Increased Enhancer-Promoter Interactions during Developmental Enhancer Activation in Mammals
Zhuoxin ChenValentina SnetkovaGrace BowerSandra JacintoBenjamin ClockAtrin DizehchiIros BarozziBrandon J. MannionAna Alcaina‐CaroJavier López-Rı́osDiane E. DickelAxel ViselL PennacchioEvgeny Z. Kvon
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Abstract:
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.Keywords:
Enhancer RNAs
Enhancer trap
CpG site
The HS2 enhancer in the beta-globin locus control region regulates transcription of the globin genes 10-50 kb away. How the HS2 enhancer acts over this distance is not clearly understood. Earlier studies show that in erythroid cells the HS2 enhancer initiates synthesis of intergenic RNAs from sites within and downstream of the enhancer, and the enhancer-initiated RNAs are transcribed through the intervening DNA into the cis-linked promoter and gene. To investigate the functional significance of the enhancer-initiated transcription, here we inserted the lac operator sequence in the intervening DNA between the HS2 enhancer and the epsilon-globin promoter in reporter plasmids and integrated the plasmids into erythroid K562 cells expressing the lac repressor protein. We found that the interposed lac operator/repressor complex blocked the elongation of enhancer-initiated transcription through the intervening DNA and drastically reduced HS2 enhancer function as measured by the level of mRNA synthesized from the epsilon-globin promoter. The results indicate that the tracking and transcription mechanism of the HS2 enhancer-assembled transcriptional machinery from the enhancer through the intervening DNA into the cis-linked promoter can mediate enhancer-promoter interaction over a long distance.
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We have developed a system to study transcriptional regulation of the λ immunoglobulin gene in a natural setting λ light chain producing lymphoid cells. This assay system has allowed the detection of an enhancer element located 3' of the λ gene coding sequence. The enhancer can stimulatetranscription from the λ promoter as well as from other imnunoglobulin and unrelated promoters. Like all enhancers, the λ enhancer can function in either orientation with respect to a promoter, but it is significantly more active in one orientation than in the other. The λ enhancer is unusual in spanning at least 4000 bp of DNA sequence and containing several distinct subelements that haveindependent enhancer activity. The enhancer is also remarkable because it functions in λ light chain producing cells but not in K chain producing cells. This fact can be interpreted to support a model of immunoglobulin gene rearrangement In which rearrangement follows and depends on transcriptional activation.
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In the human genome, the erythroid-specific hypersensitive site HS2 enhancer regulates the transcription of the downstream beta-like globin genes 10-50 kilobases away. The mechanism of HS2 enhancer function is not known. The present study employs RNA protection assays to analyze the transcriptional status of the HS2 enhancer in transfected recombinant chloramphenicol acetyltransferase (CAT) plasmids. In erythroid K562 cells in which the HS2 enhancer is active, the HS2 sequence directs the synthesis of long enhancer transcripts that are initiated apparently from within the enhancer and elongated through the intervening DNA into the cis-linked CAT gene. In nonerythroid HL-60 cells in which the HS2 enhancer is inactive, long enhancer transcripts are not detectable. Splitting the HS2 enhancer between two tandem Ap1 sites abolishes the synthesis of a group of long enhancer transcripts and results in loss of enhancer function and transcriptional silencing of the cis-linked CAT gene. In directing the synthesis of RNA through the intervening DNA and the gene by a tracking and transcription mechanism, the HS2 enhancer may (i) open up the chromatin structure of a gene domain and (ii) deliver enhancer binding proteins to the promoter sequence where they may stimulate the transcription of the gene at the cap site.
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Previous studies have identified an enhancer (enhancer I) at nucleotides (nt) 1074 to 1234 in the genome of the human hepatitis B virus (HBV), which locates immediately upstream from the X gene. By analysis of the expression of the chloramphenicol acetyltransferase gene driven by a heterologous simian virus 40 early promoter, we describe the identification of a second enhancer (enhancer II) at nt 1636 to 1741, which locates downstream of enhancer I and immediately upstream of the core gene. With various deletions at the 5' end of enhancer II, a positive regulatory element was identified at nt 1636 to 1690 (the II-A element), with the 5' boundary between nt 1636 and 1671. The II-A element alone did not have an enhancer function, but the enhancer activity was achieved by the concomitant presence of the sequence from nt 1704 to 1741 (the II-B element). The II-B element alone did not have enhancer activity. These results indicate that cooperation between the II-A and II-B elements is required to exhibit the enhancer activity of enhancer II. We also show that enhancer II stimulates the transcriptional activity of both the SPI and SPII promoters of the surface gene. Therefore, the SPI promoter activity is regulated by the proximal HNF-1 binding element and the distal enhancers I and II. These results indicate that multiple regulatory elements scattered over the whole viral genome are involved in the regulation of expression of each individual HBV gene and that the same regulatory element controls the expression of different HBV genes. The relative positions of these regulatory elements in the HBV genome suggest that they may control the expression of HBV genes in a coordinate and cooperative manner.
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A transcriptional enhancer has been identified in the first intron of the mouse alpha 2 (type I) collagen gene in a region between +418 and +1524 base pairs from the transcriptional start site. The enhancer functions both when it is located 5' and 3' to the promoter that it activates and is independent of the orientation of the element. The enhancer stimulates both the homologous alpha 2 type I [alpha 2(I)] collagen promoter and the heterologous early simian virus 40 promoter. In transient expression experiments, enhancer-dependent transcription from the alpha 2(I) collagen promoter utilizes the same transcriptional start site as the one used in the endogenous alpha 2(I) collagen gene. The enhancer activates transcription at a distance of at least 3 kilobase pairs from the transcriptional start site. The alpha 2(I) collagen enhancer displays cell specificity, since it is functional in NIH 3T3 fibroblasts but completely inactive in a lymphoid cell line, in contrast to two immunoglobulin gene enhancers that show the opposite behavior. We find several areas of sequence homology with viral enhancers, particularly the enhancer of simian virus 40.
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Identical genes in the same cellular environment are sometimes expressed differently. In some cases, including the immunoglobulin heavy chain (IgH) locus, this type of differential gene expression has been related to the absence of a transcriptional enhancer. To gain additional information on the role of the IgH enhancer, we examined expression driven by enhancers that were merely weakened, rather than fully deleted, using both mutations and insulators to impair enhancer activity. For this purpose we used a LoxP/Cre system to place a reporter gene at the same genomic site of a stable cell line. Whereas expression of the reporter gene was uniformly high in the presence of the normal, uninsulated enhancer and undetectable in its absence, weakened enhancers yielded variegated expression of the reporter gene; i.e., the average level of expression of the same gene differed in different clones, and expression varied significantly among cells within individual clones. These results indicate that the weakened enhancer allows the reporter gene to exist in at least two states. Subtle aspects of the variegation suggest that the IgH enhancer decreases the average duration (half-life) of the silent state. This analysis has also tested the conventional wisdom that enhancer activity is independent of distance and orientation. Thus, our analysis of mutant (truncated) forms of the IgH enhancer revealed that the 250 bp core enhancer was active in its normal position, ∼1.4 kb 3′ of the promoter, but inactive ∼6 kb 3′, indicating that the activity of the core enhancer was distance-dependent. A longer segment – the core enhancer plus ∼1 kb of 3′ flanking material, including the 3′ matrix attachment region – was active, and the activity of this longer segment was orientation-dependent. Our data suggest that this 3′ flank includes binding sites for at least two activators.
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Long non-coding RNAs (lncRNAs) have gained widespread interest in the past decade owing to their enormous amount and surprising functions implicated in a variety of biological processes. Some lncRNAs exert function as enhancers, i.e., activating gene transcription by serving as the cis-regulatory molecules. Furthermore, recent studies have demonstrated that many enhancer elements can be transcribed and produce RNA molecules, which are termed as enhancer RNAs (eRNAs). The eRNAs are not merely the by-product of the enhancer transcription. In fact, many of them directly exert or regulate enhancer activity in gene activation through diverse mechanisms. Here, we provide an overview of enhancer activity, transcription of enhancer itself, characteristics of eRNAs, as well as their roles in regulating enhancer activity and gene expression.
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Using transgenic mice, we have defined novel gene regulatory elements, termed ''facilitators.''These elements bilaterally flank, by up to 1 kb, a 200-bp T-cell-specific enhancer domain in the human adenosine deaminase (ADA) gene.Facilitators were essential for gene copy-proportional and integration site-independent reporter expression in transgenic thymocytes, but they had no effect on the enhancer in transfected T cells.Both segments were required.Individual segments had no activity.A lack of facilitator function caused positional susceptibility and prevented DNase I-hypersensitive site formation at the enhancer.The segments were required to be at opposed ends of the enhancer, and they could not be grouped together.Reversing the orientation of a facilitator segment caused a partial loss of function, suggesting involvement of a stereospecific chromatin structure.trans-acting factor access to enhancer elements was modeled by exposing nuclei to a restriction endonuclease.The enhancer domain was accessible to the 4-cutter DpnII in a tissue-and celltype-specific fashion.However, unlike DNase I hypersensitivity and gene expression, accessibility to the endonuclease could occur without the facilitator segments, suggesting that an accessible chromatin domain is an intermediate state in the activational pathway.These results suggest that facilitators (i) are distinct from yet positionally constrained to the enhancer, (ii) participate in a chromatin structure transition that is necessary for the DNase I hypersensitivity and the transcriptional activating function of the enhancer, and (iii) act after cell-type-specific accessibility to the enhancer sequences is established by factors that do not require the facilitators to be present.
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