Current Biology 23, 223–228, February 4, 2013 ª2013 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2012.11.061
Mask Proteins Are Cofactors
of Yorkie/YAP in the Hippo Pathway
Clara M. Sidor,1Ruth Brain,1and Barry J. Thompson1,*
1Cancer Research UK, London Research Institute,
44 Lincoln’s Inn Fields, London WC2A 3LY, UK
The Hippo signaling pathway acts via the Yorkie (Yki)/Yes-
associated protein (YAP) transcriptional coactivator family
to control tissue growth in both Drosophila and mammals
[1–3]. Yki/YAP drives tissue growth by activating target
gene transcription, but how it does so remains unclear.
Here we identify Mask as a novel cofactor for Yki/YAP. We
show that Drosophila Mask forms a complex with Yki and its
binding partner, Scalloped (Sd), on target-gene promoters
and is essential for Yki to drive transcription of target genes
and tissuegrowth. Furthermore,the stabilityand subcellular
localization of both Mask and Yki is coregulated in response
to various stimuli. Finally, Mask proteins are functionally
conserved between Drosophila and humans and are
coexpressed with YAP in a wide variety of human stem/
progenitor cells and tumors.
Results and Discussion
pathway in Drosophila [4–11] and mice [12–14]. The key
effector of the Hippo pathway is the Drosophila Yorkie (Yki)/
mammalian Yes-associated protein (YAP) (reviewed in [1–3]).
Yki/YAP is a transcriptional coactivator whose nuclear locali-
zation and activity is inhibited upon phosphorylation by the
Wts kinase [15–17]. Yki/YAP is an oncogenic component of
the pathway that is sufficient to drive tissue overgrowth
when overexpressed in either Drosophila tissues or mouse
tissues such as liver and intestine [15, 18–21]. In addition,
YAP is nuclear in human cancer cell lines but translocates to
the cytoplasm upon contact inhibition of cell proliferation at
factors, such as Scalloped (Sd), Homothorax, and Teashirt, to
activate the expression of genes promoting cell proliferation
and survival, such as cyclin E, myc, DIAP1, and bantam
[5, 23–28], as well as genes encoding upstream components
of the Hippo pathway, such as expanded, merlin, kibra, and
four-jointed [29–31]. Thus, Yki promotes tissue growth by
regulating the transcription of target genes, but how it does
so is poorly understood.
In an in vivo RNAi screen based on the Vienna Drosophila
RNAi Center library, we identified the mask (CG33106) gene
as having a strong undergrowth phenotype in the wing and
eye when silenced by expression of an inverted-repeat (IR)
hairpin RNAi transgene (mask-IR), as opposed to the effect
of wts-IR, which causes tissue overgrowth (Figures 1A–1I).
To confirm these results, we used a null mutation in the
mask gene mask10.22 to generate homozygous mutant
eyes with the eyeless.flp FRT Minute system. mask10.22mutant
eyes were much smaller than controls (Figures 1J and 1K). In
the developing larval wing imaginal disc, mask10.22mutant
clones proliferated poorly, growing to only 10% of the size of
their wild-type ‘‘twin-spot’’ clones (Figures 1L–1N). These
data show that mask is essential for cell proliferation and
tissue growth. The mask loss-of-function phenotype is similar
to that caused by mutants in the Hippo pathway component
yki  but distinct from that of other signaling pathways
such as the Ras pathway (see Figure S1 available online).
We next examined whether mask is required for the expres-
sion of Yki target genes. Silencing of mask in the posterior
compartment of the wing disc by expression of mask-IR with
hh.gal4 led to a significant reduction in the expression of
four-jointed.lacZ and DIAP1.lacZ reporters (Figures 2A–2F).
In mask10.22mutant clones, the levels of expression of
four-jointed.lacZ, DIAP1.lacZ, and expanded.lacZ were also
reduced compared to their levels in wild-type surrounding
cells (Figures 2G–2I). These results show that mask is required
for the expression of Yki target genes.
The mask gene encodes a very large protein of 4,001
amino acids containing two highly conserved ankyrin-repeat
identified in the hnRNP K protein, which was originally found in
association with RNA in ribonucleoprotein particles but later
found to bind to DNA via its KH domain and to act as a tran-
scriptionalcofactor forp53byfacilitatingassembly ofthetran-
scription factor complex on DNA . Similarly, the NF-kB
transcription factor must form a complex with the KH-domain
protein RPS3 on certain target genes to drive transcription
. We therefore tested whether Mask could interact with
Yki on target-gene promoters. In immunoprecipitation experi-
ments, we find that the ankyrin-repeat domains of Mask are
tion, we performed a DNA pull-down experiment with a biotin-
tagged 583 bp fragment of the DIAP1 promoter that contains
multiple Sd binding sites. When the DNA is pulled down with
streptavidin-coated beads from S2 cells transfected with Yki
and Sd, we find that endogenous Mask binds with Yki and
Sd to the DIAP1 promoter, but not to a negative control actin
sequence (Figure 2L). RNAi knockdown of Sd reduced the
binding of Mask to the DIAP1 promoter (Figure 2M). These
results indicate that Mask is able to complex with Yki/Sd on
We next performed genetic epistasis experiments. Overex-
pression of Yki in clones of cells is sufficient to cause a strong
overproliferation phenotype (Figures3A and 3B). In contrast,
when Yki is overexpressed in mask10.22mutant clones, it is
no longer able to induce overproliferation (Figures 3C–3E).
Overexpressed Yki accumulates in the cytoplasm and nucleus
of both wild-type and mask10.22mutant cells, indicating that
Mask is not essential for Yki to enter the nucleus (Figures 3F
and 3G). The overgrowth of clones mutant for wts or express-
ing nonphosphorylatable or nuclear-localized Yki is also
partially suppressed by mutation of mask (Figures 3H–3O; Fig-
ure S2). Furthermore, the activation of Yki target-gene tran-
scription in wts mutant clones is suppressed by mutation of
mask (Figures 3Q–3S). These results show that Mask is not
required for activation or nuclear localization of Yki but is
instead required for Yki to normally activate transcription of its
target genes. However, Yki still retains some activity in the
absence of Mask, as indicated by the fact that wts, mask
double mutants grow larger than mask single mutant clones.
Drosophila Mask has two human homologs, which we name
Mask1 (also called ANKHD1) and Mask2 (also called
ANKRD17) (Figure 2J). We find that both Mask1 and Mask2
can coimmunoprecipitate with FLAG-tagged YAP (Figure 4A).
YAP5SAstrongly induces CTGF expression in human cells, but
not when the cells are cotransfected with siRNAs targeting
either YAP or both Mask1 and Mask 2 (Figure 4B). Mask1
and Mask2 colocalize with YAP in the nucleus of sparsely
plated HEK293 cells and cytoplasm of densely confluent
HEK293 cells (Figures 4B and 4C). Translocation of Mask1,
Mask2, and YAP was also observed in Caco2 cells (Figure 4D).
These results show that human Mask proteins bind to and
colocalize with YAP to promote target-gene expression. We
observe a similar coregulation of Mask and Yki localization
and stability in Drosophila (Figure S3). Finally, we examined
the expression of YAP, Mask1, and Mask2 in human epithelial
tissue sections. We find that the three proteins are strongly ex-
progenitor cells are located (Figures 4E–4G). A systematic
survey of YAP and Mask1 expression confirms that they are
coexpressed in a wide variety of human tissues and tumors
(Figure S4). Note that similar results are described by
Sansores-Garcia et al. in this issue of Current Biology .
whose function is conserved between Drosophila and hu-
mans. Mask acts in a similar fashion to other KH-domain
cofactors for NF-kB and p53, being coregulated with their
bling with their cognate transcription factor on promoters to
drive transcription [33, 34] (Figure 4H). Thus, KH-domain
cofactors represent a novel class of control mechanism for
signal-regulated transcription factors. Given the essential
role of Mask in Yki/YAP function and the increasing evidence
implicating human YAP in stem cell proliferation and tumori-
genesis, human Mask proteins are candidate targets for new
Supplemental Information includes four figures and Supplemental Experi-
mental Procedures and can be found with this article online at http://dx.
Figure 1. The mask Gene Is Required for Tissue Growth in the Drosophila Wing and Eye
(A) Control adult Drosphila wing (MS1096.G4).
(B) RNAi knockdown of mask results in an abnormally small wing.
(C) RNAi knockdown of wts results in an abnormally large wing.
(D) Control adult Drosophila eye (ey.G4 GMR.G4).
(E) RNAi knockdown of mask results in an abnormally small eye.
(F) RNAi knockdown of wts results in an abnormally large eye.
(G) Section through an adult eye showing multiple ommatidia.
(H) RNAi knockdown of mask causes occasional loss of pigment cells and moderate disorganization of the ommatidia but does not cause loss of photore-
(I) RNAi knockdown of the wts gene causes a strong increase in pigment cells.
(J) Control (white) adult eye. Top view is shown in (J0).
(K) A mask10.22homozygous mutant eye is small and rough. Top view is shown in (K0).
(L) Third-instar wing imaginal disc containing control (GFP-negative) mitotic recombination clones and sister twin-spot clones (23 GFP) of similar size.
(M) Third-instar wing imaginal disc containing mask10.22homozygous mutant clones (GFP-negative) that are drastically smaller than their sister twin spots
(N) Quantification of the ratio between clone and twin sizes in (L) and (M).
Current Biology Vol 23 No 3
We thank Ken Irvine, Georg Halder, and Mike Simon for fly stocks. We thank
Nic Tapon for many discussions and reagents. This work was funded by
Cancer Research UK.
Received: June 22, 2012
Revised: October 1, 2012
Accepted: November 26, 2012
Published: January 17, 2013
1. Harvey, K., and Tapon, N. (2007). The Salvador-Warts-Hippo pathway -
an emerging tumour-suppressor network. Nat. Rev. Cancer 7, 182–191.
2. Halder, G., and Johnson, R.L. (2011). Hippo signaling: growth control
and beyond. Development 138, 9–22.
3. Pan,D.(2010).Thehipposignalingpathwayindevelopment andcancer.
Dev. Cell 19, 491–505.
4. Justice, R.W., Zilian, O., Woods, D.F., Noll, M., and Bryant, P.J. (1995).
The Drosophila tumor suppressor gene warts encodes a homolog of
human myotonic dystrophy kinase and is required for the control of
cell shape and proliferation. Genes Dev. 9, 534–546.
5. Tapon, N., Harvey, K.F., Bell, D.W., Wahrer, D.C., Schiripo, T.A., Haber,
D.A., and Hariharan, I.K. (2002). salvador Promotes both cell cycle exit
and apoptosis in Drosophila and is mutated in human cancer cell lines.
Cell 110, 467–478.
6. Kango-Singh, M., Nolo, R., Tao, C., Verstreken, P., Hiesinger, P.R.,
Bellen, H.J., and Halder, G. (2002). Shar-pei mediates cell proliferation
arrest during imaginal disc growth in Drosophila. Development 129,
7. Harvey, K.F., Pfleger, C.M., and Hariharan, I.K. (2003). The Drosophila
Mst ortholog, hippo, restricts growth and cell proliferation and
promotes apoptosis. Cell 114, 457–467.
Figure 2. mask Is Required for Yki Target-Gene Expression and Forms a Complex with Yki and Sd on Target-Gene Promoter DNA
(A) Control third-instar wing imaginal disc stained with an anti-Mask antibody (green).
(B) Expression pattern of the four-jointed.lacZ reporter gene in a control wing disc.
(C) Expression pattern of the DIAP.lacZ reporter gene in a control wing disc.
(D) RNAi knockdown of mask in the posterior compartment (dotted line) reduces the level of Mask protein expression.
(E) RNAi knockdown of mask in the posterior compartment (dotted line) reduces the level of four-jointed.lacZ expression.
(F) RNAi knockdown of mask in the posterior compartment (dotted line) reduces the level of DIAP.lacZ expression.
(G) A mask10.22homozygous mutant clone (nlsGFP-positive) shows reduced expression of DIAP.lacZ (G0) compared to control neighboring cells.
(H) A mask10.22homozygous mutant clone (nlsGFP-positive) shows reduced expression of expanded.lacZ (H0) compared to control neighboring cells.
(I) A mask10.22homozygous mutant clone (nlsGFP-positive) in the center of the wing pouch shows reduced expression of four-jointed.lacZ (I0).
(J) Schematic diagram of the Mask protein domain structure.
(K) CoIP of GFP-tagged Yki with FLAG-tagged Mask ankyrin-repeat domains.
(L) DNA pull-down with either actin or DIAP1 DNA sequences from extracts of S2 cells transfected with Yki-HA and Sd-FLAG. Mask, Yki, and Sd bind to
DIAP1, but not to actin.
Mask Is A Cofactor for Yorkie/YAP
8. Wu, S., Huang, J., Dong, J., and Pan, D. (2003). hippo encodes a Ste-20
family protein kinase that restricts cell proliferation and promotes
apoptosis in conjunction with salvador and warts. Cell 114, 445–456.
9. Pantalacci, S., Tapon, N., and Le ´opold, P. (2003). The Salvador partner
Hippo promotes apoptosis and cell-cycle exit in Drosophila. Nat. Cell
Biol. 5, 921–927.
10. Udan, R.S., Kango-Singh, M., Nolo, R., Tao, C., and Halder, G. (2003).
Hippo promotes proliferation arrest and apoptosis in the Salvador/
Warts pathway. Nat. Cell Biol. 5, 914–920.
11. Xu, T., Wang, W., Zhang, S., Stewart, R.A., and Yu, W. (1995). Identifying
tumor suppressors in genetic mosaics: the Drosophila lats gene
encodes a putative protein kinase. Development 121, 1053–1063.
12. Song, H., Mak, K.K., Topol, L., Yun, K., Hu, J., Garrett, L., Chen, Y., Park,
O., Chang, J., Simpson, R.M., et al. (2010). Mammalian Mst1 and Mst2
kinases play essential roles in organ size control and tumor suppres-
sion. Proc. Natl. Acad. Sci. USA 107, 1431–1436.
13. Lu, L., Li, Y., Kim, S.M., Bossuyt, W., Liu, P., Qiu, Q., Wang, Y., Halder,
G., Finegold, M.J., Lee, J.S., and Johnson, R.L. (2010). Hippo signaling
Figure 3. Mask Is Required for Yki-Driven Cell Proliferation and Target-Gene Transcription
(A) Control clones (GFP-positive) in a third-instar wing imaginal disc.
(B) Overexpression of Yki-V5 drives overproliferation of cells, leading to large clones.
(C) Mutation of mask inhibits cell proliferation, resulting in small clones.
(D) Overexpression of Yki-V5 in mask mutant cells fails to drive overproliferation.
(E) Quantification of clone sizes in (A)–(D).
(F) Yki-V5 localizes to both cytoplasm and nucleus.
(G) Yki-V5 localizes to both cytoplasm and nucleus in mask mutant cells.
(H) Control mosaic analysis with a repressible cell marker (MARCM) clones (GFP-positive) in a third-instar wing imaginal disc.
(I) wtsX1mutant clones overproliferate, leading to enlarged clone sizes.
(J) mask10.22mutant clones proliferate slowly, leading to small clone sizes.
(K) mask10.22wtsX1double mutants are smaller than wtsX1mutants.
(L) Quantification of clone sizes in (H)–(K).
(M–P) Scanning electron micrographs of control (M), mask mutant (N), wts mutant (O), and mask, wts double mutants (P).
(Q) wtsX1mutant clones show upregulation of expanded.lacZ expression.
(R) mask10.22wtsX1double mutants fail to upregulate expanded.lacZ expression.
(S) mask10.22wtsX1double mutants fail to maintain four-jointed.lacZ expression.
Current Biology Vol 23 No 3
lian liver. Proc. Natl. Acad. Sci. USA 107, 1437–1442.
14. Zhou, D., Conrad, C., Xia, F., Park, J.S., Payer, B., Yin, Y., Lauwers, G.Y.,
Thasler, W., Lee, J.T., Avruch, J., and Bardeesy, N. (2009). Mst1 and
Mst2 maintain hepatocyte quiescence and suppress hepatocellular
carcinoma development through inactivation of the Yap1 oncogene.
Cancer Cell 16, 425–438.
15. Huang, J., Wu, S., Barrera, J., Matthews, K., and Pan, D. (2005). The
Hippo signaling pathway coordinately regulates cell proliferation and
apoptosis by inactivating Yorkie, the Drosophila Homolog of YAP. Cell
Figure 4. Human Homologs of Mask
(A) Coimmunoprecipitation of GFP-tagged YAP with FLAG-tagged Mask1 or Mask2 in the presence of crosslinker in HEK293 cells.
(B)FLAG-tagged YAPS5Adrivestranscriptional activationofCTGFinquantitativeRT-PCRassaysfromMCF10Acells.Knockdown ofbothMask1andMask2
reduces YAPS5A-dependent transcription of CTGF, similarly to knockdown of YAP itself.
(C) Human Mask1 and YAP colocalize in the nucleus of sparse HEK293 cells.
(D) Human Mask2 and YAP colocalize in the cytoplasm of dense HEK293 cells.
(E) Human Mask1, Mask2, and YAP show similar redistribution from the nucleus to the cytoplasm upon contact inhibition in Caco2 cells.
(F) YAP is mainly nuclear in the basal stem cell layer of human esophageal epithelia.
(G) Mask1 is both cytoplasmic and nuclear in the basal stem cell layer of human esophageal epithelia.
(H) Mask2 is mainly nuclear in the basal stem cell layer of human esophageal epithelia, but it is also weakly expressed and nuclear in other cells of the
(I) Model showing Mask as a novel cofactor for Yki/YAP. The localization of the two proteins is coregulated. Mask and Yki/YAP form a complex with
transcription factors such as Sd/TEAD to induce transcription of target genes.
Mask Is A Cofactor for Yorkie/YAP