🔥New work from the Crocker group @justinmcrocker.bsky.social @embl.org on the mechanistic and evolutionary basis of dominance from cis-regulatory variation in Drosophila Congratulations to Noa @ottilie.bsky.social and all the authors 🥳
Come listen to my talk about how enhancers evolve and how promoters emerge from randomly synthesized DNA sequences!
New Preprint! When one enhancer allele changes another's regulatory output, is that bad? We found it could be a feature — interallelic cis-regulatory dominance buffers outputs AND enables evolutionary innovation. Two for one!
Led by @ottilie.bsky.social from @embl.org
doi: doi.org/10.64898/202...
Thanks so much Luisa! ☀️
Thanks so much Tim! 💙
Many thanks to my co-authors @timothyfuqua.bsky.social, @fabianruperti.bsky.social and @justinmcrocker.bsky.social. Please let me know if you have feedback, or want to chat more about this work! (18/18)
These results may explain how seemingly fragile cis-regulatory elements preserve their function yet remain evolutionary flexible. (17/18)
In this way, gene expression is maintained in the face of potentially disruptive mutations (robustness), while concurrently allowing the exploration of new phenotypic effects in a cryptic or compartmentalized way (innovation). (16/18)
We believe these interallelic hubs can act as evolutionary stepping stones: rather than having a single, uniform phenotypic effect, each enhancer allele can experience a distinct dominance relationship depending on cellular environment and developmental stage. (15/18)
Multiple factors may underly the cell type-specificity in dominance strength, such as interallelic pairing strengths, chromatin accessibility, post-transcriptional regulation, competition for TFs, and the chemical properties of the TFs in an interallelic hub. (14/18)
Although transvection is dependent on somatic homolog pairing, which has predominantly been characterised in Diptera and budding yeast, interallelic and interchromosomal transcriptional hubs are increasingly recognized across metazoans. (13/18)
On the top left, a stage 15 embryo resulting from a cross of the wildtype E3N-lacZ line, integrated at attP2 (chr.3L) with ectopic E3N mutant 145-2 driving lacZ, integrated at attP2 (chr.3L). RNA of both the lacZ and dsRed reporter genes are labeled by HCR RNA-FISH. On the right to it, a violin plot showing the difference in Ubx concentration between colocalized (green) and non-colocalized (purple) transcription sites of lacZ and dsRed. Colocalized sites exhibit significantly higher Ubx intensity compared to non-colocalized loci. A pie chart summarizes the proportion of observations in each category. On the left bottom, a close-up of a nucleus (in blue, DAPI) in the ventral zone, highlighted by a dotted-striped rectangle in the image above it. Separation of the different channels reveals colocalization of lacZ and dsRed nascent RNA at a shared transcription site, accompanied by a local enrichment of Ubx protein. On the right to this, a 3D surface plot of Ubx protein levels from antibody staining of the nucleus mentioned before. Ubx signal is shown in arbitrary units (AU) on a 0-255 scale, and presence of lacZ and dsRed signal is depicted in magenta and green, respectively. On the right to that, a schematic of the interallelic transcriptional hub for the genotype described before.
Using a two-color reporter assay we then showed colocalization of the two transcription sites in a "dominant" cell type, which was accompanied by a higher concentration of the activator Ubx. This suggests that interallelic transcription hubs underlie the interactions between the alleles. (12/18)
So what is the mechanism underlying this regulatory dominance? By removing, replacing and transporting different cis-regulatory elements we showed proximity-dependent, transvection-like interactions between the enhancers alleles. (11/18)
Overall, we see cell type-specific differences in the degrees of dominance within an embryo, where in some cell types mutant activity is observed, and in others it is fully repressed. This type of dominance allows for a mosaic regulatory outcome that promotes robustness and innovation. (10/18)
On the left, representative images of a homozygous wildtype E3N-lacZ, homozygous mutant 145-2-lacZ, and a heterozygous embryo at stage 15. The embryo is stained with a β-gal antibody against the lacZ reporter protein β-gal. Marked in a mustard-yellow box is the region of abdominal stripe 2 (A2) where β-gal reporter intensity was measured across embryos. Marked in blue circles are the wing and haltere discs where β-gal reporter intensity was measured across embryos. On the right, boxplots of the mean fluorescence intensity of the β-gal reporter within the A2 stripe, and the wing and haltere discs, haltere disc of different embryos of the 145-2 E3N variant at stage 15.
In one homozygous variant, the embryo gains ectopic enhancer activity in wing and haltere discs, while losing activity in the native ventral zone. In the heterozygous embryo, ventral activity is fully restored to WT levels, while we only see partial dominance of the WT allele in the discs. (9/18)
We then asked, could a wild-type allele also decrease pleiotropic expression caused by enhancer mutations? For this, we looked at multiple synthetic E3N enhancer variants we previously created. (8/18)
A comparison of one of the evolutionary trajectories from mel to sim when every step is homozygous (represented as circles) versus heterozygous (squares), where one allele remains mel up until the last step to sim. Below are representative embryos from some of the variants shown.
We tested two scenarios: in one, mutations accumulate in a homozygous manner from mel to sim. In the other, mutations accumulate on only one allele, and the other remains "mel" until the end. We observed dominance among heterozygous mutational steps that masked expression-reducing mutations. (7/18)
Since new mutations arise in single copies, natural selection initially acts on heterozygous genotypes in diploids. Therefore, we wondered how this genotype-phenotyope landscape might be navigated via heterozygous intermediates. (6/18)
Example of epistasis in the evolutionary trajectory of E3N. Variant 01000 has a significant negative effect on the nuclear β-gal intensity of mel (00000) (ε1 = -0.278, p = 0.016), whereas variant 00001 has no significant effect (ε1 = -0.089, p = 0.132) on mel. When the two variants are combined, there is a positive epistatic effect on the nuclear β-gal intensity of mel (ε2 = 0.468, p = 0.005). On the left there is a barplot showing this, and on the right there are representative embryos from each line.
In addition, we also observe non-additive effects between combinations of different mutational events (epistasis), both pairwise and higher-order, which has been suggested to constrain enhancer evolution. (5/18)
On the left representative embryos for every line in the combinatorial E3N library showing the differences in the striped gene expression pattern of the lacZ reporter. On the right the combinatorial evolutionary paths showing the possible intermediate forms from the genetic E3N sequence of melanogaster (mel) to simulans (sim). Shown colors represent the average nuclear intensity
Many mutational combinations in this genotype-phenotype map markedly alter enhancer activity, which is in stark contrast to the limited natural variation and conserved regulatory output observed in the D. melanogaster E3N enhancer. (4/18)
Sequence alignment of E3N in D. melanogaster and D. simulans, and below an evolutionary tree of the melanogaster species subgroup showing the changes in the E3N enhancer since the divergence of D. melanogaster and D. simulans 1.4-3.4 Mya, and the divergence of erecta complex 3.4-12 Mya. Here, ACD is the most likely ancestral version of the enhancer given the modern-day sequence of D. erecta.
We examined the evolutionary trajectories of a fragile, pleiotropic enhancer by testing the enhancer activity of all the intermediate sequences between the D. melanogaster and D. simulans E3N enhancer, who diverged 1-3 Mya. (3/18)
Enhancers often exhibit high degrees of mutational fragility, yet across evolution many of their expression patterns remain conserved: how can enhancers accumulate mutations and acquire new functions without compromising developmental programs? (2/18)
Excited to share my first preprint from my PhD w/ @justinmcrocker.bsky.social. We show that cell type-specific regulatory dominance promotes robustness and evolutionary innovation through interallelic transcriptional hubs, potentially expanding the mutational paths available to diploids. (1/18)
New preprint from the lab! We identify the ZnF protein Mulberry as a condensation-dependent structural regulator of genome topology that organizes “multi-way regulatory hubs” in early Drosophila embryos.
www.biorxiv.org/content/10.6...
Just discovered the wonderful covers of 'Genes to Cells', the journal of the Molecular Biology Society of Japan @mbsj-official.bsky.social – absolutely beautiful!
here some examples inspired by mitosis, CRISPR, the DNA helix, and plant pigments
🧬✨ New 2-part review on the evolution of regulatory DNA (enhancers & promoters)! What started as conversations between Gasper Tkačik @istaresearch.bsky.social and our group @embl.org grew into a broader synthesis.
Preprints here:
Part 1: arxiv.org/abs/2601.19681
Part 2: arxiv.org/abs/2601.21480
✨This week at the DB unit seminar @embl.org, Noa Ottilie Borst, a PhD student in the Crocker group @justinmcrocker.bsky.social presented her work on cell-type-specific interallelic interactions, and how they simultaneously allow for robustness and evolutionary innovation.
Hey y'all, grant-pending, I may be unemployed starting from October. If you have any leads on biotech / postdoc positions in the German-speaking part of Switzerland, I'd greatly appreciate a message. I'm great with Data Analysis, Molecular Biology, and Teaching. (Plz RT!)
More at timothyfuqua.com
#drosophila @flybase.bsky.social request emergency funding:
"As it stands, by the end of July, 2025, there will be no future updates to FlyBase, and in the worst case scenario access to the website will also be lost" => please donate!
www.philanthropy.cam.ac.uk/give-to-camb...
