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Enhancer cooperativity can compensate for loss of activity over large genomic distances

Henry Thomas, Songjie Feng, Marie Huber, Vincent Loubiere, Daria Vanina, Mattia Pitasi, Alexander Stark, Christa Buecker

Posted on: 10 June 2024 , updated on: 11 June 2024

Preprint posted on 7 December 2023

In order to understand complex gene regulatory landscapes, build them from scratch!

Selected by Milan Antonovic

Background

The development of multicellular organisms is the result of a wide range of dynamic and complex processes that require the generation and coordinated interaction of numerous cell types. In this sea of complexity, patterns emerge through precise regulation of processes from the tissue to the molecular level. At the genomic level, cis regulatory elements are one of the key regulators of gene expression. These include elements such as enhancers, insulators and promoters. Enhancers contain multiple transcription factor binding sites, marks of active chromatin, and they are several kb or even Mbs away from genes whose expression they regulate. Enhancer-promoter interactions are thought to be crucial for the assembly of the transcriptional machinery and the initiation of transcription.
The complex spatio-temporal expression patterns of developmental genes are mirrored by a complex form of regulation; often, these genes are regulated by multiple enhancers at the same time. The large genomic regions containing these enhancers are called regulatory landscapes (Bolt et al, 2020). Inside these regions, promoters interact with several enhancers at the same time and enhancers interact with each other, thereby increasing transcription levels in an additive or synergistic manner. There are still many aspects of enhancer interaction and cooperativity within regulatory landscapes that are not understood. One of the major reasons for this is that manipulating large regions of the genome is quite demanding. Because of this, there is a huge interest in the novel field of synthetic regulatory genomics, where approaches from synthetic biology are used to study gene regulation (Brosh,2023; Pinglay 2022).
In this preprint the authors sought help from the synthetic biology toolbox to develop a platform to study the activity of individual and multiple enhancers within a neutral environment.

Key findings

1. Development of a synthetic locus to study enhancer-promoter interactions
One of the highlights of this study is the generation of reporter cell lines containing a synthetic locus composed of four main components: the mCherry reporter gene with a minimal TK promotor and three landing pads (LP) positioned at different distances from the promotor (1,5kb, 25kb and 75kb) (Fig 1). Within the LPs any chosen enhancer sequence can be inserted by using the Cre/Lox and FRT recombination system. The synthetic regulatory region was generated inside the b-Globin locus, inert in mouse embryonic stem cells, assuring that only the enhancers inserted within the LPs were activating transcription from the promotor. This elegant design of the regulatory region allows testing the effects of both enhancer-promotor distance and different enhancer combinations on transcription, with a simple and straightforward readout (mCherry fluorescence intensity measured via FACS).

2. Enhancer strength determines the effect that genomic distance to the promotor has on transcriptional output
Having generated synthetic loci, the authors wanted to utilize them by inserting five different enhancers (eNanog, Rybp E2, Map4k3 E1, Map4k3 E2 and the enhancer cluster Sox2 SCR), all with varying strength, into LPs at three genomic distances from the promotor. Overall, mCherry expression was reduced with increased enhancer-promotor distance, but at all three distances the order of enhancer strength (strongest to weakest) did not change (Fig 1). This indicates that although the ability of enhancers to activate transcription diminishes with distance from the promotor, stronger enhancers are less affected by this than weak ones.

Figure 1. Structure of the synthetic landscape (above) and quantification (below) of mCherry signal intensity induced by five different enhancers inserted at three different distances from the promotor. Adapted from Thomas et al bioRxiv 2023.

3. Presence of additional (weak) enhancers rescues transcriptional output in case of increased enhancer-promotor distance
Surprisingly, the weakest enhancer Map4k3 E2 couldn’t activate transcription when positioned 75 kb from the promotor, although it activates Map4k3 transcription from a similar distance in its native locus. This inspired the authors to asses if the presence of other enhancers is necessary to overcome the reduced activity of enhancers when they are at greater distances from their respective promotors. They inserted Map4k3 E2 in combination with other stronger enhancers at different distances and in different order and measured mCherry fluorescence as before (Fig 2). Although strong enhancers – like eNanog – could activate transcription even at a bigger distance from the promotor, cooperativity with a weak enhancer allowed them to overcome this hindrance even more. However, this cooperative effect was only observed when the weak enhancer was placed between the strong enhancer and the promotor. The location within this region – between the strong enhancer and promotor – didn’t seem to be very important, since Map4k3 E2 helped boost the transcription activity of eNanog when placed both at 1,5 and 25kb.

Figure 2. Comparison of different enhancer combinations, at different distances and enhancer order. Adapted from Thomas et al bioRxiv 2023.

Why I chose this paper:
The reason why this paper sparked my interest is because it shows the power of using synthetic biology when trying to answer questions in regulatory genomics. With an elegant design of the new locus and a readout that is easy to interpret, the authors managed to broaden our understanding of the complex regulatory grammar of gene regulation.

Questions for the authors:
1. eNanog and Sox 2 SCR are two of the strongest enhancers tested in this paper, eNanog being the weaker of the two. What would happen if eNanog instead of Map4K3 E2 was placed between Sox2 SCR and the promotor? Would a strong enhancer like eNanog assume the role of the synergistic “weak” enhancer when positioned next to a stronger enhancer?

2. The authors have inserted Map4k3 E2 between strong enhancers and the promotor at 25 and 1,5 kb and haven’t observed a striking difference in the synergistic effect. However, in their first line of experiments they could show that the enhancers’ ability to activate transcription weakens with distance, with the most drastic drop observed between 25 and 75 kb. What would happen if Map4k3 E2 was inserted together with a strong enhancer at position 75kb? Would a weak enhancers’ synergistic effect also become smaller at this bigger distance?

References
Bolt CC, Duboule D. The regulatory landscapes of developmental genes. Development. 2020 Feb 3;147(3):dev171736. doi: 10.1242/dev.171736
Brosh R, Coelho C, Ribeiro-Dos-Santos AM, Ellis G, Hogan MS, Ashe HJ, Somogyi N, Ordoñez R, Luther RD, Huang E, Boeke JD, Maurano MT. Synthetic regulatory genomics uncovers enhancer context dependence at the Sox2 locus. Mol Cell. 2023 Apr 6;83(7):1140-1152.e7. doi: 10.1016/j.molcel.2023.02.027
Pinglay S, Bulajić M, Rahe DP, Huang E, Brosh R, Mamrak NE, King BR, German S, Cadley JA, Rieber L, Easo N, Lionnet T, Mahony S, Maurano MT, Holt LJ, Mazzoni EO, Boeke JD. Synthetic regulatory reconstitution reveals principles of mammalian Hox cluster regulation. Science. 2022 Jul;377(6601)

 

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Author's response

Christa Buecker shared

1. eNanog and Sox 2 SCR are two of the strongest enhancers tested in this paper, eNanog being the weaker of the two. What would happen if eNanog instead of Map4K3 E2 was placed between Sox2 SCR and the promotor? Would a strong enhancer like eNanog assume the role of the synergistic “weak” enhancer when positioned next to a stronger enhancer?

Answer: We are currently testing similar combinations, but we have not yet received the results. But we can certainly speculate: eNanog at 25kb still has about 80% strength compared to 1.5kb. If we combine it with the Sox2 SCR, the resulting expression will likely be higher than either the Sox2 SCR at 75kb or the eNanog at 25kb alone. However, we do not think that it will be strongly synergistic. The promoter itself might be a limiting factor, and the combination of very strong enhancers might lead to sub-additive behaviour. We do not know what determines the limitation of individual promoters, but we are looking forward to testing different promoters in our system in the future.

2. The authors have inserted Map4k3 E2 between strong enhancers and the promotor at 25 and 1,5 kb and haven’t observed a striking difference in the synergistic effect. However, in their first line of experiments they could show that the enhancers’ ability to activate transcription weakens with distance, with the most drastic drop observed between 25 and 75 kb. What would happen if Map4k3 E2 was inserted together with a strong enhancer at position 75kb? Would a weak enhancers’ synergistic effect also become smaller at this bigger distance?

That is another excellent question, one that we have yet to explore. We believe that the integration of the weak enhancer between the strong enhancer and the promoter could be the key to synergy, potentially altering the 3D interaction landscape and generating microcompartments, as described by Anders Hansen’s lab. If there is no physical distance between the enhancer and the promoter, synergy might not be as pronounced. This aligns with observations from the Higgs and Kassouf lab, who identified facilitator elements that work in a position-dependent manner at the alpha-globin locus. Whether all weak enhancers can function as facilitator elements and how they ultimately contribute to gene expression remains unclear.

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