Histone Acetyltransferase 7 (KAT7) - Dependent Intragenic Histone Acetylation Regulates Endothelial Cell Gene Regulation

JBC Papers in Press. Published on February 6, 2018 as Manuscript RA117.
001383
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.RA117.001383
Role of KAT7 in EC Gene Expression
Histone acetyltransferase 7 (KAT7)-dependent intragenic histone acetylation regulates endothelial cell

gene regulation
Matthew S. Yan1‡§, Paul J. Turgeon2¶§, Hon Sum Jeffrey Man3†§, Michelle K. Dubinsky2†§, Jr Jyun
David Ho4⌘v, Suzan El-Rass†§, You-Dong Wang†§, Xiao-Yan Wen†§, Philip A. Marsden5‡†§
From the ‡Department of Medical Biophysics, ¶Department of Laboratory Medicine and Pathobiology,
†Institute of Medical Science, and §Keenan Research Centre in the Li Ka Shing Knowledge Institute, St.
Michael’s Hospital, Department of Medicine, University of Toronto, Toronto, Ontario M5B 1T8, Canada,
the ⌘ Department of Biochemistry and Molecular Biology, Miller School of Medicine, University of
Miami, Miami, FL 31336, USA and vSylvester Comprehensive Cancer Center, Miller School of
Medicine, University of Miami, Miami, FL 31336, USA
Running Title: Role of KAT7 in EC Gene Expression
Downloaded from http://www.jbc.org/ at UNIVERSIDADE DO PORTO on October 9, 2018

To whom correspondence should be addressed: Philip A. Marsden, St. Michael's Hospital, 209 Victoria
St., Toronto, Ontario M5B 1T8, Canada. Tel.: 416-847-1736; Fax: 416-864-5813; E-mail:
p.marsden@utoronto.ca
Keywords: chromatin immunoprecipitation (ChIP), chromatin modification, endothelial cell, gene

regulation, histone acetylation, vascular biology, zebrafish
Abstract perturbed EC-enriched gene expression, especially

Although the functional role of chromatin marks at the VEGFR-2 homologs, contributed to these
promoters in mediating cell-restricted gene vascular defects. Mechanistically, KAT7
expression has been well characterized, the role of participates in VEGFR-2 transcription by
intragenic chromatin marks is not well understood, mediating Pol II binding, H3 lysine 14, and H4
especially in endothelial cell (EC) gene acetylation in its intragenic region. Collectively,
expression. Here, we characterized the histone H3 our findings support the importance of differential
and H4 acetylation profiles of 19 genes with EC- histone acetylation at both promoter and intragenic
enriched expression via loci-wide chromatin regions of EC genes and reveal a previously
immunoprecipitation followed by ultra high underappreciated role of KAT7 and intragenic
resolution (5 bp) tiling array analysis in ECs histone acetylation in regulating VEGFR-2 and
versus non-ECs throughout their genomic loci. endothelial function.
Importantly, these genes exhibit differential EC
enrichment of H3 and H4 acetylation in their Introduction
promoter in ECs versus non-ECs. Interestingly, Histone acetylation is a chromatin modification
VEGFR-2 and VEGFR-1 show EC-enriched involved in transcriptional activation and it is
acetylation across broad intragenic regions and are dynamically regulated by histone
upregulated in non-ECs by histone deacetylase acetyltransferases (KATs) and histone
inhibition. It is unclear which histone deacetylases (HDACs) (1). Although promoter
acetyltransferases (KATs) are key to EC histone acetylation is commonly associated with
physiology. Depletion of KAT7 reduced VEGFR- transcriptional activation, intragenic histone
2 expression and disrupted angiogenic potential. acetylation also plays a pivotal regulatory role (1).
Microarray analysis of KAT7-depleted ECs In yeast cells lacking the KATs Gcn5 and Elp3,
identified 263 differentially regulated genes, many histone H3 hypoacetylation in the intragenic
of which are key for growth and angiogenic region is associated with transcriptional inhibition
potential. KAT7 inhibition in zebrafish embryos (2). Gcn5 deficiency causes transcriptional
disrupted vessel formation and caused loss of inhibition by preventing nucleosome eviction and
circulatory integrity, especially hemorrhage, all of transcription elongation (3). Also, intragenic
which were rescued with human KAT7. Notably, histone acetylation levels are critical for
1
cotranscriptional spliceosome assembly (4). mammalian development that disrupted somites,

However, the importance of intragenic histone mesenchyme, and possibly blood vessel formation
acetylation is not well characterized in mammals. (12). KAT7 is thought to form multisubunit
A functional role for epigenetic processes MYST complexes containing various subunits,
in endothelial cell (EC) biology has been argued. including JADE1/2/3, ING4/5, hEAF6, and
Our gene-specific studies on the EC-enriched Brpf1/2/3 to mediate its regulatory activity (13-
expression of NOS3 (eNOS) showed that DNA 17). Interestingly, KAT7-containing MYST
hypomethylation, acetylated H3 lysine 9 complexes have been noted to localize to the
(AcH3K9), AcH4K12, and trimethylated H3 promoter and intragenic region of target genes
lysine 4 (H3K4me3) enrichment at the eNOS suggesting a functional role at these sites (13,18).
proximal promoter regulated its EC-enriched However, it is unknown if KAT7 regulated
expression (5,6). Also, physiologically relevant intragenic histone acetylation has consequences in
stimuli, including hypoxia, regulated eNOS cell-specific gene expression and phenotype.
expression by altering its epigenetic profile (1,7). We found that KAT7 localized to the

Additionally, HDAC inhibition reduced VEGFR-2 locus and in vitro KAT7 depletion in
angiogenesis and EC migration (8). human ECs perturbed EC gene expression,
To further our understanding on the including VEGFR-2. The reduced VEGFR-2
epigenetic regulation of EC-enriched genes (EC expression level in KAT7-depleted EC was
genes), especially locus-wide chromatin marks, we mediated transcriptionally by reducing AcH4,
characterized the pan-AcH3 and -AcH4 profiles of AcH3K14, and Pol II occupancy in the VEGFR-2
human umbilical vein endothelial cells (HUVECs) intragenic region. Perturbed EC gene expression in
and a non-EC, human aortic vascular smooth KAT7-depleted ECs was associated with deficient
muscle cells (HuAoVSMCs), at 19 EC-enriched EC function. Parallel findings were made in vivo.
genes (EC genes) via loci-wide chromatin Importantly, KAT7 morpholino (MO) inhibition in
immunoprecipitation followed by ultra high zebrafish embryos (3 dpf) resulted in aberrant
resolution (5 bp) tiling array analysis. Almost all vessel formation with compromised circulatory
of the EC genes were differentially H3 and H4 integrity, especially hemorrhage, that could be
acetylated and these profiles were highly rescued by human KAT7 RNA. Furthermore,
correlated with their expression and RNA these zebrafish embryos showed reduced
Polymerase II (Pol II) ChIP-seq profiles. Similar expression of EC-enriched genes that were
findings were evident with low resolution ChIP- similarly perturbed in human KAT7-depleted ECs.
seq AcH3K9 and AcH3K27 profiles of HUVECs Expression of a KAT7-regulated gene, the
and normal human epithelial keratinocytes VEGFR-2 homolog KDRL, in KAT7-depleted
(NHEK) from the Encyclopedia Of DNA zebrafish salvaged aberrant vessel formation and
Elements (ENCODE) database (9). Interestingly, partly restored the disrupted circulatory integrity
some notable genes, including vascular endothelial in these zebrafish. Overall, our findings suggest
growth factor receptor-1 and -2, (VEGFR-1 and that histone acetylation is important for EC-
VEGFR-2) exhibited broad differential intragenic enriched gene expression and that KAT7 plays a
histone acetylation. Consistent with this significant role in endothelial function, in part, by
observation, HDAC inhibition with trichostatin A regulating intragenic histone acetylation at
(TSA) in HuAoVSMCs increased VEGFR-1 and VEGFR-2.
VEGFR-2 expression.
Select KATs have been implicated in Results
intragenic histone acetylation modifications (1,10). Differential histone acetylation occurs at genes
Our attention focused upon KAT7 with endothelial cell-enriched expression – In our
(MYST2/HBO1), a member of the MYST KAT previous studies, we observed that differential
family. KAT7 can regulate transcription, DNA histone acetylation at eNOS is functionally
replication, and cell survival via its H3K14, H4K5, important for its EC-enriched expression (6). To
H4K8, and H4K12 acetylation activity (11-13). determine if this finding extends to other genes
Importantly, KAT7 deficient mice are embryonic with preferential EC expression, we designed an
lethal at E10.5 due to defects in post-gastrulation ultra high resolution tiling DNA microarray at 5
2
base pair (bp) resolution for 34 genes comprising due to greater global histone acetylation levels in
of: i) 19 EC genes, ii) 6 broadly expressed genes, EC, as noted previously (6). Furthermore, the
and iii) 9 EC-excluded genes (supplemental Table broadly expressed PPIA (cyclophilin A) showed
S1). These genes were tiled by an average of 24 similar high histone acetylation levels in both cell
distinct DNA probes per bp. Both DNA strands types (Fig. 5A). Also, the non-expressed CDH1
were examined. While prior studies have focused (E-cadherin) showed sparse histone acetylation
on promoters, intragenic modifications are gaining levels in the 2 cell types (Fig. 5B). Taken together,
attention. Therefore, we were also interested in genes with enriched expression are preferentially
intragenic modifications across cell types for EC histone acetylated in ECs versus non-expressing
genes. Thus, our tiling arrays probed the 34 genes cells.
at the genomic regions that are upstream of
transcription initiation (50kb), intragenic regions, Comparison of histone acetylation ChIP-chip data
and downstream of the last transcript exon (50 kb). with ChIP-seq data from the ENCODE consortium
Using the tiling arrays, we assessed for differential – Whole genome mapping of AcH3K9, AcH3K27,

pan-AcH3 (AcH3K9, AcH3K14) and pan-AcH4 H3K36me3, and Pol II has previously been
(AcH4K5, AcH4K8, AcH4K12, AcH4K16) conducted on HUVECs and NHEKs by the
enrichment at these genes in HUVEC and ENCODE consortium and represents a
HuAoVSMC via ChIP-chip analysis. Of the 19 EC comprehensive, yet relatively low resolution,
genes, 15 were preferentially acetylated at H3, H4 ChIP-seq dataset (9). These data allowed us to
or both in ECs with 12 showing EC-enriched compare our high-resolution pan-AcH3 to
differential histone acetylation at their promoters AcH3K9 and AcH3K27 profiles to gain further
(Fig. 1A; Fig. 2A; Fig. 3A; Fig. 4, A-B and D-E; insight in the histone acetylation status of the 19
and supplemental Table S1). Importantly, we EC genes. Regions of differential AcH3K9 and
recapitulated our previous findings at eNOS and AcH3K27 enrichment were noted for all 19 EC
found differential H3 and H4 acetylation in its genes in ECs versus NHEKs (Fig. 1B; Fig. 2B;
proximal promoter region in ECs versus non-ECs Fig. 3B; and supplemental Table S1). Importantly,
(Fig. 3A) (6). For the 15 preferentially histone our pan-AcH3 data mirrored the AcH3K9 and
acetylated EC genes (e.g. eNOS and VEGFR-2), AcH3K27 datasets (Fig. 1-3 and supplemental
their mRNA expression in ECs versus Table S2). However, regions of differential pan-
HuAoVSMCs was ≥ 3-fold and their HuAoVSMC AcH3, but not AcH3K9 and AcH3K27,
basal expression was low (19). The 4 non- enrichment were detected, which might reflect
preferentially histone acetylated EC genes showed AcH3K14 enrichment (Fig. 1-3 and supplemental
only modestly higher mRNA expression in ECs Table S2). Regions that were exclusively
(e.g. TFPI and EphB4), because they also differentially AcH3K9 and/or AcH3K27 enriched
evidenced modest HuAoVSMC basal expression. were also observed (Fig. 1-3, supplemental Table
Thus, genes exhibited differential histone S1; and supplemental Table S2). Aside from
acetylation when their mRNAs were robustly and SELP and VCAM1, which exhibited low basal
selectively expressed in ECs versus non-ECs. expression, EC genes were differentially
Focal differential histone acetylation occurred in H3K36me3 enriched in their intragenic region in
the intragenic regions of many EC genes (e.g. ECs versus NHEKs with 15 exhibiting widespread
eNOS). Interestingly, some genes, notably KDR differential enrichment (Fig. 1B; Fig. 2B; Fig. 3B;
(VEGFR-2) and FLT1 (VEGFR-1), showed broad and supplemental Table S1). This observation is
differential histone acetylation across their in contrast to select EC genes that are
transcriptional units (Fig. 1A; Fig. 2A; Fig. 4; and differentially acetylated throughout their loci.
supplemental Table S1). This was not expected. Pol II ChIP-seq profiles in HUVECs and
We were able to validate differential histone NHEK showed that Pol II selectively engaged EC
acetylation at VEGFR-2 by ChIP-qPCR (Fig. 4, genes in ECs versus NHEKs (Fig. 1B; Fig. 2B;
A-B). Importantly, normalization for histone H3 Fig. 3B; and supplemental Table S1). We
density, which addresses nucleosomal density, did confirmed the Pol II enrichment across the
not affect this conclusion (Fig. 4, C-E). VEGFR-2 locus by ChIP-qPCR (Fig. 4F).
Differential histone acetylation at EC genes is not Importantly, Pol II enrichment overlaps the
3
widespread regions of differential pan-AcH3 and assessed the expression and role of KAT7 in ECs.
pan-AcH4 enrichment in VEGFR-2 (Fig. 1 and We found KAT7 was expressed ubiquitously in
supplemental Table S1). Aside from SELP, which various primary cell types and was unaffected by
exhibits low basal EC mRNA expression, EC environmental stimuli, such as hypoxia (<1% O2;
genes with differential pan-AcH3 and -AcH4 Fig. 7A and supplemental Fig. S1). To assess if
profiles were also enriched for Pol II in ECs (19). KAT7 affects VEGFR-2 EC expression, we
This observation suggests that differential pan- depleted KAT7 by RNAi and found it reduced
AcH3 and -AcH4 enrichment is associated with VEGFR-2 mRNA and protein expression by 36%
transcription at these genes (Fig. 1; Fig. 2; Fig. 3; (±11% SEM, n = 4, p ≤ 0.05) and 65% (±9%
and supplemental Table S1). SEM, n = 4, p ≤ 0.05), respectively (Fig. 7, B-C,
E). In contrast, Tie1, Tie2, and eNOS expression
The effects of TSA treatment on VEGFR-1 and were not significantly altered (Fig. 7, B and E). To
VEGFR-2 – In our previous studies, we noted that determine if KAT7 regulated other genes involved
differential promoter DNA methylation was not in EC physiology, gene expression analysis was

observed at VEGFR-1 and VEGFR-2 between conducted. Of the 26,109 analyzed protein coding
ECs and non-ECs suggesting that it did not transcripts, 263 were differentially regulated with
regulate their EC-enriched expression (19). 117 downregulated and 146 upregulated by KAT7
Indeed, DNA methyltransferase (DNMT) depletion (p ≤ 0.05, ≥2-fold change, n = 4) (Fig.
inhibitors did not affect VEGFR-1 and VEGFR-2 7C; supplemental Table S3). However, we were
expression. This is in contrast to EC genes, such as unable to find an effect of KAT7 depletion on EC
eNOS, CD31, vWF, and VE-cadherin, that expression of VEGF-A itself. Interestingly, gene
exhibited differential proximal promoter DNA ontology (GO) analysis of mRNA species that are
methylation across cell types and showed downregulated by KAT7 depletion are enriched
increased mRNA expression upon DNMT for biological processes associated with EC
inhibition in non-expressing cell types where their physiology, including inflammatory response,
promoters are normally methylated (19). Since vasodilation, response to wounding, regulation of
broad differential histone acetylation occurred at blood vessel size, regulation of tube size, and
VEGFR-1 and VEGFR-2, we assessed the role of positive upregulation of cholesterol efflux (Fig.
histone acetylation on their expression by treating 7D). In contrast, GO analysis of mRNA species
HUVECs and HuAoVSMCs acutely (4h) with upregulated by KAT7 is enriched for genes
trichostatin A (TSA), a class I and II HDAC associated with cell cycle and mitosis
inhibitor. Both genes were upregulated by TSA in (supplemental Fig. S2A; supplemental Table S3).
HuAoVSMCs, a non-EC that does not basally Using independent HUVEC isolates and
express either gene (Fig. 6A). In contrast, high quantitative RT-qPCR, we confirmed the
basal VEGFR-1 and VEGFR-2 expression did not regulation of 11 select genes with roles in vessel
change significantly in HUVECs (Fig. 6B). Taken tone regulation (ACE1 and ASS1), vessel
together, histone acetylation plays a functional formation (CASZ1, CEACAM-1, CXCL16,
role in the EC-enriched expression of VEGFR-1 RGS4, VEGFR-1,-2,-3), EC activation (E-
and VEGFR-2. selectin), and cholesterol efflux (ABCG1) (Fig.
7E) (20-28). Thus, these in vitro studies suggest
KAT7 depletion disrupts the expression of that KAT7 is important in EC gene expression.
VEGFR-2 and other genes involved in EC
physiology – Since differential histone acetylation KAT7 mediates chromatin-based transcriptional
occurred broadly across VEGFR-2 and HDAC regulation of VEGFR-2 – To determine if KAT7
inhibition in HuAoVSMC upregulated VEGFR-2, regulated VEGFR-2 expression transcriptionally in
we pursued the hypothesis that broad histone human ECs, we assessed Pol II occupancy
acetylation in the VEGFR-2 locus is important for throughout the VEGFR-2 locus (Fig. 8A). Aside
its EC expression. Since KAT7-containing MYST from a genomic region at the 3' end of the
complexes can be recruited broadly across target VEGFR-2 genomic locus (+28760/+28706), Pol II
genes, KAT7 is a candidate for regulating broad occupancy at the assessed genomic regions
histone acetylation at VEGFR-2 (13). Thus, we throughout the locus was reduced in KAT7-
4
depleted ECs (Fig. 8B). We then examined the contrast, KAT7 depletion blunted EC migration
AcH3 and AcH4 residues that are EC enriched at activity in response to VEGF-A (Fig. 10C). We
the VEGFR-2 locus to determine if they also observed an appreciable increase in cell
correspond to KAT7 catalytic activity. Indeed, migration activity in KAT7-depleted ECs relative
AcH3K9, AcH3K14, and AcH4K8 were found to to control siRNA treated ECs in the absence of
be differentially EC-enriched across the VEGFR-2 VEGF-A (Fig. 10C). This increase in the cell
locus in HUVEC versus HuAoVSMC migration activity of KAT7-depleted ECs might be
(supplemental Fig. S3). To determine if KAT7 due to the effects of KAT7 on other regulators of
directly regulated the chromatin structure of the cell migration basally. However, this difference
VEGFR-2 locus, we assessed the histone was not statistically significant. Furthermore, as
acetylation status at VEGFR-2 upon KAT7 noted earlier, KAT7 depletion did not affect basal
depletion. Consistent with KAT7 catalytic activity, expression of VEGF-A in ECs. With respect to
pan-AcH4 and AcH3K14 levels were reduced at signal transduction activity of ECs in response to
many, particularly central, VEGFR-2 intragenic VEGF-A, we found that VEGFR-2 dependent

regions that were assessed in KAT7-depleted ECs signal transduction activity is disrupted in KAT7-
(Fig. 8, C and D). To determine if KAT7 can depleted ECs as shown by reduced VEGFR-2
localize to the VEGFR-2 locus, ECs were tyrosine 1175 (Y1175) phosphorylation at 5
transduced with KAT7-expressing lentiviruses and minutes in response to VEGF-A induction, even
KAT7 enrichment at the VEGFR-2 genomic locus after taking account of reduced VEGFR-2 protein
was assessed by ChIP-qPCR analysis (Fig. 9). expression (Fig. 10D). The physiology of VEGFR-
KAT7 showed enriched localization at the 2 Y1175 phosphorylation in response to VEGF-A
VEGFR-2 genomic locus in ECs transduced with administration has been implicated in VEGF-A
KAT7-expressing lentiviruses versus ECs induced increase in cell migration and
transduced with His-eGFP-expressing lentiviruses proliferation (29,30). The blunted VEGF-induced
(Fig. 9B). In contrast, KAT7 was not enriched at migratory response is consistent with the change
the eNOS promoter in ECs that are transduced in biochemical signaling in KAT7-depleted ECs.
with KAT7 and His-eGFP expressing lentiviruses KAT7 can also affect cell proliferation
(Fig. 9B). Taken together, KAT7 affects VEGFR- and survival in other cell types, perhaps due to
2 transcriptional activity, in part, by localizing to effects on DNA replication (11,31). We also found
the VEGFR-2 genomic locus and regulating that KAT7 regulated genes involved in cell cycle,
intragenic histone acetylation. cell division, and DNA replication (supplemental
Fig. S2A; supplemental Table S3) Thus, we
KAT7 regulates EC phenotype and survival – To conducted cell proliferation and apoptosis assays
determine if KAT7 regulates EC phenotype, we on KAT7-depleted ECs. We found reduced cell
conducted broadly accepted assays of EC proliferation, but no changes in apoptosis
phenotypes, namely matrigel and wound healing (supplemental Fig. S2, B-C). Thus, these in vitro
assays. We noted fewer branch points, a decreased studies suggest that KAT7 is important in EC
number of branches in EC networks, and a greater cellular phenotype and highlights its specific role
portion of branches outside of a network in in VEGFR-2 regulation and VEGF-A response.
matrigel assays of KAT7-depleted ECs (Fig. 10A).
Furthermore, KAT7 depletion reduced the surface KAT7 inhibition results in defective vascular
area recovered in wound healing assays (Fig. structure and circulation in zebrafish – Previous
10B). studies showed that KAT7-deficient mouse
Since a major target of KAT7 in EC is the embryos were developmentally arrested at the 10-
VEGF axis, we determined whether KAT7 somite stage due to defects in post-gastrulation
depletion perturbs the VEGF-A response of ECs. mammalian development, which disrupted
Therefore, we assayed cell migration and signal somites, mesenchyme, and possibly blood vessel
transduction activity of ECs in response to VEGF- formation (12). However, functional defects in the
A. We found that VEGF-A significantly induced vasculature were not addressed and the abnormal
cell migration activity of control siRNA treated vascular structure might be confounded by
ECs in a chemoattractant assay (Fig. 10C). In embryonic defects that affect developmental
5
progression (12). Thus, we characterized KAT7 RNA, which is insensitive to the E1ATG MO (Fig.
function in the zebrafish vasculature, in which 12). Perturbations in the closed cardiovascular
structure and function can be monitored in real system might reflect blood vessel
time and a functioning cardiovascular system is underdevelopment as suggested by the decreased
not required for viability at relatively advanced ISV EC numbers in KAT7-depleted tg(fli1:nGFP)
stages of embryogenesis. We deduced that zebrafish (3 dpf) (Fig. 13). Our assessments of
zebrafish have two predicted KAT7 orthologs, time-based imaging indicated that the decreased
namely KAT7a and KAT7b (Zebrafish Genome ISV EC numbers of KAT7-depleted embryos did
Build GRCz10), which encode orthologs with not reflect augmented migration in and out of the
13% versus 86% amino acid sequence identity to vessels (Fig. 11, C-D), as described for other EC
human KAT7, respectively. We focused on genes (33). Taken together, KAT7 is important for
KAT7b, which is predicted to have the conserved forming a functional closed cardiovascular
Zinc Finger and MYST domains (13). To network.
determine the expression profile of KAT7b in

zebrafish embryos, tg(kdrl:eGFP) zebrafish KAT7 regulation of EC gene expression is critical
embryos were sorted for EC-enriched GFP+ and for forming a functional cardiovascular network in
EC-depleted GFP- cells. Consistent with the zebrafish – To determine if KAT7 affects EC gene
ubiquitous expression of human KAT7 in multiple expression in zebrafish development, MO-treated
cell types, KAT7b expression is expressed in both tg(kdrl:eGFP) zebrafish (3 dpf) were subjected to
GFP+ and GFP- cells (Fig. 11A). This observation fluorescence activated cell-sorting (FACS) to
is consistent with the non tissue-specific isolate GFP+ and GFP- cells that are EC-enriched
expression of KAT7b that was noted previously by and EC-depleted, respectively. We noted robust
others in zebrafish embryos between 0 to 60 h expression of the VE-cadherin (CDH5), VEGFR1
(32). To characterize zebrafish KAT7 function, we (FLT1), and VEGFR-2 (KDR) orthologs and the
depleted KAT7b in tg(fli1:nGFP) zebrafish using VEGFR-2 paralog (KDRL) in GFP+ versus GFP-
morpholinos (MO) and assessed initial cells (Fig. 14, A and C-E) (34). Importantly,
intersegmental vessel (ISV) formation (24-34 KDR, KDRL, and FLT1 mRNA expression was
hours post fertilization), an early stage of vascular reduced in KAT7-depleted GFP+ versus control
development (Fig. 11, B-D; supplemental Fig. S4, GFP+ cells (Fig. 14, C-E). Consistent with these
A-B). KAT7 depletion reduced EC numbers in observation, KDRL and FLT1 protein expression
zebrafish ISV throughout early ISV formation, were reduced in KAT7-depleted embryos relative
suggesting the process is impeded (Fig. 11, C-D). to control MO-treated embryos (Fig. 14F). In
We further characterized KAT7 function by contrast, CDH5, which is EC-enriched, and NOS1,
depleting KAT7 using independent morpholinos which in zebrafish is not EC-enriched, were not
(E1ATG MO and E3I3 MO) in tg(kdrl:eGFP, affected by KAT7 depletion (Fig. 14, A-B). Thus,
gata1:dsRED) zebrafish embryos to as with human ECs, KAT7 is important for
simultaneously visualize EC vascular patterning zebrafish EC-enriched gene expression.
and circulating erythrocytes at 3 days post Since KAT7 regulates EC gene expression
fertilization (dpf) (Fig. 12). KAT7-depleted in zebrafish embryos, we assessed if the abnormal
zebrafish exhibited abnormal vessel formation as phenotypes of KAT7-depleted zebrafish can be
shown by the disrupted vascular plexus of the rescued by the exogenous expression of a target
subintestinal vein (SIV) (Fig. 12, A-B). Although gene, namely KDRL. KDRL RNA was able to
overall vascular architecture appeared grossly restore the disrupted SIV of KAT7-depleted
intact, the circulatory integrity of KAT7-depleted zebrafish (Fig. 15, A-B). Moreover, the reduced
zebrafish was compromised. Specifically, we blood circulation was restored, in part, with
observed reduced blood circulation in the ISVs, exogenous KDRL mRNA in KAT7-depleted
dorsal aorta, caudal vein, and SIV; and blood zebrafish as shown by similar levels of blood
accumulated in the yolk sac and trunk (Fig. 12, A circulation in the ISVs, dorsal aorta, caudal vein,
and C). Hemorrhage was also prominent in the and SIV as control MO-treated zebrafish (Fig. 15.
brain (Fig. 12, A and D). Importantly, these A and C). However, hemorrhages were
phenotypes were rescued with human KAT7 prominently observed in the brain of KAT7-
6
depleted zebrafish even with the exogenous (12,35). Intragenic histone acetylation can regulate
expression of KDRL suggesting that the disrupted gene expression via the co-transcriptional
expression or activity of other KAT7-regulated assembly of the spliceosome and transcription
genes might be responsible for this phenotype elongation (2,3,36,37). In particular, intragenic
(Fig. 15, A and D). Taken together, KAT7 histone acetylation can facilitate transcription
regulation of EC gene expression is important for elongation by promoting histone eviction and
a functional cardiovascular network in zebrafish. recruiting bromodomain-containing proteins
(3,37). We found that KAT7 can localize to the
VEGFR-2 genomic locus to mediate broad
Discussion differential AcH3K14 and pan-AcH4 enrichment,
The importance of intragenic epigenetic especially in the central intragenic genomic
modifications is beginning to be appreciated. region. These KAT7-mediated modifications
Intragenic histone modifications and DNA possibly promote transcription elongation based
methylation are thought to mediate gene on the overall reduced broad Pol II occupancy in

expression by affecting transcriptional processivity KAT7-depleted ECs (3,37). To our knowledge, we
and alternate mRNA transcript expression (1). are the first to demonstrate that broad differential
However, their role in cell-enriched gene intragenic histone acetylation occurs at unique EC
expression has received modest attention, in part, genes and that this intragenic mark is functionally
because epigenomic studies need to be performed important for EC-enriched expression. Our
in a cell-by-cell type fashion. Here, we used an findings can be compared with broad intragenic
ultra high-resolution and high throughput H3K36me3, which reflects active transcription
approach to analyze the pan-AcH3 and pan-AcH4 across a transcriptional unit. Here, the H3K36me3
profiles in the genomic loci of 19 EC genes in modification is a generalized component of gene
HUVEC versus HuAoVSMC and compared these transcription, instead of a feature of specific genes
findings to widely expressed genes and EC (1). The one nuance would be the intragenic
excluded genes. We also integrated this data with signature of both H3K27me3 and H3K36me3,
ENCODE cell-specific ChIP-seq data for which is thought to reflect chromatin marks at
AcH3K9, AcH3K27, H3K36me3 and Pol II (9). inactive and active alleles, respectively, of
Many transcriptional regulation studies have monoallelically expressed autosomal genes (38).
focused on chromatin modifications at promoters VEGFR-2 and VEGFR-1 might
and enhancers (1,9). Consistent with these studies specifically be regulated by KAT7-mediated broad
and our eNOS studies, we report that preferential intragenic histone acetylation as they are important
histone acetylation enrichment at EC gene in establishing the endothelial and hematopoietic
promoters in expressing ECs versus non-ECs is a lineages during early embryonic development.
general feature (6). Importantly, we argue that Previous studies have suggested that a subset of
differential intragenic histone acetylation is critical developmental genes is not completely silent in
to EC gene regulation. We noted unexpected broad embryonic stem cells (ESCs) and their modest
EC-enriched histone acetylation in the intragenic expression is thought to give ESCs the potential to
regions, especially at VEGFR-1 and VEGFR-2, differentiate into various lineages (39). These
that was not noted at other EC-enriched genes (e.g. genes show the bivalent H3K4me3 and
eNOS, CD31). Notably, VEGFR-1 and VEGFR-2 H3K27me3 signature at their promoter in murine
expression increased in non-expressing ESCs (39,40). This suggests that they are
HuAoVSMC following acute HDAC inhibition. transcriptionally poised in murine ESCs. This
We further implicated KAT7 as being raises the possibility that their robust expression
mechanistically and functionally important in may be regulated at the transcription elongation
maintaining VEGFR-1 and VEGFR-2 expression stage upon lineage commitment (39). Since both
in human ECs and in vivo in zebrafish ECs. VEGFR-1 and VEGFR-2 show a bivalent
Broad enrichment of acetylated histone H3K4me3 and H3K27me3 signature at their
residues in the intragenic regions has been noted promoters in human ESCs, their expression might
before, but its functional contribution to cell- be regulated in a similar manner (9). Thus, KAT7
specific gene expression has not been studied might catalyze broad intragenic histone acetylation
7
of VEGFR-2 in ECs in order to facilitate efficient ECs. The latter observation contradicts the
RNA polymerase II transcription elongation to common paradigm of genes replicating early and
establish its robust expression (supplemental Fig. late in S phase in expressing and non-expressing
S5). Consistent with the role of KAT7 in cells, respectively. In contrast, VEGFR-1 and
contributing to the robust expression of target VEGFR-2 were not differentially DNA methylated
genes, we observed reduced, but appreciable, and followed the common paradigm of replication
VEGFR-1 and VEGFR-2 expression even after timing (19). Here, we demonstrate that broad
KAT7 depletion. The loss of KAT7 therefore differential intragenic histone acetylation also
results in RNA polymerase II pausing and, distinguishes VEGFR-1 and VEGFR-2 from other
ultimately, transcription termination. Indeed, we EC-enriched genes.
noted overall reduced RNA polymerase II Although we argue that KAT7 acts on EC
occupancy across the VEGFR-2 genomic locus physiology through VEGFR-2 regulation, KAT7’s
after KAT7 knockdown (supplemental Fig. S5) role in DNA replication may contribute to its
(41). effects on EC physiology via specific effects on

Similar to VEGFR-2, the expression of VEGFR-1 and VEGFR-2 replication. KAT7 was
other KAT7 target genes in ECs might also be noted to have a role in replication licensing during
regulated at the transcription elongation level. the G1-S phase transition (31). Yet, no studies
Additionally, KAT7 regulated genes in ECs could have found replication timing defects at specific
be the common targets of transcription factors. genes following KAT7 disruption. Future studies
Indeed, it is suggested that KAT7 can be recruited should address this possibility, especially since
by trans factors, including NF-κB and the KAT7 depletion in ECs and HeLa cells in this
androgen receptor, to regulate transcription study and others, respectively, reduced cell
(42,43). Alternatively, KAT7 may indirectly proliferation and dysregulated genes with roles in
regulate the expression of some genes in ECs by cell proliferation, cell cycle, and DNA replication
acting on other upstream regulators. (18). In particular, we noted an increase in mRNA
Regarding our key finding of broad species associated with cell cycle and mitosis even
differential intragenic histone acetylation at select though cell proliferation is reduced. The increased
EC genes, it is likely not due to higher nucleosome expression of these mRNA species may represent
density. In VEGFR-2, differential AcH3 and a frustrated cellular response to cell cycle
AcH4 enrichment is still observed after impairment. Interestingly, only 9 mRNAs were
normalizing for histone H3 density. Instead, it is shared between KAT7-depleted ECs and HeLa
likely dependent on the competing activity of cellsi. This data highlight the role of KAT7
KAT and HDAC that can localize to intragenic function and cell-specific gene expression.
regions (3,10). Indeed, we showed that KAT7 KAT7 has been noted to have cell-specific
could localize to VEGFR-2, but not eNOS, to functions. Here, we argue that KAT7 regulates EC
mediate broad differential AcH3K14 and pan- gene expression and physiology. Previously,
AcH4 enrichment in its intragenic region. others have suggested that Brpf2-containing
Furthermore, our TSA studies suggest that class I KAT7 complexes play a role in murine fetal liver
and/or II HDACs might be regulating differential erythropoiesis and T lymphocyte development
broad histone acetylation at VEGFR-1 and (15,45). KAT7 is also implicated to be involved in
VEGFR-2. However, H3K36me3-dependent the early stages of adipocyte differentiation by
HDAC recruitment is unlikely to regulate broad being required for the process of mitotic clonal
differential histone acetylation at select EC genes expansion (46). Surprisingly, we noted that KAT7
as H3K36me3 occurred at all expressed genes is ubiquitously expressed in multiple human cell
(44). types and zebrafish embryos (32). Thus, the cell-
We previously analyzed the DNA specific functions of KAT7 may instead be
methylation profiles of key EC-enriched genes and imparted by the unique composition of cell-
found differential promoter DNA methylation at a specific KAT7 complexes (14,15,17,45). Indeed,
subset of these genes (e.g. eNOS). This finding JADE and Brpf proteins can act as interchangeable
was associated with their EC-enriched mRNA scaffolding subunits of KAT7 complexes that
expression and early S phase replication in non- determine their residue preference for histone
8
acetylation(14). Future studies should address the more prominent role (27,48). Although SIV were
composition of KAT7 complexes in different cell disrupted in both KAT7-depleted and KDRL-
types and how their composition can affect gene deficient zebrafish, KAT7-depleted zebrafish did
expression. not exhibit truncated ISVs (48). Also, they did not
Previous studies suggest that KAT7 is display aberrant ISV hyperbranching observed in
required for early embryonic development. KAT7- FLT1-depleted zebrafish (49). We infer that the
deficient mice have gross abnormalities in their phenotypic differences are due to the
immature vascular architecture, but developmental dysregulation of other genes in KAT7-depleted
arrest at the 10-somite stage and embryonic zebrafish. Alternatively, the respective phenotypes
lethality (E10.5), precluded later assessments (12). observed by deficiency in the VEGFR-1 and
Here, we found that KAT7 depletion perturbed EC VEGFR-2 orthologs might have been neutralized
function based on cell-based assays. Moreover, by their combined reduced expression. Regardless,
KAT7-depleted zebrafish showed disrupted SIV the ISVs of KAT7-depleted zebrafish were
formation and compromised circulatory integrity, perturbed based on reduced EC numbers in these

especially hemorrhage. Importantly, these defects vessels (3 dpf). Furthermore, initial ISV sprouting
could be rescued with human KAT7 RNA. appeared dysregulated suggesting a defect in cell
Overall, our study complements and extends the migration and proliferation that is consistent with
findings in the KAT7-deficient mice by invaluably reduced wound healing, cell migration, VEGFR-2
offering further insight into KAT7’s role in mature tyrosine 1175 phosphorylation, and cell
ECs and EC of the vasculature at advanced stages proliferation activity of KAT7-depleted ECs (50).
of embryonic development (47). The compromised circulatory integrity in KAT7-
Consistent with the vascular phenotype of depleted zebrafish might be due to vessel
KAT7-depleted zebrafish, KAT7 disrupted human underdevelopment in the ISVs. Alternatively, it
and zebrafish EC gene expression. Indeed, mRNA might reflect disrupted hematopoiesis as suggested
species that are downregulated by KAT7 depletion by KAT7’s role in murine fetal liver
are enriched for biological processes associated erythropoiesis (15). However, this is unlikely as
with EC physiology, including the regulation of others had previously reported that primitive and
blood vessel size. In particular, we found that definitive haematopoiesis were only mildly
KAT7 depletion has a major effect on VEGF affected in KAT7-depleted zebrafish (51).
biology in vitro and in vivo. Although basal Furthermore, hemorrhage and blood accumulation
VEGF-A expression was not affected by KAT7 in the trunk occurred in KAT7-depleted zebrafish.
knockdown in human ECs, we found that KAT7 In summary, our findings support the
depletion disrupted VEGFR-1 and VEGFR-2 importance of differential histone acetylation at
expression in human ECs and that this regulation both the promoter and intragenic regions of EC
was conserved in zebrafish based on the reduced genes. Although previous studies have shown a
expression of their homologs, FLT1, KDRL, and gene regulatory function of intragenic histone
KDR. Furthermore, acute VEGF-A signaling and modifications, their role in EC gene regulation has
VEGF-A EC phenotypic responses are blunted in not been noted (1). We posit that differential
cell culture. Importantly, exogenous KDRL intragenic histone acetylation plays a critical role
expression rescued the vascular plexus in the SIV in EC gene regulation and physiology and reveal a
and reduced blood circulation was restored in previously underappreciated role for KAT7 in
KAT7-depleted zebrafish. These findings further these processes, especially as it relates to VEGFR-
support KAT7 as a major regulator of EC 2. Also, evidence suggests that known intragenic
physiology and that it plays a major role in VEGF- histone modifying enzymes contributes to vascular
A biology in vitro and in vivo. development and function (52,53). These
Surprisingly, KAT7-depleted zebrafish emerging findings may suggest that intragenic
showed a perturbed vascular phenotype that was chromatin modifiers may be viable therapeutic
distinct from gene specific depletion of either targets for treating cardiovascular disease and
KDRL, KDR, or FLT1 alone. KDRL and KDR warrant further study (1).
regulate the formation and function of vessels
generated by angiogenesis with KDRL playing a Experimental Procedures
9
Cell Culture – Human umbilical vein endothelial Technologies) was added. Cells were cultured for
cells (HUVEC) were isolated from multiple another 48 h prior to use in matrigel, wound
independent donors and maintained as described healing, BrdU incorporation cell proliferation, and
previously (5-7). Studies were conducted with Caspase-3 apoptosis assays. For all other assays
early passage HUVEC (passage 3-5). Human that use siRNA transfected HUVECs, cells were
aortic smooth muscle cells (HuAoVSMC) cultured for another 72 h instead. Hypoxia
(ScienCell), human dermal microvascular ECs treatments of HUVECs (<1% O2) were conducted
(HMVEC) (Lonza), human hepatocytes (Lonza), as previously described for 0, 4 and 24h using a
normal human neonatal foreskin dermal temperature- and humidity-controlled incubator
fibroblasts (NHDF) (Lonza), and normal human with a sealed anaerobic system (ThermoForma,
epidermal keratinocytes (NHEK) (Lonza) were Model 1025) using a high purity anaerobic gas
cultured according to the supplier’s instructions. mixture (5% CO2, 10% H2, 85% N2; Linde).
Cells were maintained at 37 °C in 5% CO2 in a VEGF-A induction studies were performed with
humidified Steri-Cycle incubator (ThermoForma, recombinant human VEGF165 (293-VE) from R &

Model 370). Total cellular RNA was extracted D systems. HUVECs were induced with VEGF-A
from cultured cells as previously described in (7) for 3, 5, 10, and 30 min at a final concentration of
using the Solution D RNA extraction method (54). 50 ng/ml after serum starvation for 1h with Media
Total cellular protein was extracted from cultured 199 (Thermo Fisher Scientific) in the VEGF-A
cells using RIPA cell lysis buffer (Cell Signaling signal transduction studies.
Technology) according to the manufacturer’s
instructions. Protease/phosphatase inhibitor Lentiviral Transduction – His-eGFP and KAT7
cocktail (Cell Signaling Technology) was added in (UHN Vector Core Facility) lentivirus were used
the RIPA cell lysis buffer for total cellular protein to transduce HUVECs. Specifically, 30,000 to
extraction in VEGF-A signal transduction studies. 60,000 HUVECs were transduced with the
respective lentivirus at 10 multiplicity of infection
Cell Treatments – Trichostatin A (1 µM; Sigma) (MOI) in the presence of protamine at a final
studies were performed on HUVECs and concentration of 8 µg/ml for 24 h.
HuAoVSMCs as previously described for 4h with
an equivalent amount of ethanol added to control Antibodies Used – The following antibodies were
plates (6). siRNA knockdowns were conducted used in ChIP experiments: pan-acetyl histone H3
with 40 nM of siGENOME human KAT7 siRNA - (K9 and K14) (06-599), pan-acetyl histone H4
SMART pool (M-017668-00), which is composed (K5, K8, K12, and K16) (06-866), acetyl histone
of 4 unique KAT7 siRNAs (D-017668-01, D- H3 (K9) (06-942), acetyl histone H3 (K14) (07-
017668-02, D-017668-03, D-017668-04) and non- 353), acetyl histone H4 (K5) (06-759), acetyl
targeting siRNA (D-001210-03) from Dharmacon histone H4 (K8) (06-760), acetyl histone H4 (K12)
as previously described (7,55). The pool of KAT7 (06-761), acetyl histone H4 (K16) (07-329) from
siRNAs was used to reduce potential off-target Millipore; histone H3 (ab1791) from Abcam;
effects, without jeopardizing KAT7 specific RNA Polymerase II (Pol II) (N-20; sc-899),
knockdown (56). Potential off-target effects are normal rabbit IgG (sc-2027) and normal rabbit
reduced as a result of the siRNA pool being serum (sc-2338) from Santa Cruz Biotechnology.
composed of individual siRNAs at low Primary antibodies used in immunoblot analysis
concentrations (56). Briefly, siRNA transfections are as follows: eNOS (sc-654) and VEGFR-2 (sc-
were conducted on 90% confluent HUVECs 504) from Santa Cruz Biotechnology,
grown on 60-mm tissue culture plates using 33 µl KDRL/FLK-1 (RB-1526) and FLT-1 (RB-1527)
of Oligofectamine transfection reagent (Thermo antibodies from Lab Vision; phospho-VEGF
Fisher Scientific) in a total volume of 2000 µl. receptor 2 (Y1175) (19A10) from Cell Signaling
After 4 h of transfection at 37 °C in Opti-MEM Technology; and α-tubulin (T9026) from Sigma-
medium (Thermo Fisher Scientific), M-199 Aldrich. Mouse anti-rabbit IgG-HRP (sc-2004)
medium (Thermo Fisher Scientific) containing from Santa Cruz Biotechnology and rabbit anti-
fetal bovine serum (Hyclone), heparin, and mouse IgG-HRP (ab6728) from Abcam were used
endothelial cell growth supplement (Biomedical as appropriate secondary antibodies for
10
immunoblot analysis. The KAT7/HBO1 peaks using the moving average (MA) mode. The
(ab70183) and His-tags (ab9108) antibodies from MA algorithm uses a modified t-statistic averaged
Abcam were used in both ChIP and immunoblot over a sliding 400 bp window of adjacent probes
analysis. (58). False Discovery Rate (FDR) calculations are
based on a left tail distribution estimate of the MA
ChIP – ChIP was performed as previously test statistics. We used a MA cutoff of 3.0 for
described with the aforementioned antibodies peak selection. Based on the average probe
using ~1x 106 cells per ChIP, except for the KAT7 spacing on the custom Agilent arrays, a window
and His-tag ChIPs (5-7). The KAT7 and His-tag size of 3 was chosen for the tilemap configuration
ChIPs used ~3x 106 cells per ChIP instead. parameter file. Various choices for the
Samples were purified using the QIAquick PCR configuration of tilemap were tested during the
purification kit (QIAGEN) and analyzed by course of experiments to optimize peak finding
quantitative PCR (qPCR) as previously reported using both real and simulated data (58). Resulting
with Power SYBR Green Master Mix (Thermo peaks in each analysis were visualized with the

Fisher Scientific) using primers in Table 1(5-7). Integrated Genome Browser (IGB) (59). Genomic
regions with a log(2) fold change difference
ChIP- Custom High Resolution Tiling Array and between experimental and control ChIPs in
ChIP-seq data Analysis – ChIP samples were HUVEC that were ≥0.6 versus in HuAoVSMC
processed for ChIP-chip analysis by the UHN were considered EC-enriched. The ChIP-chip data
microarray centre (Toronto, Canada) using custom for pan-AcH3 (GSE93868) and pan-AcH4
tiling microarrays. The microarrays were designed (GSE93962) are deposited in the NCBI GEO
to tile across the non-repetitive DNA sequences of database. ChIP-seq signal enrichment tracks of
34 genes and the 50 kb regions flanking upstream AcH3K9, AcH3K27, and Pol II for HUVEC and
of their transcriptional start site and downstream NHEK were obtained from the ENCODE database
of their polyadenylation and cleavage signal. The and visualized using IGB (9,59).
genes included: i) 19 EC genes, ii) 6 broadly
expressed genes, and iii) 9 EC-excluded genes Gene Expression Microarray Analysis – Total
(supplemental Table S1). Both DNA strands of the RNA of HUVEC samples treated with control
genes were tiled by an average of 24 distinct DNA siRNA or KAT7 siRNAs (n = 4) were processed
probes per 5 bp on a 1M Agilent feature array with by Arraystar Inc for gene expression microarray
60-mers oligonucleotides. The oligonucleotides analysis. The microarray used for sample
were designed with the Agilent’s eArray program hybridization is the human LncRNA microarray
using the UCSC HG18 March 2006 human V3.0, a custom 8 x 60K Agilent array containing
genomic assembly. The data were processed 58944 probes that detect 26109 mRNA transcripts
according to a predefined pipeline. Briefly, and 30586 long noncoding RNA transcripts.
quantified data files from Agilent Feature Differentially regulated genes had > 2-fold change
Extraction software (Agilent) were first collected in expression between conditions (p≤ 0.05). The
into a single directory. Each file contained sense microarray data (GSE93608) is deposited in the
and corresponding antisense tiled probes to NCBI GEO database.
genomic regions surrounding genes of interest
(supplemental Table 1). Mean expression Quantitative Reverse Transcription-Polymerase
measurements (cy5 and cy3 channels) for each of Chain Reaction (qRT-PCR) – First strand cDNA
the strand probes were extracted and sorted into an synthesis was conducted on total cellular RNA
ascending chromosomal order as defined by the using the SuperScript® III First-Strand Synthesis
mid-point of the probe location in a format Supermix for qRT-PCR kit (Thermo Fisher
acceptable for input into the Tilemap command Scientific). cDNA was quantified by qPCR as
line software using custom scripts written in PERL previously described using primers in Table 1 (5-
(57). Preprocessing of the data consisted of 7). For the TSA experiments, VEGFR-1 and
quantile normalization followed by taking the log VEGFR-2 mRNA expression was determined by
base 2. Bar files were then created for input to the absolute quantification with plasmid standard
Tilemap v2 program to find pulled down fragment curves and subsequent normalization to
11
cyclophilin A mRNA levels. For other qRT-PCR succession with an approximate 10% overlap
experiments on human ECs, relative mRNA between adjacent tiles/images. The overlaps were
expression was determined by either absolute used to stitch individual tiles/images to generate
quantification with plasmid standard curves or the composite images for samples using the Zen Blue
comparative Ct method using 18s rRNA 2012 software.
expression for normalization. For qRT-PCR
experiments on zebrafish embryo samples, mRNA Wound Healing Assay – Wound healing assays
expression was determined by comparative Ct were performed in HUVECs grown on 6-well
using zebrafish b-actin for normalization. plates 48 h post-siRNA transfection as previously
described in (61). Composite images were
Immunoblots – Immunoblots were conducted as acquired from the Zeiss AxioObserver.Z1
previously described (7,55). Briefly, total protein microscope at 0 h and 20 h at 10X magnification
extracts were size fractionated with NuPAGE (numerical aperture: 0.3). The microscope was
Novex 4-12% Bis-Tris or 3-8% Tris acetate gels programmed to image a 3x15 tile region that

(Thermo Fisher Scientific) using the XCell represents most of the scratched area of a sample
SureLock Mini-Cell (Thermo Fisher Scientific) in rapid succession with an approximate 10%
and transferred onto 0.45-µm nitrocellulose overlap between adjacent tiles/images. The
membranes using the XCell II Blot Module overlaps were used to stitch individual tiles/images
according to the manufacturer’s recommendations to generate composite images for samples using
(Thermo Fisher Scientific). Immunoreactive bands the Zen Blue 2012 software. Images were
were detected using the aforementioned primary analyzed using a commercial imaging service
and secondary antibodies and visualized with the (Wimasis).
Amersham ECL Prime Western Blotting Detection
Reagent (GE Healthcare). Signal quantification Cell Migration Assay – HUVECs treated with
was performed using NIH ImageJ and normalized control siRNA or KAT7 siRNAs were serum
with α-tubulin. starved for 3h with Media 199 (Thermo Fisher
Scientific) after 72 h of siRNA transfection and
Matrigel Assay – Growth factor-reduced matrigel subsequently seeded (50 000 cells/well) on
(BD Biosciences) was plated onto 96-well plates fibronectin coated (50 µg/ml; Sigma Aldrich) 8.0
(50 µl/well), incubated at 37 °C for 30 minutes, µm pore polycarbonate transwell membrane
and seeded with HUVECs at 16,250 cells/well 48 inserts (Corning Inc.). Cell migration was
h post-siRNA transfection. Images were captured conducted for 4h in the presence of 0.1% BSA or
after 8 h using a Nikon Eclipse TS100 for VEGF165 (25 ng/mL) at the bottom of each well.
quantification. Specifically, one central image was Non-migrated cells were removed and inserts were
captured from each well for a total of 3 wells for subsequently fixed with 10% formalin (Thermo
each condition at 4X magnification (numerical Fisher Scientific) for 15 minutes. Inserts were then
aperture: 0.13). Images were analyzed using a washed and stained with 0.5% crystal violet
commercial imaging service (Wimasis; (Bioshop) and dried overnight. Membranes from
https://www.wimasis.com/en/) to obtain the the inserts were removed and mounted on glass
number of branch points and branches per field. slides with Permount (Thermo Fisher Scientific).
The number of isolated and network branches Membranes were visualized using an Olympus
were determined for the images with the ImageJ upright BX50 microscope equipped with a DP72
angiogenesis analyzer using default parameters camera and 5 unique fields of view at 10 X
(60). These experiments were conducted on three magnification (numerical aperture: 0.2) per
biological replicates. Representative composite membrane were captured for each sample. The
images were acquired using a Zeiss total number of cells in the 5 fields of view were
AxioObserver.Z1 microscope with environmental quantified by manual counting.
control (37 °C, 5 % CO2 with humidity) at 10X
magnification (numerical aperture: 0.3). The BrdU-Incorporation Cell Proliferation Assay –
microscope was programmed to image a 4x4 tile HUVEC treated with control siRNA or KAT7
region in a well that represents a sample in rapid siRNAs were subjected to BrdU cell proliferation
12
assays (Millipore) at 48 h post-treatment following dissection microscope (Leica M205 FA) for GFP
the manufacturer’s instructions in 96 well plates and red fluorescent protein (dsRED) fluorescence
(20 000 cells/ well). Colorimetric measurements signal after manual alignment of zebrafish. Images
were conducted using the SpectraMax M5e of living tg(fli1:nEGFP)y7 embryos were also
microplate reader (Molecular Devices). taken using a confocal microscope (Zeiss LSM
700). Embryos were anesthetized with 0.16 mg/ml
Caspase-3 Apoptosis Assay – Caspase-3 assays tricaine methanesulfonate (Finquel) and embedded
were conducted on HUVECs treated with control in 2.5% methyl cellulose (Sigma) or 1% low
siRNA or KAT7 siRNAs using the EnzChek® melting agarose (Bioshop) in the desired
Caspase-3 assay kit #1 (Thermo Fisher Scientific) orientation prior to imaging with the dissection or
at 48 h post-treatment according to the confocal microscope respectively. Total cellular
manufacturer’s instructions. The volume of cell protein was extracted from zebrafish embryos (3
lysates used in the assays was adjusted according dpf) with RIPA cell lysis buffer (Cell Signaling
to the amount of protein present in each sample. Technology) containing dissolved mini protease

Fluorescence measurements were conducted using inhibitor cocktail tablets (Roche) upon
the SpectraMax M5e microplate reader (Molecular homogenization with a microfuge pestle. To sort
Devices). GFP+ and GFP- cells from tg(kdrl:eGFP)
zebrafish embryos (3 dpf), embryos were
Zebrafish Husbandry and Ethics Statement – dechorionated, deyolked, and digested with
Zebrafish were housed in the Li Ka Shing TrypLE (Thermo Fisher Scientific) for 1 h at 28.5
Knowledge Institute research vivarium (Toronto, °C. The dissociated cells were resuspended in
Canada) and maintained and staged as previously PBS-1% BSA and sorted using a BD FACS Aria I
described (62). All zebrafish (Danio rerio) cell sorter as previously described (63). RNA was
experiments were conducted under the approved purified from pre-sorted, GFP+, and GFP- cells
protocol ACC403 of St. Michael’s Hospital using the PicoPure® RNA isolation kit (Thermo
Animal Care Committee (Toronto, Canada). Fisher Scientific) according to the manufacturer’s
Briefly, all fish strains were housed under a 14 h instructions. Zebrafish RNA was also used for
light: 10 h dark cycle at 28 °C. Pairs of appropriate standard RT-PCR to confirm KAT7 inhibition by
zebrafish strains were mated to produce embryos the E3I3 MO. It was performed with the following
that were raised in 1x E3 embryo medium (5 mM KAT7 primers: KAT7 ex3-ex4 amplicons: 5’-TTC
NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 GGA TGA CTC TGG AGA TC-3’ and 5’-TCC
MgSO4). TGG GAG GAG ACT CAT CT-3’; and KAT7
ex3-in3 amplicons: 5’-AAG CCA ACC AGC
Zebrafish Experiments – Tg(kdrl:eGFP), GCA ATA AC-3’ and 5’-CAG CCT GGC CTT
tg(gata1:dsRED), and tg(fli1:nEGFP)y7 zebrafish TTT TCT GT-3’ under the following PCR
strains were used. The following morpholinos conditions: 94 °C denaturation for 5 min., 35
(MO) were designed for KAT7 inhibition: E1ATG cycles of 94 °C for 30 s, 57.9 °C and 55.6 °C for
MO (5¢-CATGTTCCCTCCGATAAATCCAATC- 30 s respectively, and 72 °C for 30 s, and 72 °C
3¢) and E3I3 MO (5¢- final extension for 15 min.
TGAGCTGTGTGTTTTTACCTTGAGA-3¢). The
MOs (GeneTools) (2-4 nl of 0.100 mM solution) Statistical Analysis – Statistical analyses were
were injected into embryos at the 1 cell stage. 5¢- performed using a Student’s t-test or analysis of
capped human KAT7 RNA and 5¢-capped variance and Newman-Keuls post hoc test, as
zebrafish KDRL RNA were synthesized using the appropriate. A p-value ≤0.05 was considered
mMESSAGE mMACHINE kit according to the statistically significant.
manufacturer’s instructions (Thermo Fisher
Scientific). A total of 200 pg KAT7 and 20 pg Acknowledgements
1
KDRL RNA were injected into embryos at the 1 M.S.Y. is a recipient of a CIHR Frederick
cell stage for rescue experiments, respectively. Banting and Charles Best Canada graduate
Images were taken from living embryos with a scholarship. 2P.J.T. and M.K.D. are recipients of
Queen Elizabeth II Graduate Scholarship in
13
Science and Technology. 3H.S.J.M. is a recipient

of a CIHR Training Program in Regenerative Author contributions
Medicine Fellowship. 4J.J.D.H. is a recipient of a Contributions: M.S.Y, P.J.T., H.S.J.M., M.K.D.,
CIHR Fellowship. 5This work was supported by J.J.D.H., S.E, and Y.D.W. performed experiments
grant MOP-1423017 from CIHR to P.A. Marsden. and collected data; X.Y.W. provided reagents and
gave critical intellectual input; and M.S.Y. and
Conflict of Interest P.A.M. designed the research and wrote the paper.
The authors declare no competing financial
interest.
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Footnotes
i
Unpublished observations.
ii
The abbreviations used are: KAT, histone acetyltransferase; HDAC, histone deacetylase; EC, endothelial
cell; HUVEC, human umbilical vein endothelial cell; HuAoVSMC, human aortic vascular smooth muscle
cell; EC genes, EC-enriched genes; Pol II, RNA polymerase II; NHEK, normal human epithelial
keratinocytes; ENCODE, Encyclopedia of DNA Elements; VEGFR, vascular endothelial growth factor
receptor; TSA, trichostatin A; qPCR, quantitative PCR; ISV, intersegmental vessel; dpf, days post
fertilization; SIV, subintestinal vein; HMVEC, human dermal microvascular endothelial cell; NHDF,
normal human neonatal foreskin dermal fibroblasts.
18
Table 1. Primers used in qRT-PCR and ChIP-qPCR analysis

Primer name Primer sequence
18S rRNA transcript 5’- AGG AAT TGA CGG AAG GGC AC -3’
5’- GGA CAT CTA AGG GCA TCA CA -3’
ABCG1 transcript 5’- GCT GGA GCT GGT GAA CAA C-3’
5’- GGT GGA TGG TGC AAA TGA T -3’
ACE1 transcript 5’- AGG CCA ACT GGA ACT ACA A -3’
5’- TCT GCA ACT GGT TCA CAT C -3’
ASS1 transcript 5’- GCC CGC AAA CAA GTG GAA AT -3’
5’- CAT CCT CCA GGG AGC AAT GAC -3’
CASZ1 transcript 5’- CCT CCC TGT CCT TCA ACA CT -3’
5’- TGA CGG CTG GTT TAT CTG TG -3’
CD34 transcript 5’- CAG GCA TCA GAG AAG TGA AAT T -3’
5’- CCC TCT CCC CTG TCC TTC TTA AA -3’
CEACAM1 transcript 5’- CAG CCC CAC TTC ACA GAG TG -3’
5’- GCG GGT TCC AGA AGG TTA GA -3’
CXCL16 transcript 5’- CAT GGG TTC AGG AAT TGA TGA -3’
5’- GGG GCT GGT AGG AAG TAA ATG -3’
Cyclophilin A transcript 5’- GAC GGC GAG CCC TTG G -3’
5’- TCT GCT TTT GGG ACC TTG T -3’

eNOS transcript 5’- GGC ATC ACC AGG AAG AAC ACC -3’
5’- TCA CTC GCT TCG CCA TCA C -3’
E-Selectin transcript 5’- AGC TGT GAG GAG GGA TTT GAA TTA -3’
5’- ACT GCC AGG CTT GAA CAT TTT A -3’
KAT7 transcript 5’- AGA GGC AGC TTC GAT ATA AG -3’
5’- TCG CTT GTC AGG TTT TCT A -3’
Luciferase transcript 5’- ACT CCT CTG GAT CTA CTG GTC -3’
5’- GTA ATC CTG AAG GCT CCT CA -3’
RGS4 transcript 5’- TTC CCA CAA CAA GAA GGA CAA -3’
5’- CCA GCC CAC ATT CAT GAC TAA -3’
TIE1 transcript 5’- GGC GGG TGC CCC CTT TCT TG -3’
5’- GCC AGC AGC GTC AGG TCC AC -3’
TIE2 transcript 5’- CAG CTT GCT CCT TTC TGG AAC T -3’
5’- CTA TGG TGA TGG GCT CAT GG -3’
VEGFR-1 transcript 5’- CAG TGT GAG CGG CTC CCT TAT G -3’
5’- CAC AGT CCG GCA CGT AGG TGA T -3’
VEGFR-2 transcript 5’- TTA CTA TTC CCA GCT ACA TGA TCA G -3’
5’- AGA CGG ACT CAG AAC CAC ATC ATA A -3’
VEGFR-3 transcript 5’- CCG CCA GCT CCT ACG TGT TC -3’
5’- CGG GGA TGG ACA CCA GAC AG -3’
VEGFR-2 +45/-12 5’- CGC AGC GAC CAC ACA TTG ACC -3’
5’- TCC CAC CCT GCA CTG AGT C -3’
VEGFR-2 +1928/+1786 5’- CAA GCT TGA GCT GGA CTC TT -3’
5’- GAG CTG GAA GGG TAA CAG TG -3’
VEGFR-2 +5092/+5020 5’- GTG GGG AAA GAT GCA TTC CAG -3’
5’- CTG AGC GCA TGA TCT ACA TAA -3’
VEGFR-2 +15671/+15524 5’- AAC CAA GCC AAC AGT GTC AAC -3’
5’- ATT GCC CAC CTG GTA CAC A -3’
VEGFR-2 +28760/+28706 5’- GGG AAG TGT AGC AGT CCA TAC AG -3’
5’- GGC TGA TGG CAC TAA GAC TAT GT -3’
VEGFR-2 +46097/+46024 5’- AAG CCT CTT GGA TAG ACT CAG -3’
5’- AAC CAG GCA ATG TAA GTG TT -3’
eNOS promoter 5’- GTG GAG CTG ACG CTT TAG AGC -3’
5’- TTT CCT TAG GAA GAG GGA GGG -3’
Zebrafish β-actin 5’- CCC GTC CTG CTC ACA GAG G -3’
5’- CGG GGG TGT TGA AGG TCT C -3’
Zebrafish CDH5 5’- TGG AAA TGG GAT AAA CTG TAT G -3’
5’- GCT CCA TCT CCT TTG AGA ATA T -3’
Zebrafish KDRL 5’- CAT CCA TTC CTC AAT GTT ACT C -3’
5’- GGC CAC TCC ATC TTT GTA C -3’
Zebrafish FLT1 5’- CAA GCA AGA CCA AGA GAA TG -3’
5’- CAA GCT GCT TCT CAG TAA CAT C -3’
Zebrafish KDR 5’- CGG TCC AGC ACT TCT GTC T -3’
5’- AGG TTG GAG GAT CAG GTA CAA -3’
Zebrafish NOS1 5’- GGG CCA GAG GGT CAT CGA TA -3’
5’- GTT CTG TCC ATT TCC CAC ACC -3’
Zebrafish KAT7 E3E4 5’- TTC GGA TGA CTC TGG AGA TC -3’
5’- TCC TGC GAG GAG ACT CAT CT -3’
Zebrafish KAT7 E3I3 5’- AAG CCA ACC AGC GCA ATA AC -3’
5’- CAG CCT GGC CTT TTT TCT GT -3’
19
Figure Legends
Figure 1. Histone modification and RNA polymerase II profiles of KDR/VEGFR-2. A, histone H3
(pan-AcH3; AcH3K9, AcH4K14) and H4 (pan-AcH4; AcH4K5, AcH4K8, AcH4K12, AcH4K16)
acetylation profiles of KDR/VEGFR-2 in human umbilical vein endothelial cells (HUVEC) and human
aortic vascular smooth muscle cells (HuAoVSMC). The histone acetylation level at each genomic
location is defined as the fold difference between the pan-AcH3/pan-AcH4 ChIP (Cy5) and the control
rIgG ChIP (Cy3) hybridization signals. Only fold differences between pan-AcH3/pan-AcH4 ChIP and
isotype control ChIP that are ≥2-fold are shown. Biological replicates of pan-AcH3 (n=2) and pan-AcH4
(n=3) ChIP-chip analysis showed similar results. B, AcH3K9, AcH3K27, H3K36me3 and RNA
polymerase II (Pol II) ChIP-seq profiles of KDR/VEGFR-2 in HUVECs and normal human epithelial
keratinocytes (NHEK). The HUVEC and NHEK-associated ChIP-seq profiles were obtained from the
ENCODE database (9). The enrichment levels of AcH3K9, AcH3K27, H3K36me3, and Pol II are
represented by normalized intensities.

Figure 2. Histone modification and RNA polymerase II profiles of FLT1/VEGFR-1. A, histone H3
(pan-AcH3; AcH3K9, AcH4K14) and H4 (pan-AcH4; AcH4K5, AcH4K8, AcH4K12, AcH4K16)
acetylation profiles of FLT1/VEGFR-1 in human umbilical vein endothelial cells (HUVEC) and human
aortic vascular smooth muscle cells (HuAoVSMC). The histone acetylation level at each genomic
location is defined as the fold difference between the pan-AcH3/pan-AcH4 ChIP (Cy5) and the control
rIgG ChIP (Cy3) hybridization signals. Only fold differences between pan-AcH3/pan-AcH4 ChIP and
isotype control ChIP that are ≥2-fold are shown. Biological replicates of pan-AcH3 (n=2) and pan-AcH4
(n=3) ChIP-chip analysis showed similar results. B, AcH3K9, AcH3K27, H3K36me3 and RNA
polymerase II (Pol II) ChIP-seq profiles of KDR/VEGFR-2 in HUVECs and normal human epithelial
ENCODE database (9). The enrichment levels of AcH3K9, AcH3K27, H3K36me3, and Pol II are
Figure 3. Histone modification and RNA polymerase II profiles of NOS3/eNOS. Representative pan-
AcH3 and -H4 acetylation profiles throughout (A) NOS3 in human umbilical vein endothelial cells
(HUVEC) and human aortic vascular smooth muscle cells (HuAoVSMC). The histone acetylation level at
each genomic location is defined as the fold difference between the pan-AcH3/pan-AcH4 ChIP (Cy5) and
the isotype control ChIP (Cy3) hybridization signals. Only fold differences between pan-AcH3/pan-
AcH4 ChIP and isotype control ChIP ≥2-fold are shown. Biological replicates of pan-AcH3 (n=2) and
pan-AcH4 (n=3) ChIP-chip analysis showed similar results. ChIP-seq data profiles of AcH3K9,
AcH3K27, H3K36me3 and Pol II throughout (B) NOS3 in HUVEC and normal human epithelial
ENCODE database (9). The enrichment levels of AcH3K9, AcH3K27, H3k36me3, and Pol II are
Figure 4. Pan-AcH3, pan-AcH4, H3 and Pol II profiles of select genomic regions in VEGFR-2.
Select regions in the VEGFR-2 genomic locus were validated for their endothelial enrichment of (A) pan-
AcH3, (B) pan-AcH4, and (F) Pol II by ChIP-qPCR in human umbilical vein endothelial cells (HUVEC)
and human aortic vascular smooth muscle cells (HuAoVSMC). The positions of the analyzed amplicons
are numbered relative to the VEGFR-2 transcription start site. C, The histone H3 profiles at select
genomic regions of VEGFR-2 were determined by ChIP-qPCR. The (D) pan-AcH3 and (E) pan-AcH4
profiles at select genomic regions of VEGFR-2 after normalizing for H3 enrichment at their respective
genomic regions to account for nucleosomal density. Data represent mean ± S.E. (error bars) of three
independent experiments. * denotes statistical significance at p≤0.05 compared to HUVECs.
Figure 5. Pan-AcH3 and Pan-AcH4 profiles of PPIA, and CDH1. Representative pan-AcH3 and -H4
acetylation profiles throughout (A) PPIA and (B) CDH1 in human umbilical vein endothelial cells
20
(HUVEC) and human aortic vascular smooth muscle cells (HuAoVSMC). The histone acetylation level at
each genomic location is defined as the fold difference between the pan-AcH3/pan-AcH4 ChIP (Cy5) and
the isotype control ChIP (Cy3) hybridization signals. Only fold differences between pan-AcH3/pan-
AcH4 ChIP and isotype control ChIP ≥2-fold are shown. Biological replicates of pan-AcH3 (n=2) and
pan-AcH4 (n=3) ChIP-chip analysis showed similar results.
Figure 6. The effects of TSA on VEGFR-1 and VEGFR-2 steady state mRNA levels. Steady state
mRNA levels of VEGFR-1 and VEGFR-2 were determined at 0 and 4 h post-TSA (1 µM) treatment in
(A) human aortic vascular smooth muscle cells (HuAoVSMC) and (B) human umbilical vein endothelial
cells (HUVEC), respectively. Values are shown relative to 0 h TSA treatment. Data represents the mean ±
S.E. (error bars) of three independent experiments. * denotes a statistical significance of p≤0.05 compared
with 0 h of TSA treatment.
Figure 7. KAT7 depletion perturbs VEGFR-2 and endothelial cell gene expression. A, steady state

mRNA levels of KAT7 in 2 endothelial cell types and 4 non-endothelial cell types were determined by
qRT-PCR. The 2 EC types are human umbilical vein endothelial cells (HUVEC) and human
microvascular endothelial cells (HMVEC). The 4 non-endothelial cell types are human aortic vascular
smooth muscle cells (HuAoVSMC), hepatocytes, normal human dermal fibroblasts (NHDF), and normal
human epithelial keratinocytes (NHEK). Data represent mean ± standard error (error bars) of three
independent experiments. * denotes statistical significance at p≤0.05 for HMVEC compared to
hepatocyte. B, representative immunoblot of KAT7, VEGFR-2, eNOS, and a-tubulin in control and
KAT7-depleted endothelial cells (ECs). Quantification of KAT7, VEGFR-2, and eNOS immunoblots are
shown. Data represent mean ± S.E. (error bars) of three independent experiments. * denotes a statistical
significance of p≤0.05 compared with control siRNA. C, volcano plot of mRNA in control vs KAT7-
depleted ECs (n=4). D, Gene ontology (GO) analysis of biological processes that are enriched for genes
downregulated by KAT7 depletion. E, relative expression levels of differentially regulated mRNA in
control vs KAT7-depleted ECs that are highlighted (red) in (C), eNOS, TIE1, and TIE2 in control and
KAT7-depleted ECs. Data represent mean ± S.E. (error bars) of three independent experiments. * denotes
a statistical significance of p≤0.05 compared with control siRNA.
Figure 8. KAT7 depletion disrupts chromatin-based transcriptional regulation of VEGFR-2. A,

Schematic of the VEGFR-2 locus with genomic regions that are analyzed in blue. ChIP-qPCR analysis of
(B) RNA polymerase II, (C) pan-AcH4, and (D) AcH3K14 levels at highlighted VEGFR-2 regions in
control and KAT7-depleted human endothelial cells. Histone acetylation levels are normalized for H3
enrichment. The analyzed regions are numbered with respect to the VEGFR-2 transcription start site. Data
represents mean ± S.E. (error bars) of three independent experiments. * denotes statistical significance at
p≤0.05 compared to control siRNA.
Figure 9. KAT7 localizes to the VEGFR-2 locus. A, Representative immunoblots of KAT7 and His-
eGFP on His-eGFP and KAT7-transduced human endothelial cells (ECs). Quantification of KAT7 and
His-tagged eGFP immunoblots of His-eGFP (white) and KAT7 (black)-transduced ECs are shown. Data
represent mean ± S.E. (error bars) of three independent experiments. * denotes a statistically significant
difference at p≤0.05 for protein expression between His-tagged eGFP- and KAT7-transduced ECs. B,
Representative ChIP-qPCR analysis of His-eGFP and KAT7 localization at the VEGFR-2 genomic locus
and the eNOS promoter in His-tagged eGFP and KAT7 lentivirus-transduced ECs. Biological replicates
show similar results (n=3).
Figure 10. KAT7 depletion disrupts endothelial cell activity. A, representative images of matrigel
assays on control and KAT7-depleted ECs. The proportion of isolated and network branches, number of
branch points, and total number of branches in matrigel assays on KAT7 siRNAs vs control siRNA
21
treated HUVECs are quantified. Data represent mean ± S.E. (error bars) of three replicates in a
representative experiment of the matrigel assay. Biological replicates show similar results (n = 3). *
denotes a statistical significance of p≤0.05 compared with control siRNA. B, representative images of
wound healing assay on control and KAT7-depleted ECs with the quantification of the scratch area
recovered. Data represents mean ± S.E. (error bars) of three independent wound healing experiments. *
denotes a statistical significance of p≤0.05 compared with control siRNA. Representative composite
images of the matrigel and wound healing assays were acquired from a Zeiss/AxioObserver.Z1
microscope (magnification: 10X, numerical aperture: 0.3) equipped with an EC Plan-Neofluar objective
lens and environmental control (37 °C and 5% CO2 with humidity) using a Hamamatsu ORCA-R2
camera. Composite images were extracted with the Zen Blue 2012 software. The black dotted lines in the
composite images of the matrigel assay and grey dotted lines in the composite images of the wound
healing assays denote the splice sites between stitched individual images. C, representative images of cell
migration assays conducted on control and KAT7-depleted ECs in the presence of VEGF-A165. The
number of migrated cells is quantified. Representative images of the cell migration assays were acquired

from an Olympus upright BX50 microscope (magnification: 10X, numerical aperture: 0.2) equipped with
a DP72 camera. Images were extracted using the CellSens software. Data represents mean ± S.E. (error
bars) of four independent experiments. * denotes a statistical significance of p≤0.05 compared with
control siRNA in the absence of VEGF-A165. D, representative immunoblot of VEGFR-2 Tyrosine 1175
Phosphorylation (P-VEGFR-2 Y1175), VEGFR-2, KAT7, and a-tubulin in control and KAT7-depleted
endothelial cells (ECs) treated with VEGF-A165 for 0, 3, 5, 10, and 30 min. Gaps that represent empty
single lanes between the control and KAT7-depleted samples in the P-VEGFR2 Y1175, KAT7, and a-
tubulin immunoblots were removed from the images. Quantification of VEGFR-2 tyrosine 1175 (Y1175)
phosphorylation immunoblots after normalizing for VEGFR-2 expression. Data represent mean ± S.E.
(error bars) of three independent experiments. * denotes a statistical significance of p≤0.05 compared
with control siRNA.
Figure 11. KAT7 morpholino (MO) inhibition in zebrafish embryos impedes initial intersegmental
vessel (ISV) formation. A, KAT7 mRNA expression profile of zebrafish embryos. KAT7 mRNA
expression was determined in tg(kdrl:eGFP) zebrafish embryos (3 days post fertilization; 3 dpf) prior to
fluorescence activated cell-sorting (FACS; Pre-sort) and post-FACS in accordance with GFP expression.
KAT7 mRNA levels in endothelial cell (EC)-enriched GFP+ and EC-depleted GFP- cells are expressed
relative to their mRNA levels in corresponding Pre-sort samples. Data represents mean ± S.E. (error
bars) of three independent experiments. * and § denote statistical significance at p≤0.05 for Pre-sort
versus GFP- cells and GFP+ versus GFP- cells, respectively. B, representative KAT7 and a-tubulin
immunoblots of control, E1ATG, and E3I3 MO treated zebrafish embryos (3 dpf). Immunoblots of KAT7
are quantified. Data represents mean ± S.E. (error bars) of three independent experiments. * denotes a
statistical significance of p≤0.05 compared with control MO. C, Quantification of the average number of
nuclei observed in 10 similarly located ISVs of tg(fli1:nGFP) zebrafish embryos (24-34 hours post
fertilization; 24-34 hpf). Quantification was conducted by manual nuclei counting. Data represent mean ±
S.E. of 12 control and 13 E1ATG MO-treated zebrafish, respectively. * denotes statistical significance of
p≤0.05 compared to Control MO. Experiments were conducted 3 times with similar results. D,
Representative composite fluorescence images of control and E1ATG MO-treated tg(fli1:nGFP)
zebrafish embryos between 24 to 34 hpf (magnification: 73.2, numerical aperture: 0.20). All zebrafish
were manually aligned prior to imaging. All fluorescence images were acquired using a Leica M205 FA
dissecting microscope equipped with a Leica DFC 365 FX digital camera and extracted with the Leica
Application Suite Advanced Fluorescence software.
Figure 12. KAT7 morpholino (MO) inhibition in zebrafish embryos disrupts vascular structure and
circulation. A, quantification of the abnormal phenotypes in zebrafish embryos treated with control MO,
E1ATG MO, E1ATG MO with human KAT7 RNA, and E3I3 MO. Data represents mean ± S.E. (error
22
bars) of three independent experiments. Each experiment consists of 30-130 and 20-50 embryos for the
measurements of SIV defects and other phenotypes, respectively. Specifically, a total of 147 control MO
treated zebrafish were used for assessing the frequency of all phenotypes. A total of 229, 125, and 140
E1ATG MO treated zebrafish were used for assessing the frequency of the SIV, circulatory integrity
(circulation) and hemorrhage phenotypes, respectively. A total of 251, 142, and 132 E1ATG MO + KAT7
RNA treated zebrafish were used for assessing the frequency of the SIV, circulatory integrity (circulation)
and hemorrhage phenotypes, respectively. A total of 101, 156, and 149 E3I3 MO treated zebrafish were
used in determining the frequency of the SIV, circulatory integrity (circulation), and the hemorrhage
phenotypes, respectively. * denotes a statistical significance of p≤0.05 compared with control MO, while
¶ represents a statistically significant difference between E1ATG MO vs E1ATG MO + KAT7 RNA.
Representative fluorescence images of tg(kdrl:eGFP, gata1:dsRED) zebrafish embryos (3 dpf) treated
with MOs and human KAT7 RNA showing effects on (B) Subintestinal vein (SIV) formation
(magnification: 111X, numerical aperture: 0.10), (C) circulatory integrity (magnification: 38.3, numerical
aperture: 0.12), and (D) hemorrhage (see arrow; magnification: 119X, numerical aperture: 0.25). The

dorsal aorta, intersegmental vessels, caudal vein, and subintestinal vein are labeled as DA, ISV, CV, and
SIV, respectively. The images in (C) are composite fluorescence images. All zebrafish were manually
aligned prior to imaging. All fluorescence images were acquired using a Leica M205 FA dissecting
microscope equipped with a Leica DFC 365 FX digital camera using the Leica Application Suite
Advanced Fluorescence software.
Figure 13. KAT7 morpholino (MO) inhibition in zebrafish embryos reduces the number of
endothelial cells in the intersegmental vessels of zebrafish embryos. Representative fluorescence
images (magnification: 38X, numerical aperture: 0.08) of tg(fli1:nGFP) zebrafish embryos (3 dpf) that
were injected with the control, E1ATG MO, and E3I3 MO. Fluorescence images (magnification: 10X,
numerical aperture: 0.45) on magnified subsets of intersegmental vessels (ISV) (boxed) are also shown.
The average number of nuclei observed in an ISV of the MO-treated tg(fli1:nGFP) zebrafish embryos (3
dpf) were quantified by manual nuclei counting. Specifically, they were counted and averaged across 10
similarly located ISVs of each embryo. Data represents mean ± S.D. of 19 control MO-treated zebrafish
embryos and 17 E1ATG and 17 E3I3 MO-treated zebrafish embryos. * denotes a statistical significance at
p≤0.05 compared to control MO. Fluorescence images, except for the magnified subsets of ISVs, were
acquired using a Leica M205 FA dissecting microscope equipped with a Leica DFC 365 FX digital
camera and extracted with the Leica Application Suite Advanced Fluorescence software. The images of
the magnified subsets of ISVs were acquired using a Zeiss LSM 700 confocal microscope equipped with
a point scanner and extracted as maximum intensity projections with the Zen Black 2012 software.
Figure 14. KAT7 morpholino (MO) inhibition in zebrafish embryos disrupts endothelial cell gene
expression. mRNA expression profiles were determined for (A) CDH5, (B) NOS1, (C) KDRL, (D) KDR,
and (E) FLT1 in control MO and KAT7 MO treated tg(kdrl:eGFP) zebrafish embryos (3 dpf) that were
sorted according to their GFP expression. All mRNA levels of genes in endothelial cell (EC)-enriched
GFP+ and EC-depleted GFP- cells were expressed relative to their expression levels in corresponding
whole zebrafish embryos. Data represents mean ± S.E. (error bars) of three independent experiments. *,
§, ¶ denote statistical significance at p≤0.05 for control MO treated GFP+ compared to KAT7 MO treated
GFP+ cells, control MO treated GFP- cells, and KAT7 MO treated GFP- cells, respectively. F,
representative immunoblot of KDRL, FLT1, KAT7, and a-tubulin in control and KAT7 MO treated
tg(kdrl:eGFP) zebrafish embryos (3 dpf). Quantification of KDRL, FLT1, and KAT7 immunoblots are
shown. Data represent mean ± S.E. (error bars) of three independent experiments. * denotes a statistical
significance of p≤0.05 compared with control siRNA.
Figure 15. Reduced KDRL expression contributes to the disrupted vascular structure and
circulation of KAT7-depleted embryos. A, quantification of the abnormal phenotypes in zebrafish
23
embryos treated with control MO, E1ATG MO, E1ATG MO with zebrafish KDRL RNA. Data represents
mean ± S.E. (error bars) of 4-5 independent experiments. Each experiment was conducted with 20-130
Control MO, 70-140 E1ATG, and 60-130 E1ATG MO + KDRL RNA treated zebrafish embryos for the
measurements of all phenotypes. Specifically, a total of 239 and 289 control MO treated zebrafish were
used for assessing the frequency of SIV defects and other phenotypes, respectively. A total of 520 and
425 E1ATG MO treated zebrafish were used for assessing the frequency of SIV defects and other
phenotypes, respectively. A total of 461 and 412 E1ATG MO + KDRL RNA treated zebrafish were used
for assessing the frequency of SIV and other phenotypes, respectively. * denotes a statistical significance
of p≤0.05 compared with control MO, while ¶ represents a statistically significant difference between
E1ATG MO vs E1ATG MO + KDRL RNA. Representative fluorescence images of tg(kdrl:eGFP,
gata1:dsRED) zebrafish embryos (3 dpf) treated with MOs and zebrafish KDRL RNA showing effects on
(B) Subintestinal vein (SIV) formation (magnification: 74.7X, numerical aperture: 0.10), (C) circulatory
integrity (magnification: 23.9, numerical aperture: 0.05), and (D) hemorrhage (see arrow; magnification:
74.7X, numerical aperture: 0.10). The dorsal aorta, intersegmental vessels, caudal vein, and subintestinal

vein are labeled as DA, ISV, CV, and SIV, respectively. All fluorescence images were acquired using a
Leica M205 FA dissecting microscope equipped with a Leica DFC 365 FX digital camera using the Leica
Application Suite Advanced Fluorescence software.
24
A
4
[ log2(ChIP/IgG)]
Pan-AcH3
Probe Intensity
1
HUVEC 4
Pan-AcH4 1
4
HuAo Pan-AcH3 1
VSMC Pan-AcH4 4
1
20 kb
B
100
AcH3K9
Normalized Intensity (Arbitrary Units)
0
500
AcH3K27 0
HUVEC 40
H3K36me3 0
50
Pol II 0
100
AcH3K9 0
500
AcH3K27 0
NHEK 40
H3K36me3 0
50
Pol II 0
20 kb
25
Figure 1.
A
4
Pan-AcH3
1
[ log2(ChIP/IgG)]
HUVEC
Probe Intensity 4
Pan-AcH4
1
4
Pan-AcH3
HuAo 1
VSMC 4
Pan-AcH4
1
50 kb
B
200
AcH3K9
0
500
Normalized Intensity (Arbitrary Units)
AcH3K27
0
HUVEC 40
H3K36me3
0
50
Pol II
0
200
AcH3K9
0
500
AcH3K27
0
NHEK 40
H3K36me3
0
50
Pol II
0
50 kb
26
Figure 2.
A
4
Pan-AcH3
[ log2(ChIP/IgG)]
1
Probe Intensity
HUVEC 4
Pan-AcH4
1
4
Pan-AcH3
HuAo 1
VSMC 4
Pan-AcH4
1
10 kb
B
80
AcH3K9
Normalized Signal (Arbitrary Units)
0
100
AcH3K27
HUVEC 0
20
H3K36me3 0
30
Pol II
0
80
AcH3K9
0
100
AcH3K27 0
NHEK 20
H3K36me3 0
30
Pol II 0
10 kb
27
Figure 3.
A
D
H3 normalized IP DNA
IP DNA (Arbitrary Units)
(Arbitrary Units)
0
2
4
6
8
0
2
4
6
8
10
12
14
16
10
12
14
16
*
Pan-AcH3
Pan-AcH3/H3
E
B
IP DNA (Arbitrary Units)

0
3
6
9
12
15
18
21
24
*
Pan-AcH4
Pan-AcH4/H3
F
C
IP DNA (Arbitrary Units) IP DNA (Arbitrary Units)

0
3
6
9
12
15
18
21
24
0
1
2
3
4
5
6
7
8

H3
Pol II
28
Figure 4.
A
4
Pan-AcH3
[ log2(ChIP/IgG)]
HUVEC Probe Intensity 1
4
Pan-AcH4
1
4
Pan-AcH3
HuAo 1
VSMC 4
Pan-AcH4
1
20 kb
B
4
Pan-AcH3
1
[ log2(ChIP/IgG)]
HUVEC
Probe Intensity
4
Pan-AcH4
1
4
Pan-AcH3
HuAo 1
VSMC 4
Pan-AcH4
1
20kb
29
Figure 5.
A B
HuAoVSMC HUVEC
5 5
Relative Quantity
Relative Quantity
4 * 4
*
3 3
VEGFR-1
2 2 VEGFR-2
1 1
0 0
0h 4h 0h 4h
TSA treatment TSA treatment
30
Figure 6.
E
A
C
Relative Quantity
# of copies per
0.2
0.4
0.6
0.8
1.2
0
1
K 1 ug RNA
AT
A
*
B 7
C
10000
15000
20000
25000
5000
0
G H
1 U
*
A VE
C C
E1 H
*
A H MV
SS uA E
C 1
*
oV C
A H S
SZ ep MC
1
*
at
C CD oc
EA 3 yt
*
C 4 e
*
A N
C M1 H
*
XC EK
L1 N
6 H
*
D
ES
VE E F
G L
*
F
VE R-
G 1
*
F
D
B
VE R-
G 2
*
FR
-3
*
KAT7
α-tubulin
VEGFR-2
eNOS
0
1
2
3
4
5
6
7
R
G
S
*
eN 4
O
S
TI
E1
50
60
80
60
110
160
160
260
260
kDa
TI
E2
Relative Expression
0.2
0.4
0.6
0.8
1.2
1.4
0
1
T7
VE
*
*
31
KA GFR eN
-2 OS
Figure 7.
B
A
Relative Pol II
normalized
Normalized IP DNA
0.5
1.5
2.5
0
1
2
+45/-2
+1 +45
92 /-2
*
7/
+5 + 1
09 7 86
*
+1 2/
56 +50
+1927/+1786
71 20
*
+2 /+
87 155
Pol II
60 24
*
/+
+5092/+5020
+4 2
60 8
97 7 06
/+
46
02
9
*
C
Relative AcH4/H3
Normalized
normalized IPIP DNA
DNA
0.5
1.5
0
1
2
+4
5
+15671/+15524
+1
9 27
/-2
/
+5 +17
09 86
+1 2/
56 +50
71 20
*
/+
KDR
+2
87 155
60 24
+4 /+
60 287
Pan-AcH4
97 06
*
/+
46
02
9
+28760/+28706
Relative AcH3K14/H3
normalized DNA
NormalizedIPIPDNA
0
0.5
1
1.5
2
+4
+1 5/
92 -2
7 /+
+5 17
09 86
*
+1 2
/+
567 5 02 Downloaded from http://www.jbc.org/ at UNIVERSIDADE DO PORTO on October 9, 2018
0
*
1/
+2 +1
87 5
60 524
*
+4 /+
AcH3K14
60 28 7
5 kb
97 06
*
/+
46
32
+46097/+46029
02
9
Figure 8.
A
kDa
80 25 His-eGFP
KAT7 *
Relative Expression
60 20 .06 KAT7
40
15
His 30
10
5
15
60 0 *
α-tubulin
50 His KAT7
B
0.6 0.5
IP DNA (Arbitary Units)
IP DNA (Arbitary Units)
0.5 0.4 anti-His, His-eGFP HUVEC

0.4 anti-KAT7, His-eGFP HUVEC
0.3
0.3 anti-His, KAT7 HUVEC
0.2 0.2 anti-KAT7, KAT7 HUVEC
0.1 0.1
0
0
/- 12 5 02
0
60 2
9
+ 45 9 2 /+ /+4 eNOS
+ 50 0 97
+ 46 promoter
33
Figure 9.
A Control siRNA KAT7 siRNAs
200 μm 200 μm
Isolated *
branches
Network
branches
B Control siRNA KAT7 siRNAs

C Control siRNA KAT7 siRNAs
0h -VEGF-A
20 h +VEGF-A
200 μm 200 μm 200

200 μm
m
D Control siRNA KAT7 siRNAs

VEGF - + + + + - + + + + kDa
P-VEGFR-2 260
Y1175
160
260
VEGFR-2
160
80
KAT7
60
60
α-tubulin 34
50
Time (min) 0 3 5 10 30 0 3 5 10 30 Figure 10.
A B C
kDa
80
KAT7 60
60
α-tubulin 50
40
D 24 hpf 26 hpf 28 hpf
Control
MO
E1ATG
MO
30 hpf 32 hpf 34 hpf
Control
MO
E1ATG
MO
100 μm 35
Figure 11.
A B
100 **
Embryos (%)80 * Control MO
Abnormal
* **
60 ¶
40 ¶
20 E1ATG MO
0
ol G 7 I3
o ntr 1AT KAT E3 E1ATG MO
C E +
T G
E1
A +KAT7
SIV morphology
Blood Circulation E3I3 MO
Hemorrhage
250 μm
C
Control
MO
E1ATG
MO
E1ATG
MO+KAT7
E3I3
MO
500 μm
D
Control MO E1ATG MO E1ATG MO+KAT7 E3I3 MO
75 μm
36
Figure 12.
* *
37
Figure 13.
A D
B E
C F
kDa
260 1.2
Relative Expression
KDRL 1
160 0.8
260 *
0.6 * *
FLT1
160 0.4
80 0.2
KAT7
60 0
60
α-tubulin 50 RL LT1 AT7
40 KD F K
38
Figure 14.
A B
100
Embryos (%)
80 * Control MO
Abnormal
* *
60
*
40 ¶ ,¶
*
20
0 E1ATG MO
nt ro l AT
G
DR
L
Co E1 + K
A TG
E1 E1ATG MO
SIV morphology +KDRL
Blood Circulation
Hemorrhage 100 μm
Control
MO
E1ATG
MO
E1ATG
MO+KDRL
500 μm
D
Control MO E1ATG MO E1ATG MO+KDRL
100 μm
39
Figure 15.
Histone acetyltransferase 7 (KAT7)-dependent intragenic histone acetylation regulates
endothelial cell gene regulation
Matthew S. Yan, Paul J. Turgeon, Hon Sum Jeffrey Man, Michelle K. Dubinsky, Jr Jyun
David Ho, Suzan El-Rass, You-Dong Wang, Xiao-Yan Wen and Philip A. Marsden
J. Biol. Chem. published online February 6, 2018
Access the most updated version of this article at doi: 10.1074/jbc.RA117.001383
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Histone Acetyltransferase 7 (KAT7) - Dependent Intragenic Histone Acetylation Regulates Endothelial Cell Gene Regulation

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Histone Acetyltransferase 7 (KAT7) - Dependent Intragenic Histone Acetylation Regulates Endothelial Cell Gene Regulation

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JBC Papers in Press. Published on February 6, 2018 as Manuscript RA117.

Histone acetyltransferase 7 (KAT7)-dependent intragenic histone acetylation regulates endothelial cell

Running Title: Role of KAT7 in EC Gene Expression

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Keywords: chromatin immunoprecipitation (ChIP), chromatin modification, endothelial cell, gene

Abstract perturbed EC-enriched gene expression, especially

cotranscriptional spliceosome assembly (4). mammalian development that disrupted somites,

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Science and Technology. 3H.S.J.M. is a recipient

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Table 1. Primers used in qRT-PCR and ChIP-qPCR analysis

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Figure 8. KAT7 depletion disrupts chromatin-based transcriptional regulation of VEGFR-2. A,

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IP DNA (Arbitrary Units)

IP DNA (Arbitrary Units) IP DNA (Arbitrary Units)

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0.5 0.4 anti-His, His-eGFP HUVEC

B Control siRNA KAT7 siRNAs

200 μm 200 μm 200

D Control siRNA KAT7 siRNAs

D 24 hpf 26 hpf 28 hpf

30 hpf 32 hpf 34 hpf

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