Supplementary Materials
The shadow enhancer of short gastrulation also directs its expression in the ventral midline of
the Drosophila embryo
Dong-Hyeon Shin and Joung-Woo Hong*
Graduate School of East-West Medical Science, Kyung Hee University, Yongin, 446-701, South Korea
*Corresponding Authors:
Joung-Woo Hong, Ph.D.
Graduate School of East-West Medical Science, Kyung Hee University
Yongin, 446-701, South Korea
E-mail: jwhong46@khu.ac.kr
Phone: +82-31-201-3853
Fax: +82-31-204-8119
Table S1. DNA oligonucleotide sequences used in this study.
Enhancer Name
Sequence (5’ to 3’ direction)1
sog primary enh
F: GTTGCCAATGCCATTGCGCATACGCCGTGTCG
R:GCTTTATGGTCCATGGTCCATACCAC
Fig. 1
0.88 kb (full length)
F1:GAGAAGGAGGAGAAGTTGGT
R1:ATTTAATCGAAGGACTGCAA
Fig. 3
0.73 kb
F2:TACAGCGTATGGCGATTT
R1:ATTTAATCGAAGGACTGCAA
Fig. 3
0.616 kb
F3:AGAGCACTCTCACGCATC
R1:ATTTAATCGAAGGACTGCAA
Fig. 3
0.40 kb
F4:GCTCCTTATCCTTGCACA
R1:ATTTAATCGAAGGACTGCAA
Fig. 3
0.24 kb
F5:TATAGCCACATGTGTATGGTG
R1:ATTTAATCGAAGGACTGCAA
Fig. 3
0.75 kb
F1:GAGAAGGAGGAGAAGTTGGT
R6:GTTTCAGCGGAACAGGTAG
Fig. 3
0.54 kb
F1:GAGAAGGAGGAGAAGTTGGT
R7:CGCCATATCTGCTATTCCTA
Fig. 3
0.37 kb
F1:GAGAAGGAGGAGAAGTTGGT
R8:ATTCTACCTGTCCTGGGAAT
Fig. 3
0.68 kb
F1-1:GATTCAGCAGTTCCACAGAA
R6:GTTTCAGCGGAACAGGTAG
Fig. 3
0.61 kb
F1-1:GATTCAGCAGTTCCACAGAA
R6-1:GTGCGAAAACAGATGCAG
Fig. 3
0.50 kb
F2:TACAGCGTATGGCGATTT
R6-1:GTGCGAAAACAGATGCAG
Fig. 3
1
Note2
All primer sequences are presented in the 5’ to 3’ direction relative to the physiological
orientation of short-gastrulation (sog) transcription.
2
Indicates section(s) in the manuscript where the primer was used.
Key: F = forward; R = reverse.
2
Table S2. DNA sequences of primary and shadow enhancers that direct sog expression in the
neurogenic ectoderm of the early Drosophila embryo.
Enhancer DNA Sequence (5’ to 3’ direction)
>sog_primary_392bp
GTTGCCAATGCCATTGCGCATACGCCGTGTCGTCTATATGGCTATATGGCTATATGGCTGTATGGTGCGGGGAAATC
CCCGTAATCGCAGGTAGAATTCCAGCCGGTGCCGAGGCGGGACCTGCTCGCACCTCTAATCCCGCCAGGGTTTTCGG
GACATGGGATATTCCCGACGGCACAGCATAGCACTCCGTTTTCTTTTTTTTTTTTTATTATTATTGTGTCCAGTTTT
AATCCGGAAAGCGGGAATTCCCTTCCGCTCGCTGCCTGCACTGCGCTGCGCAGACGCATCGGCGTCCGTAAGCCGCT
TACCAAAAAGATACGGGTATACCCAAATGGATGCCTGCCCATGTATATAGACCATTGGGTGGTATGGACCATGGACC
ATAAAGC
>sog_shadow_884bp
GAGAAGGAGGAGAAGTTGGTTGAGAGGTCATCGTTGCGATTCTGCGATTCAGCAGTTCCACAGAAGGTGTCGTAATC
CTGGACGCAAGGGTGCACGGACCAACTGACAGGGGCAAGTGCGTCCTGTGCCACCAGATGACGCACGATGCGGCCGG
AAAAACCCAAAATCAAAAACCGAAAACCGAAAACCTGGTCAGAGTTTCCGAAAACCAAAGAGCCAACATCGAATGCG
GCACAATAACCCGATTGTCTGCGAATACCCACGATGATCTAGAATCGCACGGAGAGCACTCTCACGCATCCGTGGCC
ATATGGGTGCGGCCAAATCGGAAATTCCCAGGACAGGTAGAATGCATTGGATATACGGGTATACGGATTGGAATTGG
GATTGGGATTGGGACTAGCACCAGGTTGCAACGCCCGCCAAGAAGCCAATTTAAATAAGCAGCATAAACAAAAGCGA
CAGCGTTTTATGATCCCCGCTCCTTATCCTTGCACAAGGATATCGCCATGGCCACGCAGGTAGGAATAGCAGATATG
GCGGCAATGATGCGCCAACCGCACTGCTTCGTCCTGGTCCTGGTCGGATGGGCTTTTCCCACGCAACCGCGACCTTA
TCTGCGCCCCTTTTATGAGGCTGCATCTGTTTTCGCACCTCGATGCCGTTGGCATTATAGCCACATGTGTATGGTGG
GAATTTCCGATCGACCAGCCTACCTGTTCCGCTGAAACCCGGGAATCTGTCCATCCTGAGCTTCCACACACACACAC
ACACACACACAGGTCAGTCGGCATCAATTGGCTGCCATAAACATATAACAATCAATATTGAATCCTTTATCGTAGAA
TTTGTTGTATATGCCCATTGCAGTCCTTCGATTAAAT
Primer sequences used for genomic PCR amplification are underlined. DNA binding sites for
Dorsal (Dl) and Zelda (Zld) in the shadow enhancer are identified in green and red, respectively.
DNA binding sites for Snail (Sna) are wave-underlined. Genomic coordinates for primary and
shadow enhancers are X: 15,622,698-15,627,089 and X: 15,647,477-15,646,594, respectively.
3
Table S3. DNA sequences of distal and proximal elements required for midline enhancer activity
in the sog shadow enhancer.
Enhancer DNA Sequence (5’ to 3’ direction)
>distal element_107bp
GATTCAGCAGTTCCACAGAAGGTGTCGTAATCCTGGACGCAAGGGTGCACGGACCAACTGACAGGGGCAAGTGCGTC
CTGTGCCACCAGATGACGCACGATGCGGCC
>proximal element_77bp
CTCGATGCCGTTGGCATTATAGCCACATGTGTATGGTGGGAATTTCCGATCGACCAGCCTACCTGTTCCGCTGAAAC
Genomic coordinates for distal and proximal elements are X: 15,645,326-15,649,432 and X:
15,644,747-15,648,823, respectively. Trithorax-like (Trl)-binding motifs (GAGA elements) and
Zld-binding motifs are labeled in blue and red, respectively.
4
Table S4. DNA sequence of the 0.68-kb minimal region of the shadow enhancer that drives sog
expression in the ventral midline.
Enhancer DNA Sequence (5’ to 3’ direction)
>sog shadow enhancer_686 bp
GATTCAGCAGTTCCACAGAAGGTGTCGTAATCCTGGACGCAAGGGTGCACGGACCAACTGACAGGGGCAAGTGCGTC
CTGTGCCACCAGATGACGCACGATGCGGCCGGAAAAACCCAAAATCAAAAACCGAAAACCGAAAACCTGGTCAGAGT
TTCCGAAAACCAAAGAGCCAACATCGAATGCGGCACAATAACCCGATTGTCTGCGAATACCCACGATGATCTAGAAT
CGCACGGAGAGCACTCTCACGCATCCGTGGCCATATGGGTGCGGCCAAATCGGAAATTCCCAGGACAGGTAGAATGC
ATTGGATATACGGGTATACGGATTGGAATTGGGATTGGGATTGGGACTAGCACCAGGTTGCAACGCCCGCCAAGAAG
CCAATTTAAATAAGCAGCATAAACAAAAGCGACAGCGTTTTATGATCCCCGCTCCTTATCCTTGCACAAGGATATCG
CCATGGCCACGCAGGTAGGAATAGCAGATATGGCGGCAATGATGCGCCAACCGCACTGCTTCGTCCTGGTCCTGGTC
GGATGGGCTTTTCCCACGCAACCGCGACCTTATCTGCGCCCCTTTTATGAGGCTGCATCTGTTTTCGCACCTCGATG
CCGTTGGCATTATAGCCACATGTGTATGGTGGGAATTTCCGATCGACCAGCCTACCTGTTCCGCTGAAAC
Primer sequences used for genomic PCR amplification are underlined. DNA binding sites for
Sim-Tgo, Trl (GAGA elements) and Zld are identified in pink, blue and red, respectively.
Genomic coordinates for the 0.68-kb enhancers are X: 15,646,747-15,647,432.
5
Figure S1
sim-tgo Position Frequency Matrix
A:
C:
G:
T:
0.227
0.182
0.591
0.000
0.273
0.000
0.091
0.636
1.000
0.000
0.000
0.000
0.000
1.000
0.000
0.000
0.000
0.000
1.000
0.000
0.000
0.000
0.000
1.000
0.000
0.000
1.000
0.000
0.864
0.136
0.000
0.000
0.000
0.000
1.000
0.000
0.632
0.158
0.053
0.158
sim-arnt Position Frequency Matrix
A:
C:
G:
T:
0.158
0.053
0.632
0.158
0.105
0.211
0.105
0.579
0.053
0.000
0.947
0.000
0.000
1.000
0.000
0.000
0.000
0.000
1.000
0.000
0.000
0.000
0.000
1.000
Figure S1. Position frequency matrixes (PFMs) of DNA binding motifs to which heterodimers of
Single-minded (Sim)-Tango (Tgo) and Sim-Aryl hydrocarbon receptor nuclear translocator
(Arnt) bind are shown. PFM of Sim-Tgo was retrieved from the FlyFactorSurvey database, in
which the binding site preferences of transcription factors in Drosophila melanogaster are
described based on the Bacterial One-Hybrid system (1). PFM of Sim-Arnt was retrieved by
systematic evolution of ligands by exponential enrichment (SELEX) performed with a random
double-stranded oligonucleotide library and recombinant Sim and Arnt (2).
6
Figure S2.
A 0.50-kb region within the shadow enhancer completely failed to direct sog
expression in the neurogenic ectoderm and the ventral midline. Top vertical lines indicate
distances from the transcription start site of the sog gene. All constructs were prepared by
placing a PCR-amplified genomic fragment just upstream of a 142-bp even-skipped (eve)
minimal promoter fused with the lacZ open reading frame. LacZ expression in the transgenic
embryos was visualized by in situ hybridization with antisense lacZ RNA probe. LacZ patterns
directed by fragments of the sog shadow enhancer in the ventral midline are shown to the right of
each construct. Initially identified 0.88-kb shadow enhancer is referred to as the ‘full-length’
shadow enhancer hereafter. The 0.88-kb construct directed lacZ expression comparable to that of
endogenous sog expression in the neurogenic ectoderm and the ventral midline. The 0.50-kb
construct completely failed to drive expression of the lacZ fusion gene in the neurogenic
ectoderm and the ventral midline. These results suggest that the distal and proximal elements are
required to direct sog expression in the neurogenic ectoderm and the ventral midline. “st”
indicates the developmental stage during Drosophila embryogenesis.
7
REFERENCES
1. Zhu, L. J., Christensen, R. G., Kazemian, M., Hull, C. J., Enuameh, M. S., Basciotta, M. D.,
Brasefield, J. A., Zhu, C., Asriyan, Y., Lapointe, D. S., Sinha, S., Wolfe, S. A. and Brodsky,
M. H. (2011) FlyFactorSurvey: a database of Drosophila transcription factor binding
specificities determined using the bacterial one-hybrid system. Nucleic Acids Res. 39, D1117.
2. Swanson, H. I., Chan, W. K. and Bradfield, C. A. (1995) DNA binding specificities and
pairing rules of the Ah receptor, ARNT, and SIM proteins. J. Biol. Chem. 270, 2629226302.
8