No evidence for transvection in vivo by a superenhancer:promoter pair integrated into identical open chromatin at the Rosa26 locus

Long-range associations between enhancers and their target gene promoters have been shown to play critical roles in executing genome function. Recent variations of chromosome capture technology have revealed a conprehensive view of intra- and inter-chromosomal contacts between specific genomic sites. The locus control region of the β-globin genes (β-LCR) is a super-enhancer that is capable of activating all of the β-like globin genes within the locus in cis through physical interaction by forming DNA loops. CTCF helps to mediate loop formation between LCR-HS5 and 3’HS1 in the human β-globin locus, in this way thought to contribute to the formation of a “chromatin hub”. The β-globin locus is also in close physical proximity to other erythrocyte-specific genes located long distances away on the same chromosome. In this case, erythrocyte-specific genes gather together at a shared “transcription factory” for co-transcription. Theoretically, enhancers could also activate target gene promoters on different chromosomes in trans, a phenomenon originally described as transvection in Drosophilla. Although close physical proximity has been reported for the β-LCR and the β-like globin genes when integrated at the mouse homologous loci in trans, their structural and functional interactions were found to be rare, possibly because a lack of suitable regulatory elements that might facilitate trans interactions. Therefore, we re-evaluated presumptive transvection-like enhancer-promoter communication by introducing CTCF binding sites and erythrocyte-specific transcription units into both LCR-enhancer and β-promoter alleles, each inserted into the mouse ROSA26 locus on separate chromosomes. Following cross-mating of mice to place the two mutant loci at the identical chromosomal position and into active chromation in trans, their transcriptional output was evaluated. The results demonstrate that there was no significant functional association between the LCR and the β-globin gene in trans even in this idealized experimental context.


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such as GATA-1, NF-E2 and EKLF are essential for efficient globin genes transcription 89 through binding to both the LCR and globin gene promoters. It is therefore presumed that 90 they participate somehow in long-range enhancer-promoter interactions. In fact, both 91 GATA-1 and NF-E2 are essential for LCR and βmaj-globin proximity in murine erythroid cells 92 [16,17], as well as for LCR and γ-globin proximity in human erythroid cells [18]. Similarly, 93 EKLF is required for loop formation between the LCR and β-globin promoter sites, but not for 94 LCR-HS5 and 3'HS1 sites [19]).

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Interestingly, CTCF binding was found around the HSSs at both ends of the locus, 7 96 i.e. LCR-HS5 and the 3'HS1 regions ( Fig. 1A; [13]). Although 3C assays revealed proximal 97 positioning of these sites in the nucleus, it was not confined to erythroid cells. In globin 98 expressing cells, the LCR and 3'HS1 regions are further located proximally to the actively 99 expressed β-globin genes, which structure has been termed an active chromatin hub ( Fig. 1A; 100 [13,20]). Therefore, transcriptional activation of the β-like globin genes is predicted to be a 101 multi-step process, in which HS5-3'HS1 interaction may help to bring LCR enhancer 102 sequences within close proximity of the β-globin promoter, thus facilitating their productive 103 interaction (Fig. 1A).

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It has been reported that non-genic LCR sequences are transcribed in erythroid cells 105 [21,22]. Therefore, LCR and -globin gene may be co-transcribed in the same RNA 106 polymerase II (PolII) factory, which then aids their physical association and transcriptional 107 activation of the -globin gene by the LCR enhancer. In accord with this notion, the β-globin 108 gene locus on mouse chromosome 7 was found to colocalize with erythroid specific genes 109 located 20 Mb away on the same chromosome in erythroid cell nuclei [6]. Furthermore, the

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In their experimental design, though, CTCF-assisted or co-transcriptionally 126 mediated mechanisms were not considered. We therefore decided to re-evaluate transvection 127 in mammals by incorporating well-characterized β-globin cis elements at the ROSA26 locus.

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Firstly, LCR-HS5 and 3'HS1 sequences were introduced into enhancer and promoter alleles, 129 respectively, in expectation that CTCF factors bound at these sites might promote the 130 formation of an interchromosomal bridge, which would in turn facilitate functional interactions 131 between the LCR enhancer elements (HS4~1) and the -globin promoter. In addition,

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In the absence of a cis-linked LCR, the human -globin gene locus becomes 148 heterochromatinized (e.g. in the Hispanic thalassemia patient; [28,29]) and the expression of all 149 the -like globin genes is reduced in transgenic mice [30,31]. Therefore, a -globin transgene 150 without an LCR enhancer in cis on the promoter allele is expected to be heterochromatinized

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In other words, it appears that at least 90% of -globin+3'HS1 gene transcription in the 208 spleen initiates from the -globin gene promoter.

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To test for CTCF binding to well-established CTCF binding sites in LCR-HS5 and 3'HS1 of

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Our observations reported here are consistent with their results (LCR x β(γ) in Fig. 4C).

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Because enhancer-promoter looping in the β-globin gene locus is dependent on

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Despite their significant expression in erythroid cells (Fig. 3A & B), however, we did not 366 observe increased reporter gene expression when compared to that in the absence of a paired 367 enhancer allele in trans (Fig. 4E & F).

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Mix with gDNA Remover (TOYOBO). One-fortieth of the reaction mixture was subjected to 487 quantitative PCR amplification using the KOD SYBR qPCR Mix (Toyobo) and thermal Cycler

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Following fixation with 1% formaldehyde for 10 min at room temperature. Nuclei (2 x 10 7 509 cells) were digested with 12.5 units/ml of micrococcal nuclease at 37°C for 20 min. The