Temporal dissection of an enhancer cluster reveals distinct temporal and functional contributions of individual elements

TL;DR: In this paper, the functions and interdependencies of five enhancer elements that together activate Fgf5 expression during exit from naive murine pluripotency were dissected.

About: This article is published in Molecular Cell.The article was published on 2021-03-04 and is currently open access. It has received 36 citations till now. The article focuses on the topics: Enhancer & Super-enhancer.

Summary

Introduction

• The search for Majorana fermions in superconducting nanowires is both rewarding and difficult.
• The authors consider a one-dimensional chain of magnetic nanoparticles (magnetic dots17) on a superconducting substrate (see Fig. 1).
• In the next section the authors introduce the model of a chain of magnetic nanoparticles on a superconductor, and then in Sec. III they use scattering theory22 to calculate the topological quantum number of a finite chain, including the competing effects of magnetic and hopping disorder.

B. Single-band limit

• In the large magnetization regime B0 |μ| 0,tn the electron spin on the nth nanoparticle is nearly polarized along Bn.
• The Hamiltonian can then be projected onto the lowest spin band, with electron operator ψn.
• In what follows the authors will use the full Hamiltonian (2.2), valid to all orders in 1/B0.

C. Disorder

• The authors distinguish the localizing effect of disorder in the hopping energy tn, which localizes the electrons without opening an excitation gap, from the gap opening effect of disorder in the orientation bn = Bn/B0 of the magnetic moments.
• For the magnetic moments the authors take a dipolar ferromagnetic correlation of the unit vectors bn on neighboring nanoparticles, according to the distribution P (b1,b2, . . .

III. SCATTERING MATRIX

• To identify the topologically nontrivial phase of a finite disordered chain of N nanoparticles it is more efficient to work with the scattering matrix than the Hamiltonian.
• The waves can come in from the left end or from the right end of the chain, in two spin directions and as electron or hole, so S is an 8 × 8 unitary matrix.
• (3.8) Unitarity together with particle-hole symmetry (3.2) ensure that the determinants of R and R′ are identical real numbers.

IV. PHASE DIAGRAM

• Results of their search for a topologically nontrivial phase are shown in Figs. 2–4.
• The authors have kept B0 = 2t0 fixed and varied the material parameters 0,μ as well as the disorder parameters δb,δt .
• These are plots for a single disorder realization and a single system size (N = 6000).
• Disorder in the hopping energies or in the magnetic moments causes localization, which reduces the nontrivial region (mainly on the small 0 side).
• For a system without disorder in the hopping energies (δt = 0) and with random and uncorrelated directions of the magnetic moments (δb = ∞).

V. CONCLUSION

• In conclusion, the authors have proposed to create Majorana fermions by depositing magnetic nanoparticles on a superconducting substrate.
• The superconductor has conventional s-wave pairing and need not have any spin-orbit coupling.
• For 10-nm size nanoparticles the level spacing is comparable to the superconducting gap, so the authors need only consider a single electronic orbital per nanoparticle.
• The spacing of the nanoparticles should therefore be uniform on the scale of the wavelength of the electronic orbital (typically a few nm).
• The authors predict level crossings at the Fermi energy (E = 0) upon variation of the spacing of the two magnetic atoms, as a signature of the switch from an even to an odd number of electrons in the ground state.

Figures

Table 3: RT-qPCR primers

Figure 6: High levels of Pol II accumulate at the PE element

Figure 2: The intergenic enhancers E1-E4 mediate induction of Fgf5 upon differentiation

Table 4: ChIP-qPCR primers

Figure 5: PE is not activated earlier than E1-E4 and does not primarily function by removing H3K27me3 from the Fgf5 promoter or by facilitating activation of the intergenic enhancers E1-E4

Figure 1: The Fgf5 locus as a model to study collaborative enhancer action

Table 5: PRO- and PRO-Cap-Seq primers and adapters

Figure 3: PE amplifies Fgf5 expression levels at every time point, yet has little canonical enhancer activity in luciferase assays

Figure 4: PE and E1-E4 regulate Fgf5 transcription in super-additive fashion

Table 1: gRNAs

Table 2: PCR primers

Frequently asked questions

Q1. What are the contributions mentioned in the paper "Detailed temporal dissection of an enhancer cluster reveals two distinct roles for individual elements" ?

1: Max Perutz Labs Vienna, University of Vienna, Vienna Biocenter, Dr Bohr Gasse 9, 1030 Vienna 5 2: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria 6 3: Department of Chemical and Systems Biology and Department of Developmental Biology, Stanford 7 University School of Medicine, Stanford, CA 94305, USA 8 4: Institute of Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, 9 Stanford, CA 94305, USA 10 5: Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305, USA 11 *: corresponding author: christa. 

Q2. What was used to sort and index the resulting bam files?

SAMtools 1.5 (H. Li et al., 15 2009) was used to sort and index the resulting bam files, as well as for extracting uniquely mapping reads. 

Q3. How long after starting the differentiation was the luciferase 2 activity measured?

24 or 40 h after starting the differentiation, luciferase 2 activity was measured using the Dual-Glo® Luciferase Assay System (Promega). 

Q4. What was used to convert the sam files to bam files?

SAMtools 1.5 (H. 13 Li et al., 2009) was used to convert the resulting sam files to bam files, to sort and index the bam files as 14 well as for extracting uniquely mapping reads. 

Q5. How many times was the pre-warmed Amplifier Mix added to the cells?

After washing cells three times in 20 wash buffer – again including the 2 min incubation before removing the buffer – Amplifier Mix diluted 21 1:25 in pre-warmed Amplifier Diluent QF was added. 

Q6. How did the authors map reads to 20 custom-made bowtie indexes?

To account for the genetic changes in the KI cell lines, the authors mapped reads to 20 custom-made bowtie indexes, in which PE had been removed from its endogenous position, and instead 21 had been reintroduced upstream of the promoter in either sense or antisense orientation. 

Q7. What is the effect of distance on enhancer activity?

While enhancer activity is 11 generally believed to be independent of genomic distance and large distances can be overcome by 12 enhancer-promoter loops (Furlong & Levine, 2018), recent studies suggest that enhancer-promoter 13 distance can indeed have an effect on expression levels (Carleton et al., 2017; Scholes et al., 2019). 

Q8. How many enhancers could act at the same promoter?

Previous studies in Drosophila have suggested that 21 multiple weak enhancers could act simultaneously at a promoter to achieve higher or super-additive 22 transcription initiation rates compared to individual enhancers (Bothma et al., 2015; Carleton et al., 23 2017). 

Q9. How many reads were sequenced to a depth of 30-60 million reads?

Sequencing was performed at the VBCF NGS Unit. 13Data analysis 14PRO-Cap-Seq libraries were sequenced to a depth of 22-30 million reads while PRO-Seq libraries were 15 sequenced to a depth of 30-60 million reads (both: single-end, 50 bp). 

Q10. How many AMPure XP beads were used to remove primers and adapters?

If necessary, 13 additional purification with AMPure XP beads was performed to remove primers and adapters 14 (purification with 1x AMPure XP beads; the supernatant was discarded and the DNA bound to the beads 15 subsequently eluted) or to exclude DNA fragments of more than 1 kb (purification with 0.54 x AMPure 16 XP beads; the high molecular weight fragments bound to the beads and were discarded, while the library 17 enriched for smaller DNA fragments remained in the supernatant). 

Q11. How was the SV40 promoter removed from the luciferase plasm?

For assays with the endogenous 14 promoter, the SV40 promoter was removed from the luciferase-enhancer plasmids by restriction digestion 15 with BglII and HindIII-HF (both NEB). 

Q12. What is the reason why the luciferase assays showed low enhancer?

The 5 individual elements of the Fgf5 enhancer cluster showed very low enhancer activity in classical luciferase 6 assays, even when combined with the endogenous promoter. 

Q13. How many cycles for amplification of the ChIP-Seq libraries?

The number of cycles for optimal 25 PCR amplification was estimated to be 9-14 in total as described above for the ChIP-Seq libraries. 

Q14. How does the effect of PE on expression levels in Drosophila be relieved?

When 28 placing PE outside of the intron and upstream of the promoter, this attenuating effect should be relieved 29 and the resulting expression levels should be higher than in a WT cell line. 

Q15. What was the protocol for a biotin RNA enrichment?

In addition to this, the authors included a buffer exchange with a P-30 column - as 7 described in the PRO-Seq protocol - before the very first biotin-enrichment with Streptavidin beads. 

Q16. Who has granted bioRxiv a license to display the preprint in perpetuity?

32.CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 

Q17. How did the authors determine whether H3K27ac accumulation at the E1 enhancer was?

loss of E1 affected H3K27ac deposition at the E2 enhancer (Fig 5C), 15 and the authors observed reduced H3K27ac levels at the E1 enhancer upon E2 deletion (although not significant, 16 p-value=0.06).