Multi-enhancer transcriptional hubs confer phenotypic robustness

We previously showed in Drosophila melanogaster embryos that low-affinity Ultrabithorax (Ubx)-responsive shavenbaby (svb) enhancers drive expression using localized transcriptional environments and that active svb enhancers on different chromosomes tended to colocalize (Tsai et al., 2017). Here, we test the hypothesis that these multi-enhancer ‘hubs’ improve phenotypic resilience to stress by buffering against decreases in transcription factor concentrations and transcriptional output. Deleting a redundant enhancer from the svb locus led to reduced trichome numbers in embryos raised at elevated temperatures. Using high-resolution fluorescence microscopy, we observed lower Ubx concentration and transcriptional output in this deletion allele. Transcription sites of the full svb cis-regulatory region inserted into a different chromosome colocalized with the svb locus, increasing Ubx concentration, the transcriptional output of svb, and partially rescuing the phenotype. Thus, multiple enhancers could reinforce a local transcriptional hub to buffer against environmental stresses and genetic perturbations, providing a mechanism for phenotypical robustness.


28
"Come together, right now, over me" 29 -The Beatles (who may, or may not, have been singing about transcription) 30 During embryogenesis, transcriptional regulation controls the expression of genes that  how kinetically inefficient binding sequences are able to achieve robust transcriptional activation 52 in developing embryos. 53 We have previously shown in living Drosophila melanogaster embryos that Ubx 54 transiently but repeatedly explores the same physical locations in a nucleus, which are likely 55 hotspots with clusters of binding sites (Tsai et al., 2017). We have additionally shown that  To understand the mechanistic implications of having multiple enhancers in a shared 73 microenvironment, here we examined the transcriptional robustness of the svb locus to 74 temperature-induced stress in flies harboring the wild-type allele or one containing a deletion 75 (Df(X)svb 108 ) that includes the ventral svb enhancer DG3. When embryos were raised at high 76 temperatures, we observed phenotypical defects in ventral trichome formation for the DG3-77 3 deletion svb allele but not for the wild-type. At the molecular level, Ubx concentrations around 78 transcription sites of the DG3-deletion allele decreased. The transcriptional output of svb without 79 DG3 also decreased. To test the hypothesis that shared microenvironments modulate 80 transcriptional output and provide buffering under stress, we sought to rescue the DG3-deletion 81 allele through inserting the complete svb cis-regulatory region on a BAC (svbBAC) on a different 82 chromosome. We observed that Ubx concentration around active transcription sites of the DG3-83 deletion allele and their transcriptional output increased when the svbBAC is physically nearby.

84
Moreover, we found that trichome formation was partially rescued at high temperature. As a result, 85 our findings support the hypothesis that shared microenvironments provide a mechanism for 86 phenotypic robustness.

88
The DG3 enhancer responds specifically to Ubx in the A1 segment unexplored. Therefore, we tested the response of the DG3 enhancer to Ubx by altering Ubx levels 98 and measuring the transcriptional output with a reporter gene (lacZ). In wild-type embryos, the 99 DG3 reporter gene was expressed ventrally in stripes along segments A1-A7, in addition to narrow 100 thoracic stripes ( Figure 1C). In the absence of Ubx, DG3 reporter expression was almost 101 completely lost on the ventral side of A1 and significantly reduced between A2-A7 ( Figure 1D Figure 1E). In summary, we showed that DG3 105 responds specifically to Ubx for driving gene expression, which is consistent with our previous   the T1 trichomes were missing in larvae homozygous for the deletion (Df(X)svb 108 ) allele (compare 120 Figure 2B and 2C), which we subsequently used as a recessive marker to select for embryos 121 carrying the deletion allele when crossing Df(X)svb 108 flies to other lines. Also, we observed 122 defects in trichome formation in the dorsal edges of the stripe pattern, which are exclusively output of svb, we adopted the same approach, but in the svb RNA FISH channel ( Figure 3D). conditions. Therefore, we tested the capacity of a DNA sequence containing the full svb cis-  Figure 4B).

171
To test the rescue, Df(X)svb 108 embryos or larvae with a svbBAC-dsRed crossed into them 172 were incubated at 32 °C. We observed that the introduction of the svb regulatory region was able

186
Regarding phenotype, ventral trichome formation on the A1 segment ( Figure 5A-C), which 187 is reduced with the DG3-deletion allele, is also rescued by the introduction of svbBAC ( Figure 5D).

188
Interestingly, the loss of the outer edge trichomes in A1 (in the black brackets, as shown in Figure   189 5A-C, where only DG3 provides coverage) with the DG3-deletion allele was not rescued with 190 svbBAC. Additionally, introducing only the DG3 enhancer as opposed to svbBAC did not rescue 191 trichome formation under heat-stress ( Figure 5D).   222 We previously observed that transcription sites of reporter genes driven by minimal svb 223 enhancers tended to colocalize with the endogenous svb locus when it is transcriptionally active 224 (Tsai et al., 2017). This is true also at the scale of entire cis-regulatory regions, as we observed that 225 the svb locus does the same with svbBAC, which implies that they potentially share a common  It is possible that such long range interactions are driven, or reinforced, through shared 232 microenvironments. 233 We were able to partially rescue the DG3-deletion svb allele with svbBAC, which contains                 were: 25 between diBAC-gfp and wild-type svb, 25 between svbBAC-dsRed and wild-type svb and 553 26 between svbBAC-dsRed and Df(X)svb 108 . We analyzed 3 embryos for each genotype. One-sided 554 Student's t-test was applied for each individual comparison. In box plots, center line is mean, 555 upper and lower limits are standard deviation and whiskers show 95% confidence intervals.