Assay of insulator enhancer-blocking activity with the use of transient transfection
3
Citation
41
Reference
10
Related Paper
Citation Trend
Keywords:
Transient (computer programming)
Plasmid preparation
Agarose gel electrophoresis
Strain (injury)
T-DNA Binary system
Transduction (biophysics)
Cite
Citations (0)
Abstract Enhancers are short DNA sequences that activate their target promoter from a distance; however, increasing the genomic distance between the enhancer and the promoter decreases expression levels. Many genes are controlled by combinations of multiple enhancers, yet the interaction and cooperation of individual enhancer elements is not well understood. Here, we developed a novel synthetic platform that allows building complex regulatory landscapes from the bottom up. We tested the system by integrating individual enhancers at different distances and revealed that the strength of an enhancer determines how strongly it is affected by increased genomic distance. Furthermore, synergy between two enhancer elements depends on the distance at which the two elements are integrated: introducing a weak enhancer between a strong enhancer and the promoter strongly increases reporter gene expression, allowing enhancers to activate from increased genomic distances.
Cooperativity
Enhancer trap
Enhancer RNAs
genomic DNA
Cite
Citations (5)
Cite
Citations (77)
Gene expression is determined by genomic elements called enhancers, which contain short motifs bound by different transcription factors (TFs). However, how enhancer sequences and TF motifs relate to enhancer activity is unknown, and general sequence requirements for enhancers or comprehensive sets of important enhancer sequence elements have remained elusive. Here, we computationally dissect thousands of functional enhancer sequences from three different Drosophila cell lines. We find that the enhancers display distinct cis -regulatory sequence signatures, which are predictive of the enhancers’ cell type-specific or broad activities. These signatures contain transcription factor motifs and a novel class of enhancer sequence elements, dinucleotide repeat motifs (DRMs). DRMs are highly enriched in enhancers, particularly in enhancers that are broadly active across different cell types. We experimentally validate the importance of the identified TF motifs and DRMs for enhancer function and show that they can be sufficient to create an active enhancer de novo from a nonfunctional sequence. The function of DRMs as a novel class of general enhancer features that are also enriched in human regulatory regions might explain their implication in several diseases and provides important insights into gene regulation.
Enhancer RNAs
Sequence motif
Cite
Citations (137)
Abstract IncX4 plasmids are one of the most epidemiologically successful vehicles for mcr-1 spread. Here we found that the IncX4 plasmids carried two different replication proteins encoded by genes pir-1 and pir-2 , respectively, but mcr-1 was only carried by IncX4 plasmid encoding pir-1 . The copy number of pir-2 encoding plasmids (3.15±0.9 copies) are higher than that of pir-1 encoding plasmids (0.85±0.5 copies). When mcr-1 was cloned into IncX4 plasmid encoding pir-2 , the higher copy number of these plasmids resulted in increased expression of mcr-1 and a greater fitness burden on their host cells. However, these plasmids exhibited a lower rate of invasion into the bacterial population compared to mcr-1 positive plasmids encoding the pir-1 gene. These findings collectively explain the absence of mcr-1 in all IncX4 plasmids encoding pir-2 . Our results further confirmed that low-copy numbers are important for the spread of mcr-1 plasmid from the perspective of natural evolution.
Cite
Citations (0)
Abstract Genomic enhancers are key transcriptional regulators which, upon the binding of sequence-specific transcription factors, activate their cognate target promoters. Although enhancers have been extensively studied in isolation, a substantial number of genes have more than one simultaneously active enhancer, and it remains unclear how these cooperate to regulate transcription. Using Drosophila melanogaster S2 cells as a model, we assay the activities of more than a thousand individual enhancers and a million enhancer pairs towards housekeeping and developmental core promoters with STARR-seq. We report that housekeeping and developmental enhancers show distinct modes of enhancer-enhancer cooperativity: while housekeeping enhancers are additive such that their combined activity mirrors the sum of their individual activities, developmental enhancers are synergistic and follow a multiplicative model of cooperativity. This developmental enhancer synergy is promiscuous and neither depends on the enhancers’ endogenous genomic contexts nor on specific transcription factor motif signatures, but it saturates for the highest levels of enhancer activity. These results have important implications for our understanding of gene-regulation in complex multi-enhancer loci and genomically clustered housekeeping genes, providing a rationale for strong and mild transcriptional effects of mutations within enhancer regions.
Housekeeping gene
Cooperativity
Enhancer RNAs
Transcription
Housekeeping
Enhancer trap
Cite
Citations (3)
Remote enhancers are thought to interact with their target promoters via physical proximity, yet the importance of this proximity for enhancer function remains unclear. Here, we investigate the 3D conformation of enhancers during mammalian development by generating high-resolution tissue-resolved contact maps for nearly a thousand enhancers with characterized in vivo activities in ten murine embryonic tissues. 61% of developmental enhancers bypass their neighboring genes, which are often marked by promoter CpG methylation. The majority of enhancers display tissue-specific 3D conformations, and both enhancer-promoter and enhancer-enhancer interactions are moderately but consistently increased upon enhancer activation in vivo. Less than 14% of enhancer-promoter interactions form stably across tissues; however, these invariant interactions form in the absence of the enhancer and are likely mediated by adjacent CTCF binding. Our results highlight the general significance of enhancer-promoter physical proximity for developmental gene activation in mammals.
Enhancer RNAs
Enhancer trap
CpG site
Cite
Citations (8)
IncX4 plasmids are one of the most epidemiologically successful vehicles for mcr-1 spread. Here we found that the IncX4 plasmids carried two different replication proteins encoded by genes pir-1 and pir-2, respectively, but mcr-1 was only carried by IncX4 plasmid encoding pir-1. The copy number of pir-2 encoding plasmids (3.15±0.9 copies) are higher than that of pir-1 encoding plasmids (0.85±0.5 copies). When mcr-1 was cloned into IncX4 plasmid encoding pir-2, the higher copy number of these plasmids resulted in increased expression of mcr-1 and a greater fitness burden on their host cells. However, these plasmids exhibited a lower rate of invasion into the bacterial population compared to mcr-1 positive plasmids encoding the pir-1 gene. These findings collectively explain the absence of mcr-1 in all IncX4 plasmids encoding pir-2. Our results further confirmed that low-copy numbers are important for the spread of mcr-1 plasmid from the perspective of natural evolution.
Replicon
Cite
Citations (0)
Enhancer elements regulate the tissue- and developmental-stage-specific expression of genes. Recent estimates suggest that there are more than 50,000 enhancers in mammalian cells. At least a subset of enhancers has been shown to recruit RNA polymerase II transcription complexes and to generate enhancer transcripts. Here, we provide an overview of enhancer function and discuss how transcription of enhancers or enhancer-generated transcripts could contribute to the regulation of gene expression during development and differentiation.
Enhancer RNAs
Transcription
RNA polymerase II
Cite
Citations (8)
Gene transcription can be regulated by promoter competition when multiple promoters are available for a single enhancer. The converse, however, is largely undescribed. Here we report an occurrence of several enhancers competing for one promoter (enhancer interference) and propose its underlying mechanism. The promoter targeting sequence from the Abdominal-B locus of the Drosophila bithorax complex overcomes the enhancer-blocking activity of insulators in transgenic embryos. It may facilitate the long-range enhancer–promoter communication in Abdominal-B by bypassing insulator elements such as Frontabdominal-7 and Frontabdominal-8 . In transgenic embryos, the anti-insulator activity allowed both an insulator-blocked enhancer and a nonblocked enhancer to contact the same promoter. We found that these two enhancers exhibited mutual inhibition or mutual exclusion in cells where both are transcriptionally active. This enhancer interference occurred at the level of enhancer–promoter communication. It occurred only between enhancers on opposite sides of an insulator and depended on enhancer activity. We hypothesize that enhancer interference limits the interaction of the Abdominal-B promoter to the enhancer(s) from only one regulatory domain in a specific abdominal segment.
Enhancer trap
Enhancer RNAs
Transcription
Cite
Citations (21)