visualizing the role of boundary elements in enhancer ...in brief yokoshi et al. employ quantitative...
TRANSCRIPT
Article
Visualizing the Role of Bou
ndary Elements inEnhancer-Promoter CommunicationGraphical Abstract
Highlights
d Intra-domain enhancer-promoter interaction occurs in the
absence of TADs
d Topological boundaries increase transcriptional output
independently of TAD formation
d TAD formation is essential for domain-skipping activity of
distal enhancers
Yokoshi et al., 2020, Molecular Cell 78, 224–235April 16, 2020 ª 2020 Elsevier Inc.https://doi.org/10.1016/j.molcel.2020.02.007
Authors
Moe Yokoshi, Kazuma Segawa,
Takashi Fukaya
In Brief
Yokoshi et al. employ quantitative live-
imaging methods to visualize impacts of
TAD formation on enhancer-promoter
communication. They show that intra-
domain enhancer-promoter interaction
occurs independently of TAD formation.
In contrast, domain-skipping activity of
distal enhancers is lost upon TAD
disruption, suggesting that intra-domain
and inter-domain enhancer-promoter
interactions are differentially regulated by
chromosome topology.
Molecular Cell
Article
Visualizing the Role of Boundary Elementsin Enhancer-Promoter CommunicationMoe Yokoshi,1 Kazuma Segawa,2 and Takashi Fukaya1,3,4,*1Laboratory of Transcription Dynamics, Research Center for Biological Visualization, Institute for Quantitative Biosciences, The University of
Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan2Department of Bioinformatics and Systems Biology, Faculty of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan3Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan4Lead Contact
*Correspondence: [email protected]
https://doi.org/10.1016/j.molcel.2020.02.007
SUMMARY
Formation of self-associating loop domains is afundamental organizational feature of metazoan ge-nomes. Here, we employed quantitative live-imagingmethods to visualize impacts of higher-order chro-mosome topology on enhancer-promoter communi-cation in developing Drosophila embryos. Evidenceis provided that distal enhancers effectively producetranscriptional bursting from target promoters overdistances when they are flanked with boundary ele-ments. Importantly, neither inversion nor deletion ofa boundary element abrogates this ‘‘enhancer-as-sisting activity,’’ suggesting that they can facilitateintra-domain enhancer-promoter interaction andproduction of transcriptional bursting independentlyof topologically associating domain (TAD) formation.In contrast, domain-skipping activity of distal en-hancers was lost after disruption of topological do-mains. This observation raises a possibility thatintra-domain and inter-domain enhancer-promoterinteractions are differentially regulated by chromo-some topology.
INTRODUCTION
Enhancers are regulatory DNAs that control spatiotemporal ON/
OFF pattern of gene expression in response to developmental
timing and environmental signals. Whole-genome chromatin
immunoprecipitation (ChIP) studies estimated that the human
genome contains ~400,000 enhancers (ENCODE Project Con-
sortium, 2012), suggesting that a typical human gene is regu-
lated by approximately 20 enhancers. A number of evidences
have been provided that non-coding variations at enhancers
are a major source of phenotypic polymorphism in a population
(e.g., Guenther et al., 2014; Chan et al., 2010). Recent live-imag-
ing studies suggested that enhancers drive bursts of de novo
transcription, or transcriptional bursting, from their target core
promoters to control the level of gene activities in developing
Drosophila embryos (Fukaya et al., 2016; Bothma et al., 2014).
224 Molecular Cell 78, 224–235, April 16, 2020 ª 2020 Elsevier Inc.
Single-molecule fluorescence in situ hybridization assay also
concluded that enhancers modulate bursting parameters in
mammalian cells (Bartman et al., 2016, 2019). More recently,
allele-sensitive single-cell RNA sequencing method further sup-
ported the idea that enhancer control of transcriptional bursting
is a general mechanism of gene regulation conserved among
species (Larsson et al., 2019).
Importantly, recent chromosome conformation capture
methods revealed that the genome consists of a series of self-
associating loop domains, or topologically associating domains
(TADs) (Dixon et al., 2012; Nora et al., 2012; Sexton et al., 2012).
In vertebrates, it is thought that chromatin extruding cohesin, a
ring-shaped structural maintenance of chromosomes (SMC)
complex, stops at the convergent CCCTC-binding factor
(CTCF) sites to establish TADs (Fudenberg et al., 2016; Sanborn
et al., 2015; Rao et al., 2014). One of the key insights obtained
from these studies is that enhancers and their target genes typi-
cally reside in a same TAD, suggesting that regulatory interac-
tions mainly occur within individual topological domains (re-
viewed in Long et al., 2016). However, it remains largely
unclear how TAD formation itself influences dynamics of
enhancer-promoter communication and resulting transcriptional
bursting.
In this study, we successfully visualized impacts of genome to-
pology on transcription dynamics by using MS2/MCP live-imag-
ing method in developing Drosophila embryos (Garcia et al.,
2013; Lucas et al., 2013). Evidence is provided that large
enhancer-promoter distance significantly diminishes the level
of gene activities by affecting the timing and the size of transcrip-
tional bursting. Intriguingly, bursting profiles were recovered
when distal enhancer and target promoter were flanked with a
pair of topological boundaries. This ‘‘enhancer-assisting activ-
ity’’ was not lost even after inversion or deletion of the CTCF-
binding site at one side, giving rise to a possibility that boundary
elements can facilitate distal enhancers to induce transcriptional
bursting independently of TAD formation. This idea was further
supported by genome editing and live-imaging analysis of highly
structured endogenous fushi-tarazu (ftz) locus. Although intra-
domain enhancer-promoter interaction can take place even after
deletion of topological boundary, quantitative image analysis re-
vealed that TAD formation per se mediates subtle increase
(~10%–20%) in total RNA production, which may contribute to
robust gene expression in a natural population. In contrast,
A
24x MS2
Drosophila SysntheticCore Promoter
yellowsna shadow
Enhancer-Promoter distance: 6.5 kb
enhancer
C Enhancer-Promoter distance: 9 kb
24x MS2 yellowsna shadowenhancer
Drosophila SysntheticCore Promoter
0
0.2
0.4
0.6
0.8
1.0
Cum
ulat
ive
frac
tion
0 6 8 10 122 4 x105
Total RNA produced (AU)
Distance: 9 kb (363)Distance: 6.5 kb (356)
G
P <10-71
EMid nuclear cycle 14
His2Av-mRFP Most ventral nuclei
Onset of gastrulation
5.0 min 19.8 min 39.9 min
His2Av-mRFP Cumulative RNA production
B Distance: 6.5 kb D
5.0 min 19.8 min 39.9 min
His2Av-mRFP Cumulative RNA production
Distance: 9 kb
0 10 20 30 40Time (min)
F
Mea
n cu
mul
ativ
eR
NA
pro
duct
iuon
(A
U)
0
2
3
4
5
6
1
Distance:6.5 kb
Distance:9 kb
x105
0
0.5
1.0
1.5
2.0
0 10 20 30 40Time (min)
Distance: 6.5 kb
MS
2 in
tens
ity (
AU
) x104
H
0
0.5
1.0
1.5
2.0
0 10 20 30 40Time (min)
Distance: 9 kb
MS
2 in
tens
ity (
AU
) x104
I
OFF
ON
0 10 20 30 40Time (min)
Bur
st
J
0 10 20 30 40Time (min)
OFF
ON
Bur
st
K
Distance: 6.5 kb Distance: 9 kb
Num
ber
of b
urst
Frequency
0
2
4
6
8
10
P
median 67
RN
A pr
oduc
ed p
er b
urst
(AU
)
Burst size
0
5
10
15x10
4
L
56median
Duration
Tim
e (m
in)
0
2
4
6
8
10
M
median 86
N
% of 6.5 kbreporter
Tim
e (m
in)
Onset of first burst
0
10
30
40
20
O
median 247
Amplitude
02468
141210
x103
MS
2 in
tens
ity (
AU
)
median 66
(legend on next page)
Molecular Cell 78, 224–235, April 16, 2020 225
analysis of the neighboring Hox geneSex comb reduced (Scr) re-
vealed that gene-skipping activity of distal T1 enhancer is strictly
dependent on TAD formation, suggesting that intra-domain and
inter-domain enhancer-promoter interactions are differentially
regulated by chromosome topology. Our study provides a first
glimpse into previously uncharacterized roles of topological
boundaries in the control of enhancer-promoter communication
during animal development.
RESULTS
Enhancer-Promoter Distance Significantly ImpactsTranscriptional OutputTo quantitatively visualize how enhancer-promoter distance in-
fluences the dynamics of transcriptional activities, the well-
defined snail (sna) shadow enhancer was positioned in two
different locations of the synthetic MS2 reporter gene, 6.5 kb
or 9 kb downstream of the core promoter sequence (Figures
1A and 1C). The ~1.5-kb sna shadow enhancer drives expres-
sion in the presumptive mesoderm cells and plays an essential
role in the formation of ventral furrow by delineating the boundary
from neurogenic ectoderm (Dunipace et al., 2011; Perry et al.,
2010). To quantify transcription activities in living Drosophila em-
bryos, production of nascent MS2 transcripts was visualized us-
ing maternally provided MCP-GFP fusion protein during nuclear
cycle (nc) 14 (Video S1). In either configuration, sna shadow
enhancer mediated expression at the ventral region. However,
there was significant diminishment in the level of nascent RNA
production when enhancer-promoter distance became larger
(Figures 1B and 1D; Video S2). To unambiguously compare
expression profiles of two different reporter genes at the same
dorsal-ventral (DV) position, we quantified transcription activities
at themost ventral nuclei defined by the location of ventral furrow
formation during onset of gastrulation (Figure 1E). Consistent
with whole ventral view (Figures 1B and 1D), there was clear
(more than 50%) reduction in the total amount of RNA synthesis
Figure 1. Enhancer-Promoter Distance Impacts Transcriptional Outpu
(A) Schematic representation of the yellow reporter gene containing the 155-bp D
shadow enhancer, and 243 MS2 RNA stem loops (Bertrand et al., 1998) within t
(B) False coloring of yellow reporter gene expression in the embryo containing D
colored nuclei is proportional to the level of cumulative RNA production at given tim
in gray. Images are oriented with anterior to the left and ventral view facing up. S
(C) Schematic representation of the yellow reporter gene containing the 155-bpDS
50 UTR. The 2.5-kb linker sequence derived from bacterial DNA was inserted in b
(D) False coloring of yellow reporter gene expression in the embryo containingDS
colored nuclei is proportional to the level of cumulative RNA production at given tim
in gray. Images are oriented with anterior to the left and ventral view facing up. S
(E) Snapshot of a gastrulating embryo (left) and false coloring of most ventral nucle
in gray. Images are oriented with anterior to the left and ventral view facing up. S
(F) Mean cumulative RNA production per nucleus. Shades represent standard de
for 6.5-kb reporter; 363 nuclei for 9-kb reporter) from three independent embryo
(G) A cumulative plot showing fraction of most ventral nuclei (y axis) and total RN
(H and I) A representative trajectory of transcriptional activity of theMS2 reporter g
(J) Binarized burst profile obtained from the analysis of raw trajectory shown i
shown in (I).
(L–P) Boxplots showing the distribution of burst size (L), burst duration (M), ampli
lower (25%) and upper (75%) quantiles, and the solid line indicates the median. W
363 most ventral nuclei, respectively, were analyzed from three individual embr
Relative median values normalized to the 6.5-kb reporter were shown in bottom.
226 Molecular Cell 78, 224–235, April 16, 2020
when the distance became larger (Figures 1F and 1G). These
data indicate that the size of enhancer-promoter separation
has a significant impact on gene activities. Supporting this
view, transcriptional output was further diminished when dis-
tance was extended from 9 kb to 11 kb (Figure S1A).
To explore the mechanism underlying this ‘‘distance effect,’’
we obtained MS2 signal trajectories from over 300 nuclei at the
most ventral region per reporter. In both 6.5-kb and 9-kb re-
porters, sna shadow enhancer produced intermittent bursts of
de novo transcription, or transcriptional bursting (Figures 1H
and 1I), but their profiles look quite different. To characterize
quantitative differences in detail, we computationally detected
individual bursting events (Figures 1J and 1K) and analyzed func-
tional parameters, including frequency, timing, and burst size
(Figure S1B). We found that burst size (i.e., number of nascent
transcripts produced per burst) became nearly half when the dis-
tance got larger (Figure 1L). This reduction seems to be mainly
attributed to lower amplitude rather than shorter duration of indi-
vidual bursting events (Figures 1M and 1N). Moreover, there was
significant delay in the onset of transcriptional bursting (Fig-
ure 1O), which leads to overall diminishment of bursting fre-
quency (Figure 1P). Consistent with this, difference in output at
the first 15 min was much larger than difference in total output
(Figure S1C). Overall, our data suggest that enhancer-promoter
distance contributes to the control of gene activities by changing
the size and the timing of transcriptional bursting.
Boundary Elements Facilitate Enhancer-PromoterInteractionOur results showed that large enhancer-promoter distance di-
minishes the level of gene activities (Figure 1). However, regula-
tory interactions in this distance range are thought to commonly
take place in the Drosophila genome because whole-genome
enhancer survey estimated median enhancer-promoter separa-
tion to be ~10 kb (Kvon et al., 2014). Then how do enhancers
overcome large distances from their target promoters?
t
rosophila synthetic core promoter (DSCP) (Pfeiffer et al., 2008), the 1.5-kb sna
he 50 UTR.SCP-MS2-yellow-sna shadow enhancer (distance: 6.5 kb). Intensity of false-
e in a given nucleus. Themaximumprojected image of His2Av-mRFP is shown
cale bar indicates 25 mm.
CP, the 1.5-kb sna shadow enhancer, and 243MS2RNA stem loopswithin the
etween yellow and sna shadow enhancer.
CP-MS2-yellow-linker-sna shadow enhancer (distance: 9 kb). Intensity of false-
e in a given nucleus. Themaximumprojected image of His2Av-mRFP is shown
cale bar indicates 25 mm.
i at mid-nc 14 (right). Themaximum projected image of His2Av-mRFP is shown
cale bar indicates 25 mm.
viation of the mean across all analyzed nuclei at most ventral region (356 nuclei
s.
A production (x axis). The p value of Wilcoxon rank-sum test was shown.
enewith 6.5-kb (H) and 9-kb enhancer-promoter distance (I) in individual nuclei.
n (H). (K) Binarized burst profile obtained from the analysis of raw trajectory
tude (N), onset of first burst (O), and burst frequency (P). The box indicates the
hiskers extend to the most extreme, non-outlier data points. A total of 356 and
yos for the reporter gene with 6.5-kb and 9-kb enhancer-promoter distance.
See also Figure S1.
0
0.4
0.8
1.2
1.6
0 10 20 30 40
Time (min)
MS
2 in
tens
ity (
AU
)
No boundary
x104
I
0
0.4
0.8
1.2
1.6
MS
2 in
tens
ity (
AU
)
0 10 20 30 40
Time (min)
Nhomie/Homie
x104
J
0
0.4
0.8
1.2
1.6
MS
2 in
tens
ity (
AU
)
0 10 20 30 40
Time (min)
Nhomie/Homieinv
x104
KN
umbe
r of
bur
st
0
2
4
6
8
10
Tim
e (m
in)
0
10
30
40
20
Tim
e (m
in)
0
2
4
6
8
10
0
5
10
15
MS
2 in
tens
ity (
AU
)
FrequencyPL Onset of first burst
ODurationM AmplitudeN
x103
Nhomie/Homie Nhomie/HomieNo boundary inv
median 125 125median 273 198 median 146 126 median 181 160 median 45 57 % ofNo boundary
RN
A pr
oduc
ed p
er b
urst
(AU
)
0
5
15
25
20
10
Burst size
x104
6,390 6,410 6,430 6,450 6,470 (kb)
6,39
06,
410
6,43
06,
450
6,47
0 (
kb)
Color Range [0-472]
A
Reporterlanding site
9,94
09,
960
9,98
010
,000
10,0
20 (
kb)
Color Range [0-474]
9,940 9,960 9,980 10,000 10,020 (kb)
Nhomie
Homie
B
eve TAD
Nhomie Homie
Rad21CTCFPol II
eve TER94CG12134
[0-1132]
[0-3]
[0-2000]
C
inv
F G
0
0.2
0.4
0.6
0.8
1.0
Cum
ulat
ive
frac
tion
0 6 8 10 122 4 x105
Total RNA produced (AU)
H
Nhomie/Homie (350)
Nhomie/Homie (359)inv
P <10-70P <10
-90No boundary (363)
P <10-12
Nhomie/Homie
01234567
0 10 20 30 40
Time (min)
x105
Mea
n cu
mul
ativ
eR
NA
pro
duct
ion
(AU
)
Nhomie/Homieinv
01234567
0 10 20 30 40
Time (min)
x105
Mea
n cu
mul
ativ
eR
NA
pro
duct
ion
(AU
)
E No boundary
x105
01234567
0 10 20 30 40
Time (min)
Mea
n cu
mul
ativ
eR
NA
pro
duct
ion
(AU
)
D
Nhomie Homie Homieor
Enhancer-Promoter distance: 9 kb
24x MS2 yellowsna shadow
enhancer
Figure 2. Boundary Elements Augment Transcriptional Output
(A and B) Hi-C map of the reporter gene landing site used in this study (A) and the endogenous eve locus (B). Hi-C data from a WT embryonic cell line (Cubenas-
Potts et al., 2017) was visualized with Juicebox browser (Durand et al., 2016).
(C) Organization of the endogenous eve locus. Rad21 ChIP sequencing (ChIP-seq) data from a WT embryonic cell line (Van Bortle et al., 2014), CTCF ChIP-chip
data from 0- to 12-h WT embryos (Roy et al., 2010), and Pol II ChIP-seq data from 2- to 3-h WT embryos (Sun et al., 2015) were visualized with Integrative
Genomics Viewer (IGV).
(D) Schematic representation of the yellow reporter gene containing the 1,328-bp Nhomie, the 155-bp DSCP, the 2.5-kb linker sequence, the 1.5-kb sna shadow
enhancer, and 243MS2RNA stem loopswithin the 50 UTR. The 367-bp Homie was placed in the same orientation as in endogenous eve locus (Homie) or inverted
orientation (Homieinv).
(legend continued on next page)
Molecular Cell 78, 224–235, April 16, 2020 227
Intriguingly, when we looked at Hi-C profile (Cubenas-Potts
et al., 2017), we noticed that our reporter gene landing site lacks
formation of TADs (Figure 2A). On the other hand, many of devel-
opmental patterning genes are known to be located in topolog-
ical domains (e.g., Stadler et al., 2017; Ulianov et al., 2016). A
well-characterized pair-rule gene even-skipped (eve) is also
located within a clear TAD structure (Figure 2B). Previous genetic
studies showed that the eve TAD is shaped by pairing of
well-characterized boundary elements, Nhomie and Homie
(Fujioka et al., 2009, 2013), both of which contain high level of
CTCF and Rad21 binding (Figure 2C). This interaction is thought
to be strictly orientation dependent because inversion of Nhomie
or Homie disrupts long-range interaction in cis and trans (Fujioka
et al., 2009, 2016). We have also examined orientation
dependence of Homie elements by analyzing trans-homolog
enhancer-promoter interaction or transvection (Lim et al.,
2018b). Because transvection strictly requires pairing of bound-
ary elements across homologous chromosomes, we utilized this
system to functionally characterize their interaction properties.
Our data showed that inversion of Homie is sufficient to eliminate
transvection activities, supporting the idea that they act as an
orientation-dependent boundary element in early embryos.
To explore the possibility that TAD formation may increase
an efficiency of intra-domain enhancer-promoter interaction,
Nhomie and Homie were inserted into the 50 and 30 locations of
synthetic MS2 locus as a loop anchor (Figure 2D). Live-imaging
analysis revealed substantial increase in RNA production when
these boundary elements were placed in the same orientation
as in endogenous eve locus (Figures 2E and 2F; Video S3).
Intriguingly, however, even after inversion of Homie, transcrip-
tional upregulation was still observed (Figures 2E and 2G; Video
S4), giving rise to a possibility that boundary elements can
somehow facilitate enhancer-promoter interaction indepen-
dently of TAD formation. Supporting this view, transcriptional
output of the inverted Homie reporter remained to be compara-
ble even after deletion of Homie at the 30 location (Figures S2A–
S2E). Importantly, the reporter gene containing Homie produced
even higher output than the one with inverted Homie (Figure 2H),
implicating that the Nhomie/Homie pairing can further increase
transcription activities. Overall, these observations are consis-
tent with the idea that boundary elements employ pairing-
dependent and independent mechanisms to facilitate distal
enhancers to activate target promoters from remote locations.
We also suggest that boundary elements can exert their function
in various genomic context because Nhomie/Homie facilitated
(E–G) Mean cumulative RNA production per nucleus. Embryos containing the MS2
with Nhomie and Homieinv (G) were analyzed. Shade represents standard deviatio
boundary reporter; 350 nuclei for Nhomie/Homie reporter; 359 nuclei for Nhomie/
same as the plot in Figure 1F (distance: 9 kb).
(H) A cumulative plot showing fraction of nuclei (y axis) and total RNA production (x
is the same as the plot in Figure 1G (distance: 9 kb).
(I–K) A representative trajectory of transcriptional activity of the MS2 reporter gen
and Homieinv (K) in individual nuclei.
(L–P) Boxplots showing the distribution of burst size (L), burst duration (M), ampli
lower (25%) and upper (75%) quantiles, and the solid line indicates the median. W
and 359 most ventral nuclei, respectively, were analyzed from three individual e
Nhomie/Homieinv. Plots of no boundary are the same as the plots in Figures 1L–
boundary elements were shown in bottom. See also Figure S2.
228 Molecular Cell 78, 224–235, April 16, 2020
enhancer-promoter interaction separated by a shorter genomic
distance (Figures S2F–S2I).
When we analyzed bursting profiles after placing boundary
elements, there was clear upregulation of burst production
(Figures 2I–2K). Significant changes were seen for burst size
and timing. Nhomie/Homie increased overall frequency by pro-
moting rapid induction of transcriptional bursting (Figures 2O,
2P, and S1D). In addition, when Nhomie and Homie were
aligned as in eve locus, burst size became more than 2.5-fold
bigger (Figure 2L). Burst size remained to be nearly twice
bigger even after Homie inversion (Figure 2L). In either case,
amplitude was substantially increased with slightly extended
burst duration when compared with the control (Figures 2M
and 2N). These results are consistent with the idea that pair-
ing-dependent and independent mechanisms contribute to
induction of transcriptional bursting by distal enhancers.
Because bursts are thought to occur by recruiting a series of
RNA polymerase II (Pol II) clusters through the formation of
transcription ‘‘hubs’’ or transcriptional ‘‘condensates’’ (Boija
et al., 2018; Cho et al., 2016, 2018; Chong et al., 2018; Lim
et al., 2018b; Sabari et al., 2018), it might be possible that
topological boundaries act as a scaffold to increase local
concentration of active transcription machineries at nearby
enhancers (see Discussion).
Deletion of SF1 Topological Boundary EliminatesExpression of Scr, but Not ftzTo further explore the role of boundary elements in the context of
endogenous genome, we decided to focus on another well-char-
acterized pair-rule gene fushi-tarazu (ftz) because it is located in
the highly structured Hox gene cluster, Antennapedia complex.
Recent Hi-C study revealed that ftz transcription unit and its
regulatory elements are embedded in a same topological
domain as in eve locus (Stadler et al., 2017). ftz TAD has been
shown to be bordered by well-characterized boundary elements
SF1 and SF2 (Li et al., 2015b; Belozerov et al., 2003), both of
which are enriched for CTCF and Rad21 as well as other archi-
tectural proteins, including Su(Hw), CP190, Mod(mdg4), and
TFIIIC (Figure 3A). To monitor ftz transcription in living embryos,
we first inserted 243 MS2 sequence into the 30 UTR by using
CRISPR/Cas9 (Lim et al., 2018b; Ren et al., 2013). Homozygotes
of ftz-MS2 allele were fully viable and phenotypically indistin-
guishable from wild-type (WT) (Lim et al., 2018a), suggesting
that MS2 stem loops do not impede either transcription or
translation. Subsequently, SF1 was removed by the second
reporter gene without boundary elements (E), with Nhomie and Homie (F), and
n of the mean across all analyzed nuclei at most ventral region (363 nuclei for no
Homieinv reporter) from three independent embryos. Plot of no boundary is the
axis). The p values of Wilcoxon rank-sum test were shown. Plot of no boundary
e without boundary elements (I), with Nhomie and Homie (J), and with Nhomie
tude (N), onset of first burst (O), and burst frequency (P). The box indicates the
hiskers extend to the most extreme, non-outlier data points. A total of 363, 350,
mbryos for the reporter gene with no boundary element, Nhomie/Homie, and
1P (distance: 9 kb). Relative median values normalized to the reporter without
A
B C
D E F
Figure 3. SF1 Deletion Eliminates Expres-
sion of Scr, but Not ftz
(A) Organization of the endogenous Scr-ftz locus.
Rad21 and TFIIIC ChIP-seq data from a WT em-
bryonic cell line (Van Bortle et al., 2014), CTCF,
Su(Hw), CP190, Mod(mdg4), and BEAF-32 ChIP-
chip data from 0- to 12-h WT embryos (Roy et al.,
2010), and Pol II ChIP-seq data from 2- to 3-h WT
embryos (Sun et al., 2015) were visualized with IGV
browser. Approximate locations of stripe 1, stripe
2, and T1 enhancers were shown in orange.
(B and C) Double fluorescent in situ hybridization of
endogenous ftz (upper panel) and Scr (lower panel)
in WT ftz-MS2 (B) and ⊿SF1 ftz-MS2 homozygote
embryos (C). Embryos at late nc 14 were shown.
Imageswere cropped and rotated to align embryos
(anterior to the left and posterior to the right). In
⊿SF1 embryos, there was faint ectopic expression
of Scr that colocalized with anterior ftz stripes.
Scale bars indicate 50 mm.
(D) False coloring of nuclei at stripes 1 and 2
region (yellow) and inter-stripe region (blue). The
maximum projected image of His2Av-mRFP is
shown in gray. Images are oriented with anterior to
the left and ventral to the bottom. Scale bar in-
dicates 25 mm.
(E and F) A cumulative plot showing fraction of
nuclei (y axis) and total RNA production (x axis) in
stripe 1 (E) and stripe 2 nuclei (F). The p value of
Wilcoxon rank-sum test was shown. Change in
median value after SF1 deletion was shown in up-
per left. See also Figure S3.
round of genome editing. Deletion has been confirmed by PCR
analysis of genomic DNA (Figures S3A and S3B).
We then performed in situ hybridization to examine spatial
patterning of ftz expression. Even after SF1 deletion, ftz re-
mained to be expressed in seven stripes (Figures 3B and 3C),
suggesting that TAD formation is not a prerequisite for the estab-
lishment of ftz stripes. We also examined expression of the
neighboring Hox gene, Sex comb reduced (Scr). Scr is ex-
pressed at the posterior head segment whose transcription is
driven by the distal T1 enhancer located 30 of the ftz gene and
~25 kb away from the Scr promoter (Figure 3A; Gindhart et al.,
1995). Because ftz TAD is located in between T1 enhancer and
Scr promoter, this interaction is thought to require skipping of
an intervening topological domain. Indeed, recent Hi-C analysis
captured inter-domain T1-Scr interaction in earlyDrosophila em-
bryos (Stadler et al., 2017). Contrary to stripe enhancers, T1
enhancer failed to mediate correct Scr expression in the
absence of SF1 (Figures 3B and 3C), suggesting that pairing be-
tween SF1 and SF2 is essential for the domain-skipping activity
of distal T1 enhancer. Although Scr expression in nc 14 was lost
Mol
in ⊿SF1 mutant embryos, there was no
reduction in the number of sex comb teeth
(Figure S3C), suggesting that the remain-
ing intronic enhancer compensates Scr
expression in later development (Eksi
et al., 2018). Indeed, we detected Scr
expression in both WT and SF1 deletion
mutant after germband extension (Figures S3D and S3E). Impor-
tantly, we reproducibly detected weak ectopic Scr expression at
anterior region of mutant embryos (Figure 3C). Double fluores-
cent in situ hybridization revealed that ectopic Scr pattern over-
laps with anterior ftz stripes (Figures S3F and S3G), suggesting
that loss of SF1 leads to crosstalk between Scr promoter and
neighboring stripe enhancers. Staining of ectopicScr expression
in ⊿SF1 mutant embryos was much weaker than in WT (Figures
3B and 3C), which is consistent with previous observation that ftz
enhancers exhibit intrinsic preference for TATA-containing ftz
promoter over TATA-less Scr promoter (Calhoun and Levine,
2003; Calhoun et al., 2002; Ohtsuki et al., 1998). Overall, our
data suggest that intra-domain and inter-domain enhancer-pro-
moter interactions have differential dependence on chromo-
some topology.
Topological Regulation of ftz Stripe FormationNext, we performed live-imaging analysis to explore quantitative
differences during ftz stripe formation in WT and ⊿SF1 embryos
(Video S5). We particularly focused on stripes 1 and 2 because
ecular Cell 78, 224–235, April 16, 2020 229
0
0.5
1.0
1.5
2.0
0 10 20 30 40
Time (min)
MS
2 in
tens
ity (
AU
)
2.5
WT Stripe 1A
0
0.5
1.0
1.5
2.0
0 10 20 30 40
Time (min)
MS
2 in
tens
ity (
AU
)
2.5
WT Stripe 2E
0
0.5
1.0
1.5
2.0
0 10 20 30 40
Time (min)
MS
2 in
tens
ity (
AU
)
2.5
⊿SF1 Stripe 1B
0
0.5
1.0
1.5
2.0
0 10 20 30 40
Time (min)
MS
2 in
tens
ity (
AU
)2.5
⊿SF1 Stripe 2F
0 10 20 30 40
Time (min)
Mea
n in
tens
ity (
AU
)
0
0.4
0.8
1.2
1.6
2.0 ⊿SF1WTStripe 1D
⊿SF1WTStripe 2
Mea
n in
tens
ity (
AU
)
0
0.4
0.8
1.2
1.6
2.0
H
0 10 20 30 40
Time (min)
0 10 20 30 40
Total ON duration (min)
0 10 20 30 40
Total ON duration (min)
0
0.2
0.4
0.6
0.8
1.0
Cum
ulat
ive
frac
tion
0
0.2
0.4
0.6
0.8
1.0
Cum
ulat
ive
frac
tion
C
G
Stripe 1
Stripe 2
WT (249)⊿SF1 (236)
P < 10-8
WT (232)⊿SF1 (246)
P < 10-4
x104
x104
x104
x104
27.5 min23.1 min
25.6 min23.0 min
x104
x104
Figure 4. Dynamics of ftz Stripe Formation in SF1 Deletion Mutant
(A and B) A representative trajectory of transcriptional activity of ftz-MS2 in WT (A) and ⊿SF1 allele (B) in individual nuclei at the stripe 1 region.
(C) A cumulative plot showing fraction of nuclei (y axis) and total active duration (x axis) in nuclei at stripe 1 region. The p value of Wilcoxon rank-sum test was
shown. Median values were shown in upper left.
(D) Mean MS2 spot intensity per nucleus. Shades represent standard deviation of the mean across all analyzed nuclei at stripe 1 region (249 nuclei for WT; 236
nuclei for ⊿SF1) from three independent embryos.
(E and F) A representative trajectory of transcriptional activity of ftz-MS2 in WT (E) and ⊿SF1 allele (F) in individual nuclei at the stripe 2 region.
(G) A cumulative plot showing fraction of nuclei (y axis) and total active duration (x axis) in nuclei at stripe 2 region. The p value of Wilcoxon rank-sum test was
shown. Median values were shown in upper left.
(H) Mean MS2 spot intensity per nucleus. Shades represent standard deviation of the mean across all analyzed nuclei at stripe 2 region (232 nuclei for WT; 246
nuclei for ⊿SF1) from three independent embryos. See also Figure S4.
enhancers responsible for each stripe were separately posi-
tioned at downstream and upstream of the ftz transcription unit
(Figure 3A; Schroeder et al., 2011; Calhoun and Levine, 2003), al-
lowing us to monitor impacts of SF1 deletion on enhancers at
different locations. In addition, it provides a nice system to visu-
alize transcriptional repression because formation of inter-stripe
region requires silencing by the product of gap gene repressor
Kr€uppel (Kr) (Calhoun and Levine, 2003). We observed slight
diminishment (~10%–13%) in the level of total RNA production
at stripes 1 and 2 after SF1 deletion (Figures 3D–3F), suggest-
ing that TAD formation can facilitate both 50 and 30 enhancers to
efficiently interact with ftz promoter. Importantly, however,
reduction was not as drastic as seen for double deletion of
Nhomie and Homie in our reporter system (more than 60%
change; Figures 2E and 2F), implicating that the remaining
SF2 facilitates enhancer-promoter interaction independently
of TAD formation as in the case of Homie inversion or single
deletion at synthetic locus (Figures S2A–S2E). We suggest
that, although TAD formation per se can increase overall tran-
scriptional output, it has a limited contribution on intra-domain
enhancer-promoter communication and spatial patterning of
gene expression. Supporting this view, recent whole-genome
analysis of Drosophila balancer chromosome showed that
expression for the majority of genes is not altered even after
large-scale chromosome rearrangements and misregulation of
TAD structures (Ghavi-Helm et al., 2019). We also suggest
that transcriptional repression is not dependent on the higher-
order chromosome topology because nuclei at inter-stripe re-
230 Molecular Cell 78, 224–235, April 16, 2020
gion did not exhibit derepression after SF1 deletion (Fig-
ure S4A). This result is consistent with previous observation
that Kr acts as a short-range repressor that mediates direct
repression of promoters and transcriptional activators in a close
linkage (Gray and Levine, 1996).
To examine how SF1 deletion influences transcription dy-
namics, we next analyzed individual MS2 trajectories. In WT em-
bryos, transcription generally occurs in burst both at stripe 1 and
stripe 2 (Figures 4A and 4E; Video S5), but individual bursting
events were hard to be discerned due to continuity of bursting
activities. In contrast, each burst was clearly separated at in-
ter-stripe region (Figure S4B). We speculate that ftz locus is opti-
mized to achieve a high level of gene expression at stripe regions
by producing next burst before clearance of previous bursts.
Intriguingly, ftz bursts became less continuous when SF1 was
deleted (Figures 4B and 4F). When we quantified total active
duration to avoid ambiguity to define individual bursting event,
it turned out that this trend was true for entire nuclei both at stripe
1 and stripe 2 (Figures 4C and 4G). In addition, there was slight
reduction in the mean fluorescent intensity of ftz transcription
foci (Figures 4D and 4H). These data suggest that, although
TADs are not a prerequisite for transcriptional activation, they
can facilitate intra-domain enhancer-promoter interaction to
achieve highest level of RNA production. We speculate that sub-
tle increase mediated by TAD formation can contribute to robust
gene expression under various stress conditions in natural envi-
ronment, which may give selective advantages in a population.
At the inter-stripe region, there appears to be no repression lag
Inter-domaininteraction
Enhancer
Boundaryelement Boundary
element
Intra-domaininteraction
Enhancer
Increase local concentration oftranscription machinaries
B
EnhancerBoundaryelement Boundary
element
Assembly of transcription hub
A
TADformation
Figure 5. Roles of Boundary Elements in
Enhancer-Promoter Communication
(A) Intra-domain enhancer-promoter interaction
can take place in the absence of TADs (left). In
contrast, TAD formation is required for inter-
domain enhancer-promoter interaction (right).
(B) Boundary elements promote distal enhancers
to activate target promoters independently of TAD
formation. Not only cohesin and CTCF but also
many other regulatory proteins are highly enriched
at TAD boundaries. They may contribute to as-
sembly of transcription ‘‘hub’’ by facilitating
recruitment of transcription factors (yellow and
orange ovals) and co-activators (pink oval) at
nearby enhancers and Pol II complexes (blue
ovals) at core promoters. See also Figure S5.
even after SF1 deletion (Figures S4B–S4D), supporting the idea
that short-range repressors can inhibit burst production inde-
pendently of TAD formation. Finally, we examined activity of
SF1 and SF2 boundary elements in the synthetic reporter
system (Figure S4E). They increased the level of total RNA pro-
duction (Figures S4F and S4G) but with different degree from
Nhomie and Homie (Figures 2E–2H), suggesting that each pair
of boundary elements has distinct capability of facilitating
enhancer-promoter communication to differentially control tran-
scriptional activities at individual loci.
DISCUSSION
In this study, we presented evidence that boundary elements
help distal enhancers to induce transcriptional bursting from
their target promoters. Our data suggest that TAD formation is
not a prerequisite for facilitating intra-domain enhancer-pro-
moter interaction because inversion or deletion of Homie did
not abrogate this ‘‘enhancer-assisting activity.’’ Supporting this
view, deletion of SF1 boundary element from the endogenous lo-
cus did not eliminate ftz stripe formation. In contrast, analysis of
the neighboring Hox geneScr revealed that domain-skipping ac-
tivity of distal T1 enhancer relies on pairing between SF1 and SF2
boundary elements, suggesting that intra-domain and inter-
domain enhancer-promoter interactions have differential depen-
dence on TAD formation (Figure 5A).
Roles of TADs in Transcriptional RegulationIt is well established that TAD formation is mainly regulated by
a Zn-finger DNA-binding protein CTCF and the ring-shaped
SMC complex cohesin in vertebrates. When CTCF or Rad21,
a kleisin subunit of cohesin, is acutely removed by auxin-induc-
ible degron (Natsume et al., 2016), essentially all TADs are lost
in mammalian cultured cells (Nora et al., 2017; Rao et al., 2017).
Consistent with this, knockout of cohesin-loading factor Nipbl
leads to loss of TADs in mouse liver (Schwarzer et al., 2017).
Importantly, these studies agree well in a point that elimination
Mol
of TADs has a modest influence on over-
all transcription activities. Thus, topolog-
ical boundaries and resulting TAD struc-
tures have been implicated to play an
auxiliary role in enhancer-promoter communication and gene
expression. In this study, we combined quantitative live imag-
ing and genome-editing methods to directly visualize impacts
of higher-order genome topology on enhancer-promoter inter-
action. Our data suggest that TAD formation itself has minor in-
fluence on intra-domain enhancer-promoter interaction and
spatial patterning of gene expression because we observed
only ~20% reduction in the total output after Homie inversion
(Figure 2; Nhomie/Homie versus Nhomie/Homieinv) and
~10%–13% reduction after SF1 deletion (Figure 3). Consistent
with this, recent whole-genome sequencing study of balancer
chromosomes that contain massive rearrangements of genome
topology revealed that alternation of TAD structures has minor
impacts on overall level and specificity of gene expression in
Drosophila (Ghavi-Helm et al., 2019). Similarly, in mammalian
system, genome-editing analysis of Sox9 locus showed that
TAD disruption results in only ~10%–15% reduction of Sox9
expression and no detectable phenotypes in developing mouse
limb buds (Despang et al., 2019), again suggesting non-essen-
tial role of TADs in gene expression. Supporting this view, it
was recently reported that Drosophila CTCF-null mutant can
progress through embryogenesis (Gambetta and Furlong,
2018). Moreover, it is known that C. elegans and Arabidopsis
lack CTCF and TAD-like structures (Crane et al., 2015; Feng
et al., 2014; Grob et al., 2014), yet they achieve productive
gene expression during development. We speculate that
TAD-mediated intra-domain interactions confer robustness of
transcriptional program under environmental fluctuations,
such as temperature change and nutrient availability, thereby
providing selective advantages in a population. Contrary to
intra-domain regulatory interactions, our data clearly showed
that inter-domain T1-Scr interaction was compromised in
⊿SF1 embryos (Figure 3), suggesting that pairing of boundary
elements is required for gene-skipping activity of distal en-
hancers (Figure 5A). Given an essential role of SF1 in correct
Scr expression, it might be possible that ftz TAD is formed as
a consequence of bringing distal T1 enhancer and Scr
ecular Cell 78, 224–235, April 16, 2020 231
promoter together by looping out an intervening ftz transcrip-
tion unit. Importantly, ⊿SF1 mutant embryos developed nor-
mally even without early Scr expression (Figures 3B, 3C, and
S3C–S3E), suggesting that enhancer redundancy acts as an
alternative mechanism to achieve reliable gene expression un-
der various genetic perturbations, including misregulation of
TAD formation.
Enhancer-Assisting Activity of Boundary ElementsOur data suggest that boundary elements are capable of facili-
tating enhancer-promoter communication independently of
TAD formation because the level of gene activities remained to
be substantially higher even after Homie inversion or deletion
(Figures 2 andS2A–S2E). As inmammalian genome, TADbound-
aries are enriched for CTCF and Rad21 in Drosophila (Van Bortle
et al., 2014). In past studies, much effort has been focused on
functional analysis of these proteins because CTCF and cohesin
have been long known to play a fundamentally important role in
genome organization (reviewed in Ong and Corces, 2014). How-
ever, these are not the only proteins enriched at boundaries.
Whole-genome ChIP assays revealed that so-called insulator
proteins, such as Su(Hw),Mod(mdg4), CP190, andBEAF-32, co-
localize with CTCF-binding sites in the Drosophila genome (Ne-
gre et al., 2010). Moreover, other regulatory proteins, such as
Fs(1)h, DREF, Chromator, L3mbt, Z4 (also known as Putzig),
TFIIIC, and condensin subunits (CAP-H2 and Barren), are shown
to be a major constituent of the complex at TAD boundaries (Cu-
benas-Potts et al., 2017; Van Bortle et al., 2014; Kellner et al.,
2013). Indeed, Nhomie and Homie used in this study are highly
enriched for these components (Figure S5). Among these, Fs(1)
h is known as aDrosophila homolog of bromodomain-containing
protein Brd4 (Kellner et al., 2013). Recent studies suggested that
Brd4 undergoes dynamic liquid-liquid phase separation through
the conserved intrinsically disordered region at C-terminal
domain, producing transcriptional condensates or transcription
hubs that colocalize with the Mediator and Pol II clusters (Cho
et al., 2018; Sabari et al., 2018; Shin et al., 2018). In addition,
Brd4 is known to interact with the positive transcription elonga-
tion factor P-TEFb (Bisgrove et al., 2007), a complex of Cdk9
and its regulatory subunit cyclin T1. Intriguingly, P-TEFb is also
suggested to undergo liquid-liquid phase separation though the
conserved histidine-rich domain of cyclin T1 to increase proces-
sivity of Ser2 phosphorylation at Pol II CTD (Guo et al., 2019; Lu
et al., 2018). Thus, it might be possible that boundary-enriched
Fs(1)h acts as a scaffold to increase local concentration of tran-
scription factors, co-activators, elongation factors, and Pol II
complexes (Figure 5B), thereby affecting the timing and the size
of transcriptional bursting independently of TAD formation.
Importantly, other boundary-enriched proteins DREF, Chroma-
tor, and Z4 are also suggested to be involved in recruitment of
Pol II into asubset of genes (Haberle et al., 2019; Zabidi andStark,
2016), giving rise to a possibility that these proteins further
contribute to assembly of transcription hubs at nearby enhancers
(Figure 5B). It appears that this mechanism is particularly
important for distal enhancers to overcome large genomic
separation because extension of enhancer-promoter distance
from 6.5 kb to 9 kb leads to significant (more than 50%) diminish-
ment in the level of total output when boundary elements
232 Molecular Cell 78, 224–235, April 16, 2020
are not present (Figure 1). We speculate that this is a common
regulatory mechanism in the Drosophila genome because
typical enhancer-promoter separation is estimated to be ~10
kb (Kvon et al., 2014). Future functional studies should elucidate
the molecular mechanism underlying ‘‘enhancer-assisting
activity’’ of boundary elements.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d LEAD CONTACT AND MATERIALS AVAILABILITY
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
d METHOD DETAILS
B Site specific transgenesis by phiC31 system
B Genome editing by CRISPR/Cas9
B Fly strain
B Genomic DNA extraction and PCR analysis
B cDNA synthesis
B In situ hybridization
B Quantification of sex comb teeth numbers
B MS2 Live imaging
B Plasmids
B Visualization of genomics data
B Image analysis
B Nuclei segmentation and tracking
B Recording MS2 signal
B Detection of transcriptional bursting
B Description of bursting properties
B Quantification of total active duration
B False-coloring by cumulative RNA production
B False-coloring by instantaneous MS2 signals
d DATA AND CODE AVAILABILITY
SUPPLEMENTAL INFORMATION
Supplemental Information can be found online at https://doi.org/10.1016/j.
molcel.2020.02.007.
ACKNOWLEDGMENTS
We thank Bomyi Lim and Tyler Heist for sharing nuclei segmentation and
tracking code; Hitomi Takishita, Mai Nakabayashi, Yuko Maeyama, and Mis-
ako Sato for fly husbandry; University of Tokyo Olympus Bioimaging Center
(TOBIC) for helping acquisition of preliminary imaging data not used in this
study; the Tabata laboratory for use of P-97 needle puller and help for quanti-
fication of sex comb teeth; and the Bloomington Drosophila Stock Center for
fly strains. We are also grateful to members of the Fukaya laboratory for dis-
cussions. M.Y. is supported by the Grants-in-Aid for Young Scientists (B)
(JP17K17834) from the Japan Society for the Promotion of Science and the
Employment Stability Support for Young Researchers from the University of
Tokyo. This study was funded by the Grants-in-Aid for Research Activity
Start-up (JP18H06040), the Grants-in-Aid for Scientific Research (B)
(JP19H03154), and the Grants-in-Aid for Challenging Research (Exploratory)
(JP19K22378) from the Japan Society for the Promotion of Science; the
Grants-in-Aid for Leading Initiative for Excellent Young Researchers from the
Ministry of Education, Culture, Sports, Science and Technology in Japan;
and research grants from the Molecular Biology Society Japan, the Mochida
Memorial Foundation for Medical and Pharmaceutical Research, the Nakajima
Foundation, the Inamori Foundation, the Takeda Science Foundation, and the
Sumitomo Foundation.
AUTHOR CONTRIBUTIONS
M.Y. and T.F. designed and performed the experiments. K.S. produced SF1
deletion mutant with help of T.F. T.F. performed image analysis and wrote
the manuscript. All the authors discussed the results.
DECLARATION OF INTERESTS
The authors declare no competing interests.
Received: August 23, 2019
Revised: December 19, 2019
Accepted: February 5, 2020
Published: February 27, 2020
SUPPORTING CITATIONS
The following reference appears in the Supplemental Information: Li
et al. (2015a).
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Molecular Cell 78, 224–235, April 16, 2020 235
STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
Sheep Anti-Digoxigenin-AP Fab fragments Roche Cat# 11093274910; RRID: AB_514497
Mouse Anti-Biotin Invitrogen Cat# 03-3700; RRID: AB_2532265
Alexa Fluor 555 donkey anti-sheep IgG Invitrogen Cat# A21436; RRID: AB_2535857
Alexa Fluor 488 donkey anti-mouse IgG Invitrogen Cat# A21202; RRID: AB_141607
Bacterial and Virus Strains
DH5a Competent Cells This study N/A
Chemicals, Peptides, and Recombinant Proteins
DIG RNA Labeling MIx, 10x conc. Roche Cat# 11277073910
Biotin RNA Labeling MIx, 10x conc. Roche Cat# 11685597910
ProLong Gold antifade reagent Thermo Fisher Cat# P36930
Western Blocking Buffer Roche Cat# 11921681001
Modified Silicone Fluids Shin-Etsu Silicone FL-100-1000CS
Modified Silicone Fluids Shin-Etsu Silicone FL-100-450CS
polylap (polyethylene membrane) Ube Film N/A
KOD One PCR Master Mix Takara Cat# KMM-101
TRIzol Reagent Invitrogen Cat# 15596026
PrimeScript 1st strand cDNA Synthesis Kit Takara Cat# 6110A
in vitro Transcription T7 Kit Takara Cat# 6140
Experimental Models: Organisms/Strains
D. melanogaster nos > MCP-GFP, His2Av-mRFP/CyO This study N/A
D. melanogaster DSCP-MS2-yellow-sna shadow enhancer This study N/A
D. melanogaster DSCP-MS2-yellow-linker-sna shadow enhancer This study N/A
D. melanogaster DSCP-MS2-yellow-long linker-sna shadow
enhancer
This study N/A
D. melanogaster Nhomie-DSCP-MS2-yellow-linker-sna shadow
enhancer-Homie
This study N/A
D. melanogaster Nhomie-DSCP-MS2-yellow-linker-sna shadow
enhancer-HomieinvThis study N/A
D. melanogaster Nhomie-DSCP-MS2-yellow-linker-sna shadow
enhancer
This study N/A
D. melanogaster Nhomie-DSCP-MS2-yellow-sna shadow
enhancer-Homie
This study N/A
D. melanogaster ftz-MS2 Lim et al., 2018b N/A
D. melanogaster ⊿SF1 ftz-MS2 This study N/A
D. melanogaster SF1-DSCP-MS2-yellow-linker-sna shadow
enhancer-SF2
This study N/A
D. melanogaster y[1] M{vas-int.Dm}ZH-2A w[*]; PBac{y[+]-attP-
3B}VK00033
Venken et al., 2006 BDSC
Bischof et al., 2007 # 24871
D. melanogaster y[1] M{vas-int.Dm}ZH-2A w[*]; PBac{y[+]-attP-
3B}VK00001
Venken et al., 2006 BDSC
Bischof et al., 2007 # 24861
Oligonucleotides
Primers are listed in STAR Methods. This study N/A
Recombinant DNA
Plasmids are listed in STAR Methods. N/A N/A
(Continued on next page)
e1 Molecular Cell 78, 224–235.e1–e5, April 16, 2020
Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Software and Algorithms
MATLAB 2019a MathWorks https://www.mathworks.com
Fiji Schindelin et al., 2012 https://fiji.sc
IGV Broad Institute https://software.broadinstitute.org/
software/igv/
Juicebox Durand et al., 2016 https://github.com/aidenlab/Juicebox/
wiki/Download
Deposited Data
Original images of fluorescent in situ hybridization This study https://dx.doi.org/10.17632/yypbkpxkx3.1
LEAD CONTACT AND MATERIALS AVAILABILITY
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Takashi
Fukaya ([email protected]).
EXPERIMENTAL MODEL AND SUBJECT DETAILS
In all imaging experiments, we studied Drosophila melanogaster embryos at nuclear cycle 14 unless otherwise noted. The following
fly lines were used in this study: nos > MCP-GFP, His2Av-mRFP/CyO (this study), DSCP-MS2-yellow-sna shadow enhancer (this
study),DSCP-MS2-yellow-linker-sna shadow enhancer (this study),DSCP-MS2-yellow-long linker-sna shadow enhancer (this study),
Nhomie-DSCP-MS2-yellow-linker-sna shadow enhancer-Homie (this study), Nhomie-DSCP-MS2-yellow-linker-sna shadow
enhancer-Homieinv (this study), Nhomie-DSCP-MS2-yellow-linker-sna shadow enhancer (this study), Nhomie-DSCP-MS2-yellow-
sna shadow enhancer-Homie (this study), ftz-MS2 (Lim et al., 2018b), ⊿SF1 ftz-MS2 (this study), SF1-DSCP-MS2-yellow-linker-
sna shadow enhancer-SF2 (this study), y[1] M{vas-int.Dm}ZH-2A w[*]; PBac{y[+]-attP-3B}VK00033 (Bloomington Drosophila Stock
Center #24871), y[1] M{vas-int.Dm}ZH-2A w[*]; PBac{y[+]-attP-3B}VK00001 (Bloomington Drosophila Stock Center #24861).
METHOD DETAILS
Site specific transgenesis by phiC31 systemAll reporter plasmids were integrated into a unique landing site on the third chromosome using VK00033 strain (Venken et al., 2006).
PhiC31wasmaternally provided using vas-phiC31 strain (Bischof et al., 2007). Microinjection was performed as previously described
(Ringrose, 2009). In brief, 0-1 hour embryoswere collected and dechorionatedwith bleach. Aligned embryoswere dried with silica gel
for ~7 min and covered with FL-100-1000CS silicone oil (Shin-Etsu Silicone). Subsequently, microinjection was performed using
FemtoJet 5247 (Eppendorf) and DM IL LED invertedmicroscope (Leica) equipped withM-152Micromanipulator (Narishige). Injection
needle was prepared with P-97 or P-1000 needle puller (Shutter). Injection mixture contains ~500 ng/ml plasmid DNA, 5 mM KCl,
0.1 mM phosphate buffer, pH 6.8. mini-White marker was used for subsequent screening.
Genome editing by CRISPR/Cas9pCFD3 gRNA expression plasmids, pBS-3xP3-GFP donor plasmid and pBS-hsp70-Cas9 plasmid (addgene #46294) were co-in-
jected to homozygote ftz-MS2 embryos (Lim et al., 2018a; Lim et al., 2018b). Microinjection was performed as described in previous
section. Injection mixture contains 450 ng/ml pCFD3 gRNA expression plasmids, 450 ng/ml pBS-3xP3-GFP donor plasmid, 450 ng/ml
pBS-hsp70-Cas9 plasmid, 5 mM KCl, 0.1 mM phosphate buffer, pH 6.8. 3xP3-GFP marker was used for subsequent screening.
Fly strainThe nanos >MCP-GFP, His2Av-mRFP expression plasmid (Lim et al., 2018b) was integrated into a unique landing site on the second
chromosome using VK00001 strain (Venken et al., 2006) to obtain maternal expression of the MCP-GFP and His2Av-mRFP fusion
proteins. The resulting transgenic allele was balanced over CyO.
Genomic DNA extraction and PCR analysisGenomic DNAwas extracted from 20 adults of WT ftz-MS2 and ⊿SF1 ftz-MS2 homozygotes. Flies were homogenized with 250 mL of
DNA extraction buffer (0.1 M Tris-HCl pH 9.0, 0.1 M EDTA, 1% SDS), and then incubated at 70 �C for 30 min. Subsequently, 35 mL of
5 M KOAc was added and incubated for 30 min on ice. After centrifugation at 20,000 g for 30 min at 4 �C, supernatant was collected.
Genomic DNA was further purified by phenol-chloroform extraction and recovered by isopropanol precipitation. PCR analysis of
Molecular Cell 78, 224–235.e1–e5, April 16, 2020 e2
purified DNA was done by using following primers: primer A (50-TAC AAC AGC CAC AAC GTC TAT ATC-30), primer B (50-ACT TAG
CTT CCC ACA AAC CAT AAC C-30), primer C (50-AAG TGG GAG AAT GAG ACT CG-30), primer D (50-TAG CGG CTG AAG CAC TGC
AC-30), primer E (50-ACGGAA TCC TTC TAT TGCAGT TCCG-30) and primer F (50-GCA TGT ACTCAA ATA AGGCAAG-30). KODOne
PCR Master Mix (Takara) was used for the reaction. All PCR products were then verified by sanger sequencing.
cDNA synthesisTotal RNA was extracted from 40 adults of Oregon-R using TRIzol reagent (Thermo Fisher) followed by chloroform purification and
isopropanol precipitation. Three mg of total RNAwas subjected to reverse transcription using PrimeScript 1st strand cDNA Synthesis
Kit (Takara).
In situ hybridizationEmbryos were dechorionated and fixed in fixation buffer (0.5x PBS, 4% formaldehyde and 50% Heptane) for ~25 min at room tem-
perature. Antisense RNA probes labeled with digoxigenin (DIG RNA Labeling Mix 103 conc, Roche) and biotin (Biotin RNA Labeling
Mix 10 3 conc, Roche) were used. Template DNA for ftz probe was PCR amplified from genomic DNA using primers (50-CGT AAT
ACG ACT CAC TAT AGG GTG GGG AAG AGA GTA ACT GAG CAT CGC-30) and (50-ATT CGC AAA CTC ACC AGC GT-30). Template
DNA for Scr probe was PCR amplified from cDNA using primers (50-CAG TCAAAG TGAGACATTGGCGC-30) and (50-CGT AAT ACG
ACT CAC TAT AGG GCC GAC CAA TTG CAT TGA ACA CAC-30). Hybridization was performed at 55 �C overnight in hybridization
buffer (50% formamide, 5x SSC, 50 mg/ml Heparin, 100 mg/ml salmon sperm DNA, 0.1% Tween-20). Subsequently, embryos
were washed with hybridization buffer at 55 �C and incubated with Western Blocking Buffer (Roche) at room temperature for one
hour. Then, embryos were incubated with sheep anti-digoxigenin (Roche) and mouse anti-biotin primary antibodies (Invitrogen) at
4 �C for overnight, followed by incubation with Alexa Fluor 488 donkey anti-sheep (Invitrogen) and Alexa Flour 555 goat anti-mouse
(Invitrogen) fluorescent secondary antibodies at room temperature for one hour. DNA was stained with DAPI, and embryos were
mounted in ProLong Gold Antifade Mountant (Thermo Fisher Scientific). Imaging was performed on a Zeiss LSM 900 confocal mi-
croscope. Plan-Apochromat 20x / 0.8 N.A. objective was used. Images were captured in 16-bit. Maximum projections were obtained
for all z sections, and resulting images were shown. Brightness of images was linearly adjusted using Fiji (https://fiji.sc).
Quantification of sex comb teeth numbersAdult males of WT ftz-MS2 and ⊿SF1 ftz-MS2 homozygotes were used for counting numbers of sex comb teeth. Forelegs were
viewed with a Leica MZ16F stereomicroscope and images were acquired with a LUMIX DMC-GH3 camera (Panasonic). Sex
comb teeth were then manually quantified.
MS2 Live imagingMCP-GFP, His2Av-mRFP/CyO virgin females were mated with homozygous males carrying the MS2 reporter gene. The resulting em-
bryoswere dechorinated andmounted between a polyethylenemembrane (Ube Film) and a coverslip (18mmx18mm), and embedded
in FL-100-450CS (Shin-Etsu Silicone). Embryos were imaged using a Zeiss LSM 800 (Figures 1, 2, and S1B–S1D) or Zeiss LSM
900 (Figures 3, 4, S1A, S2, S3, and S4). Temperature was kept in between 24.0 to 25.0 �C during imaging. Plan-Apochromat 40x /
1.4 N.A. oil immersion objective was used. At each time point, a stack of 26 images separated by 0.5 mm was acquired. Typical
time resolution of resulting maximum projection was 16.5 s. In subsequent image analysis, all movies were considered to have
same time resolution (16.5 s/frame). Images were captured in 16-bit. Images have been typically taken from the end of nc 13 to the
onset of gastrulation at nc 14. During imaging, data acquisition was occasionally stopped for seconds to correct z-position, and
data were concatenated afterward. For each cross shown inmain figures, three biological replicateswere taken. The same laser power
and microscope setting were used for a set of experiments.
Plasmidspbphi-DSCP-MS2-yellow
Two DNA oligos (50-TTT CCC TCG AGG AGC TCG CCC GGG GAT CGA GCG CAG CGG TAT AAA AGG GCG CGG GGT GGC TGA
GAG CAT CAG TTG TGA ATG AAT GTT CGA GCC GAG C-30) and (50-GGA AAG GAT CCG TTT GGT ATG CGT CTT GTG ATT CAA
AGT TGG CTT ATT CAA AGG ATA TTA ACG AAG GCA GCG GCA CGT CTG CTC GGC TCG AAC AT-30) were annealed and blunt-
ended by PCR. Resulting DNA fragment was inserted between XhoI and BamHI sites in pbphi-snaPr-MS2-yellow plasmid devoid of
intronic modification (Fukaya et al., 2016).
pbphi-DSCP-MS2-yellow-sna shadow enhancer
A DNA fragment containing snail shadow enhancer was purified from pbphi-snail shadow enhancer (Lim et al., 2018b) by digesting
with HindIII and NheI. The resulting fragment was inserted between HindIII and NheI sites in pbphi-DSCP-MS2-yellow.
pbphi-DSCP-MS2-yellow-linker-sna shadow enhancer
A DNA fragment containing partial sequence of LacZ was amplified using primers (50-TTT CCA AGC TTC GGT TAC GAT GCG CCC
ATC T-30) and (50-GGA AAA AGC TTC AAT GGC AGA TCC CAG CGG T-30) and digested with HindIII. The resultant fragment was
inserted into the unique HindIII site in the pbphi-DSCP-MS2-yellow-sna shadow enhancer as a linker DNA.
e3 Molecular Cell 78, 224–235.e1–e5, April 16, 2020
pbphi-DSCP-MS2-yellow-long linker-sna shadow enhancer
A DNA fragment containing partial sequence of LacZ was amplified using primers (50-AAA TTG ACG TCT CGT TGC TGC ATA AAC
CGA C-30) and (50-AAT TTG ACG TCT CGC TCG CCA CTT CAA CAT C-30) and digested with AatII. The resultant fragment was in-
serted into the unique AatII site in the pbphi-DSCP-MS2-yellow-linker sna shadow enhancer to extend the linker length.
pbphi-Nhomie-DSCP-MS2-yellow-linker-sna shadow enhancer
ADNA fragment containing Nhomie was amplified from genomic DNA using primers (50-TTT AAGCGGCCGCGA TAT CACCTCGTT
GCGGTT CC-30) and (50-TTA AAG CGGCCGCGC TAGCTT GTGGGA TGGCCAGGGGG-30) and digested with NotI. The resultant
fragment was inserted into the unique NotI site in the pbphi-DSCP-MS2-yellow-linker-sna shadow enhancer. Orientation of Nhomie
insertion was confirmed by sequencing.
pbphi-Nhomie-DSCP-MS2-yellow-linker-sna shadow enhancer-Homie
A DNA fragment containing Homie was amplified from genomic DNA using primers (50-TTT AAT CTA GAA ATA CTA AAA AGT TTT
TAC G-30) and (50-TTA AAT CTA GAG ATT ACA CGC TGC GAT GGT TTG-30) and digested with XbaI. The resultant fragment was
inserted into the unique XbaI site in the pbphi-Nhomie-DSCP-MS2-yellow-linker-sna shadow enhancer. Orientation of Homie inser-
tion was confirmed by sequencing.
pbphi-Nhomie-DSCP-MS2-yellow-linker-sna shadow enhancer-Homieinv
A DNA fragment containing Homie was amplified from genomic DNA using primers (50-TTT AAT CTA GAA ATA CTA AAA AGT TTT
TAC G-30) and (50-TTA AAT CTA GAG ATT ACA CGC TGC GAT GGT TTG-30) and digested with XbaI. The resultant fragment was
inserted into the unique XbaI site in the pbphi-Nhomie-DSCP-MS2-yellow-linker-sna shadow enhancer. Orientation of Homie inser-
tion was confirmed by sequencing.
pbphi-Nhomie-DSCP-MS2-yellow-sna shadow enhancer-Homie
The pbphi-Nhomie-DSCP-MS2-yellow-linker-sna shadow enhancer-Homie was digestedwith HindIII to remove linker DNA, followed
by self-ligation of resulting linearized plasmid.
pbphi-SF1-DSCP-MS2-yellow-linker-sna shadow enhancer
A DNA fragment containing SF1 was amplified from genomic DNA using primers (50-ACA TGG CGG CCG CGA ATT CGG TTT TCG
AAG CCG GAG C-30) and (50-AAT TTG CGG CCG CGG ATT CCC CAT CCT ATA CCC TTC-30) and digested with NotI. The resultant
fragment was inserted into the unique NotI site in the pbphi-DSCP-MS2-yellow-linker-sna shadow enhancer. Orientation of SF1
insertion was confirmed by sequencing.
pbphi-SF1-DSCP-MS2-yellow-linker-sna shadow enhancer-SF2
A DNA fragment containing SF2 was amplified from genomic DNA using primers (50-ACA TGT CTA GAC CAT CCT CTT GTG AGG
CTG GG-30) and (50-AAT TTT CTA GAC TGA TTG ACG AAT TGC GTG CG-30) and digested with XbaI. The resultant fragment was
inserted into the unique XbaI site in the pbphi-SF1-DSCP-MS2-yellow-linker-sna shadow enhancer. Orientation of SF2 insertion
was confirmed by sequencing.
pCFD3-dU6-SF1 gRNA-1
Two DNA oligos (50-GTC GCA ATA GAA GGA TTC CGT TGG-30) and (50-AAA CCC AAC GGA ATC CTT CTA TTG-30) were annealed
and inserted into the pCFD3-dU6:3gRNA vector (addgene # 49410) using BbsI sites.
pCFD3-dU6-SF1 gRNA-2
Two DNA oligos (50-GTCGGAGGGCAA AAT CTG TGAGG-30) and (50-AAA CCC TCA CAG ATT TTG CCC TC-30) were annealed and
inserted into the pCFD3-dU6:3gRNA vector (addgene # 49410) using BbsI sites.
pBS-50SF1-loxP-3xP3-GFP-loxP-30SF1ADNA fragment containing 50SF1was amplified from genomic DNA using primers (50-GGGGGCTCGAGAAGGAGCAGTAAGGCA
CGA G-30) and (50-AAA AAG ATA TCT GGC GGC GCG CCG CAG GAC G-30) and digested with XhoI and EcoRV. The resultant frag-
ment was inserted between XhoI and EcoRV sites in pBS-loxP-3xP3-GFP-loxP (Lim et al., 2018a). Subsequently, a DNA fragment
containing 30SF1 was amplified from genomic DNA using primers (50-GGG GGA CTA GTA GGC GGG GAT GTT GGC GCC T-30)and (50-AAA AAG CGG CCG CCG GTG ATG GGT GAA ACG AAG-30) and digested with SpeI and NotI. The resultant fragment
was inserted between SpeI and NotI sites in pBS-50SF1-loxP-3xP3-GFP-loxP.
Visualization of genomics dataFor visualization of Hi-C data, Juicebox (version 1.11.08; Durand et al., 2016) was used (https://github.com/aidenlab/Juicebox/wiki/
Download). Hi-C data pre-implemented in the software (Kc167 DpnII HinfI; Cubenas-Potts et al., 2017) was shown. For visualization
of chromatin immunoprecipitation profile, previously reported ChIP-seq data was downloaded from the Gene Expression Omnibus
(GEO) database. Processed files were obtained from the following sources: Fs(1)h-L (GEO: GSE42086), DREF (GEO: GSE63518),
L3mbt (GEO: GSE63518), Z4 (GEO: GSE63518), Chromator (GEO: GSE54529), TFIIIC (GEO: GSE54529), Rad21 (GEO:
GSE54529), CAP-H2 (GEO: GSE54529), Barren (GEO: GSE54529), and Pol II (GEO: GSE65441). ChIP-chip data (CTCF, Su(Hw),
Mod(mdg4), CP190, BEAF-32) was obtained from the modENCODE database (http://data.modencode.org). All data was visualized
using IGV (version 2.4.19).
Image analysisAll the image processing methods and analysis were implemented in MATLAB (R2019a, MathWorks).
Molecular Cell 78, 224–235.e1–e5, April 16, 2020 e4
Nuclei segmentation and trackingFor each time point, maximum projections were obtained for all 26 z sections per image. His2Av-mRFP was used to segment nuclei.
Nuclei-labeled channel images were pre-processed with Gaussian filtering, top-hap filtering, and adaptive histogram equalization, in
order to enhance the signal-to-noise contrast. Processed imageswere converted into binary images using a threshold value obtained
from Otsu’s method. Nuclei were then watershedded to further separate and distinguish from neighboring nuclei. Subsequently, bi-
nary images were manually corrected by inspecting all individual time frames using Fiji (https://fiji.sc). The number and the position of
separate components within a frame was obtained, where each component serves as a mask for individual nuclei. Nuclei tracking
was done by finding the object with minimal movement across the frames of interest. In Figures S1A, S2, S4F, and S4G, 512 3 512
maximum projection images were initially cropped into 430 3 430 to remove nuclei at the edge, and used for subsequent analysis.
Recording MS2 signalMaximum projections of MCP-GFP images were used to record fluorescent intensities. Using nuclei segmentation files as a mask for
each nucleus, fluorescence intensities within each nucleus were extracted. the signal of MS2 foci was determined by taking an
average of the top three pixels (Figures 1, 2, S1, S2, S4F, and S4G) or top two pixels (Figures 3, 4, and S4A–S4D) with the highest
fluorescence intensity within each nucleus. After obtaining MS2 trajectories, median fluorescence intensities within a nucleus were
subtracted as a nuclear background. Subsequently, minimum MS2 intensity was determined for individual trajectories and sub-
tracted to make the baseline zero.
Detection of transcriptional burstingA transcriptional burst was defined as a local change in fluorescence intensity. First, MS2 trajectories were smoothed by the moving
averagemethod.When a nucleus had above-threshold transcriptional activity, burst was considered to beON. Burst was considered
to be ended when the intensity dropped below 55% of the local peak value. When the burst duration is less than 5 time frames, it was
considered as a false-positive derived from detection noise. When signal trace exhibits continuous decreasing at the beginning of
burst detection, it was also considered as a false-positive. Location of defined burst was then moved two time frames afterward
to better capture the center of individual bursting event. Same method and threshold value were used throughout the analysis.
Description of bursting propertiesFrom each trajectory, number of bursts, amplitude and duration of each burst, integrated signal of each burst (burst size), onset of first
burst, cumulative integrated signal (cumulative RNA production) and total integrated signal (total RNA production) produced by each
nucleus were measured. To determine amplitude, the peak value during the burst was measured using trajectories after moving
average. The duration was determined by measuring the length of each burst. Burst size was measured by taking the area under
a single burst using rawMS2 intensities. Cumulative and total RNA production weremeasured by taking the area under the trajectory
using rawMS2 intensities. The amplitude, duration, and burst sizewere determined by taking average of all analyzed bursts in a single
nucleus.
Quantification of total active durationTo determine threshold, the maximum MS2 signal throughout all trajectories was determined for each embryo. The 10% of the
maximum was used as a threshold. Total duration above the threshold was measured for each nucleus.
False-coloring by cumulative RNA productionCumulative RNA production at each nucleus was measured as described in the previous section. Using segmentation mask, individ-
ual nuclei were false-colored with the pixel intensity proportional to the level of cumulative RNA production at given time in a given
nucleus. Resulting image was then colored and layered over the maximum projected image of His2Av-mRFP.
False-coloring by instantaneous MS2 signalsMS2 signal intensity at each nucleus wasmeasured as described in the previous section. Using segmentation mask, individual nuclei
were false-colored with the pixel intensity proportional to the instantaneous MS2 signal at given time in a given nucleus. Resulting
image was then colored and layered over the maximum projected image of His2Av-mRFP.
DATA AND CODE AVAILABILITY
Original imaging data of fluorescent in situ hybridization has been deposited to Mendeley Data (https://dx.doi.org/10.17632/
yypbkpxkx3.1)
e5 Molecular Cell 78, 224–235.e1–e5, April 16, 2020
Molecular Cell, Volume 78
Supplemental Information
Visualizing the Role of Boundary Elements
in Enhancer-Promoter Communication
Moe Yokoshi, Kazuma Segawa, and Takashi Fukaya
0 10 20 30 40Time (min)
6.5 kb
enhancerM
ean
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Figure S1
00.51.01.52.0
0 10 20 30 40Time (min)
MS2
inte
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(AU) x104
size
duration
ampli
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onset offirst burst
frequency
B
Total
median 20 46median % of6.5 kb reporter
RN
A pr
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)
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10 x105 First 15 min Total
median 441 334 median 276 223 % ofNo boundary
RN
A pr
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(AU
)
0
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8
10 x105C DFirst 15 min
Distance: 6.5 kb Distance: 9 kb Nhomie/Homie Nhomie/HomieNo boundary inv
Figure S1. Delayed burst induction leads to lower output, related to Figure 1. (A) Mean cumulative RNA production per nucleus. Shade represents standard deviation
of the mean across all analyzed nuclei at most ventral region (84 nuclei for the 6.5-kb
reporter, 87 nuclei for the 9-kb reporter, 84 nuclei for the 11-kb reporter).
(B) Trajectory of transcriptional activity shown in Figure 1H. Amplitude, duration and
burst size were measured for every single bursting event. Frequency was determined by
counting the total number of bursts during analysis. Onset of first burst was defined as a
time when the first burst started.
(C, D) Boxplots showing the distribution of RNA production at first 15min (left), and
total RNA production (right). The box indicates the lower (25%) and upper (75%)
quantile and the solid line indicates the median.�Whiskers extend to the most extreme,
non-outlier data points. In (C), a total of 356 and 363 most ventral nuclei, respectively,
were analyzed from three individual embryos for the reporter gene with 6.5-kb and 9-kb
enhancer-promoter distance. In (D), a total of 363, 350 and 359 most ventral nuclei,
respectively, were analyzed from three individual embryos for the reporter gene with no
boundary element, Nhomie/Homie and Nhomie/Homieinv. Plots of no boundary in (D) are
the same as the plots in (C) (distance: 9 kb).
�
Nhomie alone (101)Nhomie/Homie (80)inv
P > 0.5No boundary (96) P < 10-9
0
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0 6 8 10 122 4 x105
Total RNA produced (AU)
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Nhomie/Homieinv
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Nhomie alone
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0 10 20 30 40Time (min)
x105
No boundary
inv
A
Nhomie Homie
Enhancer-Promoter distance: 9 kb
24x MS2 yellowsna shadow
enhancer
24x MS2 yellowsna shadow
enhancer
Deletion
B C D
E
Figure S2
F
Nhomie Homie
Enhancer-Promoter distance: 6.5 kb
24x MS2 yellowsna shadow
enhancer
G
Mea
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Distance: 6.5 kbNo boundary
x105
II
0 6 12 180
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Nhomie/Homie (91)P < 10-12
x1050 10 20 30 40Time (min)
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Distance: 6.5 kbNhomie/Homie
x105
H
Figure S2. A single boundary element can increase transcriptional output, related to Figure 2. (A) Homie was removed from the 3´ location of the yellow reporter gene.
(B-D) Mean cumulative RNA production per nucleus. Shade represents standard
deviation of the mean across all analyzed nuclei at most ventral region (80 nuclei for the
Nhomie/Homieinv reporter, 101 nuclei for the Homie-deletion reporter, 96 nuclei for the
no boundary reporter).
(E) A cumulative plot showing fraction of most ventral nuclei (y-axis) and total RNA
production (x-axis). The p values of Wilcoxon Rank-Sum test were shown.
(F) Schematic representation of the 6.5-kb reporter gene containing Nhomie and Homie.
(G, H) Mean cumulative RNA production per nucleus. Shade represents standard
deviation of the mean across all analyzed nuclei at most ventral region (84 nuclei for the
the no boundary reporter, 91 nuclei for the Nhomie/Homie reporter). Plot of no boundary
is the same as the plot in Figure S1A (distance: 6.5 kb).
(I) A cumulative plot showing fraction of most ventral nuclei (y-axis) and total RNA
production (x-axis). The p value of Wilcoxon Rank-Sum test was shown.
A
homologyarm
3xP3-GFP
SF1
gRNA gRNA
homologyarm
WTScr-ftz locus
Donor plasmid
⊿SF1Scr-ftz locus 3xP3-GFP
Homology-directedrepair
Primer F
Primer E
Primer A
Primer BPrimer D
Primer C
Scr ftz-MS2
Scr ftz-MS2
4000—
1500—2000—
1000—
(bp) WT⊿SF1
Primer A+B Primer C+D Primer E+F
WT⊿SF1 WT⊿SF1
B
D
E
WT
⊿SF1
12
10
8
6
4
2
0Num
ber
of s
ex c
omb
teet
hpe
r leg
WT ⊿SF1
n=58 n=48
C
Scr
1 2 3 4 5ftz
stripe1 2 3 4 5ftz
stripe
ectopicScr expression
WT ⊿SF1F G
Figure S3
Figure S3. Characterization of SF1 deletion mutant, related to Figure 3. (A) Schematic representation of SF1 deletion from the endogenous Scr-ftz locus. Two gRNA expressing plasmids were co-injected with the Cas9 expressing plasmid and the
3xP3-GFP donor plasmid. Approximate annealing sites for primers used in (B) were shown. (B) PCR analysis of genomic DNA purified from WT ftz-MS2 and ⊿SF1 ftz-MS2
homozygotes. SF1 deletion was further confirmed by sanger sequencing of resulting PCR products. (C) Boxplots showing the distribution of numbers of sex comb teeth per first leg for WT
and homozygote ⊿SF1 mutant males. The box indicates the lower (25%) and upper (75%) quantile and the solid line indicates the median. Whiskers extend to the most extreme, non-outlier data points. A total of 58 and 48 legs, respectively, were analyzed for WT and
⊿SF1. Number of sex comb teeth was not reduced in homozygote ⊿SF1 mutant adults. (D, E) Fluorescent in situ hybridization of endogenous Scr in WT ftz-MS2 (D) and homozygote ⊿SF1 ftz-MS2 embryos (E). Embryos after germ band extension were shown.
Images were cropped and rotated to align embryos (anterior to the left and posterior to the right). Scale bar indicate 50 μm. (F, G) Merged images of endogenous ftz (blue) and Scr (gray) in WT ftz-MS2 (F) and
⊿SF1 ftz-MS2 homozygote embryos (G) shown in Figure 3B and C. Anterior part of embryo was zoomed in. Images were cropped and rotated to align embryos (anterior to the left and posterior to the right).
D⊿SF1
0
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0 10 20 30 40Time (min)
x104 WT
Figure S4
00.51.01.52.0
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) 2.5
B
0 10 20 30 40Time (min)
WT interstripex104
Repression
00.51.01.52.0
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⊿SF1 interstripex104
Repression
Interstripe
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0 8 12 164 x105
Total RNA produced (AU)
median 102% of WT
Interstripe
E
SF1 SF2
Enhancer-Promoter distance: 9 kb
24x MS2 yellowsna shadow
enhancer
F G
0 4 8 12Total RNA produced (AU)
Mea
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0.20.40.60.81.0
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ion
No boundary (87)SF1/SF2 (96)
P < 10-4
No boundary SF1/SF2
median 139% of No boundary
x105 x105
x105
Figure S4. Dynamics of transcriptional repression at inter-stripe region, related to Figure 4. (A) A cumulative plot showing fraction of nuclei (y-axis) and total RNA production (x-axis) in inter-stripe nuclei. The p value of Wilcoxon Rank-Sum test was shown. Change in median value after SF1 deletion was shown in upper right. (B, C) A representative trajectory of transcriptional activity of WT ftz-MS2 (B) and ⊿SF1 ftz-MS2 (C) in individual nuclei at the inter-stripe region. (D) Mean MS2 spot intensity per nucleus. Shades represent standard deviation of the mean across all analyzed nuclei at inter-stripe region (241 nuclei for WT, 235 nuclei for ⊿SF1) from three independent embryos. (E) Schematic representation of the yellow reporter gene containing the 2231-bp SF1 and the 1993-bp SF2. Orientation of SF1 and SF2 is same as in endogenous Scr-ftz locus. (F) Mean cumulative RNA production per nucleus. Shade represents standard deviation of the mean across all analyzed nuclei at most ventral region (87 nuclei for no boundary reporter, 96 nuclei for SF1/SF2 reporter). Plot of no boundary is the same as the plot in Figure S1A (distance: 9 kb). (G) A cumulative plot showing fraction of most ventral nuclei (y-axis) and total RNA production (x-axis). The p value of Wilcoxon Rank-Sum test was shown.
Pol IIBEAF-32
CP190Mod(mdg4)
Su(Hw)CTCF
BarrenCAP-H2
Rad21
Z4L3mbt
ChromatorDREF
Fs(1)h-L
TFIIIC
eveCG12134 TER94
[0-2000]
[0-5]
[0-5]
[0-4]
[0-5]
[0-3]
[0-60]
[0-1226]
[0-1132]
[0-335]
[0-400]
[0-166]
[0-132]
[0-269]
[0-253]
Figure S5
Nhomie Homie
Figure S5. ChIP-seq and ChIP-chip profiles of endogenous eve locus, related to Figure 5. Not only CTCF and Rad21, but also other regulatory proteins are highly enriched at
topological boundaries. Fs(1)h-L ChIP-seq data from a WT embryonic cell line (Kellner, et al., 2013), DREF, L3mbt and Z4 ChIP-seq data from a WT embryonic cell line (Li, et al., 2015a), Chromator, TFIIIC, Rad21, CAP-H2 and Barren ChIP-seq data from a WT
embryonic cell line (Van Bortle, et al., 2014), CTCF, Su(Hw), Mod(mdg4), CP190 and BEAF-32 ChIP-chip data from 0-12 h WT embryos (Roy, et al., 2010), and Pol II ChIP-seq data from 2-3 h WT embryos (Sun, et al., 2015) were visualized with IGV browser.