Science - USA (2022-02-04)

fig. S2, A to C; and table S1). Insulators are

characterized by H3 lysine 4 trimethylation

(H3K4me3) and the binding of canonical

insulator proteins (CTCF, CP190), whereas

tethers are distinguished by H3K4 mono-

methylation (H3K4me1) and the binding of

pioneer factors Trithorax-like (Trl), grainyhead

(grh), and zelda (zld; fig. S2, A and B). There

are 103 focal contacts (33%) that connect

promoters of protein-coding genes to“orphan”

intergenic sequences, which we term distal

tethering elements (DTEs; Fig. 1D); others

connect different genes together. These con-

tacts typically span tens of kilobases (mean

43.5 kb; Fig. 1D and table S2) and are observed

at many critical developmental loci, includ-

ingvestigialandcut(fig. S3). Because DTEs

generally display no enhancer activity in the

early embryo (Fig. 1E and fig. S2D), we hy-

pothesized that they might be organizational

elements dedicated to fostering long-range

SCIENCEscience.org 4 FEBRUARY 2022•VOL 375 ISSUE 6580 567

Fig. 1. Hierarchical genome organization: Bound-

aries and focal contacts.(A) ANT-C organization

(Dfd-Antpinterval). The following are shown from top

to bottom: Micro-C contact map showing TADs and

focal contacts (arrows); Hox genes (black); other

genes (gray); regulatory elements; and chromatin

immunoprecipitation (ChIP) data for Trl and CP190.

(B) Tethers and boundaries are physically distinct.

(C) Epigenetic signatures of tethers and boundaries.

DNase, deoxyribonuclease I. (D) Fraction of contacts

connecting gene promoters to“orphan”DTEs

and a histogram of loop spans (black, all loops).

(E) Enhancer activity, by functional class (***p< 10−^7

versus Zld peaks, Bonferroni-corrected chi-square

test; n.s., not significant). (F) Image of a live embryo

showing transcription ofDfd,Antp(cyan), andScr

(yellow, image enhanced), with nuclei in purple.

0

70

% Active

Zld Peaks Distal TEsBounda

ries

Zld (

Loops) All Tiles

n.s.

E Enhancer activity

D Focal contacts (N=313)

Loop span (kb)

0 200

Density (x10

-2)^2

0

Gene-DTE

Others

C Chromatin profiles

Boundaries

Tethers0 50

DNase

0 4

K4me3

0 1.5

K4me1

08

Tr l

0 1.5

CP190

B Distinct regulatory classes

Boundaries

Tethering

Elements

2,034 620

71

A ANT-C Organization: Boundaries & Tethering Elements

miR-10 ftz Antp P2

Dfd Scr Antp P1

TADs

[ 400 bp resolution ]

0.0

0.2 100 kb

Micro-C

Trl

CP190

Contacts

Dfd Scr Antp

Enhancer Boundary Tether

F Live imaging of transcription

Dfd Scr Antp (P2)Antp (P1)

Anterior cephalic furrow Posterior

Dorsal

Ventral

Fig. 2. Tethering elements foster enhancer-

promoter interactions and control activation

kinetics.(A) Micro-C forScrDTE mutant embryos.

(The triangle indicates the location of the deletion.)

Virtual 4C (v4C) shows decreased interactions

of the EE enhancer with the promoter upon DTE

deletion (arrow) and increased interactions with

regions beyond the DTE (asterisk). The focal contact

persists inDScrEEembryos (inset). (B) Live mea-

surements of endogenousScrtranscription show

delayed activation inDScrDTEembryos. A-P,

anterior-posterior; FU, fluorescence units; N, number

of embryos; shading, ±SEM).

BLive transcription measurements

0

10

20

30

% Nuclei Active

10 30 50 70

Time after Metaphase 13 (min)

Scr WT (N=9)

ΔScrDTE (N=8)

Output / Nucleus (x10

3 ) 20

0

10

Relative A-P position (μm)

-30 0 30

Mean Intensity (FU)

500

0

10 30 50 70

Time after Metaphase 13 (min)

ASpatial organization of the Scr TA D

v4C

0.012

Interact. Freq.0.000

WT

ΔScrDTE

CP190

Tr l

TE DTE

Scr ftz Scr EE Antp

ΔScrDTE

ΔScrEE

WT

50 kb [ 200 bp ]

0.0

0.1

RESEARCH | REPORTS