Received 7 April 2016; accepted 15 July 2016; published 18 July 2016

ABSTRACT

In previous work it was shown that mutation of site 1 in the downstream enhancer sequence (DE) led to ablation of enhancement. Mutation of the Wilms tumour factor element (Wt), situated in the Amh promoter between the tata box and the start of translation (TSS), also led to ablation of enhancement. This suggested that these sites may be the anchor points for a specific duplex factor bridging remote DNA elements to the promoter. Mutation analysis of the DNA sequence between sections 1 and 2 of DE was carried out by site directed mutagenesis. It is reported here that site 4 lying between DE1 and DE2, plays a key role in controlling the level of enhancement.

Keywords:

Enhancement-, Super-Enhancement, Amh, Gene Expression

1. Introduction

AMH (Amh) (Anti-Müllerian hormone), a member of the TGF β (BMP) family of growth factors, has been widely studied with regard to its role in sexual development [1] . SMAT-1, a prepubertal line of mouse Sertoli cells [2] , transfected with a plasmid where a prokaryotic reporter gene (d2EGFP) is driven and controlled by Amh promoter and enhancer sequences, has helped an understanding of control of gene expression. While the use of such a reporter may be somewhat indirect to the expression of the eukaryotic gene in its normal physiological environment, results obtained using the EGFP reporter largely agree with previous work where Amh expression was measured directly [3] - [7] : thus providing support for the view that reporter gene analysis can make valid contributions to understanding the driving and control of specific gene expression. It is assumed that SMAT cells contain an Amh gene in its normal genomic setting together with a full set of the requisite transcription factors present in a prepubertal Sertoli cell. Figure 1(a) is a summary of key factors thought to influence Amh expression. The role of promoter elements in Amh (EGFP) expression is summarised in the legend to Figure 1(b). The prospective downstream enhancer region (DE) was divided into 3 subregions on the basis of a comparison with human AMH.

2. Methods

SMAT-1 cells [2] were grown adherently to tissue culture plasticware in DMEM-F12 medium containing glutamax (Gibco), 10% FCS and antibiotics (penicillin at 10 U/ml; streptomycin at 10 µg/ml)―for an assay 10⁵ cells were established in individual wells of Costar 24-well plates, one day prior to transfection (in the absence of antibiotics) by LipofectAmine 2000 with 800 ng plasmid DNA per well. After a further 2 days of culture the green EGFP expression was measured using a flow cytometer. Fluorescence emission by individual cells was recorded as red and green so autofluorescence could be excluded. Expressed d2EGFP has a half-life of 2 hours in vivo, so measurement is mainly of rate not accumulation. An index of expression was calculated as geometric mean brightness per cell (G_m) x% cells in the green window.

Site directed mutations were successfully carried out in the promoter region and at DE2 in the enhancer, using the double overlap PCR method [8] [9] with Deep Vent polymerase: this method was consistently unsuccessful when applied to other enhancer sites, using oligo-nucleotide primers based on the sequence shown in Figure 1(b). The “Phusion” method (Thermopol) was successful and has been used for mutation sites 3 - 6 (see Figure 1(b)).

3. Experiments and Results

GATA-1 and GATA-4 DNA have opposite effects when added to the transfection mixture which included a plasmid vector with an Amh promoter but lacking any enhancer DNA [10] . Figure 2 shows that a proportionally similar difference is still manifested if the DE enhancer sequence is added to the vector, although the actual overall level of expression is higher. It is clear from the results summarised in this figure that control at the level of the promoter is supplemented and not overridden by enhancement. It seems that the enhancement mechanism is independent of control at the level of transcription factor-promoter interaction: however with other enhancers other interpretations are likely [14] [15] .

Previously it was shown that a DNA sequence immediately downstream of the PA signal of a mouse Amh gene, when inserted in an equivalent position in the EGFP plasmid vector, had a moderate enhancer effect on green (reporter) expression [12] . Since it seemed possible that ~23 bp of DE between DE1 and DE2, might include a key sequence for enhancement, DNA of this region was made by annealing the appropriate oligo-nu- cleotides [13] and adding it to the transfection mixture of plasmid constructs with Amh promoter sequences but lacking an intrinsic DE. This double stranded oligo-DNA and the single stranded constituent oligos, were added to the plasmid DNA-Lipofect Amine mixture at the time of transfection of the SMAT cells. The results of these experiments are summarised in Figure 3. High levels of added reagents had a profound (non-specific) inhibitory effect on green expression. A small but significant expression was seen with lower amounts of the reverse oligo- nucleotide but not with annealed oligo-DNA. Further experiments (Figure 3(b) and Figure 3(c)) confirmed these results and in addition showed that the forward oligo also showed a small but significant incremental effect.

Figure 4(a) and Figure 4(b) illustrate the effect of mutating site 4, (see Figure 1(b), Figure 5) in the prospective minor loop of DE formed when it is closely associated with the Amh promoter. This modification of DE results in “super-enhancement” of EGFP expression. It is also clear that with a simultaneous mutation of Wt super- enhancement is ablated, presumably by disruption of the Wt-DE2 bridge. The term “super-enhancer” is used here in the context of the experimental results described-there are other definitions [14] .

4. Discussion

In plasmid constructs without an enhancer sequence, mutation of the Wilms tumour factor element (Wt), either alone or in combination with mutation of other promoter elements, had no effect. However when DE is present in the construct, mutation of Wt and/or the downstream enhancer element DE2 resulted in ablation of enhancement [12] . These results suggested that the DE sequence was brought into close contact with the promoter immediately upstream of the start of translation, by a specific duplex factor, thus forming a major loop in the genomic DNA, as well as bringing the essential elements for enhancement into close juxtaposition with the promoter.

A reverse identity exists between parts of the DE and Wt sequences. The gap between the anchor points Wt and DE2, and the homology (cccacc) “sites” are adjacent in Wt but 15 nt apart in DE, suggesting that when DE is bridged (looped) to Wt, there is a bulge or minor loop in the DE DNA. Mutation of site 4 (see Figure 1(b) and Figure 5), which is in this hypothetical minor loop, was made to ascertain if this DNA played any part in enhancement. The results of mutating sites 3 - 6 are summarised in Figure 4.

A sequence consisting of 23 nt immediately upstream of DE2 was put in a NIH Blast search in the mouse genome data base, in particular with reference to chromosome 10; expected hits < 4; hits achieved > 120. Since this sequence forms part of the Amh enhancer it seems unlikely to be an example of a random insert of viral origin. Perhaps it is an example of a mechanism forming ordered 3D structure to genomic DNA, which may be related in some way and in some circumstances, to enhancement [15] .

Figure 5 is a sequence based schematic illustrating a possible interface of DE2 with the promoter between the tata box and the start of translation. One interpretation of the observation that super-enhancement occurs on mutation of site 4, is that super-enhancement is the default condition, normally down regulated (silenced) by a masking factor or organelle such as a nucleosome-like body [16] , specifically binding to this site. Competition for the site by a non-inhibitory factor such as a micro RNA or perhaps an oligo-nucleotide, could result in an easing of control over the release of the brake on the underlying default enhancement mechanism. A similar relaxation of control is achieved by mutating the site. Removal of control of default expression at critical moments in development, such as the removal of the Müllerian duct in early male development [1] , would result in a burst of hyper-expression. The high frequency of the occurrence of ~23 nt upstream of DE2 suggests that this sequence may be associated with a mechanism for imparting an ordered 3D structure to genomic DNA, which may be part of the mechanism for enhancement by distant DNA elements: a phenomenon widely acknowledged in the recent literature [17] . If enhancement is manifested by the addition of signals to the message, triggering its longevity and/or rapid recycling during translation [18] , then this might be detectable by mRNA sequence changes during super-enhancement.

Acknowledgements

I thank Dr. N. di Clemente for providing the SMAT-1 cell line; Prof. Rick Maizels for the benefit of discussion and a critical reading of a draft of this paper; and Yvonne Harcus for help in the laboratory.

Cite this paper

David W. Dresser, (2016) Super-Enhancement and Control of Amh Expression. American Journal of Molecular Biology,06,117-123. doi: 10.4236/ajmb.2016.63012

References