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. 2022 Aug 30;222(1):iyac098.
doi: 10.1093/genetics/iyac098.

Altering enhancer-promoter linear distance impacts promoter competition in cis and in trans

Jack R Bateman  1 , Justine E Johnson  1
Affiliations

Altering enhancer-promoter linear distance impacts promoter competition in cis and in trans

Jack R Bateman et al. Genetics. .

Abstract

In Drosophila, pairing of maternal and paternal homologs can permit trans-interactions between enhancers on one homolog and promoters on another, an example of a phenomenon called transvection. When chromosomes are paired, promoters in cis and in trans to an enhancer can compete for the enhancer's activity, but the parameters that govern this competition are as yet poorly understood. To assess how the linear spacing between an enhancer and promoter can influence promoter competition in Drosophila, we employed transgenic constructs wherein the eye-specific enhancer GMR is placed at varying distances from a heterologous hsp70 promoter driving a fluorescent reporter. While GMR activates the reporter to a high degree when the enhancer and promoter are spaced by a few hundred base pairs, activation is strongly attenuated when the enhancer is moved 3 kb away. By examining transcription of endogenous genes near the point of transgene insertion, we show that linear spacing of 3 kb between GMR and the hsp70 promoter results in elevated transcription of neighboring promoters, suggesting a loss of specificity between the enhancer and its intended transgenic target promoter. Furthermore, increasing spacing between GMR and hsp70 by just 100 bp can enhance transvection, resulting in increased activation of a promoter on a paired homolog at the expense of a promoter in cis to the enhancer. Finally, cis-/trans-promoter competition assays in which one promoter carries mutations to key core promoter elements show that GMR will skew its activity toward a wild-type promoter, suggesting that an enhancer is in a balanced competition between its potential target promoters in cis and in trans.

Keywords: Drosophila; Condensin; chromosome pairing; core promoter elements; enhancer–promoter specificity; transvection.

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Figures

Fig. 1.
Fig. 1.
Enhancer–promoter spacing influences strength of transcriptional activation. a) Schematic of transgenes with spacers of varying sizes inserted between GMR and the hsp70 promoter (bent arrow) driving mCherry expression. b) Third instar eye disc from flies carrying a GMR-mCherry transgene lacking a spacer. A and P, anterior and posterior, MF, the morphogenetic furrow that separates differentiated posterior cells from undifferentiated anterior cells. Dashed box indicates general field of view for higher-resolution images in (c) and (d). c, d) mCherry fluorescence in eye discs from flies carrying GMR-mCherry with no spacer (c) and GMR-3kb-mCherry (d). Images represent max-projected confocal z-stacks. e) Relative quantification of mCherry mRNA levels in third instar eye-antennal discs from flies carrying transgenes with spacers relative to those with a transgene lacking a spacer. One-way ANOVA, P<0.0001; Dunnett’s multiple comparisons test relative to no spacer control: 100 bp, P = 0.003; 200 bp, P = 0.0004; 3 kb, P < 0.0001. All transgenes were analyzed as hemizygous insertions.
Fig. 2.
Fig. 2.
Enhancer–promoter spacing influences strength of transvection. a) Schematic of genotypes to assess cis-/trans-promoter competition. Crosses were established to create progeny with an enhancerless hsp70-GFP on one homolog and GMR-mCherry constructs with or without spacers on the other homolog. b) Eye discs showing fluorescence of mCherry (representing activation in cis) and GFP (representing activation in trans) in flies carrying GMR-mCherry with no spacer (left column) or GMR-3kb-mCherry (right column). c, d) Relative quantification of mCherry (c) or GFP (d) mRNA levels in eye-antennal discs from flies carrying the experimental setup depicted in (a). Faded bars with dotted outlines in (c) represent relative mCherry mRNA levels from flies lacking the hsp70-GFP transgene on the homologous chromosome as observed in Fig. 1e. “No GMR” in (d) was generated from flies carrying hsp70-GFP on one homolog and no transgene on the other homolog, and was used as a measure of baseline GFP mRNA levels in the absence of enhancer action in trans. One-way ANOVA, P<0.001 for both (c) and (d); Dunnett’s multiple comparisons test relative to no spacer control in cis (c): 100 bp, P=0.002; 200 bp, P=0.16 (not significant); 3 kb, P<0.0001; Dunnett’s multiple comparisons test relative to no spacer control in trans (d): 100 bp, P<0.0001; 200 bp, P<0.0001; 3 kb, P=0.99 (not significant); no GMR, P<0.0001.
Fig. 3.
Fig. 3.
Increased enhancer–promoter spacing results in ectopic expression of flanking genes. a) Simplified view of the genomic locus surrounding the RMCE insertion site at polytene position 53F. Five genes and their major promoters are depicted, with the table to the right indicating the linear distances in kilobases between GMR and the nearest promoter of each gene for GMR-mCherry and GMR-3kb-mCherry insertions. Two additional small protein-coding genes were not analyzed (CG6984, encoded between GstS1 and Sply, and CG46492, encoded within an intron of CG46491), and an additional weak GstS1 promoter has been annotated to the right of the transgenic insertion site in some annotations (Wangler et al. 2015). Vertical dashed line indicates a TAD boundary; an additional TAD boundary is located roughly 5 kb to the left of CG30460 (Cubeñas-Potts and Corces 2015; Li et al. 2015; Ramírez et al. 2018). All transgenes were inserted such that transcription proceeds from right to left. A more detailed view of the locus is presented in Supplementary Fig. 1, including TAD structure, ATAC-seq/FAIRE-seq peaks, and alternative TSS/splicing of each gene. b, c) RNA in situ hybridization targeting GstS1 in eye discs from control flies lacking a transgene (w1118, b) or with an insertion of GMR-3kb-mCherry (c). d, e) Relative quantification of mRNA levels from the 5 genes depicted in (a) in larval eye-antennal discs from flies carrying GMR-3kb-mCherry relative to GMR-mCherry (d), or from flies carrying GMR-hsp70-lacZ or a promoterless GMR-(P-)lacZ relative to an enhancerless hsp70-lacZ (e). Unpaired t-tests with Holm–Sidak correction for multiple comparisons in GMR-cherry vs GMR-3kb-cherry discs (d), adjusted P<0.05 for all 5 genes; in GMR-hsp70-lacZ vs GMR-(P-)-lacZ (e), adjusted P<0.05 for GstS1, CG46491, and CG15611 only. f) Allele-specific qRT–PCR of CG46491. Top, schematic showing experimental setup based on TaqMan probes that differentiate homologs based on a polymorphic indel that differs between the transgenic chromosome (allele 2) and DGRP line 443 (allele 1). Bottom, relative quantification of mRNA levels for GMR-3kb-GFP compared to 3kb-GFP lacking an enhancer. Unpaired t-tests with Holm–Sidak correction for multiple comparisons, adjusted P<0.05 for both cis and trans expression. All transgenes were analyzed as hemizygous insertions.
Fig. 4.
Fig. 4.
The sev minimal enhancer shows reduced specificity for the transgenic hsp70 promoter in the presence of a 100-bp spacer. a) Schematic of transgenes with the sev minimal enhancer and the hsp70 promoter driving mCherry expression. b) Representative third instar eye discs from flies carrying sev-mCherry transgenes with or without a 100-bp spacer. c, d) Relative quantification of mCherry (c) or GstS1 (d) mRNA levels in third instar eye-antennal discs from flies carrying a transgene with a 100-bp spacer relative to those with a transgene lacking a spacer. Unpaired t-test, P<0.001 in both (c) and (d). Transgenes in panels (a–d) were analyzed as hemizygous insertions. e) Schematic of genotype to assess cis-/trans-promoter competition (left) and relative quantification of mCherry and GFP mRNA levels in eye-antennal discs with or without a 100-bp spacer between the sev minimal enhancer and the hsp70 promoter. Unpaired t-test, P<0.0001 for mCherry in cis, P= 0.09 (not significant) for GFP in trans. f) Eye discs showing fluorescence of mCherry (enhancer acting in cis) and GFP (enhancer acting in trans) in flies carrying sev-mCherry with no spacer (top row) or with a 100-bp spacer (bottom row).
Fig. 5.
Fig. 5.
Mutations in CPEs impact cis-/trans-promoter specificity. a) Schematic of CPEs (TATA box, Inr, and MTE) in the wt hsp70 promoter and mutations designed to generate TATAless and MTEless variants. Sequence context of the wt promoter is provided to the right, with TATA box in blue, Inr in orange, and MTE in green. The transcription start site (TSS) is boxed. b–e) Relative quantification of GFP (b, e), mCherry (c), or lacZ (d) mRNA levels from eye-antennal discs carrying transgenes with promoter variants: b) hsp70-GFP carrying promoter variants and lacking an enhancer. c) GMR-mCherry with a wild-type promoter in trans to hsp70-GFP carrying promoter variants. Variation in expression reflects the ability of the promoter in trans to compete for GMR activity. d) GMR-lacZ carrying promoter variants. e) hsp70-GFP with a wild-type promoter in trans to GMR-lacZ carrying promoter variants. Variation in expression reflects the ability of the promoter in cis to compete for GMR activity. One-way ANOVA, P<0.0002 for each of (b) through (e); Dunnett’s multiple comparisons test, P< 0.001 for each comparison of mutant promoter to wild type in (b) through (e). Transgenes in panels (b) and (d) were analyzed as hemizygous insertions. f) Eye discs showing antibody staining of beta-galactosidase (enhancer acting in cis, top row) and GFP (enhancer acting in trans, middle row) in flies carrying wild type (first column), MTEless (second column), or TATAless (third column) hsp70 promoters upstream of lacZ and a wild-type hsp70 promoter upstream of GFP. g) cis-/trans-promoter competition between GMR-hsp70-mCherry and hsp70-GFP transgenes in wild type and Cap-H20019/Df(3L)Exel6159 backgrounds. Unpaired t-test comparing wild type to mutant, P= 0.0007 for mCherry (cis), P=0.003 for GFP (trans). Note that all transgenes analyzed in Fig. 5 were constructed without spacer elements between the enhancer and promoter.
Fig. 6.
Fig. 6.
Model for influence of spacing on enhancer–promoter specificity in Drosophila. Close spacing between an enhancer and promoter results in high enhancer activity on that promoter in cis (broad arrow), and less activity on nearby potential competing promoters in cis and in trans. With greater spacing, enhancer activity increases on competing promoters (dashed arrows), potentially including nearby promoters in trans, at the expense of the target promoter.
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