Sg4 cells with antibodies against Sfmbt.

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Additional file 6: Supplementary methods. Western blotting, preparation of the nuclear extracts and immunoprecipitation.

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Abbreviations

ChIP: Chromatin immunoprecipitation; DTT: Dithiothreitol; PcG: Polycomb group; PCR: Polymerase chain reaction; qPCR: Quantitative polymerase chain reaction; EDTA: Ethylenediaminetetraacetic acid; EGTA: Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid; SDS: Sodium dodecyl sulfate.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

ME, VS, PG and DC conceived and designed the experiments. ME, AD and DC performed cloning, fly crosses and analysis, and antigen expression. ME performed affinity purification of antibodies and ChIP analysis. ME and DC performed RT-PCR experiments. AP performed embryo injections. ME, VS, PG and DC analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We are grateful to N.A. Gorgolyuk for his help in preparing the manuscript. This work was supported by the Russian Foundation for Basic Research (12-04-00195-а to D.C., 11-04-01250-а to M.E.), the Molecular and Cellular Biology Program of the Russian Academy of Sciences (to P.G.) and the National Science Foundation (7046342 to V.M.S.). Experiments were performed using the equipment of the IGB RAN facilities supported by the Ministry of Science and Education of the Russian Federation.

References

  1. Bulger M, Groudine M: Functional and mechanistic diversity of distal transcription enhancers.

    Cell 2011, 144:327-339. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  2. Palstra RJ, Grosveld F: Transcription factor binding at enhancers: shaping a genomic regulatory landscape in flux.

    Front Genet 2012, 3:195. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  3. Bertone P, Stolc V, Royce TE, Rozowsky JS, Urban AE, Zhu X, Rinn JL, Tongprasit W, Samanta M, Weissman S, Gerstein M, Snyder M: Global identification of human transcribed sequences with genome tiling arrays.

    Science 2004, 306:2242-2246. PubMed Abstract | Publisher Full Text OpenURL

  4. Kapranov P, Cheng J, Dike S, Nix DA, Duttagupta R, Willingham AT, Stadler PF, Hertel J, Hackermüller J, Hofacker IL, Bell I, Cheung E, Drenkow J, Dumais E, Patel S, Helt G, Ganesh M, Ghosh S, Piccolboni A, Sementchenko V, Tammana H, Gingeras TR: RNA maps reveal new RNA classes and a possible function for pervasive transcription.

    Science 2007, 316:1484-1488. PubMed Abstract | Publisher Full Text OpenURL

  5. Cheng J, Kapranov P, Drenkow J, Dike S, Brubaker S, Patel S, Long J, Stern D, Tammana H, Helt G, Sementchenko V, Piccolboni A, Bekiranov S, Bailey DK, Ganesh M, Ghosh S, Bell I, Gerhard DS, Gingeras TR: Transcriptional maps of 10 human chromosomes at 5-nucleotide resolution.

    Science 2005, 308:1149-1154. PubMed Abstract | Publisher Full Text OpenURL

  6. Young RS, Marques AC, Tibbit C, Haerty W, Bassett AR, Liu JL, Ponting CP: Identification and properties of 1,119 candidate lincRNA loci in the Drosophila melanogaster genome.

    Genome Biol Evol 2012, 4:427-442. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  7. Wang K, Chang H: Molecular mechanisms of long noncoding RNAs.

    Mol Cell 2011, 43:904-914. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  8. Hung T, Wang Y, Lin MF, Koegel AK, Kotake Y, Grant GD, Horlings HM, Shah N, Umbricht C, Wang P: Extensive and coordinated transcription of noncoding RNAs within cell-cycle promoters.

    Nat Genet 2011, 43:621-629. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  9. Petruk S, Sedkov Y, Riley KM, Hodgson J, Schweisguth F, Hirose S, Jaynes JB, Brock HW, Mazo A: Transcription of bxd noncoding RNAs promoted by trithorax represses Ubx in cis by transcriptional interference.

    Cell 2006, 127:1209-1221. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  10. Yang PK, Kuroda MI: Noncoding RNAs and intranuclear positioning in monoallelic gene expression.

    Cell 2007, 128:777-786. PubMed Abstract | Publisher Full Text OpenURL

  11. Heard E, Disteche CM: Dosage compensation in mammals: fine-tuning the expression of the X chromosome.

    Genes Dev 2006, 20:1848-1867. PubMed Abstract | Publisher Full Text OpenURL

  12. Rinn JL, Kertesz M, Wang JK, Squazzo SL, Xu X, Brugmann SA, Goodnough LH, Helms JA, Farnham PJ, Segal E, Chang HY: Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs.

    Cell 2007, 129:1311-1323. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  13. Schmitt S, Prestel M, Paro R: Intergenic transcription through a Polycomb group response element counteracts silencing.

    Genes Dev 2005, 19:697-708. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  14. Ørom UA, Derrien T, Beringer M, Gumireddy K, Gardini A, Bussotti G, Lai F, Zytnicki M, Notredame C, Huang Q, Guigo R, Shiekhattar R: Long noncoding RNAs with enhancer-like function in human cells.

    Cell 2010, 143:46-58. PubMed Abstract | Publisher Full Text OpenURL

  15. Kim TK, Hemberg M, Gray JM, Costa AM, Bear DM, Wu J, Harmin DA, Laptewicz M, Barbara-Haley K, Kuersten S, Markenscoff-Papadimitriou E, Kuhl D, Bito H, Worley PF, Kreiman G, Greenberg ME: Widespread transcription at neuronal activity-regulated enhancers.

    Nature 2010, 465:182-187. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  16. De Santa F, Barozzi I, Mietton F, Ghisletti S, Polletti S, Tusi BK, Muller H, Ragoussis J, Wei CL, Natoli G: A large fraction of extragenic RNA Pol II transcription sites overlap enhancers.

    PLoS Biol 2010, 8:e1000384. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  17. Kowalczyk MS, Hughes JR, Garrick D, Lynch MD, Sharpe JA, Sloane-Stanley JA, McGowan SJ, De Gobbi M, Hosseini M, Vernimmen D, Brown JM, Gray NE, Collavin L, Gibbons RJ, Flint J, Taylor S, Buckle VJ, Milne TA, Wood WG, Higgs DR: Intragenic enhancers act as alternative promoters.

    Mol Cell 2012, 45:447-458. PubMed Abstract | Publisher Full Text OpenURL

  18. Spitale RC, Tsai MC, Chang HY: RNA templating the epigenome: long noncoding RNAs as molecular scaffolds.

    Epigenetics 2011, 6:539-543. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  19. Palmer AC, Egan JB, Shearwin KE: Transcriptional interference by RNA polymerase pausing and dislodgement of transcription factors.

    Transcription 2011, 2:9-14. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  20. Mazo A, Hodgson JW, Petruk S, Sedkov Y, Brock HW: Transcriptional interference: an unexpected layer of complexity in gene regulation.

    J Cell Sci 2007, 120:2755-2761. PubMed Abstract | Publisher Full Text OpenURL

  21. Martens JA, Laprade L, Winston F: Intergenic transcription is required to repress the Saccharomyces cerevisiae SER3 gene.

    Nature 2004, 429:571-574. PubMed Abstract | Publisher Full Text OpenURL

  22. Bird AJ, Gordon M, Eide DJ, Winge DR: Repression of ADH1 and ADH3 during zinc deficiency by Zap1-induced intergenic RNA transcripts.

    EMBO J 2006, 25:5726-5734. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  23. Qian S, Varjavand B, Pirrotta V: Molecular analysis of the zeste–white interaction reveals a promoter-proximal element essential for distant enhancer–promoter communication.

    Genetics 1992, 131:79-90. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  24. Geyer PK, Corces VG: Separate regulatory elements are responsible for the complex pattern of tissue-specific and developmental transcription of the yellow locus in Drosophila melanogaster.

    Genes Dev 1987, 1:996-1004. PubMed Abstract | Publisher Full Text OpenURL

  25. Martin M, Meng YB, Chia W: Regulatory elements involved in the tissue-specific expression of the yellow gene of Drosophila.

    Mol Gen Genet 1989, 218:118-126. PubMed Abstract | Publisher Full Text OpenURL

  26. Brand AH, Perrimon N: Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.

    Development 1993, 118:401-415. PubMed Abstract | Publisher Full Text OpenURL

  27. Silicheva M, Golovnin A, Pomerantseva E, Parshikov A, Georgiev P, Maksimenko O: Drosophila mini-white model system: new insights into positive position effects and the role of transcriptional terminators and gypsy insulator in transgene shielding.

    Nucleic Acids Res 2010, 38:39-47. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  28. Kostyuchenko M, Savitskaya E, Koryagina E, Melnikova L, Karakozova M, Georgiev P: Zeste can facilitate long-range enhancer-promoter communication and insulator bypass in Drosophila melanogaster.

    Chromosoma 2009, 118:665-764. PubMed Abstract | Publisher Full Text OpenURL

  29. Moses AM, Pollard DA, Nix DA, Iyer VN, Li XY, Biggin MD, Eisen MB: Large-scale turnover of functional transcription factor binding sites in Drosophila.

    PLoS Comput Biol 2006, 2:e130. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  30. Déjardin J, Cavalli G: Chromatin inheritance upon Zeste-mediated Brahma recruitment at a minimal cellular memory module.

    EMBO J 2004, 23:857-868. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  31. Hur MW, Laney JD, Jeon SH, Ali J, Biggin MD: Zeste maintains repression of Ubx transgenes: support for a new model of Polycomb repression.

    Development 2002, 129:1339-1343. PubMed Abstract | Publisher Full Text OpenURL

  32. Shao Z, Raible F, Mollaaghababa R, Guyon JR, Wu CT, Bender W, Kingston RE: Stabilization of chromatin structure by PRC1, a Polycomb complex.

    Cell 1999, 98:37-46. PubMed Abstract | Publisher Full Text OpenURL

  33. Klymenko T, Papp B, Fischle W, Köcher T, Schelder M, Fritsch C, Wild B, Wilm M, Müller J: A Polycomb group protein complex with sequence-specific DNA-binding and selective methyl-lysine-binding activities.

    Genes Dev 2006, 20:1110-1122. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  34. Corbin V, Maniatis T: Role of transcriptional interference in the Drosophila melanogaster Adh promoter switch.

    Nature 1989, 337:279-282. PubMed Abstract | Publisher Full Text OpenURL

  35. Lefevre P, Witham J, Lacroix CE, Cockerill PN, Bonifer C: The LPS-induced transcriptional upregulation of the chicken lysozyme locus involves CTCF eviction and noncoding RNA transcription.

    Mol Cell 2008, 32:129-139. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  36. Saxena A, Carninci P: Long non-coding RNA modifies chromatin: epigenetic silencing by long non-coding RNAs.

    Bioessays 2011, 33:830-839. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  37. Yap KL, Li S, Muñoz-Cabello AM, Raguz S, Zeng L, Mujtaba S, Gil J, Walsh MJ, Zhou MM: Molecular interplay of the noncoding RNA ANRIL and methylated histone H3 lysine 27 by Polycomb CBX7 in transcriptional silencing of INK4a.

    Mol Cell 2010, 38:662-674. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  38. Kanhere A, Viiri K, Araújo CC, Rasaiyaah J, Bouwman RD, Whyte WA, Pereira CF, Brookes E, Walker K, Bell GW, Pombo A, Fisher AG, Young RA, Jenner RG: Short RNAs are transcribed from repressed polycomb target genes and interact with Polycomb repressive complex-2.

    Mol Cell 2010, 38:675-688. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  39. Birnbaum RY, Clowney EJ, Agamy O, Kim MJ, Zhao J, Yamanaka T, Pappalardo Z, Clarke SL, Wenger AM, Nguyen L, Gurrieri F, Everman DB, Schwartz CE, Birk OS, Bejerano G, Lomvardas S, Ahituv N: Coding exons function as tissue-specific enhancers of nearby genes.

    Genome Res 2012, 22:1059-1068. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  40. Ritter DI, Dong Z, Guo S, Chuang JH: Transcriptional enhancers in protein-coding exons of vertebrate developmental genes.

    PLoS One 2012, 7:e35202. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  41. Karess RE, Rubin GM: Analysis of P transposable element functions in Drosophila.

    Cell 1984, 38:135-146. PubMed Abstract | Publisher Full Text OpenURL

  42. Rubin GM, Spradling AC: Genetic transformation of Drosophila with transposable element vectors.

    Science 1982, 218:348-353. PubMed Abstract | Publisher Full Text OpenURL

  43. Spradling AC, Rubin GM: Transposition of cloned P elements into Drosophila germ line chromosomes.

    Science 1982, 218:341-347. PubMed Abstract | Publisher Full Text OpenURL

  44. Golic KG, Lindquist S: The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome.

    Cell 1989, 59:499-509. PubMed Abstract | Publisher Full Text OpenURL

  45. Siegal ML, Hartl DL: Application of Cre/loxP in Drosophila: site-specific recombination and transgene co-placement.

    Methods Mol Biol 2000, 136:487-495. PubMed Abstract | Publisher Full Text OpenURL

  46. Kyrchanova O, Toshchakov S, Parshikov A, Georgiev P: Study of the functional interaction between Mcp insulators from the Drosophila bithorax complex: effects of insulator pairing on enhancer-promoter communication.

    Mol Cell Biol 2007, 27:3035-3043. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  47. Pirrotta V: Vectors for P-mediated transformation in Drosophila.

    Biotechnology 1988, 10:437-456. PubMed Abstract OpenURL

  48. Savitskaya E, Melnikova L, Kostuchenko M, Kravchenko E, Pomerantseva E, Boikova T, Chetverina D, Parshikov A, Zobacheva P, Gracheva E, Galkin A, Georgiev P: Study of long-distance functional interactions between Su(Hw) insulators that can regulate enhancer-promoter communication in Drosophila melanogaster.

    Mol Cell Biol 2006, 26:754-761. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  49. Chetverina D, Savitskaya E, Maksimenko O, Melnikova L, Zaytseva O, Parshikov A, Galkin AV, Georgiev P: Red flag on the white reporter: a versatile insulator abuts the white gene in Drosophila and is omnipresent in mini-white constructs.

    Nucleic Acids Res 2008, 36:926-937. OpenURL

  50. Erokhin M, Davydova A, Kyrchanova O, Parshikov A, Georgiev P, Chetverina D: Insulators form gene loops by interacting with promoters in Drosophila.

    Development 2011, 138:4097-4106. PubMed Abstract | Publisher Full Text OpenURL