1. Distant acting enhancer specificity during mammalian development
Adam K. Dziulko1, Evgeny Z. Kvon2, Axel Visel2
1. Iowa State University; 2. Lawrence Berkeley National Laboratory
Abstract
Results –Knock ins
Conclusion and Future Directions
Materials and Methods
Acknowledgments
This work was supported in part by the U.S. Department of
Energy, Office of Science, Office of Workforce Development for
Teachers and Scientists (WDTS) under the Science
Undergraduate Laboratory Internship (SULI) program. Visel A,
Minovitsky S, Dubchak I, Pennacchio LA (2007). VISTA Enhancer
Browser-a database of tissue-specific human enhancers. Nucleic
Acids Res 35:D88-92. Special thanks for contribution in the
Visel/Pennacchio lab: Catherine Pickle, Ingrid Plajzer-Frick,
Elizabeth Lee, Momoe Kato, Tyler Garvin, Jennifer Akiyama, and
Veena Afzal.
Regulatory elements within our genome such as enhancers play important role in evolution, development, and disease. Despite importance of enhancers, our understanding of the mechanisms governing their function in the
genome is limited. Here we focused on one of the most distant-acting enhancers, the ZRS, which activates the expression of its target gene, Sonic Hedgehog (Shh) from ~1Mb. To test whether other limb enhancers are also able
to operate from such a long distance we replaced ZRS with three independent limb enhancers (hs1109, mm1179, and hs72). CRISPR/Cas9-generated mosaic knock-in mice containing replaced enhancer were bred with
heterozygous ZRS knock-out mice and F1 population was phenotyped in detail. Both hs1109 and mm1179 were able to activate Shh expression as was evident from limb morphology while the hs72 enhancer knock-in had a
phenotype analogous to the ZRS knock-out suggesting that enhancer cannot activate Shh. Despite the lack of activation of the Shh from hs72 at a 1Mb distance, hs72 was able to activate Shh promoter when the two were side-
by-side in a transgenic reporter construct. This result suggests that distance determines the activity of an enhancer, but how it plays a role is remained to be determined.
Forelimb Hind limb
E18.5
ZRS
hs72
hs1109
mm179
Figure 3: Limb phenotypes of ZRS replacement knock-in enhancers.
Embryos on left (E11.5) represents enhancer activity in the limb bud. The
forelimb/hind limb columns represent the resultant phenotypes of the
enhancer knock-ins with genotypes: ZRSWT/Δ (mouse), ZRShs72/Δ (human),
ZRShs1109/Δ (mouse), and ZRSmm179/Δ (human) respectively. Bottom right corner
indicates representative sample numbers. Images taken at 1x objective.
Forelimb Hind limb
Figure 4: Limb phenotype of ZRS knock-out.
Embryos collected at E 18.5. The genotype of the embryo is
ZRSΔ/Δ (mouse). Images taken at 1x objective. (will provide
scale bar)
• Enhancers hs1109 and mm179 were able to rescue Shh
expression in place of ZRS at a 1Mb distance from the Shh
promoter
• hs72 was unable to activate Shh at the 1Mb distance resulting in
a phenotype analogous to Shh-knockout
• hs72 was able to activate the Shh-promoter at a 50bp distance
suggesting that the distance of an enhancer to a promoter plays
a role in its activity
• Further experiments will focus on testing if there are tethering
elements surrounding an enhancer that contribute to the
activity at remote distances
E18.5
E11.5
Figure 1: From transgenic mouse to limb imaging.
A) Knock-in mice were generated using CRISPR/Cas9. B) Parent genotypes: ♀ZRSΔ/wt &
♂ZRS(wt/knock-in)M. C) Embryos were then harvested at 18.5 days post conception. D) After
genotyping, ZRSΔ/knock-in embryos and wt controls (ZRSΔ/wt) were put through staining
process. Alcian blue stains cartilage and Alizarin red stains bone. Images were taken on a
stereo microscope at 1x objective. Hill, M.A. (2016) Embryology Mouse-E17.5.jpg. Retrieved April 21,
2016, from https://embryology.med.unsw.edu.au/embryology/index.php/File:Mouse-E17.5.jpg
Harvest/genotype
embryos at E18.5
Extract/stain limbs
E11.5
Figure 5: hs72-Hsp68 promoter-LacZ vs hs72-Shh
promoter-LacZ.
Both embryos were harvested at E11.5. Embryo on left
contains hs72 enhancer next to Hsp68 (broad) promoter
while embryo on right contains hs72 enhancer next to Shh
(sharp) promoter. Six out of seven embryos stained
positive.
Collect/Stain
Figure 2: In vivo enhancer activity reporter.
A) Using Gibson cloning, hs72-Shh promoter-LacZ vector was created. B) Vector was
then linearized and injected into the pro-nucleus of fertilized mouse egg. C) Embryos
were then harvested at E11.5 and stained. To be defined as positive activity, results have
to show reproducible expression in the same structure in at least three independent
transgenic embryos.
Identical staining
patterns found in
both elements. This
suggests that the
Shh promoter
wasn’t the factor in
hs72 activity, but
the distance is.
Inject linearized vector
ZRS knock-out
phenotype is
analogous to hs72
knock-in phenotype.
Results – hs72 activity
Targeted
Enhancer
CRISPR Cas9
ZRS
Right Homology ArmLeft Homology Arm
Shh
A.
Shh
promoter
LacZhs72
F0 knock in founder line:
1) hs72 knock-in
2) hs1109 knock-in
3) mm1179 knock-in
B.
Create mouse knock-ins
C. D.
C.B.
A.
6/7
4/4
2/2
3/3 3/3
4/4
2/2 2/2
2/2
Radius
Ulna
Humerus
Scapula
Phalanges