Conservation of epigenetic regulation by the MLL3/4 tumour suppressor in planarian pluripotent stem cells

Abstract

Currently, little is known about the evolution of epigenetic regulation in animal stem cells. Here we demonstrate, using the planarian stem cell system to investigate the role of the COMPASS family of MLL3/4 histone methyltransferases that their function as tumor suppressors in mammalian stem cells is conserved over a long evolutionary distance. To investigate the potential conservation of a genome-wide epigenetic regulatory program in animal stem cells, we assess the effects of Mll3/4 loss of function by performing RNA-seq and ChIP-seq on the G2/M planarian stem cell population, part of which contributes to the formation of outgrowths. We find many oncogenes and tumor suppressors among the affected genes that are likely candidates for mediating MLL3/4 tumor suppression function. Our work demonstrates conservation of an important epigenetic regulatory program in animals and highlights the utility of the planarian model system for studying epigenetic regulation.

Full text

ARTICLE
Conservation of epigenetic regulation by the
MLL3/4 tumour suppressor in planarian
pluripotent stem cells
Yuliana Mihaylova 1, Prasad Abnave 1, Damian Kao1, Samantha Hughes2, Alvina Lai1, Farah Jaber-Hijazi 3,
Nobuyoshi Kosaka1& A. Aziz Aboobaker 1
Currently, little is known about the evolution of epigenetic regulation in animal stem cells.
Here we demonstrate, using the planarian stem cell system to investigate the role of the
COMPASS family of MLL3/4 histone methyltransferases that their function as tumor sup-
pressors in mammalian stem cells is conserved over a long evolutionary distance. To
investigate the potential conservation of a genome-wide epigenetic regulatory program in
animal stem cells, we assess the effects of Mll3/4 loss of function by performing RNA-seq
and ChIP-seq on the G2/M planarian stem cell population, part of which contributes to the
formation of outgrowths. We find many oncogenes and tumor suppressors among the
affected genes that are likely candidates for mediating MLL3/4 tumor suppression function.
Our work demonstrates conservation of an important epigenetic regulatory program in ani-
mals and highlights the utility of the planarian model system for studying epigenetic
regulation.
DOI: 10.1038/s41467-018-06092-6 OPEN
1Department of Zoology, Tinbergen Building, South Parks Road, Oxford OX1 3PS, UK. 2HAN University of Applied Sciences, Institute of Applied Sciences,
Laan van Scheut 2, 6525EM Nijmegen, The Netherlands. 3Beatson Institute for Cancer Research, Switchback Road, Bearsden, Glasgow G61 1BD, UK. These
authors contributed equally: Yuliana Mihaylova, Prasad Abnave Correspondence and requests for materials should be addressed to
A.A.A. (email: Aziz.Aboobaker@zoo.ox.ac.uk)
NATURE COMMUNICATIONS| (2018) 9:3633 | DOI: 10.1038/s41467-018-06092-6| www.nature.com/naturecommunications 1
1234567890():,;
Content courtesy of Springer Nature, terms of use apply. Rights reserved

The pluripotent adult stem cell population of planarian
flatworms is a highly accessible study system to elucidate
fundamental aspects of stem cell function1,2. These stem
cells, collectively known as neoblasts (NBs), bestow these animals
with an endless capacity to regenerate all organs and tissues after
amputation. Comparisons of stem cell expression profiles and
functional data between animals show that some key aspects of
stem cell biology are deeply conserved3–8, while others, like the
transcription factors that define pluripotency in mammalian stem
cells, appear not to be. Thus, studies of NBs have the potential to
inform us about the origins of fundamental stem cell
properties that underpin metazoan evolution, such as main-
tenance of genome stability9, self-renewal7,10, pluripotency11–13,
differentiation14–16, and migration17. All of these are highly
relevant to understanding human disease processes, particularly
those leading to cancer.
Currently, very little comparative data exists for the role of
epigenetic regulation in animal stem cells. Planarian NBs offer an
opportunity to ask whether the cellular and physiological roles of
different epigenetic regulators might be conserved between
mammalian and other animal stem cells. Additionally, as muta-
tions in many chromatin modifying enzymes are implicated in
cancer18–20, using NBs as a model system may provide funda-
mental insight into why these mutations lead to cancer, if epi-
genetic regulatory programs are conserved.
The genome-wide effects of chromatin modifying enzymes
make understanding how they contribute to cancer phenotypes
very challenging. Complexity in the form of tissue and cell het-
erogeneity, life history stage and stage of pathology make reso-
lution of epigenetic regulatory cause and effect relationships
in vivo very challenging. From this perspective, planarians and
their easily accessible NB population may be a very useful model
system. The planarian system could be particularly suitable for
investigating the early transformative changes in stem cells at the
onset of hyperplasia, as the NB identity of all potentially hyper-
plastic cells is known a priori.
The human MLL proteins are the core members of the highly
conserved COMPASS-like (complex of proteins associated with
Set1) H3K4 methylase complexes. An extensive research effort
has now established the evolutionary history and histone mod-
ifying activities of this protein family (Supplementary Figure 121–
32). Perturbation of MLL-mediated H3K4 methylase activity is
characteristic of numerous cancer types. While prominent
examples include the translocation events widely reported in
leukemias involving the Mll1 gene33,34, the mutation rate of Mll3
across malignancies of different origin approaches 7%, making
Mll3 one of the most commonly mutated genes in cancer19.In
attempts to model the role of Mll3 in cancer, mice homozygous
for a targeted deletion of the Mll3 SET domain were found to
succumb to ureter epithelial tumors at high frequency24, an effect
enhanced in a p53+/−mutational background. Heterozygous
deletions of Mll3 in mice also lead to acute myeloid leukemia, as
hematopoietic stem cells fail to differentiate correctly and over-
proliferate, implicating Mll3 in dose-dependent tumor suppres-
sion20. Recent studies have revealed an increasingly complicated
molecular function of MLL3, its closely related paralog MLL4,
and their partial Drosophila orthologs—LPT (Lost PHD-fingers of
trithorax-related; corresponding to the N-terminus of MLL3/4)
and Trr (trithorax-related; corresponding to the C-terminus of
MLL3/4)26. LPT-Trr/MLL3/4 proteins have a role in transcrip-
tional control via mono-methylating and/or tri-methylating
H3K4 at promoters and enhancers22,23,25,26,30,35 (Supplemen-
tary Figure 1).
Links between mutations in Mll3/4, effects on downstream
targets of MLL3/4 and human cancers remain to be elucidated. If
the role of MLL3/4 in regulating stem cells is conserved in NBs,
planarians could provide an informative in vivo system for
identifying potential candidate target genes that drive tumor
formation. Here we exploit the accessibility of NBs to identify and
investigate the role of Mll3/4 orthologs in the planarian Schmidtea
mediterranea, and show knockdown leads to NB over prolifera-
tion, tissue outgrowths containing proliferative NBs and
differentiation defects. Investigating the regulatory effects
accompanying this phenotype, we demonstrate mis-regulation of
both oncogenes and tumor suppressors, providing a potential
explanation for how tumor suppressor function is mediated by
MLL3/4.
Results
Planarian orthologs of Mll3/4 are expressed in stem cells.We
found 3 partial orthologs of mammalian Mll3 and Mll4 genes. We
named the planarian gene homologous to Drosophila LPT and
the N-terminus of mammalian Mll3/4—Smed-LPT (KX681482)
(Supplementary Figure 2a). Smed-LPT (LPT) protein contains
two PHD-fingers and a PHD-like zinc-binding domain, sug-
gesting that it has chromatin-binding properties36 (Fig. 1a). There
are two planarian genes homologous to Drosophila Trr and the C-
terminus of mammalian Mll3/4—Smed-trr-1 (KC262345) and
Smed-trr-2 (DN309269, HO004937), both previously described27.
Both SMED-TRR-1 and SMED-TRR-2 contain a PHD-like zinc-
binding domain, a FYRN (FY-rich N-terminal domain), FYRC
(FY-rich C-terminal domain) and a catalytic SET domain.
SMED-TRR-1 (TRR-1) contains only a single NR (Nuclear
Receptor) box at a non-conserved position and SMED-TRR-2
(TRR-2) has no NR boxes (Fig. 1a). This could indicate some
functional divergence exists between TRR-1 and TRR-2, where
only TRR-1 is capable of interacting with nuclear receptors26,37.
We performed whole-mount in situ hybridization (WISH) and
found that LPT,trr-1 and trr-2 are broadly expressed across many
tissues and organs. Gamma irradiation to remove cycling cells in
S. mediterranea revealed that the three transcripts are also likely
to be expressed in NBs (Fig. 1b). A double fluorescent in situ
hybridization (FISH) with the pan-stem cell marker Histone 2B
(H2B) confirmed that over 90% of all NBs co-express LPT,trr-1
and trr-2 (Fig. 1c, d). The genes also showed clear expression in
the brain, pharynx and other differentiated tissues (Fig. 1b).
Mll3/4 RNAi causes regeneration defects and outgrowths.In
order to study the function of planarian Mll3/4, we investigated
phenotypes after RNAi-mediated knockdown. Following LPT
(RNAi), there was a clear failure to regenerate missing structures,
including the eyes and pharynx, with regenerative blastemas
smaller than controls (Fig. 2a, b). After 7 days of regeneration we
observed that, as well as failure to regenerate missing structures,
animals began to form tissue outgrowths (Fig. 2c). Intact
(homeostatic) LPT(RNAi) animals also developed outgrowths,
but at a lower frequency than regenerates (Fig. 2d).
Following individual knockdown of trr-1 and trr-2, milder
differentiation defects were observed compared to LPT(RNAi),
with no obvious outgrowths (Supplementary Figure 2b-d),
confirming results from an earlier study27. However, trr-1/trr-2
double knockdown recapitulated the phenotype of LPT(RNAi),
but with higher penetrance and increased severity (Supplemen-
tary Figure 2e, f). Functional redundancy between the two trr
paralogs likely accounts for the reduced severity after individual
knockdown. Double knockdown animals all developed out-
growths and started dying at day 5 post-amputation. Based on
these observations, we decided to focus our attention on the LPT
(RNAi) phenotype as regeneration defects and the formation of
tissue outgrowths were temporally distinct and could be studied
consecutively.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06092-6
2NATURE COMMUNICATIONS | (2018) 9:3633 | DOI: 10.1038/s41467-018-06092-6 | www.nature.com/naturecommunications
Content courtesy of Springer Nature, terms of use apply. Rights reserved

A more thorough study of the differentiation properties of LPT
(RNAi) animals following amputation showed that the triclad gut
structure failed to regenerate secondary and tertiary branches and
to extend major anterior and posterior branches (Fig. 2e).
Cephalic ganglia (CG) regenerated as smaller structures, the two
CG lobes did not join in their anterior ends in LPT(RNAi)
animals (Fig. 2f) and the optic chiasm and optic cups were mis-
patterned and markedly reduced (Fig. 2g, h). We found that 80%
of LPT(RNAi) animals did not regenerate any new pharyngeal
tissue (Fig. 2i). We interpreted these regenerative defects as being
indicative of either a broad failure in stem cell maintenance and/
or differentiation.
MLL3/4 controls differentiation of the epidermis and neurons.
One of the structures most severely affected following loss of
LPT function was the brain so we looked at the regeneration of
different neuronal subtypes. LPT(RNAi) animals had reduced
numbers of GABAergic (Fig. 3a), dopaminergic (Fig. 3b),
acetylcholinergic (Fig. 3c) and serotonergic (Fig. 3d) neurons.
D. melanogaster
(Arthropoda, Diptera)
PHD PHD
FYRN FYRC SET
zf-HC5HC2H2
PHD PHD FYRN FYRC SET H. sapiens
(Chordata, Vertebrata)
zf-HC5HC2H zf-HC5HC2H2
PHD PHD
FYRN FYRC SET
FYRN FYRC SET
S. mediterranea
(Platyhelminthes,
Turbellaria)
zf-HC5HC2H
zf-HC5HC2H2
zf-HC5HC2H
PHD PHD FYRN FYRC SET H. sapiens
(Chordata, Vertebrata)
zf-HC5HC2H zf-HC5HC2H2
LPT
LPT
TRR
TRR-1
TRR-2
MLL3
MLL4
PHD zf-HC5HC2H(2) FYRN FYRC SET NR box
(LLXXL/LXXLL)
SET
domain
FYRC
domain
FYRN
domain
PHD-like zinc-
binding domain
PHD-
finger
d
a
H2B
+
LPT
H2B
+
trr-1
H2B
+
trr-2
0
300
600
150
450
750
Number of cells
H2B +
Co-localized
WT
LPTtrr-1trr-2 Porcupine-1H2B
2 days PI Hoechst
Hoechst
Hoechst
LPT
trr-1
trr-2
Merge
Merge
Merge
H2B
H2B
H2B
91% H2B +
cells are LPT +
97% H2B +
cells are trr-1 +
92% H2B +
cells are trr-2 +
bc
Fig. 1 S. mediterranea has three partial Mll3/4 orthologs expressed in stem cells. aA schematic depicting the structure and domain composition of MLL3/
MLL4 proteins in D. melanogaster,H. sapiens and S. mediterranea.bgenes’expression pattern in wildtype (WT) and two days following a lethal dose (60 Gy)
of gamma irradiation (PI=post-irradiation). Porcupine-1 (expressed in the irradiation-insensitive cells of the differentiated gut) and H2B (expressed in the
irradiation-sensitive neoblasts) are used as a negative and positive control respectively. Ten worms per condition were used. Scale bar: 500 μm. cWhite
arrows point to examples of cells double-positive for Mll3/4 transcripts and H2B transcripts. The schematic shows the body area imaged. Scale bar: 50 μm.
dGraph showing the raw cell counts used for percentage estimates in (c). Green color represents all counted H2B-positive cells, yellow represents H2B-
positive cells also expressing an Mll3/4 ortholog. Error bars represent Standard Error of the Mean (SEM). Ten animals per condition were used
NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06092-6 ARTICLE
NATURE COMMUNICATIONS | (2018) 9:3633 | DOI: 10.1038/s41467-018-06092-6 | www.nature.com/naturecommunications 3
Content courtesy of Springer Nature, terms of use apply. Rights reserved

Homeostasis
GFP (RNAi)
LPT (RNAi)
Regeneration
Day1 regeneration
Amputate
at day 10
H
T
M
ab
7 days regeneration
GFP (RNAi) LPT (RNAi)
GFP (RNAi) LPT (RNAi)
11 days regeneration
cd
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
17
20
13
20
7
20
H
T
M
20
20
16
20
RNAi 9 days
M
M
ef
20
20
14
20
gh i
GFP (RNAi)LPT (RNAi)
8 days regeneration
PharynxOptic cupsOptic chiasmaGut CNS
20
20
20
20
10
10
09
10
10
10
09
10
20
20
16
20
Fig. 2 LPT(RNAi) results in differentiation defects and outgrowth formation during regeneration. aA schematic showing the amputation of RNAi worms
into head (H), middle (M) and tail (T) pieces in order to observe regeneration of different structures. The time-course of all the experiments on Mll3/4
knockdown animals is depicted underneath the worm schematic. A total of 9 days of dsRNA microinjection-mediated RNAi was followed by amputation on
the 10th day and subsequent observation of regeneration. bHead, middle and tail pieces following LPT(RNAi) or control GFP(RNAi) at day 7 of
regeneration. Yellow arrows point towards the defects in blastema formation. cHead, middle and tail pieces following LPT(RNAi) or control GFP(RNAi) at
day 11 of regeneration. Red arrows point towards outgrowths. dHomeostatic animals following LPT(RNAi) or control GFP(RNAi) at day 14 post RNAi. Red
arrows point towards outgrowths. eGut regeneration and maintenance in middle pieces following LPT(RNAi), as illustrated by RNA probe for the gene
porcupine-1 at 8 days of regeneration. fBrain regeneration in middle pieces at 8 days post-amputation following LPT(RNAi), as illustrated by anti-SYNORF-1
antibody labeling the central nervous system (CNS). gOptic chiasm recovery in tail pieces at 8 days of regeneration following LPT(RNAi), as shown by
anti-VC-1 antibody. hRecovery of optic cups and organized trail of optic cup precursor cells in tail pieces at 8 days of regeneration following LPT(RNAi), as
demonstrated by RNA probe for SP6-9.iPharynx recovery in head pieces at 8 days of regeneration following LPT(RNAi), as illustrated by RNA probe for
laminin. Number of animals with observable phenotype are recorded out of the total number of animals in the top right of each panel. Scale bars: 200 μm
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06092-6
4NATURE COMMUNICATIONS | (2018) 9:3633 | DOI: 10.1038/s41467-018-06092-6 | www.nature.com/naturecommunications
Content courtesy of Springer Nature, terms of use apply. Rights reserved

Brain defects were milder following knockdown of either trr-1
or trr-2 in agreement with the hypothesis of some functional
redundancy between these paralogs (Supplementary
Figure 3a–d).
Epidermal tissue was also affected. Both early (Prog-1+ve cells)
and late (AGAT-1+ve cells) epidermal progeny were significantly
decreased, but not entirely absent, in LPT(RNAi) regenerating
animals (Fig. 3e). No such defect was seen in individual trr-1 and
trr-2 knockdown animals (Supplementary Figure 3e).
These effects along with defects in pharynx and gut tissues
implicate broad NB differentiation defects in LPT(RNAi) animals.
LPT limits normal stem cell proliferation and tissue growth.
Aside from impairment of regeneration following LPT(RNAi),
the other major phenotype we observed were outgrowths of tissue
that appeared at unpredictable positions in regenerating pieces.
Planarian regeneration is characterized by an early burst of
increased NB proliferation, 6–12 h after wounding, and a second
peak of proliferation, 48 h after amputation38. Following LPT
(RNAi), we observed significant increases in proliferation at both
of these peaks and at 8 days post-amputation, as proliferation
failed to return to normal homeostatic levels (Fig. 4a). It was
previously reported that Trr-1(RNAi) leads to decreases in
T
T
T
T
M
8 days regeneration
GFP (RNAi) LPT (RNAi)
a
b
c
d
e
GABAergic
neurons
Dopaminergic
neurons
Acetylcholinergic
neurons
Serotonergic
neurons
Early and late epidermal
stem cell progeny
20
15
10
GAD+ cells/mm2
TH+ cells/mm2
TPH+ cells/mm2
Cells/mm2ChAT + cells/mm2
5
0
**
**
**** ****
***
****
30
20
10
0
800
600
400
0
0
100
200
300
5
10
15
20
25
200
0
GFP
(RNAi)
LPT
(RNAi)
GFP
(RNAi)
LPT
(RNAi)
GFP
(RNAi)
LPT
(RNAi)
GFP
(RNAi)
LPT
(RNAi)
GFP
(RNAi)
prog-1+AGAT-1+
LPT
(RNAi)
GFP
(RNAi)
LPT
(RNAi)
Fig. 3 LPT controls differentiation across neuronal and epidermal lineages. aQuantification of the number of GABAergic neurons (labeled by GAD),
bdopaminergic neurons (labeled by TH), cacetylcholinergic neurons (labeled by ChAT), dserotonergic neurons (labeled by TPH) and eearly (labeled
by prog-1) and late (labeled by AGAT-1) epidermal stem cell progeny at 8 days of regeneration following LPT(RNAi). Two-tailed t-test was used for all
comparisons; **p< 0.005, ***p< 0.0005, ****p< 0.0001. Lines and error bars indicate mean and SD. Five animals per condition per experiment were
assessed over the course of two separate experiments. Scale bars: 200 μm
NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06092-6 ARTICLE
NATURE COMMUNICATIONS | (2018) 9:3633 | DOI: 10.1038/s41467-018-06092-6 | www.nature.com/naturecommunications 5
Content courtesy of Springer Nature, terms of use apply. Rights reserved

mitotic cells, whereas Trr-2(RNAi) doesn’t affect NB
proliferation27.
In 8 day-regenerating LPT(RNAi) worms the observed over-
proliferation is a result of localized clusters of mitotic cells rather
than broad increase in proliferation across regenerating animals
(Fig. 4b). This is different from previously reported planarian
outgrowth phenotypes from our group and others, where
hyperplastic stem cells are evenly distributed39,40. It seems likely
that these mitotic clusters might eventually be responsible for the
formation of outgrowths. When we looked at outgrowths, we
found mitotic cells usually restricted to mesenchymal tissue, had
penetrated into epidermal outgrowths in LPT(RNAi) animals
(Fig. 4c).
In order to understand if ectopically cycling NBs represented
the breadth of known stem cell heterogeneity in planarians or
only a subset of lineages, we performed FISH for markers of the
sigma (collectively pluripotent NBs), zeta (NBs committed to the
epidermal lineage) and gamma (NBs committed to the gut
lineage) cell populations41. We found that all three NB
populations are represented in the outgrowths of LPT(RNAi)
animals (Fig. 5a–c). Sigma,zeta, and gamma NBs are not
significantly increased in pre-outgrowth LPT(RNAi) animals
(Supplementary Figure 4), suggesting that the presence of these
cells in outgrowths is not a secondary effect of increased cell
number and passive spread of these cell populations, but rather
local proliferation at outgrowth sites.
The epidermal progeny, marked by Prog-1 and AGAT-1, were
concentrated in the outgrowths of LPT(RNAi) animals, while
being in reduced numbers in non-outgrowth tissue (Supplemen-
tary Figure 5a). The observed disarray of Prog-1+and AGAT-1+
cells in outgrowths could be the result of perturbed patterning
and polarity of the epidermal layer in LPT(RNAi) animals
(Supplementary Figure 5b), as epidermal cells appear to have lost
polarity and to be no longer capable of forming a smooth
epidermal layer. Furthermore, the average epidermal nuclear size
is significantly increased in LPT(RNAi) animals compared to
*
*
*
0
6
12
24
48 72 192
1000
900
800
700
600
500
400
300
200
100
0
a
ph
ph
ph
ph
ph
ph
GFP (RNAi)LPT (RNAi)
b
M
GFP (RNAi)
LPT (RNAi)
Hours after amputation
12 h
regeneration
48 h
regeneration
192 h
regeneration
Mitotic cells/mm2
LPT (RNAi) GFP (RNAi)
Hoechst H3P
Hoechst H3P
10 days regeneration
c
M
M
Fig. 4 Over-proliferation and mitotic cell clustering precedes and accompanies the emergence of outgrowths in LPT(RNAi) animals. aQuantification of
mitotic cells (labeled by anti-H3P antibody) at different post-amputation time-points following LPT(RNAi). N=10 animals per time-point. Two-tailed t-test
was used for analysis; *p< 0.05. Error bars represent Standard Error of the Mean (SEM). bExamples of middle pieces at the time-points post-amputation
showing significant difference in mitotic cell (white) counts according to (a). ‘ph’indicates the pharynx. The red arrows point towards clusters of mitotic
cells in late stage regenerates (192 h/8 days). Scale bar: 500 μm. cMitotic cells (white) are always restricted to mesenchyme tissue in GFP(RNAi) animals
but penetrate into epidermal outgrowths in LPT(RNAi) animals. Orange line indicates the border of mesenchymal tissue. Scale bar: 50 μm
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06092-6
6NATURE COMMUNICATIONS | (2018) 9:3633 | DOI: 10.1038/s41467-018-06092-6 | www.nature.com/naturecommunications
Content courtesy of Springer Nature, terms of use apply. Rights reserved

smedwi-1 / Sigma pool / Hoechst
smedwi-1 / Zeta pool / Hoechst
smedwi-1 / Gamma pool / Hoechst
smedwi-1 Sigma pool
GFP (RNAi)
a
Merge
smedwi-1 Sigma
pool
LPT (RNAi)
Merge
smedwi-1 Zeta pool
GFP (RNAi)
b
Merge
smedwi-1 Zeta pool
LPT (RNAi)
Merge
smedwi-1 Gamma pool
GFP (RNAi)
c
Merge
smedwi-1 Gamma
pool
LPT (RNAi)
Merge
Fig. 5 Pluripotent as well as lineage restricted stem cells are present in outgrowths of LPT(RNAi) animals. aHead piece showing distribution of Sigma stem
cells in GFP(RNAi) and LPT(RNAi) animals. Sigma stem cells are double positive for smedwi-1 and the ‘Sigma pool’of RNA probes (Soxp1,Soxp2). White
arrows in LPT(RNAi) animals point towards the outgrowth. Red arrows indicate a double-positive cell magnified in red inset box. bImages showing
distribution of Zeta stem cells in GFP(RNAi) and LPT(RNAi) animals. Zeta stem cells are double positive for smedwi-1 and the ‘Zeta pool’of RNA probes
(zfp-1,Soxp3, egr-1). White arrows in LPT(RNAi) animals point towards the outgrowth. Red arrows indicate a double-positive cell magnified in red inset box.
cImages showing distribution of Gamma stem cells in GFP(RNAi) and LPT(RNAi) animals. Gamma stem cells are double positive for smedwi-1 and the
‘Gamma pool’of RNA probes (gata4/5/6,hnf4). White arrows in LPT(RNAi) animals point towards the outgrowth. Red arrows indicate a double-positive
cell magnified in red inset box. Scale bars: 50 μm
NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06092-6 ARTICLE
NATURE COMMUNICATIONS | (2018) 9:3633 | DOI: 10.1038/s41467-018-06092-6 | www.nature.com/naturecommunications 7
Content courtesy of Springer Nature, terms of use apply. Rights reserved

controls (Supplementary Figure 5c), an effect similar to the
pathology seen following knockdown of the tumor suppressor
SMG-140. The epithelial layer in LPT(RNAi) animals also appears
less well-defined than that in control animals, with a blurred
distinction between epithelium and mesenchyme. Another
feature of the LPT(RNAi) phenotype, encountered in a variety
of malignancies42, are changes in nuclear shape (Supplementary
Figure 5d).
In summary, LPT controls NB proliferation and restricts stem
cells to pre-defined tissue compartments as well as being
responsible for the successful differentiation of several lineages.
Taken together, our data demonstrate that disturbance of the
function of planarian LPT leads to development of both
differentiation and proliferation defects (Fig. 6), allowing us to
conclude that the function of LPT/trr/Mll3/4 proteins as an
epigenetic tumor suppressor function is conserved over a large
evolutionary distance.
Transcriptional changes driving proliferation in stem cells.A
key insight missing from the literature for Mll3 and Mll4 muta-
tions, is the downstream targets that are mis-regulated in disease
states, for example in hematopoietic stem cells that cause leuke-
mia26. Given the conserved tumor suppressor function in pla-
narians, we decided to focus on early regeneration when LPT
(RNAi) animals that do not yet exhibit any outgrowth phenotype,
providing the possibility to describe early regulatory changes that
are potentially causal of out growths, rather than consequential.
We performed RNA-seq on X1 (G2/M) fluorescence activated cell
sorted (FACS) NBs from LPT(RNAi) and GFP(RNAi) planarians
at 3 days of regeneration. Our analysis revealed that 540 tran-
scripts are down-regulated (fold change≤−1.5, p< 0.05) and
542 –up-regulated (fold change≥1.5, p< 0.05) in X1 stem cells
from LPT(RNAi) animals when compared to controls (Supple-
mentary Data 1).
A recent meta-analysis of all available S. mediterranea RNA-
seq data allowed classification of all expressed loci in the
planarian genome by their relative expression in FACS sorted
cell populations representing stem cells, stem cell progeny and
differentiated cells8. Superimposing the differentially expressed
genes following LPT(RNAi) onto a gene expression spectrum
reflecting FACS compartments, shows that LPT(RNAi) has a
broad effect on gene expression in X1 cells (Fig. 7a), affecting
genes normally expressed in different planarian cell types
(Fig. 7b).
Analysis of Gene Ontology (GO) terms revealed a clear
enrichment for cell cycle and cell division-associated terms in the
list of up-regulated genes (Fig. 7c), in agreement with the
observed hyper-proliferation in LPT(RNAi) phenotype. The list
of down-regulated genes is also enriched for cell cycle-related
terms, as well as cell differentiation and metabolism-related
processes (Fig. 7c). These findings suggest a broad link between
gene expression changes caused by LPT loss of function and NB
over-proliferation.
Promoter H3K4 methylation and transcription are correlated.
Previous studies tie MLL3/4/LPT-Trr function directly to mono-
and tri-methylation of H3K422–25 and indirectly to tri-
methylation of H3K27, because the H3K27me3 demethylase
Pharynx
Brain
Optic cups
Gut
Stem cells
Epidermis
- Anti-synapsin
central nervous system
- TH dopaminergic neurons
- TPH serotonergic neurons
- chat acetylcholinergic neurons
and neuronal precursors
- GAD GABAergic neurons
- Laminin (pharynx)
- Anti-H3P
- Smedwi-1/SoxP-1
pluripotent Sigma stem cells
Differentiated cells
Late neoblast progeny
Early neoblast progeny
Comitted neoblasts
AGAT-1 late epidermal
stem cell progeny
NB.21.11e early epidermal
stem cell progeny
Smedwi-1/hnf4/gata4/5/6
gut lineage stem cells
Porcupine-1
differentiated gut
Smedwi-1/zfp-1 epidermal
lineage Zeta stem cells
?
EvidenceLineage GFP (RNAi) LPT (RNAi)
- SP6-9 optic cups and
optic cup progenitors
Disrupted following LPT(RNAi)
Fig. 6 LPT(RNAi) results in a cancer-like phenotype. A summary of the differentiation and neoblast proliferation data presented, together with a simplified
flowchart illustrating the tested lineages’development under knockdown conditions. A red cross sign indicates where the defect in a lineage is detected
following LPT(RNAi)
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06092-6
8NATURE COMMUNICATIONS | (2018) 9:3633 | DOI: 10.1038/s41467-018-06092-6 | www.nature.com/naturecommunications
Content courtesy of Springer Nature, terms of use apply. Rights reserved

UTX is present in the same protein complex43. We set out to
understand potential epigenetic causes of the transcriptional
changes following LPT(RNAi) in planarians. To this end, we
performed Chromatin Immunoprecipitation sequencing (ChIP-
seq) on isolated planarian G2/M stem cells and used Drosophila
S2 cells as a spike-in control to normalize for any technical dif-
ferences between samples44. The profile of H3K4me3, H3K4me1
and H3K27me3 at the transcriptional start sites (TSSs) of genes in
control X1 cells was in agreement with their conserved roles in
transcriptional control8,25 (Fig. 8a, Supplementary Figure 6),
suggesting out methodology was robust. LPT(RNAi) led to only
subtle changes in the overall level of H3K4me3 and H3K4me1 at
TSSs throughout the genome. A decrease in H3K4me1 and
H3K4me3 was apparent proximal to the TSS in genes where
expression is normally enriched in differentiated cells (Fig. 8a).
Concomitant with this, we also observed an increase in
H3K4me1 signal downstream of the predicted TSS for genes in
enriched in stem cells (Fig. 8a). For the H3K27me3 mark, no
consistent pattern was observed as a result of LPT(RNAi) in any
group of genes subdivided by expression profiles (Fig. 8a).
We next looked specifically at the promoter histone methyla-
tion status of those genes whose transcript levels were affected by
ATPase activity
Lipid particle
Helicase activity
Cell cycle
Cell division
Methyltransferase activity
Microtubule organizing center
Cell morphogenesis
Mitotic nuclear division
Cell proliferation
Autophagy
Oxidoreductase activity
Mitochondrion organization
Kinase activity
Cell–cell signaling
Proteinaceous extracellular matrix
Extracellular region
Cell differentiation
Cell adhesion
Cytoskeleton organization
Oxidoreductase activity
Cell proliferation
Carbohydrate metabolic process
Lipid metabolic process
Cellular amino acid metabolic process
Methyltransferase activity
Lipid particle
Cell cycle
Mitochondrion
Gene expression
in wild type
Log2
fold change
Proportional expression in
G2/M stem cells (X1)
Proportional expression in G1 stem
cells and stem cell progeny (X2)
Proportional expression in
differentiated cells (X ins)
G2/M-enriched
genes (>50%) G1 stem cells and stem cell progeny-enriched genes (>50%)
Genes enriched in
differentiated cells (>50%)
a
b
49 160 86 37 174 155
X ins
(5119)
X2
(8444)
X1
(2253)
Down-regulated
(366)
Up-regulated
(295)
*White indicates significant enrichment (p-value < 0.01)
0 0.5 1.0 1.5
Average log2 fold change
–1.0 –0.5 0
Average log2 fold change
GO enrichment RNA-Seq up-regulated
genes in LPT(RNAi) G2/M stem cells
GO enrichment RNA-Seq down-regulated
genes in LPT(RNAi) G2/M stem cells
c
0
Down-regulated in LPT(RNAi)
G2/M stem cells
Up-regulated in LPT(RNAi)
G2/M stem cells
X ins
(5119)
X2
(8444)
X1
(2253)
Number of genes up/down-regulated according to different cell population
–5
5
Fig. 7 RNA-seq of G2/M stem cells following LPT(RNAi) reveals effects on genes enriched in different cell populations. aGenes were classified according
to their proportional expression in the X1 (G2/M stem cells; dark blue), X2 (G1 stem cells and stem cell progeny; light blue) and X ins (differentiated cells;
orange) FACS populations of cells. Genes were defined as enriched in certain population(s) if >50% of their expression is observed in that population in
wild type animals. Each vertical line represents a gene. Under the population expression enrichment track is a track with all the significantly up- and down-
regulated genes in G2/M stem cells following LPT(RNAi). The genes with fold change >1.5 (p< 0.05) are shown in red following a log2 fold change
transformation. The genes with fold change <−1.5 (p< 0.05) are shown in blue following a log2 fold change transformation. The Wald’s test (as part of the
Sleuth software) was used for assessing differential expression. bEnrichment for genes in each of the three classes was calculated for the up- and down-
regulated genes’list (red and blue respectively). The number of genes in each group is indicated in brackets under the group’s name. Numbers in white
represent significant enrichment (p< 0.01) according to a hypergeometric enrichment test. cGene Ontology (GO) enrichment analysis on the genes
significantly up-regulated (red) and down-regulated (blue) in G2/M stem cells following LPT(RNAi). Categories are sorted by average Log2 fold change of
the up- or down-regulated genes falling in each category. In bold are shown terms that relate to the described Mll3/4 loss of function phenotype
NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06092-6 ARTICLE
NATURE COMMUNICATIONS | (2018) 9:3633 | DOI: 10.1038/s41467-018-06092-6 | www.nature.com/naturecommunications 9
Content courtesy of Springer Nature, terms of use apply. Rights reserved

LPT(RNAi). For genes with enriched expression in NBs, we
observed a significant inverse correlation between expression
following LPT(RNAi) and the amount of TSS-proximal
H3K4me1 (Fig. 8b). This indicates that LPT(RNAi) leads to a
reduction of this repressive mark at some loci and an up-
regulation of the cognate transcript expression in stem cells,
consistent with the role of H3K4me1 as a repressive mark. For
mis-regulated genes not normally enriched in X1 NBs, we
observed instead a positive correlation between changes in
transcriptional expression (upregulation) and changes in
H3K4me3 levels at the TSS and gene bodies (Fig. 8b).
Overall, our data suggest that reductions in H3K4me1
following LPT(RNAi) cause up-regulation of some of the stem
cell genes implicated by RNA-seq data from LPT(RNAi) animals.
Our data are consistent with MLL3/4’s known role in H3K4
methylation and identify gene expression profiles following LPT
(RNAi) that are broadly correlated with the amount of H3K4me1
and H3K4me3 at gene promoters.
Mis-regulation of oncogenes and tumor suppressors. After
observing the global changes in expression and histone mod-
ification patterns following LPT(RNAi), we wanted to investigate
individually mis-regulated genes that could be major contributors
to the differentiation and outgrowth phenotypes (Figs. 9,10).
Within our list of up- and down-regulated genes, we saw mis-
regulation of tumor suppressors, oncogenes and developmental
genes (Supplementary Data 1). Detailed inspection of changes in
promoter patterns of H3K4 methylation revealed example loci
Genes enriched in G2/M stem
cells (X1)
Genes enriched in G1 stem cells
and stem cell progeny (X2)
Genes enriched in differentiated
cells (Xins)
Spearman’s rank correlation
between RNA-Seq and
ChIP-Seq data
(RNA-Seq Log2 fold change
filter ≤ –1 and ≥ 1), p<0.001
Genes enriched in X1
TSS
H3K4me3
H3K4me1
H3K4me3H3K4me1H3K27me3
–1000
–2000 2000–1000 1000TSS–2000 2000–1000 1000TSS–2000
0.4
0.3
0.2
0.4
0.3
0.2
0.4
0.2
0.0
–0.4
–0.2
1.5
0.0
0.5
1.5
0.0
0.5
0.4
0.2
0.0
–0.4
–0.2
30
20
10
0
30
20
10
0
0.4
0.2
0.0
–0.4
–0.2
2000–1000 1000
TSS
–2000 2000–1000 1000
TSS
–2000 2000–1000 1000
TSS
–2000 2000–1000 1000
TSS–2000 2000–1000 1000TSS–2000 2000–1000 1000TSS–2000 2000–1000 1000
0.5
0.0
–0.5
0.5
0.0
–0.5
0.5
0.0
–0.5
–500 TSS 500 1000–1000 –500 TSS 500 1000–1000 –500 TSS 500 1000
Genes enriched in XinsGenes enriched in X2
GFP (RNAi) LPT (RNAi) Log2 fold-change
a
b
Fig. 8 LPT(RNAi) is mainly manifested in changes in H3K4me1 and H3K4me3 around the TSS in G2/M (X1) stem cells. aGraphs presenting the average
read coverage across the genome for H3K4me3, H3K4me1 and H3K27me3 after performing ChIP-seq on X1 (G2/M stem cells). The graphs are centered
on the TSS (showing 2 kb upstream and downstream) and the data is normalized to Drosophila S2 signal spike-in. The input coverage is subtracted. Log2
fold change graphs are also shown for each histone modification, where signal above zero shows increase following LPT(RNAi) and signal below zero
represents a decrease. Three colors are used for different gene classes—dark blue (genes enriched in G2/M stem cells, i.e., X1), light blue (genes enriched
in G1 stem cells and stem cell progeny, i.e., X2), orange (genes enriched in differentiated cells, i.e., X ins). Standard deviation is shown by a faded color
around each line. bSpearman’s rank correlation between changes in RNA-seq signal following LPT(RNAi) and H3K4me1 or H3K4me3 ChIP-seq signal for
the region around the TSS of genes from different enrichment classes (only examples where a significant correlation exists are shown). The green line
shows a correlation where RNA-seq fold change data was filtered for Log2 fold changes ≤−1 and ≥+1. Faded areas of the lines represent results not
significant at p< 0.001, while darker colors represent results significant at p< 0.001
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06092-6
10 NATURE COMMUNICATIONS | (2018) 9:3633 | DOI: 10.1038/s41467-018-06092-6 | www.nature.com/naturecommunications
Content courtesy of Springer Nature, terms of use apply. Rights reserved

pitx (TF)
(asx1.1_ox1.0.loc.19147)
Enriched in X2/Xins
Fold-change
LPT(RNAi)RNA-Seq
ChIP-Seq profileGene name/loci ID
Smed-pitx + cells Smed-pitx +/smedwi-1 +
M
8 days regeneration
Smedwi-1 + cells
100 μm
TSS–1000 1000
1
0.5
0
+2.46 MedulloblastomaCerebellum
MedulloblastomaCerebellum
–10
0
H3K4me3
pim-2-like
(asx1.1_ox1.0.loc.18774)
Enriched in X1
TSS
–1000 1000
1
0.5
0
+3
H3K4me1
TSS 1000
1
0.5
0
Elf5 (TF)
(asx1.1_ox1.0.loc.02286)
Enriched in X2
H3K4me3
+9.5
ChIP-Seq signal following GFP (RNAi)ChIP-Seq signal following LPT (RNAi)
GFP/GFP GFP/LPT
Elf5/LPT
H3P in double RNAi animals
–1000
No outgrowths
*
200
150
100
50
0
ns
pitx +/smedwi-1+
cells/mm2
15
10
5
0
pitx + cells/mm2
Outgrowths
200 406080100
% Regenerating animals
Su(z)12
paralogue/LPT
ULK2-like/LPT
FoxA1/LPT
ETS4/LPT
pim-2/LPT
pim-2-like/LPT
Elf5/LPT
GFP/LPT
GFP/GFP
Su(z)12
paralogue/LPT
ULK2-like/LPT
FoxA1/LPT
ETS4/LPT
pim-2/LPT
pim-2-like/LPT
Elf5/LPT
GFP/LPT
GFP/GFP
RNAi
Cells/mm2
600
500
400
300
200
100
*
*
RNAi
GFP
(RNAi)
GFP
(RNAi)
LPT
(RNAi)
LPT
(RNAi)
log2 median-centered
intensity
Log2 median-centered
intensity
0
5
–10
–5
Mll3 expression
pitx2 expression
–8
–6
–4
–2
LPT (RNAi)
GFP (RNAi)
pim-2-like/LPT
ab
cd
ef
Fig. 9 Double knockdown with Elf5 or pim-2-like alleviates the LPT(RNAi) over-proliferation and outgrowth phenotype. aExamples of genes significantly
(p< 0.05) up-regulated in G2/M stem cells following LPT(RNAi). The ChIP-seq profile for H3K4me3 or H3K4me1 in the 2 kb region around the TSS of each
gene is presented. Purple color represents normalized signal following LPT(RNAi) and green color is used to show the normalized signal following GFP
(RNAi). The bold font of the gene names signifies a correlation between the genes’up-regulation and their respective H3K4me3/me1 profile. ‘TF’stands for
‘transcription factor’. All three genes show correlation between ChIP-seq profile and up-regulation in RNA-seq data. bin silico analysis (www.oncomine.
org;t-test, p< 0.0001) of Mll3 and pitx2 expression in normal tissue (cerebellum) and cancer tissue (medulloblastoma). Lines and error bars indicate mean
and SD. cpitx and smedwi-1 in situ hybridization at 8 days of regeneration of middle pieces following LPT(RNAi). White arrows show double-positive cells.
Lines and error bars indicate mean and SD. Two-tailed t-test used for analysis (n=5), *p< 0.05, ns is not significant. dRepresentative examples of mitotic
cells (labeled by anti-H3P antibody) in double RNAi condition at 48 h post amputation. eGraph showing number of mitotic cells in double RNAi animals at
48 h post amputation. Each dot represents average number of mitotic cells in single worm (n=5). Lines and error bars indicate mean and SD. Two-tailed
ttest: *p< 0.05. fGraph showing percentage quantification of double knockdown regenerates developing outgrowths. Scale bars: 200 μm
NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06092-6 ARTICLE
NATURE COMMUNICATIONS | (2018) 9:3633 | DOI: 10.1038/s41467-018-06092-6 | www.nature.com/naturecommunications 11
Content courtesy of Springer Nature, terms of use apply. Rights reserved

p53 (TF)
(asx1.1_ox1.0.loc.13302)
Enriched in X2 TSS–1000 1000
TSS
–1000 1000
TSS–1000 1000
TSS–1000 1000
1
0.5
0
1
0.5
0
–2
–1.7
H3K4me3 H3K4me3
1
0.5
0
RREBP1
(asx1.1_ox1.0.loc.24491)
Not enriched
–2.28
Fold-change
LPT (RNAi) RNA-Seq
ChIP-Seq profileGene name/loci ID
ChIP-Seq signal following GFP (RNAi)ChIP-Seq signal following LPT (RNAi)
cut-like1 (TF)
(asx1.1_ox1.0.loc.32364)
Enriched in X2
PRDM1-1 (TF)
(asx1.1_ox1.0.loc.09257)
Enriched in X2
–2.37
H3K4me3H3K4me3
18 days regeneration
48 h
GFP
(RNAi)
PRDM1-1
(RNAi)
cut-like1
(RNAi)
RREBP1
(RNAi)
GFP
(RNAi)
PRDM1-1
(RNAi)
cut-like1
(RNAi)
RREBP1
(RNAi)
GFP
(RNAi)
PRDM1-1
(RNAi)
cut-like1
(RNAi)
RREBP1
(RNAi)
GFP
(RNAi)
PRDM1-1
(RNAi)
cut-like1
(RNAi)
RREBP1
(RNAi)
GFP (RNAi) PRDM1-1 (RNAi)
cut-like1 (RNAi) RREBP1 (RNAi)
GFP (RNAi) PRDM1-1 (RNAi)
cut-like1 (RNAi) RREBP1 (RNAi)
18 dpa7 dpa
7 dpa
48 h
7 dpa
18 dpa
7 dpa
0
1
0.5
RREBP1 (RNAi)
PRDM1-1 (RNAi)
GFP (RNAi)
cut-like1 (RNAi)
H3P/Hoechst
smedwi-1 /prog-1 / Hoechst
smedwi-1
600 ns
ns
ns
ns
*
ns
Cells/mm2
500
400
300
200
100
600
Cells/mm2
Cells/mm2
500
400
300
200
100
3000
2000
*
*
*
**
*
1000
0
Cells/mm2
3000
2000
1000
0
prog-1
smedwi-1 prog-1
ab
c
d
Fig. 10 LPT(RNAi) down-regulates the expression of genes involved in stem cell proliferation and differentiation. aExamples of genes significantly
(p< 0.05) down-regulated in G2/M stem cells following LPT(RNAi). The ChIP-seq profile for H3K4me3 in the 2 kb region around the TSS of each gene is
presented. Bold font of a gene name illustrates an example where there is a correlation between H3K4me3 profile and down-regulation in RNA-seq data.
‘TF’stands for ‘transcription factor’.bRepresentative bright field images of 18 day regenerating animals following different RNAi. PRDM1-1(RNAi), cut-like1
(RNAi) and RREBP1(RNAi) animals show defective anterior regeneration compared to GFP(RNAi) animals. cRepresentative images of mitotic cells (labeled
by anti-H3P antibody) in different RNAi animals at 48h and 7 days post-amputation. Graphs show mitotic cell quantification. Each dot represents average
number of mitotic cells in single worm (n=5). Lines and error bars indicate mean and SD. Student’sttest: *p< 0.05, ns is not significant. dRepresentative
images showing stem cells (smedwi-1+) and early epidermal progeny (prog-1+) at 7 and 18 day regenerating animals from different RNAi conditions. Graphs
show quantification of stem cells and early epidermal progeny (n=5). Lines and error bars indicate mean and SD. Student’sttest: *p< 0.05. Scale bars:
200 μm
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06092-6
12 NATURE COMMUNICATIONS | (2018) 9:3633 | DOI: 10.1038/s41467-018-06092-6 | www.nature.com/naturecommunications
Content courtesy of Springer Nature, terms of use apply. Rights reserved

where changes in methylation status were both consistent and
inconsistent with changes in transcript levels (Figs. 9a, 10a,
Supplementary Figure 7a, Supplementary Figure 9a). For exam-
ple, we find that the up-regulated expression of the planarian
orthologs of the transcription factors Elf5 and pituitary homeobox
(pitx) are associated with increased levels of TSS-proximal
H3K4me3 signal following LPT(RNAi) (Fig. 9a). Furthermore,
up-regulation of some X1-enriched genes, such as pim-2-like,is
associated with a decrease in H3K4me1 signal on the TSS, con-
sistent with alleviated repression (Fig. 9a). On the other hand,
some transcriptional changes following LPT(RNAi), such as the
down-regulation of Ras-responsive element-binding protein 1
(RREBP1), are not correlated with the expected alterations in
histone modification patterns on promoters (Fig. 10a). Such
examples could potentially represent secondary (not related to
histone modifications) or enhancer-dependent changes in the
LPT(RNAi) phenotype. In the absence of similar RNA-seq/ChIP-
seq data in mammals, our data provide an important insight
beyond the deep evolutionary conservation of MLL3/4 function.
While mis-regulation of well-known oncogenes and tumor
suppressors, like p53 (Fig. 10a), would be expected to broadly
correlate with mis-regulation of Mll3/4 in cancer gene expression
datasets, shared correlative expression changes for some selected
genes not previously associated with Mll3/4 loss of function
provide some independent evidence of a conserved regulatory
program. In the Pomeroy Brain Oncomine dataset45,Mll3 and
p53 expression levels were both significantly down-regulated
(Supplementary Figure 10), supporting the functional link
between the two genes established by previous work24 and
emerging in our study. We identified more LPT(RNAi) mis-
regulated genes, which have some known association with growth
and/or cancer, but have not been previously implicated in Mll3/4
loss of function phenotypes. An investigation of the Brune46 and
Compagno47 lymphoma Oncomine datasets demonstrates that
these genes are mis-regulated in different cancers in a similar
manner to the mis-regulation we observe in planarians stem cells
as a consequence of a decreased LPT expression (Supplementary
Figure 10).
One gene of interest in this data set was the planarian pitx gene
ortholog. In planarians, pitx is expressed in the serotonergic
neuronal precursor cells48,49 and is required for their differentia-
tion. Thus, pitx is not directly implicated in planarian stem cell
proliferation, but rather in differentiation. Nonetheless, pitx’sup-
regulation was of great interest to us since in human
medulloblastomas down-regulation of Mll3 and over-expression
of pitx2 are co-occurrences (Pomeroy Brain Oncomine dataset45
((www.oncomine.org)) (Fig. 9b). To investigate the cellular basis
for pitx overexpression, we performed FISH for this gene in LPT
(RNAi) animals. We observed an accumulation of pitx-positive
cells in LPT(RNAi) regenerates (Fig. 9c). Given that production
of terminally differentiated serotonergic neurons is decreased
(Fig. 3d), the increase of pitx-positive cells following LPT(RNAi)
marks the accumulation of serotonergic neuronal precursors that
fail to differentiate. We conclude that planarian LPT normally
regulates pitx-mediated differentiation of serotonergic neurons
and that regulation of pitx is a possible example of a conserved
feature of MLL3/4 function that is mis-regulated in some cancer
types45.
Double RNAi validates overexpressed target genes. In the pla-
narian model, up-regulated genes in our data set provided the
opportunity to identify genes whose overexpression contributes
to the LPT/Mll3/4 loss of function phenotype using double
RNAi experiments. In addition to the transcription factor E74-like
factor 5 (Elf5), we observed up-regulation of planarian orthologs of
other developmental or cancer associated genes. Among them
were orthologs of the serine/threonine kinase oncogene pim-2
(two genes called Smed-pim-2 and Smed-pim-2-like), a paralog of
the epigenetic regulator Suppressor of zeste (Su(z)12), the tran-
scription factors ETS4 and FoxA1 and an ULK2-like serine/
threonine protein kinase (Fig. 9a, Supplementary Figure 7a). Our
ChIP-seq data suggested pim-2, ETS4 and ULK2-like are either
indirectly regulated or regulated by LPT at enhancers, while pim-
2-like, FoxA1, Su(z)12 paralog and Elf5 appear to be direct targets
of LPT as their increased expression was associated with either
reduced H3K4me1 or increased H3K4me3 signal at promoters
(Fig. 9a, Supplementary Figure 7a).
We decided to focus on these genes because they all have a
known role in a wide range of cancers. Overexpression of the cell
fate decision determinant Elf5 is a known driving force behind
breast cancer progression and metastasis50. The PIM family of
proteins is involved in the integration of growth and survival
signals51 and their overexpression has been associated with
hematological malignancies and solid tumors52. Inhibition of
PIM2 is a promising avenue for the treatment of multiple
myeloma51. Su(z)12 is part of the ubiquitous polycomb repressive
complex 2 (PRC2) and is overexpressed in numerous cancers,
including gastric cancer where Su(z)12 levels were associated with
increased metastasis and unfavorable survival prognosis53. The
ETS family of transcription factors has demonstrated significant
involvement in all stages of tumorigenesis54, while FoxA1 is
known as a “pioneer transcription factor in organogenesis and
cancer progression”55. Finally, ULK2 is an autophagy regulator
overexpressed in prostate cancer cells56. This selected panel of
genes represents some of the best candidates for major effects
amongst those genes with significant up-regulation in expression
following LPT(RNAi).
In order to test whether the up-regulated expression of any of
these genes is a potentially significant contributor to the LPT
(RNAi) outgrowth phenotype, we performed LPT(RNAi) rescue
experiments in the form of double RNAi knockdowns (Fig. 9d–f,
Supplementary Figure 7). At 48 h post-amputation, LPT(RNAi)
regenerates have a significantly increased NB proliferation
(Fig. 4a, b) and so do GFP/LPT(RNAi) double knockdown
animals (Fig. 9d, e). Whereas pim-2/LPT(RNAi), ETS4/LPT
(RNAi), FoxA1/LPT(RNAi), ULK2-like/LPT(RNAi) and Su(z)12
paralogue/LPT(RNAi) regenerates still have elevated NB prolif-
eration, both pim-2-like/LPT(RNAi) and Elf5/LPT(RNAi) regen-
erates have a significantly decreased NB proliferation compared
to GFP/LPT(RNAi) (Fig. 9d, e and Supplementary Figure 7b).
Furthermore, not only did pim-2-like/LPT(RNAi) and Elf5/LPT
(RNAi) regenerating animals show improved blastema formation
(Supplementary Figure 7c), but also less than half as many
animals in these two conditions went on to form outgrowths
compared to GFP/LPT(RNAi) (Fig. 9f), demonstrating a rescue of
the outgrowth phenotype. Importantly, individual knockdown of
Elf5 and pim-2-like did not lead to regenerative, proliferation or
outgrowth-related defects (Supplementary Figure 8). These
findings suggest that the up-regulation of both pim-2-like and
Elf5 is specifically involved in driving the LPT(RNAi) outgrowth
phenotype and demonstrate the utility of our data for validating
the role of MLL3/4 targets.
Defects caused by transcription factor down-regulation.We
chose a selection of genes down regulated by LPT(RNAi) for
further investigation to see if they might contribute to the
observed LPT(RNAi) phenotype (Supplementary Figure 9a-d,
Fig. 10a–d). LPT(RNAi) leads to the down-regulation of the
tumor suppressor p53,PRDM1-1 and cut-like1 in X1 stem cells
(Fig. 10a). These genes’down-regulation is associated with a
NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06092-6 ARTICLE
NATURE COMMUNICATIONS | (2018) 9:3633 | DOI: 10.1038/s41467-018-06092-6 | www.nature.com/naturecommunications 13
Content courtesy of Springer Nature, terms of use apply. Rights reserved

decrease in H3K4me3 levels on their promoters. On the other
hand, RREBP1’s down-regulation, does not correlate with the
amount of H3K4me3 on its promoter. While the function of p53
in planarians has already been described57, the role of PRDM1-1,
cut-like1 and RREBP1 remain unexplored in planarian biology.
Knockdown of each of these three genes resulted in impaired
regeneration, characterized mainly by the inability to form eyes
(Fig. 10b). RREBP1(RNAi) resulted in significantly increased
proliferation compared to control, while cut-like1(RNAi) and
PRDM1-1(RNAi) animals did not show a significant change in
mitotic cell numbers (Fig. 10c). All three knockdown conditions
showed a decreased number of prog-1-positive epidermal pro-
genitors, but intact smedwi-1-positive cell numbers (Fig. 10d).
These findings suggest that the observed differentiation pheno-
types are not associated with an inability to maintain the stem cell
pool following knockdown. Instead, stem cells seem to be
restricted in their ability to differentiate correctly.
Our data suggest that decreased expression of PRDM1-1,cut-
like1 and RREBP1 in LPT(RNAi) animals could be contributing
to the differentiation defects seen after perturbation of LPT/Trr/
MLL3/4 function, and that downregulation RREBP1 may be
contributing to the early over proliferation of stem cells observed
in LPT(RNAi).
Discussion
In mammals, Mll3 and Mll4 have been implicated in different
malignancy landscapes19, with clear evidence for tumor sup-
pressor roles in mammalian systems20,24. However, relatively
little is known about how these effects are mediated. Our study
demonstrates that loss of function of the planarian LPT (an Mll3/
4ortholog) also results in the emergence of an outgrowth phe-
notype characterized by differentiation and proliferation defects.
Our work also shows that LPT, TRR-1, and TRR-2 control dif-
ferentiation to form the gut, eyes, brain, and pharyngeal cellular
lineages, Future work in planarians combining ChIP-seq and
RNA-seq will allow closer investigation of these and other epi-
genetic effects on stem cell differentiation.
We found that clusters of mitotic cells preceded the appearance
of outgrowths in LPT(RNAi) regenerating animals, possibly pre-
empting where the outgrowths would subsequently form. The
observation of clusters of cells and the formation of outgrowths in
some, but not all, RNAi animals is evidence of heterogeneity in
stem cell responses to LPT(RNAi). This may reflect the stochastic
nature of the broad genome-wide epigenetic changes mediated by
MLL3/4 proteins that will lead to variability between cells after
knockdown, such that only some NBs cycle out of control and
cause outgrowths after the initial proliferative peaks associated
with regeneration. A contributory cause to outgrowth formation
in addition to proliferation could be failure of NBs to differentiate
appropriately and instead continue to cycle at inappropriate
positions. We also observed that outgrowth tissue contained
different classes of stem cells. Among these stem cells, the pre-
sence of sigma NBs, thought to include truly pluripotent stem
cells41, is of particular significance. When mis-regulated, these
cells could share fundamental similarities with cancer stem cells
(CSCs), thought to be founder cells in human malignancies58.
CSCs have been described as one of the main factors in cancer
aggressiveness and resistance to treatment59. Studying such cells
in a simple in vivo stem cell model, provided by the planarian
system, should bring further insight into important control
mechanisms that are mis-regulated in different cancers. Our work
here provides a useful example of this approach.
Our data suggest that LPT regulates expression of genes across
cell types, including some genes with enriched expression in stem
cells. Genes with significant expression differences following LPT
(RNAi) were mostly associated with cell proliferation, differ-
entiation, and metabolic processes. A subset of mis-regulated
genes where RNA-seq and ChIP-seq data correlate is likely a
direct consequence of LPT(RNAi) affecting promoter histone
methylation status. Genes with altered expression where there is
no such correlation, may represent indirect (secondary) changes
or, alternatively, may have enhancers that have altered histone
modifications as a result of LPT(RNAi). Future work will develop
the use of planarians as a model of epigenetic gene regulation and
it should also be possible to study enhancer function and
evolution.
Among mis-regulated genes, we saw many tumor suppressors
with reduced expression and oncogenes with increased expres-
sion. We also found a number of genes, including the tran-
scription factor pitx, which were similarly mis-regulated in LPT
(RNAi) planarians and human cancers with reduced Mll3
expression. Together, these data suggest that, as well as physio-
logical function in controlling stem cell proliferation, there may
be deep regulatory conservation of MLL3/4 function in animal
stem cells. One advantage of our approach is that we were able to
sample expression and histone states in NBs at an early time
point before tumors formed. This could be an advantage for the
identifying targets that act early to drive hyperplasia, rather than
later secondary regulatory changes.
As proof of principle that genes mis-regulated by LPT(RNAi)
directly contributed to the phenotype, we performed double
RNAi experiments. Planarian homologs of the oncogene pim-2,
called Smed-pim-2 and Smed-pim-2-like, that were overexpressed
in stem cells following LPT(RNAi), were chosen as likely candi-
dates, based on previous data on the roles of these genes from
mammals60. We found that double RNAi with pim-2-like, was
able to ameliorate LPT loss of function over-proliferation and
outgrowth phenotypes induced by LPT(RNAi). In addition,
double knockdown with the breast cancer oncogene Elf550
resulted in an even more dramatic rescue of the LPT loss of
function phenotype. This provides strong support for the
hypothesis that the over-expression of these two genes was sig-
nificant in driving stem cell hyperplasia. Future work can now
study how these genes function in stem cells and why over-
expression leads to over proliferation.
We also identified downstream candidates that could be con-
tributing to the lack of differentiation phenotype following LPT
(RNAi). Knockdown of PRDM1-1,cut-like and RREBP-1 (down-
regulated in LPT(RNAi) animals) indicated their mis-regulation
might contribute to the decreased epidermal differentiation
observed following LPT knockdown.
Together these experiments, demonstrate the value of our
approach for identifying potential downstream targets and
implicate regulatory interactions driving the Mll3/4 loss of
function phenotype. These targets can now be tested for con-
servation in mammalian experimental systems.
Overall, our work shows how perturbation of a conserved
physiological role of LPT leads to mis-regulation of genes well-
known to control cell proliferation, causing hyperplasia and
tumors in planarians. We find other genes that are mis-
regulated in planarians that are also similarly mis-regulated in
cancer expression studies that have reduced Mll3 expression.
Some of these, like pitx, may represent deeply conserved reg-
ulatory interactions. In the absence of similar RNA-seq/ChIP-
seq data in mammals, our data provide an important insight
into Mll3/4 loss of function, as well as revealing a deep evolu-
tionary conservation in animal stem cells. These findings
demonstrate the strength of the planarian system for under-
standing fundamental animal stem cell biology highly relevant
to cancer and the potential for investigation of epigenetic
mechanisms in stem cells.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06092-6
14 NATURE COMMUNICATIONS | (2018) 9:3633 | DOI: 10.1038/s41467-018-06092-6 | www.nature.com/naturecommunications
Content courtesy of Springer Nature, terms of use apply. Rights reserved

Methods
Animal husbandry. Asexual freshwater planarians of the species S. mediterranea
were used. The culture was maintained in 1x Montjuic salts water61. Planarians
were fed organic calf liver once a week. After every feeding, the water was changed.
Planarians were starved for 7 days prior to each experiment. They were also starved
throughout the duration of each experiment.
RNAi. Double-stranded RNA (dsRNA) was synthesized from DNA fragments
cloned in pCRII (Invitrogen) or pGEM-T Easy (Promega) vectors. T7 (Roche) and
SP6 (NEB) RNA polymerases were used for transcription of each strand. The two
transcription reactions were combined upon ethanol precipitation. RNA was
denatured at 68 °C and re-annealed at 37 °C. Quantification was performed on a
1% agarose gel and Nanodrop spectrophotometer.
For single RNAi experiment s a working concentration of 2 μg/μl was used. For
double RNAi, each gene’s RNA was at a concentration 4 μg/μl, resulting in solution
concentration of 2 μg/μl.
DsRNA was delivered via microinjection using Nanoject II apparatus
(Drummond Scientific) with 3.5″Drummond Scientific (Harvard Apparatus) glass
capillaries pulled into fine needles on a Flaming/Brown Micropipette Puller
(Patterson Scientific). Each animal received around 100 nl dsRNA each day. H2B
(RNAi) was performed for three consecutive days, as per Solana et al.7protocol.
For single and double LPT,trr-1 and trr-2 knockdown, a course of 7 days of
microinjections was performed (3 consecutive days +2 days rest +4 consecutive
days). Set1(RNAi) and utx(RNAi) were performed for 4 consecutive days. For all
other single and double knockdowns, a course of 10 days of microinjections was
performed (3 consecutive days +4 days rest +3 consecutive days).
Primers used for amplification of DNA for dsRNA synthesis can be found in
Supplementary Table 1.
In situ hybridization. RNA probes labeled with digoxigenin and fluorescein were
generated via anti-sense transcription of DNA cloned in PCRII (Invitrogen) or
PGemTEasy (Promega) vector. In situ hybridization was performed after fixation
in 4% paraformaldehyde62 for fluorescent experiments. For LPT,trr-1,trr-2,sigma,
zeta, and gamma fluorescent in situ procedures, a pooled probes method was used
using PCR products from across transcriptional units as templates for RNA
probes41. Colorimetric in situ hybridization procedures were performed using
alkaline phosphatase based detection of hybridized RNA probes63. Primers used for
amplification of DNA for RNA probe synthesis can be found in (Supplementary
Table 1).
Immunohistochemistry. Immunohistochemistry was performed after either par-
aformaldehyde of Carnoy fixative based fixation64. Antibodies used were: anti-H3P
(phosphorylated serine 10 on histone H3; Millipore; 09–797; 1:1000 dilution), anti-
VC1 (kindly provided by Prof. Hidefumi Orii (check title); 1:10,000 dilution), anti-
SMEDWI-1 (kindly provided by Prof. Jochen Rink; 1:500 dilution), anti-SYNORF-
1 (3C11; Developmental Studies Hybridoma Bank; 1:50 dilution), anti-acetylated
tubulin (Developmental Studies Hybridoma Bank; 1:200 dilution).
Imaging and image analysis. Colorimetric images were taken on Zeiss Discovery
V8 (Carl Zeiss) microscope with a Canon EOS 600D or Canon EOS 1200D camera.
Fluorescent images were taken on either Inverted Olympus FV1000 or FV1200
Confocal microscope. Cells were counted via Adobe Photoshop CS6 or FIJI soft-
ware and the count was normalized to image area in mm2.
Flow cytometry. A simple FACS protocol8,65 was used on disassociated planarians
cells using hoechst, calcein and propidium iodide to sort living cells in G2/M of the
cell cycle. A FACS Aria III machine equipped with a violet laser was used for the
sort. BD FACSDiva and FlowJo software was used for analysis and gate-setting.
Western blot. 2× Laemmli buffer (Sigma Aldrich), 1M DTT and complete pro-
tease inhibitors (Roche) were used for protein extraction from 10 to 15 animals per
condition. Protein extract was quantified with Qubit Protein Assay kit (Thermo
Fisher Scientific). NuPAGE Novex 4–12% Bis-Tris protein gels (Thermo Fisher
Scienitific) were used, followed by a wet transfer in a Mini Trans-Blot Electro-
phoretic Transfer Cell machine. Ponceau S (Sigma Aldrich) whole-protein stain
was used prior to antibody incubation. The antibodies used were: anti-H3
(unmodified histone H3; rabbit polyclonal; Abcam; ab1791; 1:10,000 dilution),
anti-H3K4me3 (rabbit polyclonal; Abcam; ab8580; 1:1000 dilution), anti-H3K4me1
(rabbit polyclonal; Abcam; ab8895; 1:1000 dilution), anti-H3K27me3 (mouse
monoclonal; Abcam; ab6002; 1:1000 dilution), anti-mouse IgG HRP-linked anti-
body (Cell Signalling; 7076P2), anti-rabbit IgG HRP-linked antibody (Cell Sig-
nalling; 7074P2). Western blot experiments were done to validate the specificity of
the histone modification antibodies used for ChIP-seq (Supplementary Figure 11).
The rationale behind these experiments was to knock down a methylase (set1), part
of a methylase complex (LPT) or a demethylase (utx) known to affect H3K4 and
H3K27 methylation levels and to observe whether global H3K4me1, H3K4me3,
and H3K27me3 levels would change in the expected way.
ChIP-seq. In total 600,000–700,000 planarian G2/M cells were FACS-sorted (using
3-day knockdown regenerates) in PBS and pelleted at 4 °C. During the pelleting, S2
cells were added (corresponding to roughly 15% of the number of planarian
×1 cells) for the purpose of downstream data normalization44. Samples were then
used to prepare Illumina sequencing libraries9. The process is summarized in
Supplementary Figure 12. The libraries were sequenced on an Illumina NextSeq
machine. Three biological replicates were prepared. The raw reads are available in
the NCBI Short Read Archive (PRJNA338116).
RNA-seq. In total 300,000 G2/M cells were FACS-sorted in RNALater (Ambion)
from knockdown and control RNAi animals at 3 days of regeneration. Cells were
pelleted at 4 °C and Trizol-based total RNA extraction was performed. The amount
of total RNA used for each library preparation was 0.8–1μg. Illumina TruSeq
Stranded mRNA LT kit was used for library preparation according to the manu-
facturers instructions. Libraries were quantified with Qubit, Agilent Bioanalyzer
and KAPA Library Quantification qPCR kit. Samples were sequenced on an Illu-
mina NextSeq machine. Two biological replicates were prepared. The raw reads are
available in the Short Read Archive (PRJNA338115).
ChIP-seq data analysis. ChIP-seq reads were trimmed with Trimmomatic 0.3266
and aligned to the S. mediterranea SmedGD asexual genome 1.160 and D. mela-
nogaster genome r6.1067 with BWA mem 0.7.12. Picard tools 1.115 was used to
remove read duplicates after mapping. Python scripts were used to filter and
separate out read pairs belonging to either genome. ChIP-seq coverage tracks were
then generated and normalized according to Orlando et al. in order to account for
any technical variation between samples44. For more in-depth methods, including
code, refer to Supplementary Software.
RNA-seq data analysis. Raw reads were trimmed with Trimmomatic 0.3266 and
pseudo-aligned to a set of asexual genome annotations8with Kallisto 0.4268. Dif-
ferential expression was subsequently performed with Sleuth 0.28.169. For more in-
depth methods, including code, refer to Supplementary Software.
Statistical methods. Wherever cell number was compared between experimental
condition and control, a two-tailed t-test assuming unequal variance was used.
Each legend states the number of specimens per condition, where relevant. Bar
graphs show the mean average and the error bars are always Standard Error of the
Mean.
For analysis of RNA-seq data, Wald’s test (as part of the Sleuth69 software) was
used for assessing differential expression. Spearman’s rank correlation was used for
assessing the correlation between RNA-seq and ChIP-seq data. Hypergeometric
tests were used for assessing gene enrichment in the RNA-seq data.
Code Availability. Code used in the analysis of data are presented in Supple-
mentary Software.
Data availability
The ChIP-seq and RNA-seq datasets are deposited in the Short Read Archive database
under the accession codes: PRJNA338116 and PRJNA338115, respectively. The ‘Pomeroy
Brain’dataset45 from the oncomine database (https://www.oncomine.com) was used for
assessing expression level of potential target genes and Mll3 in human medulloblastoma
versus normal cerebellum. All other data are made available within the article and its
supplementary information.
Received: 10 May 2017 Accepted: 13 August 2018
References
1. Aboobaker, A. A. Planarian stem cells: a simple paradigm for regeneration.
Trends Cell Biol. 21, 304–311 (2011).
2. Rink, J. C. Stem cell systems and regeneration in planaria. Dev. Genes. Evol.
223,67–84 (2012).
3. Onal, P. et al. Gene expression of pluripotency determinants is conserved
between mammalian and planarian stem cells. EMBO J. 31, 2755–2769 (2012).
4. Adamidi, C. et al. De novo assembly and validation of planaria transcriptome
by massive parallel sequencing and shotgun proteomics. Genome Res. 21,
1193–1200 (2011).
5. Labbé, R. M. et al. A comparative transcriptomic analysis reveals conserved
features of stem cell pluripotency in planarians and mammals. Stem Cells 30,
1734–1745 (2012).
6. Solana, J. et al. Conserved functional antagonism of CELF and MBNL proteins
controls stem cell-specific alternative splicing in planarians. eLife 5, 1193
(2016).
NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06092-6 ARTICLE
NATURE COMMUNICATIONS | (2018) 9:3633 | DOI: 10.1038/s41467-018-06092-6 | www.nature.com/naturecommunications 15
Content courtesy of Springer Nature, terms of use apply. Rights reserved

7. Solana, J. et al. Defining the molecular profile of planarian pluripotent stem
cells using a combinatorial RNA-seq, RNA interference and irradiation
approach. Genome Biol. 13, R19 (2012).
8. Dattani, A. et al. Epigenetic analyses of planarian stem cells demonstrate
conservation of bivalent histone modifications in animal stem cells. Preprint at
https://www.biorxiv.org/content/early/2018/08/09/122135 (2018).
9. Shibata, N. et al. Inheritance of a nuclear PIWI from pluripotent stem cells by
somatic descendants ensures differentiation by silencing transposons in
planarian. Dev. Cell. 37, 226–237 (2016).
10. Salvetti, A. DjPum, a homologue of Drosophila Pumilio, is essential to
planarian stem cell maintenance. Development 132, 1863–1874 (2005).
11. Reddien, P. W. Specialized progenitors and regeneration. Development 140,
951–957 (2013).
12. Jaber-Hijazi, F. et al. Planarian MBD2/3 is required for adult stem cell
pluripotency independently of DNA methylation. Dev. Biol. 384, 141–153 (2013).
13. Scimone, M. L., Meisel, J. & Reddien, P. W. The Mi-2-like Smed-CHD4 gene
is required for stem cell differentiation in the planarian Schmidtea
mediterranea. Development 137, 1231–1241 (2010).
14. Zhu, S. J., Hallows, S. E., Currie, K. W., Xu, C. & Pearson, B. J. A mex3
homolog is required for differentiation during planarian stem cell lineage
development. eLife 4, 304 (2015).
15. Cowles, M. W., Omuro, K. C., Stanley, B. N., Quintanilla, C. G. & Zayas, R. M.
COE loss-of-function analysis reveals a genetic program underlying
maintenance and regeneration of the nervous system in planarians. PLoS
Genet. 10, e1004746–12 (2014).
16. Barberan, S., Fraguas, S. & Cebrià, F. The EGFR signaling pathway controls
gut progenitor differentiation during planarian regeneration and homeostasis.
Development 143, 2089–2102 (2016).
17. Abnave, P. et al. Epithelial-mesenchymal transition transcription factors
control pluripotent adult stem cell migration in vivo in planarians.
Development 144, 3440–3453 (2017).
18. Parsons, D. W. et al. The genetic landscape of the childhood cancer
medulloblastoma. Science 331, 435–439 (2011).
19. Kandoth, C. et al. Mutational landscape and significance across 12 major
cancer types. Nature 502, 333–339 (2013).
20. Chen, C. et al. MLL3 is a haploinsufficient 7q tumor suppressor in acute
myeloid leukemia. Cancer Cell. 25, 652–665 (2014).
21. Shilatifard, A. The COMPASS family of histone H3K4 methylases:
mechanisms of regulation in development and disease pathogenesis. Annu.
Rev. Biochem. 81,65–95 (2012).
22. Herz, H. M. et al. Enhancer-associated H3K4 monomethylation by Trithorax-
related, the Drosophila homolog of mammalian Mll3/Mll4. Genes Dev. 26,
2604–2620 (2012).
23. Hu, D. et al. The MLL3/MLL4 branches of the COMPASS family function as
major histone H3K4 monomethylases at enhancers. Mol. Cell. Biol. 33,
4745–4754 (2013).
24. Lee, J. et al. A tumor suppressive coactivator complex of p53 containing ASC-2
and histone H3-lysine-4 methyltransferase MLL3 or its paralogue MLL4. Proc.
Natl Acad. Sci. USA 106, 8513–8518 (2009).
25. Cheng, J. et al. A role for H3K4 monomethylation in gene repression and
partitioning of chromatin readers. Mol. Cell 53, 979–992 (2014).
26. Chauhan, C., Zraly, C. B., Parilla, M., Diaz, M. O. & Dingwall, A. K. Histone
recognition and nuclear receptor co-activator functions of Drosophila Cara
Mitad, a homolog of the N-terminal portion of mammalian MLL2 and MLL3.
Development 139, 1997–2008 (2012).
27. Hubert, A. et al. Epigenetic regulation of planarian stem cells by the SET1/
MLL family of histone methyltransferases. Epigenetics 8,79–91 (2013).
28. Duncan, E. M., Chitsazan, A. D., Seidel, C. W. & Alvarado, A. S. Set1 and
MLL1/2 target distinct sets of functionally different genomic loci in vivo.
CellReports 13, 2741–2755 (2015).
29. Lee, J.-E. et al. H3K4 mono- and di-methyltransferase MLL4 is required for
enhancer activation during cell differentiation. eLife 2, 2817–2825 (2013).
30. Wang, C. et al. Enhancer priming by H3K4 methyltransferase MLL4 controls
cell fate transition. Proc. Natl Acad. Sci. USA 113, 11871–11876 (2016).
31. Denissov, S. et al. Mll2 is required for H3K4 trimethylation on bivalent
promoters in embryonic stem cells, whereas Mll1 is redundant. Development
141, 526–537 (2014).
32. Hsieh, J. J.-D., Ernst, P., Erdjument-Bromage, H., Tempst, P. & Korsmeyer, S. J.
Proteolytic cleavage of MLL generates a complex of N- and C-terminal
fragments that confers protein stability and subnuclear localization. Mol. Cell.
Biol. 23, 186–194 (2003).
33. Thirman, M. J. et al. Rearrangement of the MLL gene in acute lymphoblastic
and acute myeloid leukemias with 11q23 chromosomal translocations. N.
Engl. J. Med. 329, 909–914 (1993).
34. Sobulo, O. M. et al. MLL is fused to CBP, a histone acetyltransferase, in
therapy-related acute myeloid leukemia with a t(11;16)(q23; p13.3). Proc. Natl
Acad. Sci. USA 94, 8732–8737 (1997).
35. Lee, S. et al. Coactivator as a target gene specificity determinant for histone H3
lysine 4 methyltransferases. Proc. Natl Acad. Sci. USA 103, 15392–15397
(2006).
36. Bienz, M. The PHD finger, a nuclear protein-interaction domain. Trends
Biochem. Sci. 31,35–40 (2006).
37. Lee, S., Lee, J., Lee, S.-K. & Lee, J. W. Activating Signal Cointegrator-2 is an
essential adaptor to recruit histone H3 lysine 4 methyltransferases MLL3 and
MLL4 to the liver X receptors. Mol. Endocrinol. 22, 1312–1319 (2008).
38. Wenemoser, D. & Reddien, P. W. Planarian regeneration involves distinct
stem cell responses to wounds and tissue absence. Dev. Biol. 344, 979–991
(2010).
39. Oviedo, N. J., Pearson, B. J., Levin, M. & Sanchez Alvarado, A. Planarian
PTEN homologs regulate stem cells and regeneration through TOR signaling.
Dis. Models Mech. 1, 131–143 (2008).
40. Gonzalez-Estevez, C. et al. SMG-1 and mTORC1 act antagonistically to
regulate response to injury and growth in planarians. PLoS. Genet. 8,
e1002619–17 (2012).
41. van Wolfswinkel, J. C., Wagner, D. E. & Reddien, P. W. Single-cell analysis
reveals functionally distinct classes within the planarian stem cell
compartment. Stem Cell 15, 326–339 (2014).
42. Zink, D., Fische, A. H. & Nickerson, J. A. Nuclear structure in cancer cells.
Nat. Rev. Cancer 4, 677–687 (2004).
43. Lee, M. G. et al. Demethylation of H3K27 regulates polycomb recruitment and
H2A ubiquitination. Science 318, 447–450 (2007).
44. Orlando, D. A. et al. Quantitative ChIP-Seq normalization reveals global
modulation of the epigenome. CellReports 9, 1163–1170 (2014).
45. Pomeroy, S. L. et al. Prediction of central nervous system embryonal tumour
outcome based on gene expression. Nature 415, 436–442 (2002).
46. Brune, V. et al. Origin and pathogenesis of nodular lymphocyte-predominant
Hodgkin lymphoma as revealed by global gene expression analysis. J. Exp.
Med. 205, 2251–2268 (2008).
47. Compagno, M. et al. Mutations of multiple genes cause deregulation of NF-
kappaB in diffuse large B-cell lymphoma. Nature 459, 717–721 (2009).
48. Currie, K. W. & Pearson, B. J. Transcription factors lhx1/5-1 and pitx are
required for the maintenance and regeneration of serotonergic neurons in
planarians. Development 140, 3577–3588 (2013).
49. März, M., Seebeck, F. & Bartscherer, K. A Pitx transcription factor controls the
establishment and maintenance of the serotonergic lineage in planarians.
Development 140, 4499–4509 (2013).
50. Gallego-Ortega, D. et al. ELF5 drives lung metastasis in luminal breast cancer
through recruitment of Gr1+CD11b+myeloid-derived suppressor cells.
PLoS Biol. 13, e1002330 (2015).
51. Nair, J. R. et al. Novel inhibition of PIM2 kinase has significant anti-tumor
efficacy in multiple myeloma. Leukemia 31, 1715–1726 (2017).
52. Jiménez-García, M. P. et al. The role of PIM1/PIM2 kinases in tumors of the
male reproductive system. Sci. Rep. 6, 38079 (2016).
53. Xia, R. et al. SUZ12 promotes gastric cancer cell proliferation and metastasis
by regulating KLF2 and E-cadherin. Tumour Biol. 36, 5341–5351 (2015).
54. Sizemore, G. M., Pitarresi, J. R., Balakrishnan, S. & Ostrowski, M. C. The ETS
family of oncogenic transcription factors in solid tumours. Nat. Rev. Cancer
17, 337–351 (2017).
55. Zhang, G. et al. FOXA1 defines cancer cell specificity. Sci. Adv. 2,
e1501473–e1501473 (2016).
56. John Clotaire, D. Z. et al. MiR-26b inhibits autophagy by targeting ULK2 in
prostate cancer cells. Biochem. Biophys. Res. Commun. 472, 194–200 (2016).
57. Pearson, B. J. & Alvarado, A. S. A planarian p53 homolog regulates
proliferation and self-renewal in adult stem cell lineages. Development 137,
213–221 (2009).
58. Reya, T., Morrison, S. J., Clarke, M. F. & Weissman, I. L. Stem cells, cancer,
and cancer stem cells. Nature 414, 105–111 (2001).
59. Dean, M., Fojo, T. & Bates, S. Tumour stem cells and drug resistance. Nat.
Rev. Cancer 5, 275–284 (2005).
60. Robb, S. M. C., Gotting, K., Ross, E. & Sánchez Alvarado, A. SmedGD 2.0: the
Schmidtea mediterranea genome database. Genesis 53, 535–546 (2015).
61. Cebria, F. Planarian homologs of netrin and netrin receptor are required for
proper regeneration of the central nervous system and the maintenance of
nervous system architecture. Development 132, 3691–3703 (2005).
62. King, R. S. & Newmark, P. A. In situ hybridization protocol for enhanced
detection of gene expression in the planarian Schmidtea mediterranea. BMC
Dev. Biol. 13, 8 (2013).
63. Gonzalez-Estevez, C., Arseni, V., Thambyrajah, R. S., Felix, D. A. &
Aboobaker, A. A. Diverse miRNA spatial expression patterns suggest
important roles in homeostasis and regeneration in planarians. Int. J. Dev.
Biol. 53, 493–505 (2009).
64. Cebria, F. & Newmark, P. A. Morphogenesis defects are associated with
abnormal nervous system regeneration following roboA RNAi in planarians.
Development 134, 833–837 (2007).
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06092-6
16 NATURE COMMUNICATIONS | (2018) 9:3633 | DOI: 10.1038/s41467-018-06092-6 | www.nature.com/naturecommunications
Content courtesy of Springer Nature, terms of use apply. Rights reserved

65. Romero, B. T., Evans, D. J. & Aboobaker, A. A. Progenitor Cells Vol. 916,
167–179 (Humana Press, 2012).
66. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for
Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).
67. Attrill, H. et al. FlyBase: establishing a gene group resource for Drosophila
melanogaster.Nucleic Acids Res. 44, D786–D792 (2016).
68. Bray, N. L., Pimentel, H., Melsted, P. & Pachter, L. Near-optimal probabilistic
RNA-seq quantification. Nat. Biotechnol. 34, 525–527 (2016).
69. Pimentel, H. J., Bray, N., Puente, S., Melsted, P. & Pachter, L. Differential
analysis of RNA-Seq incorporating quantification uncertainty. Nat. Methods
14, 678–690 (2017).
Acknowledgements
We thank past and present members of the AAA lab for comments on the manuscript.
This work was funded by grants from the Medical Research Council (grant number MR/
M000133/1) and the Biotechnology and Biological Sciences Research Council (grant
number BB/K007564/1) to A.A.A.
Authors contributions
A.A.A., P.A., and Y.M. conceived and designed the study. Y.M. and P.A. performed the
experiments. D.K. performed the bioinformatics analyses. S.H. participated in the opti-
mization of the ChIP-seq protocol. A.G.L. provided technical support. F.J.H. performed
initial work on the project, including generating the first LPT(RNAi) results. N.K. pro-
vided technical support. Y.M., P.A., and A.A.A. wrote the manuscript.
Additional information
Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467-
018-06092-6.
Competing interests: The authors declare no competing interests.
Reprints and permission information is available online at http://npg.nature.com/
reprintsandpermissions/
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative
Commons license, and indicate if changes were made. The images or other third party
material in this article are included in the article’s Creative Commons license, unless
indicated otherwise in a credit line to the material. If material is not included in the
article’s Creative Commons license and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from
the copyright holder. To view a copy of this license, visit http://creativecommons.org/
licenses/by/4.0/.
© The Author(s) 2018
NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06092-6 ARTICLE
NATURE COMMUNICATIONS | (2018) 9:3633 | DOI: 10.1038/s41467-018-06092-6 | www.nature.com/naturecommunications 17
Content courtesy of Springer Nature, terms of use apply. Rights reserved

1.
2.
3.
4.
5.
6.
Terms and Conditions
Springer Nature journal content, brought to you courtesy of Springer Nature Customer Service Center GmbH (“Springer Nature”).
Springer Nature supports a reasonable amount of sharing of research papers by authors, subscribers and authorised users (“Users”), for small-
scale personal, non-commercial use provided that all copyright, trade and service marks and other proprietary notices are maintained. By
accessing, sharing, receiving or otherwise using the Springer Nature journal content you agree to these terms of use (“Terms”). For these
purposes, Springer Nature considers academic use (by researchers and students) to be non-commercial.
These Terms are supplementary and will apply in addition to any applicable website terms and conditions, a relevant site licence or a personal
subscription. These Terms will prevail over any conflict or ambiguity with regards to the relevant terms, a site licence or a personal subscription
(to the extent of the conflict or ambiguity only). For Creative Commons-licensed articles, the terms of the Creative Commons license used will
apply.
We collect and use personal data to provide access to the Springer Nature journal content. We may also use these personal data internally within
ResearchGate and Springer Nature and as agreed share it, in an anonymised way, for purposes of tracking, analysis and reporting. We will not
otherwise disclose your personal data outside the ResearchGate or the Springer Nature group of companies unless we have your permission as
detailed in the Privacy Policy.
While Users may use the Springer Nature journal content for small scale, personal non-commercial use, it is important to note that Users may
not:
use such content for the purpose of providing other users with access on a regular or large scale basis or as a means to circumvent access
control;
use such content where to do so would be considered a criminal or statutory offence in any jurisdiction, or gives rise to civil liability, or is
otherwise unlawful;
falsely or misleadingly imply or suggest endorsement, approval , sponsorship, or association unless explicitly agreed to by Springer Nature in
writing;
use bots or other automated methods to access the content or redirect messages
override any security feature or exclusionary protocol; or
share the content in order to create substitute for Springer Nature products or services or a systematic database of Springer Nature journal
content.
In line with the restriction against commercial use, Springer Nature does not permit the creation of a product or service that creates revenue,
royalties, rent or income from our content or its inclusion as part of a paid for service or for other commercial gain. Springer Nature journal
content cannot be used for inter-library loans and librarians may not upload Springer Nature journal content on a large scale into their, or any
other, institutional repository.
These terms of use are reviewed regularly and may be amended at any time. Springer Nature is not obligated to publish any information or
content on this website and may remove it or features or functionality at our sole discretion, at any time with or without notice. Springer Nature
may revoke this licence to you at any time and remove access to any copies of the Springer Nature journal content which have been saved.
To the fullest extent permitted by law, Springer Nature makes no warranties, representations or guarantees to Users, either express or implied
with respect to the Springer nature journal content and all parties disclaim and waive any implied warranties or warranties imposed by law,
including merchantability or fitness for any particular purpose.
Please note that these rights do not automatically extend to content, data or other material published by Springer Nature that may be licensed
from third parties.
If you would like to use or distribute our Springer Nature journal content to a wider audience or on a regular basis or in any other manner not
expressly permitted by these Terms, please contact Springer Nature at
onlineservice@springernature.com