BBA - Molecular Basis of Disease 1864 (2018) 1203–1215

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BBA - Molecular Basis of Disease

journal homepage: www.elsevier.com/locate/bbadis

Chemical and genetic rescue of an ep300 knockdown model for Rubinstein T

Taybi Syndrome in zebrafish
Aswini Babua,c, Mageshi Kamaraja,1, Moumita Basub,1, Debanjan Mukherjeeb, Shruti Kapoora,c,
⁎
Shashi Ranjana, Mahadeva M. Swamyb, Stephanie Kaypeeb, Vinod Scariaa,c, Tapas K. Kundub, ,
⁎⁎
Chetana Sachidanandana,c,
a
CSIR-Institute of Genomics & Integrative Biology, South Campus, New Delhi 110025, India
b
Transcription and Disease Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR), Bengaluru 560064,
India
c
Academy of Scientific and Innovative Research (AcSIR), New Delhi 110025, India

A R T I C L E I N F O A B S T R A C T

Keywords: EP300 is a member of the EP300/CBP family of lysine acetyltransferases (KATs) with multiple roles in devel-
ep300 opment and physiology. Loss of EP300/CBP activity in humans causes a very rare congenital disorder called
Rubinstein Taybi Syndrome (RSTS) Rubinstein Taybi Syndrome (RSTS). The zebrafish genome has two co-orthologs of lysine acetyltransferase
Zebrafish EP300 (KAT3B) in zebrafish viz. ep300a and ep300b. Chemical inhibition of Ep300 with C646, a competitive
Embryonic development
inhibitor and morpholino-based genetic knockdown of ep300a and ep300b cause defects in embryonic devel-
Histone acetylation
opment reminiscent of the human RSTS syndrome. Remarkably, overexpression of Ep300a KAT domain results
Rescue
Disease model in near complete rescue of the jaw development defects, a characteristic feature of RSTS in human suggesting the
dispensability of the protein-interaction and DNA-binding domains for at least some developmental roles of
Ep300. We also perform a chemical screen and identify two inhibitors of deacetylases, CHIC35 and HDACi III,
that can partially rescue the RSTS-like phenotypes. Thus, modeling rare human genetic disorders in zebrafish
allows for functional understanding of the genes involved and can also yield small molecule candidates towards
therapeutic goals.

1. Background class and is found to be involved in diverse physiological processes

Rubinstein Taybi Syndrome (RSTS; OMIM ID #180849, #613684)
[1,2] is a rare congenital disorder defined by intellectual disability,
craniofacial deformities, heart anomaly and, broad thumbs and halluces
[3]. This autosomal dominant disorder is caused by mutations in the
histone acetyltransferases, CBP or EP300. EP300 and the CREB binding
protein (CBP) are closely related and have semi-redundant functions
during development [4,5]. EP300 mutations associated with RSTS in-
cluding frameshift, missense, substitutions and indels are found
throughout the gene [2,6]. Somatic mutations and deregulation of
EP300 catalytic activity by hyperacetylation and hyper-autoacetylation
are also reported in various cancers [7–9]. Lysine acetyltransferases
(KATs) acetylate histones as well as non-histone proteins that are
broadly associated with transcriptional activation. The lysine acetyl-
transferase EP300 (KAT3B) (HGNC:3373) belongs to the EP300/CBP
through its intrinsic acetyltransferase catalytic activity as well as
transcriptional coactivator activity [10,11].
Although crystallographic studies reveal that yeast has a structural
homolog of EP300, Rtt109, it otherwise has little sequence or apparent
functional similarity with the human protein [12]. It is commonly be-
lieved that EP300/CBP KATs are ‘higher’ eukaryotic proteins which
have evolved with metazoan evolution and are responsible for im-
portant aspects of coordinated growth and development, such as cell-to-
cell communication and organ morphogenesis [13]. Chromosomal du-
plication during vertebrate evolution resulted in the EP300 gene and
the CBP gene present in opossum, chick, mice, and humans (EP300
locus 22q13 and CBP locus 16p13 in the human genome) [14].
Mouse studies reveal that homozygous ep300/cbp mutations are
embryonically lethal. Heterozygous mice develop severe defects in
various tissues, including neural tube, heart and blood vessels

⁎
Corresponding author.
⁎⁎
Correspondence to: C. Sachidanandan, CSIR-Institute of Genomics & Integrative Biology, South Campus, New Delhi 110025, India.
E-mail addresses: tapas@jncasr.ac.in (T.K. Kundu), chetana@igib.in (C. Sachidanandan).
1
Equal contribution.

https://doi.org/10.1016/j.bbadis.2018.01.029
Received 8 September 2017; Received in revised form 8 January 2018; Accepted 27 January 2018
Available online 31 January 2018
0925-4439/ © 2018 Elsevier B.V. All rights reserved.
A. Babu et al.
[5,15,16]. Conditional knock out for Ep300 in mouse have revealed
roles for Ep300 in T cell development [17]. ES cells null for EP300 had
defects in germ layer formation [18]. Combined mutations of Ep300
and CBP in mouse disrupted the photoreceptor morphology and func-
tion [19] and a recent study using chemical inhibition of Ep300/Crebbp
KATs in zebrafish showed that these proteins have a protective function
in the photoreceptor cells of zebrafish [20] also. However the role of
EP300 in embryonic development is not well understood. This
prompted us to investigate the conservation and function of the EP300/
CBP family of proteins in general, and EP300 in particular, in the ge-
netically amenable developmental model, zebrafish.
Zebrafish is a highly conserved vertebrate model and nearly 80% of
the genes involved in human diseases have zebrafish orthologs [21].
The transparent embryos of zebrafish make this system ideal for un-
derstanding the role of genes in embryonic development and for
studying developmental phenotypes of human diseases [22]. Two co-
orthologs of human EP300 have been annotated in the zebrafish
genome [23]. Here we report the embryonic functions of these co-or-
thologs in zebrafish, ep300a and ep300. We use genetic and chemical
perturbation of Ep300 to develop a model for Rubinstein Taybi Syn-
drome in zebrafish. Surprisingly, we find that overexpression of the
KAT domain alone is sufficient to rescue certain RSTS-like phenotypes.
We further use small molecule screening in zebrafish embryos and
identified two compounds, Histone deacetylase inhibitor III and
CHIC35, can rescue the phenotypes in the RSTS model in zebrafish.
2. Results
2.1. The EP300/CBP inhibitor, C646 affects normal development of
zebrafish embryo
In a zebrafish chemical screen for epigenetic modulating com-
pounds that affect embryonic development, we identified C646, a
specific inhibitor of the EP300/CBP family of histone acetyltransferases
[24]. We exposed zebrafish embryos from 8 to 24 hours post fertiliza-
tion (hpf) to 7 μM and 10 μM of C646. At 24 hpf, the embryos exhibited
general growth retardation and were smaller in size (Fig. 1). Closer
observation revealed that the brain ventricles had formed but failed to
inflate symmetrically and this defect was more severe in the 10 μM than
the 7 μM treated embryos (Fig. 1). At 4 days post fertilization (dpf) the
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BBA - Molecular Basis of Disease 1864 (2018) 1203–1215
7 μM C646 treated larvae had smaller eyes and smaller jaw compared to
control larvae, while the 10 μM treated larvae had an almost complete
absence of the jaw, very small eyes, small head and pronounced peri-
cardial edema suggesting heart defects (Fig. 1). At 24 hpf, RNA in situ
hybridization of the treated embryos revealed a loss of expression of the
jaw precursor marker sox9b suggesting defects in specification of cra-
niofacial cartilage (Fig. S1). Using the transgenic fish Tg(mylz7:cerulean)
that marks the heart with a fluorescent marker, we observed that at
3 dpf, embryos treated with 7 μM C646, showed an enlargement of at-
rium, further confirming the heart defect suggested by edema (Fig. S1).
By 4 dpf normal larvae begin inflating their swim bladder and develop a
pair of pectoral fins, which are crucial for staying afloat and swimming.
C646 treated larvae lacked a swim bladder (Fig. 1) and very few of the
larvae developed pectoral fins at this stage (Fig. S1). The inability to
swim and feed (due to lack of jaw) and the heart defects probably led to
the lethality observed, around 5–6 dpf, in most compound-treated
larvae.
C646 is a competitive inhibitor of the EP300 and CBP histone
acetyltransferases [25]. The striking developmental phenotypes ob-
served in the C646 treated embryos suggest that zebrafish has at least
one functional copy of the EP300/CBP family of histone acetyl-
transferases, which plays an important role in development.
2.2. Zebrafish possesses two co-orthologs of ep300
The ENSEMBL database of zebrafish genome lists two co-orthologs
each, of the human EP300 and CBP genes in zebrafish viz. ep300a
(ENSDARG00000100666), ep300b (ENSDARG00000061108) and
crebbpa (ENSDARG00000104609), crebbb (ENSDARG00000104148). A
phylogenetic comparison with other species revealed that members of
the ep300/cbp (KAT3B/KAT3A respectively) family are conserved from
yeast to humans (Fig. 2). The KAT3 genes of yeast, Drosophila and C.
elegans are closely related to each other. However, the cbp and the
ep300 subfamily members segregate in the vertebrates suggesting the
emergence of the two distinct subfamilies in an ancestral organism
[26]. The Ep300 protein sequence is conserved across all the vertebrate
species we compared (Fig. 2), suggesting conservation of function.
It is believed that an event of genome duplication preceded the
teleost evolution and as a result many human genes have more than one
orthologs in the zebrafish genome [27]. This view is supported by the
Fig. 1. The EP300/CBP inhibitor, C646 affects normal de-
velopment of zebrafish embryo.
(a–c) C646 treatment in embryos induced defective brain
ventricular folding at 24 hpf (illustrated with red filling in
the ventricular space). (d–f) Bright field images show
growth delay at 24 hpf. (g–i) C646 treated 4 dpf larvae
show phenotypes including small head, small eyes (e),
heart edema (solid arrow), reduced jaw (open arrow) and
loss of swim bladder (white asterisk). (a–c) Dorsal view,
anterior to the top. (d–i) Lateral view, anterior to the left.
The numbers in the bottom left corner indicate the fre-
quency of the represented phenotype in the total number of
embryos analyzed. Scale bar-100 μm.
A. Babu et al. BBA - Molecular Basis of Disease 1864 (2018) 1203–1215

Fig. 2. Zebrafish has two co-orthologues of ep300.

(a) Phylogenetic tree of ep300 and cbp across various model organisms from unicellular eukaryotes to human. (b) Predicted domains of zebrafish Ep300a and Ep300b proteins are
compared with human ep300. The KAT domains of human and zebrafish share 89.72% identity, while zebrafish Ep300a and Ep300b share 89.29% identity.

Fig. 3. Zebrafish ep300 co-orthologs are developmentally

regulated.
(a) RNA sequencing analysis of ep300a and ep300b across
different timepoints of zebrafish embryonic development
and various adult tissues from publically available data.
(b,c) Quantitative real time PCR of ep300a and ep300b
across different developmental stages of zebrafish embryos
show similar expression pattern as the RNA sequencing
analysis. (d–i) RNA in situ hybridization of ep300a on zeb-
rafish embryos at 0 hpf, 6 hpf, 10 hpf, 18ss, 24 hpf and 4 dpf
respectively. (j–o) RNA in situ hybridization of ep300b on
zebrafish embryos at 0 hpf, 6 hpf, 10 hpf, 18ss, 24 hpf and
4 dpf respectively. (d–e, j–k) Animal pole to the top. (f–g,
l–m) Lateral view, anterior to the top. (h–i, n–o) Lateral
view, anterior to the left. ss-somitic stage. Scale bar-
100 μm.

presence of more than one stretch of genome, which shows syntenic EP300 gene with the zebrafish ep300a and ep300b using the synteny
arrangement of neighboring genes in zebrafish and human [28]. We database [29]. We found that a large number of genes in the neigh-
compared the gene arrangements in the neighborhood of the human borhood of EP300 are shared between the human chromosome 22

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Fig. 4. Zebrafish ep300 knockdown models Rubinstein Taybi Syndrome-2.
(a–d) Bright field images of 4 dpf larvae with ep300a or ep300b or both knocked down.
ep300a and double morphants exhibit phenotypes such as small head, small eyes (e),
heart edema (black arrows), reduced jaw (black arrowheads) and loss of swim bladder
(white asterisk). (e–h) Alcian blue staining of 4 dpf larvae for visualizing cartilage ele-
ments. Arrows indicate lower jaw elements. Arrowheads indicate pectoral fins. (i–l) Tg
(mylz7:GFP) fishes expressing GFP in heart shows defects including enlarged atrium at
3 dpf. (a–h) anterior to the left, (i–l) anterior to the top, (a–d) lateral views, (e–l) ventral
view. The numbers in the bottom left corner indicate the actual number of embryos of the
total represented by the image. Scale bar-100 μm.
region and the zebrafish chromosomes 3 (ep300a) and 12 (ep300b) (Fig.
S2). However, the order of genes is not a perfect match between species
indicating further multiple rearrangements in the region post-duplica-
tion. The synteny of neighboring genes around EP300 in human and
zebrafish reiterates the common origins of these genes.
Further, we compared the predicted zebrafish ep300a and ep300b
proteins with the human EP300 protein sequence. Human EP300 is a
large (2414aa) multi-domain protein with KIX, NRID, Bromodomain,
CH1, CH2, CH3, IBID and the KAT domains [30]. We performed a se-
quence similarity analysis using ClustalW [31] and found that ep300a
(2679 aa) has an amino acid sequence identity of 65.6% and ep300b
(2615 aa) of 65.5% with the human EP300. The two paralogs are 69.8%
identical to each other. To understand the domain architecture of
Ep300a and Ep300b, we used conserved domain database (CDD) search
[32] and found that based on the sequence homology, the domain ar-
rangements of the fish co-orthologs match with that of the human
(Fig. 2). We also found that KAT (lysine acetyltransferase) catalytic
domains of the putative homologs are highly conserved bearing ~90%
sequence identity between human and zebrafish and between the
paralogs themselves.
2.3. Zebrafish ep300 paralogs are developmentally regulated
To understand the function of ep300 paralogs in zebrafish
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development, we must first determine the expression dynamics of the
genes during embryogenesis. We analyzed RNA sequencing data
available in the public domain for various developmental stages of
zebrafish development [SRP009426 [33], SRP008845 [34], ERP000016
(Zebrafish transcriptome sequencing project, PRJEB1986),
PRJNA207719 [35] and SRP017135 [36]] and found that the expres-
sion of ep300a and ep300b transcripts is highest in 0 hpf, much before
the zygotic transcription begins at 3–4 hpf [37], suggesting parental
RNA contribution (Fig. 3). Among adult tissues the expression of
ep300a and ep300b were detected in all tissues but the highest ex-
pression was present in the brain.
We performed quantitative RT-PCR of ep300a and ep300b in RNA
from various stages of embryonic development and found that in
agreement with the RNA sequencing data, both paralogs are expressed
at their highest levels in the early stages of development (Fig. 3). With
time the expression decreases to steady low levels in the older larvae.
To reveal the spatiotemporal regulation of the ep300 paralogs, we
performed whole mount RNA in situ hybridization of ep300a and ep300b
transcripts in the zebrafish embryo. We selected unique sequences from
ep300a and ep300b as probes to prevent any cross-reactivity (Supple-
mentary material, Table T5). ep300a and ep300b revealed very similar
expression patterns in the stages we tested (Fig. 3) and the sense probe
did not detect any expression pattern (Fig. S3). Early expression from
0 hpf to 10 hpf was ubiquitous (Fig. 3). By 24 hpf the expression in the
posterior region reduced significantly while remaining high in the
forebrain, midbrain and hindbrain region. The expression in the 4 dpf
larvae was even more restricted to the brain region (Fig. 3). This is in
agreement with studies in mouse and Xenopus where an early ubiqui-
tous expression is seen to progressively become restricted to various
organs and tissues [38,39], suggesting dynamic roles in development of
the embryo.
2.4. Zebrafish ep300 knockdown models Rubinstein Taybi Syndrome
Ep300/CBP mutations are implicated in Rubinstein Taybi Syndrome
(RSTS), a congenital developmental syndrome. We investigated the role
of ep300 co-orthologs in zebrafish development. We knocked down the
expression of ep300a and ep300b using antisense morpholino oligonu-
cleotides (MO). We used a 12 ng cocktail of ep300a-MO (4 ng) and
ep300b-MO (8 ng) at 1-cell stage (detailed methodology is described in
the Supplementary methods section, Figs. S4 and S5). We also injected
morpholinos against each gene separately to understand their in-
dividual roles. Since the primary goal was to knockdown the Ep300
activity as a whole, we will emphasize on the phenotypes of the double
morphants, although each figure contains the data for the individual
morpholinos and the double morpholino injections. There was no sig-
nificant difference in lethality observed in control and ep300 MO in-
jected embryos (data not shown). All the ep300 morphants tested were
apparently normal until 2 dpf with no major defects (Fig. S5).
At 4 dpf, the ep300 knockdown caused severe defects such as
smaller heads and smaller eyes, shortened jaw, reduced pectoral fins,
pericardial edema and absence of swim bladder when compared to the
control embryos (Fig. 4). We visualized the craniofacial cartilage with
alcian blue staining and observed the first arch structures (palatoqua-
drate and Meckel's cartilage), the second arch structures (ceratohyal
and hyosymplectic cartilage) and the arches 3–7 (ceratobranchial 1–5,
basibranchial and hypobranchial) in wildtype larvae (Fig. S1). The
ep300 double morphants had abnormal first and second arch derived
cartilages, while the structures of arches 3–7 were severely reduced
(Fig. 4). These phenotypes affected the same structures as in the RSTS
patients as they also present with microcephaly and dysmorphic facial
features.
RSTS is also known as the Broad Thumbs-Hallux Syndrome because
of the characteristic deformities in the hands and feet [40]. We in-
vestigated the pectoral fins, which are homologous to the forelimbs in
land-dwelling vertebrates, in the ep300 double morphants and found
A. Babu et al. BBA - Molecular Basis of Disease 1864 (2018) 1203–1215

Fig. 5. Jaw, fin and heart progenitor populations are affected by ep300 knockdown.
(a–d) RNA in situ hybridization of early chondrocyte marker sox9b at 24 hpf shows loss of expression in ep300a MO and double morphants. White dots mark perichondrium bands. (e–h)
RNA in situ hybridization of fin-bud cartilage marker sox9a on 2 dpf embryos is reduced severely. Arrows point to fin bud. (i–l) RNA in situ hybridization of fin-bud cartilage marker sox9b
is also reduced or completely absent on 2 dpf embryos. Arrows point to fin bud. (m–p) RNA in situ hybridization of fin-bud marker tbx5a, which is essential for fin bud formation is either
lost or reduced in morphant embryos at 36 hpf. White arrows indicate the pectoral fin bud. All images have anterior to the left, (a–d) lateral views, (e–p) dorsal view. The numbers in the
bottom left corner indicate the actual number of embryos of the total represented by the image. Scale bar-100 μm.

that the fins were either absent or severely shortened (Fig. 4), as also
was the case in C646 treated embryos (Fig. S1). In the 4 dpf ep300
double morphant embryos, we observed a prominent pericardial edema
(Fig. 4). We used the transgenic line Tg(mylz7:GFP) to visualize the
heart structure. By 2 dpf, the heart tube loops to form a distinct atrium
and a ventricle and we found that in ep300 double morphants, the or-
ientation of the heart chambers was perturbed (Fig. 4). Thus, knocking
down expression of ep300 affects the same tissues in zebrafish as in the
human Rubinstein Taybi Syndrome patients.
2.5. Jaw, fin and heart progenitor populations are affected by ep300
knockdown
The transcription factor SOX9 regulates the cartilage specification
and differentiation program, activating chondrocyte specific genes such
as Col2a1, Col11a2, Aggrecan, and CD-RAP [41]. We studied the ex-
pression of sox9a and sox9b, the zebrafish co-orthologs of human SOX9,
in ep300 morphants. RNA in situ hybridization of 24 hpf embryos for
sox9b marks the expression along the rhombomeres in the hindbrain.
This expression was severely disrupted in the ep300 double MO injected
embryos (Fig. 5). C646 treated embryos showed a similar disruption of
sox9b expression (Fig. S1). At 24 hpf, the ep300 double morphants
showed a mild increase in the sox9a expression but no change in the
expression domains (Fig. S6). The expression of dlx2a, another reg-
ulator of craniofacial cartilage remained unchanged compared to the
controls (Fig. S6), suggesting the defects in craniofacial cartilage may
be attributed primarily to the sox9b expressing population of cells.
Since the pectoral fins were severely shortened in the ep300 double
morphants, we looked at the expression of tbx5a, a marker of the fin
bud in the developing embryo. We observed a complete absence of
expression of tbx5a in the ep300 double morphants at 36 hpf (Fig. 5).
Sox9 is also known to play an essential role in the cartilage and bone
formation in the developing limb in mice [41]. During the zebrafish
pectoral fin formation, sox9a is expressed in scapulocoracoid and the
chondral bone while sox9b is expressed in distal portion of en-
dochondral disc [42]. Expression of both these genes in the fin bud was
either reduced or lost in the ep300 double morphant embryos at 2 dpf
(Fig. 5). Our data suggests a role for the Ep300 in regulating the fin
specification genes, tbx5a, sox9a and sox9b in zebrafish embryos.
The heart tube in zebrafish is marked by the expression of cardiac
transcription factor nkx2.5. We detected the expression of nkx2.5 by
RNA in situ hybridization in 24 hpf embryos and found an elongated
tube-like expression in ep300 double morphants instead of a concentric
circular structure observed in the controls, suggesting an orientation
defect during looping (Fig. S6).

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2.6. Nervous system defects in the ep300 morphant zebrafish reveals new
functions of Ep300
Cognitive impairment is a characteristic feature of RSTS [43]. The
ep300 mutant mice, both heterozygous and homozygous, develop
neural tube closure defects [5], which leads to defective brain devel-
opment. However, we did not observe any obvious defect in the neural
tube in the ep300 double morphant zebrafish embryos (Fig. S5). The
4 dpf ep300 double morphant larvae had smaller heads. To further in-
vestigate brain development, we performed RNA in situ hybridization
for markers of hindbrain, krox20 [44] expressed in the rhombomeres 3
and 5 and pax2a [45] expressed in the midbrain-hindbrain boundary
(Fig. S7). Pax2a is expressed in the optic stalk, midbrain-hindbrain
boundary and the otic vesicle in 24 hpf control embryos. Both krox20
and pax2a expression was normal in the ep300 double morphants at
24 hpf (Fig. S7) confirming the earlier observation that there were no
major gross structural defects in the brain of the morphant embryos.
NeuroD, a neuronal differentiation marker, is expressed in multiple
regions of the brain including the olfactory placode, retinal neurons,
regions of the forebrain, the midbrain, the cerebellar regions of the
hindbrain and the cranial ganglia in the 36 hpf embryos [46,47]. In the
ep300 double morphants we observed a loss of expression in the ol-
factory placode, retinal region and the cranial ganglia and a mild de-
crease in the expression in the statoacoustic ganglia (Fig. 6). We used
islet2a (isl2a) to mark the cranial motor and sensory ganglia in 72 hpf
embryos [48] but did not find any significant difference between the
control and the ep300 double morphant embryos (Fig. S7).
Glia are an important and essential component of the nervous
system. Foxd3 marks the cranial and trunk glial precursors in the zeb-
rafish embryo at 4 dpf. We found a significant increase in the foxd3
expression in cranial and trunk glial precursors in the ep300 double
morphants (Fig. 6). Intriguingly, in spite of the high foxd3 expression in
the precursor population, the expression of the myelination marker
myelin basic protein (mbp) in the hindbrain of CNS and PNS in the
cranial region of the ep300 morphants was significantly reduced
(Fig. 6), suggesting a defect in maturation of the myelinating glia.
Hypo-myelination of brain has been observed in RSTS patients using
MRI studies [49,50] and HDAC3 interaction with EP300 is reported to
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BBA - Molecular Basis of Disease 1864 (2018) 1203–1215
be essential for oligodendrocyte and astrocyte fate switch in the CNS
[51]. Here, we report that in addition to CNS, hypo-myelination is
observed in the PNS of ep300 zebrafish morphants.
2.7. Zebrafish EP300 KAT-homology domain is functionally conserved in
vitro
The phenotypes we observed in the ep300 morphant embryos were
reminiscent of the human RSTS features. This suggested that the human
and zebrafish Ep300 had significant functional conservation. Therefore,
to further investigate whether the catalytic activity of the zebrafish
Ep300 is indeed comparable to the human counterpart, the KAT domain
of zebrafish Ep300 was further characterized. The Ep300a KAT domain,
spanning from 1304 to 1705 amino was demarcated based on the re-
gion corresponding to the high sequence identity with the human
EP300 protein (1285–1664 amino acid in human EP300) (Fig. 2). The
minimal KAT domain was cloned, expressed, and purified to homo-
geneity (Figs. 7, S8) (detailed methodology is in the Materials and
Method section). The acetyltransferase activity of the recombinant
putative Ep300a KAT domain protein was tested in a radioactivity-
based in vitro histone acetyltransferase (HAT) assay where the KAT
activity of the protein was measured by its ability to acetylate the
EP300 substrate, recombinant histone H3 (Fig. 7). The recombinant
Ep300a KAT domain showed an acetyltransferase activity comparable
to the purified human EP300 KAT domain. To ascertain whether the
observed KAT activity of Ep300a was indeed due to the putative
Ep300a KAT domain, two conserved residues, Glu1444 and Tyr1445,
which are critical for human EP300 catalytic activity, were mutated to
alanine (Fig. 7). The mutant KAT domain showed a dramatic decrease
(> 90%) in acetyltransferase activity in comparison to the wild type
KAT domain. This unambiguously showed that the acetyltransferase
activity observed in Ep300a was from the cloned KAT domain. To
confirm that the Ep300a KAT domain was indeed sensitive to the EP300
inhibitor C646, a similar HAT assay was performed as described earlier,
in presence of varying concentrations of C646. The recombinant ep300a
KAT domain protein exhibited an ~85% reduction in activity in the
presence of 2.5 μM and 5 μM C646, and at 10 μM C646, a complete
abrogation in KAT activity was observed (Fig. 7). The in vitro assay
Fig. 6. Nervous system defects in the ep300 morphant
zebrafish reveals new functions of ep300.
(a–d) RNA in situ hybridization of neuroD on 36 hpf em-
bryos reveals distinct bands of expression in brain (yellow
dots) which is nearly completely lost in the ep300a and
double morphant compared to the controls. (e–h) RNA in
situ hybridization of foxd3 in 4 dpf larvae marks glial cells
and indicates upregulation of expression in the hindbrain
region (filled white arrow), in the cranial peripheral ner-
vous system (open white arrow) and in the trunk region
(black bracket). (i–l) Mbp that marks myelinated glia shows
reduced expression in the cranial PNS region in the mor-
phants (red arrowhead). All images have anterior to the
left. (a–d, i–l) dorsal views, (e–h) lateral views. The num-
bers in the bottom left corner indicate the actual number of
embryos of the total represented by the image. Scale bar-
100 μm.
A. Babu et al.
confirmed that the observed anomalies in the zebrafish embryos treated
with C646 (Fig. 1) was indeed a direct consequence of the inhibition of
Ep300 KAT activity.
We also cloned and expressed the Ep300b KAT domain spanning
1269–1576 amino acid. A radioactivity-based in vitro HAT assay per-
formed with this domain showed that the Ep300b KAT domain did not
have any detectable acetyltransferases activity (Fig. S8). This was likely
the reason we did not observe any marked developmental defects in the
ep300b morphant embryos.
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BBA - Molecular Basis of Disease 1864 (2018) 1203–1215
(caption on next page)
To understand the specificity of the zebrafish Ep300a KAT domain
we assessed its ability to acetylate different EP300-specific histone
acetylation sites in comparison to the human EP300 KAT domain
(Fig. 7). Western blotting analysis using specific antibodies against
different Ep300-mediated histone acetylation sites, such as, H3K9ac,
H3K14ac, H3K18ac, H3K27ac, H3K56ac, H2AK5ac, H4K8ac, and
H4K12ac in recombinant histones (Fig. 3g) and H3K9ac, H3K14ac,
H3K18ac and H3K56ac in reconstituted nucleosomes (Fig. 7) revealed
that the specificity exhibited by the Ep300a KAT domain was similar to
A. Babu et al. BBA - Molecular Basis of Disease 1864 (2018) 1203–1215

Fig. 7. Putative KAT domain of zebrafish Ep300a possesses potent acetyltransferase activity.
(a) Protein domain architecture of zebrafish Ep300a. The domains present are NRID, nuclear receptor-interacting domain; KIX, kinase-inducing domain (KID) binding region; Bromo,
Bromodomain; KAT, lysine acetyltransferase domain; IBiD, IRF-3 binding domain; CH1/2/3, Cysteine-Histidine rich domain. D1446A and Y1447A schematic representation shows the
position of the mutations introduced in the protein. (b) Multiple Sequence Alignment to highlight the conservation of the catalytically important residues D1446 and Y1447 of the
zebrafish Ep300a protein across human and zebrafish Ep300 proteins. Positions of the D1446A/Y1447A mutations on the zebrafish Ep300a KAT domain were highlighted in yellow. (c)
SDS PAGE profile of purified wild type and mutated (D1444A/Y1445A) zebrafish Ep300 KAT domain. Purified KAT domains were probed with poly His polyclonal antibodies. (d) Point
mutations (D1444A/ Y1445A) in zebrafish Ep300a KAT domain abrogates its acetyltransferase activity. In vitro filter binding assay was performed using KAT domains and recombinant
H3 as substrate. Abrogation of KAT activity of zebrafish Ep300 KAT domain is represented in terms of counts per minute. (Unpaired t-test was used for statistical analysis). (e) C646, the
Ep300/CBP specific small molecule inhibitor of acetyltransferase activity inhibits zebrafish Ep300 KAT activity. In vitro filter binding assay was performed using zebrafish Ep300 KAT
domain, recombinant H3 and different concentrations of C646. Acetyltransferase activity is represented in terms of percentage activity. (f) Zebrafish and human EP300 KAT domain
acetyltransferase activities were normalized by in vitro filter binding assay. HAT activity of zebrafish Ep300 KAT domain is represented in terms of counts per minute. Site-specificity of
zebrafish Ep300 lysine acetyltransferase activity on different core histones in vitro. In vitro KAT assay was performed using recombinant H2A, H3 and H4 and then loaded on 12% SDS
PAGE and probed with site-specific acetyl-lysine antibodies. H3 levels served as the loading control. Human EP300 KAT domain was taken as positive control for all the reactions. (h) Site
specificity of zebrafish Ep300 lysine acetyltransferase activity on reconstituted nucleosomes in vitro. In vitro KAT assay was performed using ~500 ng reconstituted nucleosome and loaded
on a 12% SDS PAGE and probed with site specific acetyl-lysine antibodies. H3 levels served as the loading control. Human EP300 KAT domain was taken as positive control for all the
reactions. Student's unpaired t-test was used for statistical analysis; N = 2; p ns > 0.5, p* < 0.5, p** < 0.01, p*** < 0.005.

the human EP300 KAT domain. Since the nucleosomes were assembled
using HeLa cell core histones trace amount of acetylation was observed
in no enzyme control. However, the enzymatic effect of zebrafish and
human p300 KAT domain was more prominent (Fig. 7).
The embryonic and biochemical results indicate that even though
there is an extensive evolutionary divergence from zebrafish to humans,
the important epigenetic enzyme EP300 is functionally conserved
through evolution, thus highlighting its importance in higher eu-
karyotes.
2.8. Ep300a KAT domain partially rescues RSTS-like phenotypes in the
zebrafish model
CHIC35, an inhibitor of SIRT1 lysine deacetylases. We exposed ep300
double morphant embryos from 8 hpf to 24 hpf to 10 μM of HDACi III or
10 μM of CHIC35 and observed the phenotypes. The control embryos
treated with the compounds did not have any apparent effect, while the
ep300 double morphants exposed to the compounds showed significant
recovery of craniofacial structures in few embryos, as evident in alcian
blue staining at 4 dpf. similarly, RNA in situ hybridization of sox9b at
24 hpf also suggests that there is a rescue of the cartilage precursor,
which might contribute to the cartilage rescue (Fig. 8).Thus, global
inhibition of deacetylation in the ep300 morphants could partially
rescue the cartilage phenotypes observed in the zebrafish model of
RSTS.

Since the Ep300a KAT domain has a well conserved histone acet-
ylation activity (Fig. 7) we tested if this activity was sufficient to rescue
any of the phenotypes observed in zebrafish morphants, we injected
50 pg of KAT domain RNA into the one-cell stage embryos that were
also injected with both the ep300 MOs. Interestingly, we observed that
compared to the double morphants, where the craniofacial structures
were almost completely absent (Figs. 4, 8), the KAT domain injected
morphant embryos developed distinct structures in the jaw (Fig. 8).
This improvement in jaw development was not evident in embryos
injected with the catalytically inactive mutant KAT domain RNA
(Fig. 8) indicating that the rescue was dependent on the KAT catalytic
activity of the Ep300a KAT domain. We analyzed the expression of
sox9b, the cartilage precursor and found that expression pattern was
recovered significantly in the rescued embryos (Fig. 8). This rescue of
sox9b expression was not observed with the catalytically inactive mu-
tant KAT domain RNA. Interestingly, a mild reduction in the expression
of sox9b and defects in jaw structures were observed in the control
embryos injected with the catalytically inactive mutant KAT domain,
suggesting a dominant negative effect (Fig. 8). We also analyzed the
expression of neuroD and found that there was no rescue of neuroD
expression upon KAT RNA injection (Fig. S9). Thus, the active KAT
domain of Ep300a could partially compensate for the loss of Ep300
activity in the craniofacial cartilage of zebrafish but not in the neuronal
lineages we tested.
2.9. Chemical inhibition of histone deacetylation partially rescues the
craniofacial defects in the zebrafish RSTS model
The phenotypic rescue of cartilage elements and its precursor sox9b
expression in the morphants, by overexpression of the Ep300a KAT
domain suggests that an increased global acetylation may contribute to
the rescue. So, we hypothesized that the global inhibition of Histone
deacetylases might phenocopy the effects of the ep300a KAT domain
overexpression. Hence, we performed a small molecule screening with
selected library of HDAC and Sirtuin modulators to identify compounds
that can rescue the phenotypes in the zebrafish RSTS model. Our screen
yielded two compounds, HDACi III, an amide analog of TSA and
3. Discussion
Heterozygous mutations in the EP300 or CBP genes results in the
human development disorder, Rubinstein-Taybi Syndrome (RSTS),
characterized by multiple abnormalities including intellectual dis-
ability, craniofacial dysmorphism, and broad digits [52]. Zebrafish is a
model of choice to study vertebrate development and the conservation
with human allows human diseases to be modeled effectively in the fish
[48]. Chemical inhibition of the EP300/CBP family of KATs in zebrafish
caused a range of phenotypes reminiscent of RSTS in humans viz. mi-
crocephaly, growth retardation, defects in craniofacial cartilage, pec-
toral fin, heart and brain. To develop a disease model both genetically
amenable and accessible to chemical screening, we knocked down the
two co-orthologs of human EP300 in zebrafish. Our results showed
subtle differences between the chemical inhibition and genetic knock-
down, such as the absence of brain ventricle defects in the ep300
knockdown fishes as compared to the C646 treated fish. Since C646 is a
competitive inhibitor of the KAT activity of both EP300 and CBP these
differences may illuminate the different roles of CBP and Ep300 in
vertebrate development and may be used as a tool to further parse out
the differential requirement for these proteins during development.
Homozygous ep300 null mouse embryos die in utero between E9 and
E11.5 probably due to multiple developmental defects such as defects in
neural tube closure, cell proliferation, and heart development [5] in-
dicating that ep300 plays a pivotal role in developmental programs. The
combined knock-down of the Ep300 paralogs in zebrafish also have
multiple defects involving the nervous system, heart, craniofacial car-
tilage and pectoral fins. These are the same organs that are also affected
in the RSTS patients. This suggests that the zebrafish, mouse and human
EP300 have significant functional conservation and that zebrafish may
be used effectively as a model for human RSTS.
Taking advantage of the accessibility of embryonic stages during
zebrafish development we checked the progenitor populations of some
of the affected organs. We find that the expression of dlx2a, a marker
for migrating cranial neural crest is not affected while the expression of
sox9b that gives rise to the lower jaw progenitors is severely perturbed.
This suggests that the craniofacial dysmorphy seen in RSTS patients

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A. Babu et al.
may have its origins in the defective regulation of SOX9. Although
EP300 is a known co-regulator and interacting partner of SOX9 protein
on the chromatin [53], it is not known whether EP300 can directly
regulate the expression of SOX9 gene expression. We also observed a
deregulation of the expression of sox9a and sox9b in the pectoral fin
buds. However, the expression of tbx5a, which specifies the fin bud and
precedes the expression of sox9a and sox9b was also defective. This
suggests that defects in pectoral fin, the equivalent of forelimb in land
vertebrates, could be because of an involvement of Ep300 in early
specification events. Tbx5a is also known to have a role in heart de-
velopment 9and it would be interesting to study whether the heart
looping defects in the ep300 morphants are due to deregulation of tbx5a
early in heart development.
RSTS patients have microcephaly and intellectual disability. The
neural defects observed in ep300 null mice indicate that ep300 may be
intimately associated with embryonic neurogenesis. Dominant negative
truncated ep300 mutants and brain region-specific conditional knock-
outs have validated the importance of ep300 in long-term memory
formation [54,55]. Intriguingly, the activation of ep300 acetyl-
transferase activity using small molecule activators of ep300 induces
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BBA - Molecular Basis of Disease 1864 (2018) 1203–1215
Fig. 8. Ep300 KAT domain partially rescues RSTS pheno-
types in the zebrafish model.
(a–f) Alcian blue staining of 4 dpf zebrafish larvae shows
jaw cartilage elements (black arrow) and fin cartilage ele-
ments (black arrow head). Zebrafish Ep300a KAT domain
RNA injection at 1 cell stage rescues jaw defects in mor-
phants compared to the double morphant controls. The
mutated KAT domain does not rescue the morphant phe-
notype, while causing defects in the wildtype controls. (g–l)
sox9b expression is perturbed in double morphant embryos
and KAT domain RNA injection rescues sox9b expression in
morphants. Mutated KAT domain RNA could not reverse
sox9b expression in morphants. (m–r) Alcian blue staining
of 4 dpf zebrafish larvae shows rescue of jaw and fin car-
tilage elements (black arrow and black arrow head re-
spectively) upon treatment on ep300 morphants with
HDAC inhibitors - HDACi III and CHIC35. (s–x) partial
rescue of sox9b expression in morphants embryos treated
with HDAC inhibitors. All images have anterior to the left.
(a–f,m–r) Ventral views, (g–l,s–x) lateral views. The num-
bers in the bottom left corner indicate the actual number of
embryos of the total represented by the image. Scale bar-
100 μm.
long-term memory formation and promotes adult neurogenesis in mice
[56]. The zebrafish morphants for ep300 seemed to have restricted
nervous system defects. The hindbrain markers krox20 and pax2a and
also the cranial ganglia marker islet2a appeared to have normal ex-
pression in the ep300 morphants. However, neuroD, a marker for neu-
ronal differentiation was severely affected suggesting defects in the
brain. Further studies would be required to understand the functional
consequences of this deregulation of differentiation.
We also found a decrease in myelination of peripheral neurons ac-
companied by an increase in the glial progenitors marked by foxd3.
Although there is one report of delayed myelination in the brain of
RSTS patients [46] this is the first report of an effect on peripheral
nervous system glial cells in RSTS. Our data suggests that myelination
defects may be responsible for some of the coordination and motor
difficulties reported in the patients [57].
Most of the phenotypes observed in the zebrafish model were
identical in the ep300a morphants and the double morphants while the
ep300b morphants were grossly normal. This combined with our dis-
covery that the Ep300b is a catalytically inactive lysine acetyl-
transferase strongly suggests that ep300a is the only functional paralog.
A. Babu et al.
However, we found that ep300a morphants and the double morphants
had some subtle differences especially in the expression of tbx5a in the
pectoral fin (Fig. 5), sox9a in the craniofacial cartilages (Fig. S6) and
the expression of myelin basic protein in peripheral neurons (Fig. 6).
These observations suggest that ep300b might have subtle but im-
portant roles in development and these may be predominantly depen-
dent on the co-activator function of the Ep300 protein. Further studies
may shed interesting new light on the functional compartmentalization
of paralogs in the duplicated genome structure of zebrafish.
Ep300 proteins function both as transcriptional co-activators and
lysine acetyl transferases (KAT). We sought to understand the role of
the KAT domain in the developmental function of Ep300 and to explore
if the KAT domain alone may be able to rescue some of the develop-
mental defects in the morphants. In vitro assays demonstrated that the
zebrafish Ep300a KAT domain is functionally conserved with the
human KAT. To our surprise we found that overexpression of just the
KAT domain from Ep300a could partially rescue the ep300 morphant
phenotypes, specifically, the defects in the craniofacial cartilage and the
pectoral fins. EP300/CBP proteins are one of the biggest protein in-
teraction hubs in the cell with multiple interacting partners [58]. The
rescue of complex developmental programs by the overexpression of
just the KAT domain suggests that outside of the protein-interactions
that are thought to recruit these KAT proteins to the chromatin, the KAT
domains themselves may execute independent functions.
Inspired by this apparent independent role for the KAT activity in
some of the developmental roles of Ep300, we asked if increasing
general acetylation levels in the embryo could also rescue some aspects
of the Ep300 functions. We took advantage of the ease of chemical
screening in zebrafish embryos (Basu and Sachidanandan) and per-
formed a small selective library screen. We discovered two compounds,
inhibitors of lysine deacetylases, which could bring about partial rescue
of the craniofacial cartilage and pectoral fin defects in the RSTS model.
This combined with previous studies where HDAC inhibition in a CBP-
based mouse model for RSTS could reverse some of the behavioural
aspects of the disease [59]suggest that modulation of acetylation levels
either by activating KATs, or inhibiting HDACs or even by modulating
acetyl-coA levels may be effective ways of treating diseases of acetyl-
transferases. Our studies further demonstrate that using zebrafish to
model such human diseases may be the quickest way to identify such
therapeutic opportunities for many more human genetic diseases.
4. Materials and method
4.1. Zebrafish maintenance
The zebrafish lines used in this study are wildtype (Tubingen-TU), Tg
(−4.9 sox10:egfp)ba2 [60] and Tg(eef1:GAL4-VP16, mylz7:cerulean).
The eef1:GAL4-VP16, mylz7:cerulean construct was made from the pCH
Gtwy G4VP16 plasmid (a kind gift from Michael Nonet [61]). 2531 bp
of the 5′-flanking region (chr14:26419107–26421637) of the XlEef1a1
gene [62] was PCR amplified and cloned into the pCH Gtwy G4VP16
plasmid by Gateway© cloning (Invitrogen). The construct was micro-
injected into one-cell stage zebrafish embryos to establish the line. The
line Tg(eef1:GAL4-VP16, mylz7:cerulean) was used only to visualize
heart.
The zebrafish lines were bred, raised and maintained as described
[63]. Embryos were staged according to Kimmel et al. [64]. Embryos
older than 24 hpf were treated with 0.003% PTU (1-phenyl-2-thiourea)
to inhibit pigment formation, aiding fluorescent imaging and RNA in
situ hybridization analysis.
4.2. Ethics statement
All zebrafish handling and experiments were performed in ac-
cordance to protocols approved for BSC0123 by the Institutional
Animal Ethics Committee (IAEC) of the CSIR-Institute of Genomics and
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BBA - Molecular Basis of Disease 1864 (2018) 1203–1215
Integrative Biology, India under the rules and regulations set by the
Committee for the Purpose of Control and Supervision of Experiments
on Animals (CPCSEA), Ministry of Environment, Forests and Climate
Change, Government of India.
4.3. Chemical treatment
Zebrafish embryos were exposed to compounds of interest from
8 hpf to 24 hpf in 6-well plates. At 24 hpf the embryos were washed and
transferred to embryo water. Phenotypes were assayed at indicated
times. All compounds were dissolved in DMSO, which was used as
vehicle control. The compounds C646 (Sigma Cat.no: SML0002),
HDACi III (Calbiochem Cat. No. 382149), and CHIC35 (Sigma Cat. No:
C8742) were used in the current study.
4.4. Morpholino and RNA injections
Embryos were injected with antisense oligonucleotides (MO) into
the yolk at one-cell stage. For all experiments, the minimum effective
concentration was used as determined by titration experiments. Two
different splice blocking MOs for both ep300a and ep300b have been
used for this study. Morpholino design across the ep300a and ep300b
gene sequences is shown in Supplementary material Fig. S4.
Concentrations of the various MOs used in injections: 4 ng for ep300a-
MO and 8 ng for ep300a-MO2; and 8 ng for ep300b-MO and 2.4 ng for
ep300b-MO2. A standard control sequence MO was used in all experi-
ments to control for phenotypes resulting from injection of oligonu-
cleotides. (For MO sequences, refer to Supplementary material Table
T1). For making the ep300a and ep300b morpholinos concentration
same to the double morpholinos we added control to both ep300a and
ep300b morpholinos.
For rescue experiments, the KAT domain of zebrafish Ep300a was
cloned (as described previously) and RNA was made by in vitro tran-
scription using T7-ultra mRNA synthesis kit (Ambion-Thermo Fisher).
50 pg of RNA was first injected into the cell at one-cell stage embryos,
followed by injection of morpholino into the yolk (1–4 cell stage).
4.5. Knockdown PCR
For all knockdown PCR experiments, RNA preparation was made
from a pool of 15–25 embryos at 24 hpf using Trizol® reagent
(Invitrogen). cDNA was made using Invitrogen Superscript3 kit. To
confirm the function of the splice blocking morpholinos, PCR was
performed to detect the mis-spliced products in morphants (primer
sequences are given in Supplementary material Table T4).
4.6. Whole mount RNA in situ hybridization
RNA in situ hybridization was performed as described [65]. Ribop-
robes of various genes foxd3 [66], sox9a, sox9b [42], dlx2a [67],
neuroD [68], isl2 [48], pax2a [69], mbp [70], and krox20 [71] were
used for this study. Fragments of ep300a and ep300b genes for making
riboprobes were cloned for this study. 516 bp fragment of the ep300a
gene and 847 bp fragment of the ep300b were PCR amplified using
cDNA obtained from 24 hpf embryos (Primer sequences mentioned in
the Supplementary material Table T5). The fragment was cloned into
pCR®4-TOPO® vector and was used as template for in vitro transcription
of antisense ep300a and ep300b RNA probes.
4.7. Alcian blue staining
Alcian blue staining was done to visualize the jaw cartilage as de-
scribed previously [72].
A. Babu et al.
4.8. Imaging
Zeiss (Semi 2000C) bright field microscope was used for live ima-
ging and gene expression pattern studies of zebrafish embryos. Zeiss
AxioScope A1 microscope (with AxiocamHRc) was used for fluorescent
imaging. Images were captured using Zeiss proprietary software and
processed and analyzed in Adobe Photoshop.
4.9. Cloning and mutagenesis of KAT domain
N terminally His6 tagged zebrafish Ep300 KAT domain was cloned
in the pET28b vector using PCR based cloning from total cDNA ob-
tained from 0 to 24 hpf zebrafish embryo lysate. KAT domain mutant
was generated by site directed mutagenesis (Agilent Quik Change II
Mutagenesis Kit). The primer sequences are provided in the
Supplementary material Tables T2, T3.
4.10. Recombinant protein expression and purification
The recombinant KAT domain purification protocol was adapted
from Bornhorst and Falke [73]. The Ep300a recombinant proteins were
co-expressed with Sirt2 in E. coli BL21(DE3) cells to reduce toxic effect
of KAT overexpression and induced with 0.4 mM IPTG at 30 °C for 5 h.
However, the Ep300b recombinant protein was expressed without co-
expression of Sirt2 (the Sirt2 expression was toxic to cells, most likely
because Ep300b is catalytically inactive). Cells were lysed in BC300
(20 mM Tris-Cl pH 7.4, 20% Glycerol, 0.2 mM EDTA, 300 mM KCl,
0.1% NP40, 2 mM PMSF, 2 mM beta mercaptoethanol, Protease In-
hibitor Cocktail (Sigma)) and sonicated until lysate was clear. Lysates
were incubated with Ni-NTA beads (Novagen) for 3 h and washed with
BC300. Proteins were eluted in BC100 containing 250 mM Imidazole.
4.11. In vitro filter binding assay
Filter binding HAT assay was performed using 50nCi 3H acetyl-coA
and 1 μg recombinant H3 as substrate [74] and analyzed by capturing
the acetylated products on Whatman p81 filter paper. After stringent
washes to remove residual free acetyl CoA, acetylation levels were
quantified with Wallac 1409 Liquid Scintillation Counter.
4.12. Nucleosome reconstitution
Nucleosome reconstitutions were performed by salt gradient dia-
lysis as described previously [75]. 601 DNA amplified from pGEM3z
clone (kind gift from Jonathan Widom) using appropriate primers and
HeLa core histone and free DNA in 1:1 molar ratio were incubated in
Buffer A containing 2 M NaCl Buffer A: 10 mM Tris (pH 7.9, 1 mM
EDTA, 1 mM β-Me). Reaction was step dialysed in Buffer A containing
1, 0.8, 0.6, 0.1 M NaCl. Finally reaction was dialysed against Buffer B
(10 mM Tris (pH 7.9), 0.25 mM EDTA, 10 mM NaCl) for 3 h. ~280 ng of
nucleosome were incubated with full length GTF3C1 in KAT buffer for
30 min at 30 °C and loaded onto 12% SDS PAGE and probed with his-
tone acetylation specific antibodies.
Competing interests
None of the authors have any competing interests.
Authors' contributions
AB designed, performed and analyzed the zebrafish experiments.
MK and DM performed and analyzed the zebrafish experiments. SK2,
MB cloned and characterized the ep300a and b KAT domains respec-
tively. DM created the KAT domain mutants. DM and MB performed the
recombinant protein purifications and in vitro KAT assays. DM and MB
has performed the in vitro inhibitor assays. MMS has synthesized the
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BBA - Molecular Basis of Disease 1864 (2018) 1203–1215
inhibitor for the study. SK1 and VS performed the synteny and evolu-
tionary conservation analysis. SR helped in the maintenance and hus-
bandry of zebrafish facility. TKK, CS conceived, designed and co-
ordinated the study. AB, MK, SK2, MB, TKK and CS prepared the
manuscript. All authors gave final approval for publication.
Transparency document
The Transparency document associated with this article can be
found, in online version.
Acknowledgement
We thank Aditi Pandey and Zainab Asad for the comments and
discussions. We thank Sandeep Basu for the final proof reading. We also
thank Dr. Kausik Chakraborty for all the valuable comments and sug-
gestions throughout the project.
Funding
This work was supported by Council of Scientific and Industrial
Research (CSIR), New Delhi [BSC0123 to CS], Jawaharlal Nehru Centre
for Advanced Scientific Research (JNCASR), Department of
Biotechnology (DBT) (Programme Support on ‘Chromatin and Disease’
Grant No. BT/01/CEIB/10/III/01). TKK is Sir J. C. Bose Fellow.
Data accessibility
All data pertaining to this manuscript is available in the main text of
the manuscript or in the supplementary materials.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bbadis.2018.01.029.
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