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2016 Nature America, Inc. All rights reserved.

the Mogrify predictions should similarly

improve. Second, Mogrify is based on CAGE
expression estimates from population samples
and tissues5. With the advent of single-cell RNA
sequencing, we will be able to define cell type
with finer granularity than was possible with
population-based measurements, and thus cell
fate prediction algorithms such as Mogrify will
gain even greater precision. Finally, Mogrify
specifically nominates positive regulators of
the target cell type. Although these predictions
may implicitly include repressors of alternate
fates, the fact that they do not explicitly search
for them may be problematic.
Direct conversion from a pluripotent state?
Mogrify has made significant progress in
addressing one of the fundamental barriers to
faithful cell engineering, what I refer to as the
improvement problem (Fig. 1a), in the context of direct conversion. The extent to which
these cocktails might help to improve the differentiation of pluripotent stem cells is unclear,
but Rackham et al. at least provide cocktails
predicted to convert from human embryonic
stem cells to each of the FANTOM5 tissues and
cell types. As directed differentiation usually
requires the stepwise application of cytokines

and growth factors and the modulation of

growth conditions, one could imagine attempting to reconstruct the sequence of signaling
events needed to activate the Mogrify cocktails.
Alternatively, one could introduce the factors
after initiating differentiation, in effect directly
converting from a pluripotent stem cell6.
However, this approach has not been tested.
A broader perspective on cell fate
engineering
Until recently, another major barrier was the
absence of a quantitative and comprehensive
metric to evaluate how closely engineered
populations resemble their in vivo counterparts
(Fig. 1b), what I refer to as the assessment
problem. Although the experimental demonstration of the keratinocyte- and endothelialinducing cocktails is impressive, the limited
evaluation of the induced populations, especially the absence of genome-wide molecular
characterization, means that we do not have
an answer to this assessment question. This
is especially important because several commonly reported deficiencies of directly converted cells are likely to influence their ability
to perform in any of the applications mentioned above (for example, disease modeling

or regenerative medicine). These deficiencies

include the residual expression of the programs
from the starting cell type2,7, incomplete activation of the program for the target cell type
and aberrant activation of alternate fates8.
The assessment question raises a more general issue, which is that the positive controls
used to evaluate Mogrify are only as good
as the converted populations themselves.
The Mogrify-predicted cocktails are an incredibly valuable and efficient starting point, and
they are likely to enable novel conversions to
be explored, yet future work will be required
to develop new means of overcoming these
remaining fundamental barriers to get engineered cells across the finish line.
COMPETING FINANCIAL INTERESTS
The author declares no competing financial interests.
1. Rackham, O.J.L. et al. Nat. Genet. 48, 331335 (2016).
2. Cahan, P. et al. Cell 158, 903915 (2014).
3. DAlessio, A.C. et al. Stem Cell Rep. doi:10.1016/
j.stemcr.2015.09.016 (23 October 2015).
4. Marbach, D. et al. Nat. Methods 9, 796804 (2012).
5. FANTOM Consortium and the RIKEN PMI and CLST
(DGT). Nature 507, 462470 (2014).
6. Li, V.C. & Kirschner, M.W. Proc. Natl. Acad. Sci. USA
111, 95039508 (2014).
7. Davis, R.L., Weintraub, H. & Lassar, A.B. Cell 51,
9871000 (1987).
8. Morris, S.A. et al. Cell 158, 889902 (2014).

Birth and upgrowth of the Hox topological domains

during evolution
Jacqueline Deschamps
The recently discovered chromatin compartments called topologically associating domains (TADs) are essential
for the three-dimensional organization of regulatory interactions driving gene expression. A new study documents
the emergence of a TAD flanking the amphioxus Hox cluster, prefiguring the vertebrate anterior Hox TAD and
preceding the appearance of the concurring posterior Hox TAD.
The last few years have witnessed spectacular progress in the elucidation of the threedimensional organization of the vertebrate
genome and its impact on gene expression1,2.
In particular, genes controlling embryonic
development were found to be regulated by
sets of remote cis-acting elements heavily
interacting with each other and with gene
promoters within TADs 3. In this issue,
Jos Luis Gmez-Skarmeta and colleagues 4
add a new dimension to this research field
by reconstructing how such TADs arose
during evolution.
Jacqueline Deschamps is at the Hubrecht Institute,
Developmental Biology and Stem Cell Research,
Utrecht, the Netherlands.
e-mail: j.deschamps@hubrecht.eu

Tracing back vertebrate Hox TADs

All bilaterian animals possess Hox genes in
more or less organized clusters, and vertebrates have four or more Hox clusters as a
result of multiple whole-genome duplications.
Previous work has shown that vertebrate Hox
gene clusters are regulated by two successive
waves of transcriptional enhancement elicited
from flanking genomic regions on opposing
sides of the clusters5. Early sequential expression of Hox genes in the 3 to 5 direction
closely correlates with the anterior to posterior
progression of development. This expression is
essential for shaping embryonic axial tissues,
including the proximal part of the limb buds.
It is controlled by enhancers spread over a large
genomic domain abutting the Hox clusters
on their 3 (early) side, forming an anterior

nature genetics | volume 48 | number 3 |March 2016

regulatory landscape that has not been

fully characterized so far5. A second wave
of transcription concerns the 5 Hox genes
and is initiated on the other (late) side of
the HoxA and HoxD clusters, responding to
well-characterized long-distance enhancers
forming the posterior landscape. In the mouse,
this latter regulation is functionally associated with the development of the most distal
part of the limbs and the external genitalia6.
The HoxA and HoxD anterior and posterior
regulatory landscapes in vertebrates were
shown to correspond to anterior and posterior TADs, respectively7. Acemel et al.4 studied
the origin of these chromatin topological
structures. In an attempt to trace back the
existence of the Hox TADs earlier in evolution,
they performed a syntenic analysis to compare
227

news and views

Mouse
Zebrafish

WGD
A

Vertebrate ancestor
2 WGDs
Anterior and
posterior TADs

Hox

Step 2
Amphioxus
A

Chordate ancestor
Hox

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2016 Nature America, Inc. All rights reserved.

Single TAD
Step 1

Beetle

No TAD

Sea star

Bilaterian ancestor

Figure 1 Schematic phylogenetic tree of bilaterian evolution showing the position of chordates, including the
cephalochordate amphioxus. The mouse and zebrafish represent the branches leading from the vertebrate ancestor
to tetrapods and teleosts, respectively. The beetle and sea star represent the branches leading from the bilaterian
ancestor to protostomes and deuterostomes, respectively. The single TAD in amphioxus and the two vertebrate
anterior (A) and posterior (P) Hox TADs are represented by pink triangles resembling Hi-C data1, and their stepwise
emergence is indicated along the tree. WGD, whole-genome duplication. The phylogenetic tree is not to scale.

the organizations of the genomic surroundings of the Hox clusters in a number of invertebrates and chordates, and in the vertebrates
mouse and zebrafish. They found synteny conservation exclusively between vertebrates and
the cephalochordate amphioxus, which still
exhibits many ancestral features of the nonvertebrate chordates. These results imply that
an interacting set of distant cis-acting elements
of the type involved in productive contacts
with the Hox genes in vertebrates can only be
expected to be present in chordates.
A single chordate Hox TAD
The authors investigated the existence of chromatin topological domains in the amphioxus
Hox neighborhood by mapping and quantifying the three-dimensional interactions between
Hox genes and their flanking territories using a
method called circularized chromosome conformation capture (4C) in this organism and
in zebrafish. Whereas their zebrafish data for
HoxD reproduced the bipartite chromosomal
228

domain architecture corresponding to the

bimodular regulation of this cluster7 (Fig. 1),
the amphioxus interaction profiles did not
comply with this scheme and showed no clear
sign of a bipartite architecture involving Hox
genes and their anterior and posterior surroundings. The data suggested the existence
of a single compartment of regulatory interactions between the amphioxus Hox genes
and their anterior neighborhood (Fig. 1).
This architecture was made even more visible
after converting the 4C interaction data into a
three-dimensional model of chromatin folding
and upon using an ingenious novel approach
to convert the 4C-derived three-dimensional
model into a heat map of distances analogous to
what would be obtained by Hi-C1. The results
beautifully illustrate that the amphioxus Hox
cluster resides within a single chromatin interaction domain, whereas the vertebrate HoxA
and HoxD clusters occupy a transition hinge
between two TADs. Acemel et al.4 obtained
functional validation of the newly discovered

amphioxus Hox TAD being a regulatory compartment. A number of long-distance enhancers within this TAD conferred Hox-like activity
to reporter constructs in transgenic zebrafish
embryos. These findings suggest that the
amphioxus TAD is a unit of coordinated regulation as it is in vertebrates8.
Future perspective
Acemel et al. are the first to have questioned
the existence in distantly related species of large
regulatory structures of the type found to control the key patterning Hox genes in vertebrates.
Their pioneering study adds a unique chapter to
the information on partitioning of the genome
into TADs as functional regulatory compartments. Their experimental work opens the way
to a deeper molecular analysis of a primitive
TAD. It suggests that a nucleus for the more
sophisticated anterior TAD of vertebrate Hox
clusters existed at the root of the chordates. The
boundaries of the amphioxus Hox TAD must
have changed along the way in evolution to vertebrates, and it will be of interest to examine the
chromatin architecture corresponding to the
anterior TAD of an intermediate species at the
base of vertebrates such as the lamprey, a jawless
fish that exhibits regulatory conservation within
the anterior part of the Hox clusters with jawed
vertebrates9. Another intriguing question is
whether molecular players underlying the functioning of vertebrate TADs, such as enrichment
of CTCF-binding sites at TAD boundaries10
and intervention of cohesin11,12, were already
involved in amphioxus and in the lamprey. The
posterior TAD of mouse and zebrafish, which is
not yet foreshadowed in amphioxus, must have
arisen later but before the double whole-genome
duplication at the root of vertebrates (Fig. 1).
This posterior TAD is thought to have formed a
favorable architecture, allowing recruitment of
Hox patterning information for the emergence
of evolutionary novelties such as digits and
external genital organs in tetrapods5,13.
COMPETING FINANCIAL INTERESTS
The author declares no competing financial interests.
Dixon, J.R. et al. Nature 485, 376380 (2012).
Nora, E.P. et al. Nature 485, 381385 (2012).
de Laat, W. & Duboule, D. Nature 502, 499506 (2013).
Acemel, R.D. et al. Nat. Genet. 48, 336341 (2016).
Andrey, G. et al. Science 340, 1234167 (2013).
Montavon, T. et al. Cell 147, 11321145 (2011).
Woltering, J.M., Noordermeer, D., Leleu, M. & Duboule, D.
PLoS Biol. 12, e1001773 (2014).
8. Schwarzer, W. & Spitz, F. Curr. Opin. Genet. Dev. 27,
7482 (2014).
9. Parker, H.J., Bronner, M.E. & Krumlauf, R. Nature 514,
490493 (2014).
10. Vietri Rudan, M. et al. Cell Rep. 10, 12971309 (2015).
11. Sofueva, S. et al. EMBO J. 32, 31193129 (2013).
12. Merkenschlager, M. & Odom, D.T. Cell 152, 12851297
(2013).
13. Lonfat, N., Montavon, T., Darbellay, F., Gitto, S. &
Duboule, D. Science 346, 10041006 (2014).
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volume 48 | number 3 |March 2016 | nature genetics