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mains controversial. Enhancers preferen-
tially interact with promoters within the
same TAD. Indeed, disruption of individual
TAD boundaries results in defects in gene
expression and disease ( 2 , 3 ). By contrast,
additional studies demonstrated that alter-
ing TAD boundaries at a genome level re-
sulted in minimal effects on gene expression
(4, 5). Furthermore, despite the pronounced
changes in gene expression that drive de-
velopment, TADs are notably stable over
time and between cell types ( 6 , 7 ). Because
of these conflicting observations, there has
been no overarching framework for how 3D
chromatin structure affects enhancer-pro-
moter contacts to promote gene expression.
Using Micro-C, a technique that enables
single-nucleosome maps of 3D chromatin
interactions, Batut et al. generated a de-
tailed interaction map of the genome in
Drosophila melanogaster embryos. The
high degree of resolution provided by
Micro-C allowed the authors to identify a
new class of regulatory elements, tethering
elements. Tethering elements do not acti-
vate transcription in transgenic reporter
assays and therefore are distinct from en-
hancers. Instead, they facilitate interactions
between enhancers and promoters. Similar
enhancer-promoter interactions have been
identified genome-wide in mammalian cells
( 8 ), suggesting that this is a conserved fea-
ture of genome structure. Focusing on the
well-studied homeobox (Hox) locus, Batut
et al. used live imaging of transcriptional
dynamics to demonstrate that tethering

elements promote the rapid activation of

gene expression and showed that disrup-

tion of these elements has phenotypic con-

sequences in the adult. As suggested by

prior studies ( 9 ), the authors show that TAD

boundaries within the Hox locus prevent in-

teractions between enhancers and promot-

ers located in different TADs. Disruption

of TAD boundaries affects gene expression,

but this can vary depending on the regula-

tory elements that are in the neighboring

TAD. Deletion of the genomic sequence of

either tethering elements or TAD boundar-

ies does not disrupt the formation of the

other. Thus, tethering elements and TADs

have independent contributions to chroma-

tin organization and the regulation of gene

expression (see the figure).

In addition to functional differences,

tethering elements and TAD boundaries

may be formed at discrete times. Chromatin

structure is established in early develop-

ment ( 10 , 11 ). A recent imaging-based strat-

egy to interrogate chromatin structure dem-

onstrated that chromatin loops are evident

before TAD formation, supporting the inde-

pendent formation of loops and TADs ( 12 ).

Batut et al. showed that tethering elements

and TAD boundaries are defined by a dis-

tinct repertoire of binding factors. Whereas

TAD boundaries are enriched for insulator

binding proteins, tethering elements are

bound by the pioneer transcription factors

Zelda, GAGA factor (GAF), and Grainyhead.

Pioneer factors are capable of binding

closed chromatin, promoting chromatin

accessibility and thereby activating gene

expression. Zelda and GAF are essential for

defining chromatin accessibility and acti-

vating gene expression in the early D. me-

lanogaster embryo ( 13 , 14 ). Similarly, both

factors have been implicated in the estab-

lishment of 3D chromatin structure ( 10 , 11 ).

A mechanistic connection between binding

of Zelda and tethering element function is

suggested by the finding that Zelda is es-

sential for a subset of early formed loops

between enhancers and promoters ( 12 ). The

enrichment of multiple pioneer factor bind-

ing sites in tethering elements suggests that

a shared property of these factors may be

essential for tethering.

Although it remains for future studies to

determine whether these pioneer factors

are important for tethering element func-

tion and, if so, how they promote tether-

ing, it is notable that GAF and Zelda are

not uniformly distributed in the nucleus.

Zelda forms hubs of high local concentra-

tion, which are important for its ability

to recruit other transcription factors and

potentiate gene expression ( 15 ). Thus, this

property may be important for bringing

enhancers and promoters in proximity.

The recent identification of condensates

or phase-separated domains of locally high

concentrations of transcription factors has

provided a framework to begin to under-

stand these enhancer-promoter interac-

tions. These transcription factor hubs may

bring multiple enhancers and promoters

together and, in this way, facilitate inter-

action between these cis-regulatory ele-

ments. It is not yet clear whether pioneer

factors form condensates that recruit addi-

tional factors to facilitate chromatin acces-

sibility and enhancer-promoter interaction

or if pioneer factors mediate chromatin ac-

cessibility and this then drives subsequent

hub formation. Defining these processes

will be important for understanding how

gene expression is precisely regulated to

control development. j

REFERENCES AND NOTES

1. P. J. Batut et al., Science 375 , 566 (2022).


2. D. G. Lupiáñez et al., Cell 161 , 1012 (2015).


3. M. Franke et al., Nature 538 , 265 (2016).


4. Y. Ghavi-Helm et al., Nat. Genet. 51 , 1272 (2019).


5. W. Schwarzer et al., Nature 551 , 51 (2017).


6. J. R. Dixon et al., Nature 485 , 376 (2012).


7. E. Ing-Simmons et al., Nat. Genet. 53 , 487 (2021).


8. T. S. Hsieh et al., Mol. Cell 78 , 539 (2020).


9. A. Despang et al., Nat. Genet. 51 , 1263 (2019).


10. C. B. Hug, A. G. Grimaldi, K. Kruse, J. M. Vaquerizas, Cell
    169 , 216 (2017).


11. Y. Ogiyama, B. Schuettengruber, G. L. Papadopoulos,
    J.-M. Chang, G. Cavalli, Mol. Cell 71 , 73 (2018).


12. S. M. Espinola et al., Nat. Genet. 53 , 477 (2021).


13. M. M. Gaskill, T. J. Gibson, E. D. Larson, M. M. Harrison,
    eLife 10, e66668 (2021).


14. H. L. Liang et al., Nature 456 , 400 (2008).


15. M. Mir et al., eLife 7 , e40497 (2018).

10.1126/science.ab n6380

INSIGHTS | PERSPECTIVES

Insulator

binding protein

Pioneer

factor hub

TAD

Enhancer Promoter

Tethering element

Pioneer

factor

Tiers of genome organization
Topologically associating domain (TAD) boundaries are enriched
for insulator binding proteins and prevent regulatory elements
outside the TAD from contacting promoters inside the TAD.
Tethering elements are bound by pioneer transcription factors,
possibly in hubs, bringing together enhancers and promoters
for rapid gene activation (orange arrows).

492 4 FEBRUARY 2022 • VOL 375 ISSUE 6580