Huang

etal. acta neuropathol commun (2020) 8:219

https://doi.org/10.1186/s40478-020-01092-4

RESEARCH

Eects ofH3.3G34V mutation ongenomic

H3K36 andH3K27 methylation patterns

inisogenic pediatric glioma cells

Tina Yi‑Ting Huang

, Andrea Piunti

, Jin Qi

, Marc Morgan

, Elizabeth Bartom

, Ali Shilatifard

and Amanda M. Saratsis

1,2,3*

Abstract

Histone H3.3 mutation (H3F3A) occurs in 50% of cortical pediatric high‑grade gliomas. This mutation replaces glycine

34 with arginine or valine (G34R/V), impairing SETD2 activity (H3K36‑speciﬁc trimethyltransferase). Consequently,

reduced H3K36me3 is observed on H3.3G34V nucleosomes relative to wild‑type, contributing to genomic instabil‑

ity and driving a distinct gene expression signature associated with tumorigenesis. However, it is not known if this

diﬀerential H3K36me3 enrichment is due to H3.3G34V mutant protein alone. Therefore, we set to elucidate the eﬀect

of H3.3G34V mutant protein in pediatric glioma on H3K36me3, H3K27me3 and H3.3 enrichment in vitro. We found

that the doxycycline‑inducible shRNA knockdown of mutant H3F3A encoding the H3.3G34V protein resulted in loss

of H3.3G34V enrichment and increased H3K36me3 enrichment throughout the genome. After knockdown, H3.3G34V

enrichment was preserved at loci observed to have the greatest H3.3G34V and H3K36me3 enrichment prior to

knockdown. Induced expression of mutant H3.3G34V protein in vitro was insuﬃcient to induce genomic H3K36me3

enrichment patterns observed in H3.3G34V mutant glioma cells. We also observed strong co‑enrichment of H3.3G34V

and wild‑type H3.3 protein, as well as greater H3K27me3 enrichment, in cells expressing H3.3G34V. Taken together,

our study demonstrates the eﬀects of H3.3G34V mutant protein on genomic H3K36me3, H3K27me3 and H3.3 enrich‑

ment patterns in isogenic cell lines.

Keywords: Pediatric high‑grade glioma, Post‑translational modiﬁcations, H3K36me3, Histone H3 mutations

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Introduction

Pediatric high-grade glioma (pHGG) is the number one

cause of cancer death in children, with a 5-year survival

of less than 20%. is dismal prognosis is in large part

due to an historical lack of understanding of its distinct

biology and the presumption that pHGG is biologically

identical to its adult counterpart, resulting in ineﬀective

treatment. However, with the development of next-gen

eration sequencing technologies to analyze rare tumor

specimens, knowledge of pHGG biology signiﬁcantly

increased over the past decade. Somatic missense muta

tions in genes encoding Histone H3 isoforms, including

H3F3A, HIST1H3B and HIST1H3C, were subsequently

identiﬁed in up to 50% of supratentorial hemispheric

pHGG, and 80% of pediatric diﬀuse midline gliomas

(DMG), a form of pHGG in the thalamus or brainstem

[11, 15, 20]. ese mutations are associated with distinct

tumor biology and poorer clinical outcome, and are now

understood to play a role in pediatric gliomagenesis. As a

result, determining the eﬀects of these mutations on his

tone H3 function and regulation of gene transcription, in

order to identify more eﬀective therapeutic targets, is of

great interest.

Open Access

*Correspondence: [email protected]g

Division of Pediatric Neurosurgery, Department of Surgery, Ann &

Robert H. Lurie Children’s Hospital of Chicago, 225 E Chicago Avenue,

Box 28, Chicago, IL 60611‑2991, USA

Full list of author information is available at the end of the article

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Huangetal. acta neuropathol commun (2020) 8:219

In hemispheric pHGG, somatic mutations of H3F3A

(GGG to GTG) result in a glycine 34 to arginine (G34R)

or valine (G34V) substitution on the histone H3.3 N-ter

minal tail, while H3F3A mutations in DMG result in

lysine to methionine alterations (K27M or K36M). Sev

eral studies have focused on the eﬀects of these muta-

tions on global methylation, chromatin structure, and

transcription regulation to promote tumorigenesis. For

example, mutant H3.3K27M protein exhibits higher

aﬃnity for EZH2, a H3K27-speciﬁc lysine methyltrans

ferase, compared to wildtype H3.3, resulting in EZH2

sequestration and preventing PRC2 from propagating

transcriptionally repressive H3K27 methylation [14].

Mutant H3.3K36M protein inhibits H3K36-speciﬁc

lysine methyltransferases, including NSD1, NSD2, and

SETD2, reducing global H3K36 methylation [3, 12, 21].

In contrast, less is known about the epigenetic and tumo

rigenic eﬀects of H3.3G34V/R mutations in pHGG. Sev-

eral studies comparing H3.3G34V mutant and wild type

cell lines suggest distinct epigenetic changes at H3K27

and H3K36 in association with H3.3G34V/R mutation,

as well as alteration to DNA repair pathways leading

to transcriptional upregulation, increased mutational

burden and genomic instability [2, 10, 16]. However, as

these studies did not use isogenic cell lines, the distinct

mechanism by which H3.3G34V/R mutation exerts the

observed changes is not clear. erefore, we set to better

elucidate the direct epigenetic eﬀects of H3.3G34V muta

tion in pediatric glioma invitro using isogenic cell lines.

Here, we demonstrate changes in genomic enrichment of

multiple chromatin marks after DOX-inducible knock

down of H3F3A in an H3.3G34V mutant pediatric glioma

cell line, and H3.3G34V mutation transduction in wild-

type astrocytes, providing insight on epigenetic eﬀects of

this mutation that promote tumorigenesis.

Materials andmethods

Cell lines andculture conditions

Experiments were conducted using an established

H3.3G34V mutant patient-derived pediatric high-grade

glioma cell line (KNS42), and Histone H3.3 wild-type

human astrocytes (NHAs, ScienCell #1800). KNS42 was

obtained from Rintaro Hashizume (Northwestern Uni

versity), it is well characterized as previously described

[6]. KNS42 cells were maintained in EMEM (10009CV,

Corning) with 5% FBS (16000-044, Gibco). NHA cells

were maintained in high glucose DMEM (11995-065,

Gibco) and 10% FBS, according to the cell line provider’s

recommendation. Cells were grown in an incubator with

5% CO

at 37 °C. All experiments were conducted in

accordance to institutional protocols and approvals (NU

IRB# STU00202063).

H3.3G34V mutation induction andknock‑down

Lentiviral delivery of a doxycycline-inducible RNAi

vector targeted against H3F3A was transduced to

KNS42 cells to knock down H3.3G34V protein expres

sion. e vector contains the selectable markers of

puromycin as well as a red ﬂuorescent protein (RFP)

reporter. Lentiviral vector-mediated doxycycline-

inducible cDNA encoding a c.104G > T p.(Gly34Val)

H3F3A mutation was transduced to NHAs in order to

express H3.3G34V mutant protein. is vector, pUC57-

Kan, contained a kanamycin resistant gene. A vector

containing doxycycline-inducible H3F3A for wild-type

H3.3 protein expression was used as negative control.

All vectors were purchased from Genscript. A total of

250,000 cells from each cell line were transduced with

lentivirus for 6h, rinsed in PBS, then cultured in their

respective media as described above. A second round of

lentiviral transduction was performed 24h later. After

an additional 24h, antibiotics were added at 2µg/mL.

Cells were then cultured in their respective media for

three to 5days to achieve desired conﬂuency. For dox

ycycline-induced transduction conditions, doxycycline

was added at 1:5000 every other day for 1week (days

one, three, ﬁve, seven), and on day eight cells were col

lected as a pellet for Western Blotting to conﬁrm pro-

tein expressions, or crosslinked with 1% formaldehyde

for ChIP-Seq (see below). Cells without doxycycline

treatments were cultured and collected in parallel. Flu

orescent imaging and ﬂow cytometry were performed

to select for cells with successful protein knockdown or

transduced expression.

Western blotting

Protein was extracted from cells using RIPA buﬀer

(89900, ermo Fisher Scientiﬁc). A total of 60 µg of

protein (from whole cell extract) was separated by elec

trophoresis in a 4–15% precast protein gel (4561086, Bio-

Rad) and transferred to PVDF membranes. Blocking was

subsequently performed with 5% non-fat milk in TBST,

followed by incubation with anti-H3K27Ac antibody at

1:500 dilution (8173S, Cell Signaling Technology) over

night. After 5 washes with TBST, membranes were incu-

bated with HRP-conjugated anti-Rabbit IgG antibody at

1:1000 (7074 Cell Signaling Technology) for 1h. Pierce

ECL Plus (32132, ermo Fisher Scientiﬁc) was used to

detect protein bands. Blots were then stripped (46430,

ermo Fisher Scientiﬁc) and re-probed with anti-total

H3 primary antibody at 1:1000 dilution (14269S, Cell

Signaling Technology) as a loading control. HRP-con

jugated anti-Mouse IgG antibody (7076, Cell Signaling

Technology) was used to detect total H3 signal. Densi

tometry analysis was performed with image J.

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Huangetal. acta neuropathol commun (2020) 8:219

Cell proliferation assay

Cell proliferation was measured by counting viable cells

using the TC20 Automated Cell Counter (Bio-Rad).

3 × 10

cells were seeded in cell culture dishes. At 3 and

7 days after seeding, cells were harvested, dissociated

into single cell suspension, and stained with 0.4% Trypan

Blue Solution (15250061, ermoFisher) for 5min before

counting.

Cell viability assay

Cell viability was assessed using the CellTiter-Glo Lumi-

nescent Assay (G7570, Promega). 3000 cells were seeded

in 96-well plate. Measurements were taken 1, 3, 5, and 7

days after seeding. Reagent was diluted at 1:1 ratio with

PBS to achieve optimal luminescent range. 100µL rea

gent was added to cells in 100µL media. e mix was

incubated for 10min at room temperature with gentle

shaking, followed by luminescent measurement.

Cell crosslinking andchromatin immunoprecipitation

For each immunoprecipitation, 30 million cells were

crosslinked using freshly prepared 1% formaldehyde in

complete cell medium for 10min at RT, and subsequently

quenched with 0.125M glycine for 5min. e cells were

then rinsed twice in cold PBS, gently scraped from the

plates and centrifuged in a 15mL tube (Falcon) at 1350×g

for 8min at 4 °C. Crosslinked cells were either stored

in − 80 °C or used immediately for chromatin immu

noprecipitation, as previously described [14]. Brieﬂy,

crosslinked cells were resuspended in 10mL buﬀer 1 for

10min at 4°C then centrifuged at 1350×g for 5min at

4°C. Pellet was resuspended in 10mL buﬀer 2 for 10min

at RT then centrifuged at 1350×g for 5min at 4°C. Pellet

was resuspended 1mL buﬀer 3 and transferred to a 1mL

milliTUBE (520135, Covaris). Sonication was performed

using the Covaris E220 ultrasonicator with the following

parameters: 20% duty cycle, 175 PIP, 200 cycles/burst,

for 8 min. After sonication, sample was centrifuged at

20,000×g for 15min at 4°C and supernatant containing

chromatin was collected. 50 µL of chromain were de-

crosslinked with elution buﬀer for 3h at 65°C and DNA

was extracted using PCR puriﬁcation kit (28104, Qiagen).

Puriﬁed DNA were loaded in a 1.5% agarose gel to check

for the fragment size (average range 200–500bp). A total

of 100µL ChIP dilution buﬀer was added to the remain

ing sheared chromatin. A total of 10µL of each sample

was saved at 4°C to serve as input. e remaining chro

matin was incubated with primary antibody on a nutator

overnight at 4°C (refer to Table1 for a list and dilution of

antibodies used). e following day, 60µL of 50% protein

A/G agarose beads (sc-2003, Santa Cruz Biotech) were

added to the samples and incubated for 3h on a nutator

at 4°C. Agarose beads were pelleted by centrifugation at

2500×g for 1min and washed with 1mL of RIPA buﬀer

four times followed by 1mL of 50mM NaCl in TE. e

beads and the 10µL input sample were resuspended in

200µL elution buﬀer for 30min at 65°C then centrifuged

for 2min at 15,000×g. Supernatant was transferred into a

new 1.6mL tube and de-crosslinked overnight 65°C. e

following day, DNA was extracted with PCR puriﬁcation

kit (28104, Qiagen), and eluted to a ﬁnal volume of 60µL.

A total of 45µL of each sample and input were used for

library preparation and subsequent sequencing steps. All

additional reagents (Elution buﬀers 1, 2, 3, Elution buﬀer,

10× ChIP dilution buﬀer, RIPA buﬀer, and elution buﬀer)

were prepared as previously described [14].

Library Preparation andNext‑Generation Sequencing

ChIP-Seq libraries were prepared with the KAPA Library

preparation kit (KK8234, Kapa Biosystems) and NETﬂex

DNA barcodes (514104, Bioo Scientiﬁc). A total of 10µg

DNA was used as starting material for input and immu

noprecipitation samples. Libraries were ampliﬁed with a

thermocycler for 13 cycles. Post-ampliﬁcation libraries

were size-selected at 250-45bp in length using the Agen

court AMPure XP beads (A63881, Beckman Coulter).

Libraries were validated using the Agilent High Sensi

tivity DNA Analysis Kit (5067-4626). ChIP-Seq libraries

were single-read sequenced on the Illumina NextSeq500

Sequencing System.

ChIP‑Seq data analysis

Reads were ﬁltered using the FASTX-Toolkit suite and

read quality was assessed with FastQC v0.11.5. After

removal of duplicated reads, unique reads were mapped

to human reference genome Hg38. ChIP-Seq reads were

aligned with the ENCODE pipeline. Peaks were called

with MACS2 v2.1.0 software with cutoﬀ of p < 0.01.

Enrichment values were determined as log2(Normalized

experimental read count—normalized input read count)

Table 1 List ofantibodies used

*We would like the table to be inserted following the section “Cell Crosslinking

and Chromatin Immunoprecipitation” of Materials and Methods

Antibody Company Catalog # ChIP WB

H3.3WT Millipore 09‑838 10 µg 1:1000

K36me3 Homemade na 30 µL 1:1000

G34V RevMAb 31‑1193‑00 10 µg 1:1000

K27me3 Homemade na 30 µL na

Total H3 CST 14,269 na 1:1000

Anti‑Rabbit IgG, HRP CST 7074 na 1:5000

Anti‑Mouse IgG, HRP CST 7076 na 1:5000

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Huangetal. acta neuropathol commun (2020) 8:219

in regions of interest in each ChIP specimen and cor-

responding input sample. Called peaks were annotated

with HOMER v4.10 to the nearest gene. Active enhanc

ers were deﬁned as H3K27ac peaks excluded from the

transcription start site (TSS) (2.5kb upstream and down

stream). ChIP-Seq reads density and data visualizations

were generated using Deeptools v3.1.1. Additional func

tional pathways and upstream regulator analysis was

performed on diﬀerentially enriched loci using Ingenuity

Pathways Analysis software (Qiagen, Germantown MD).

Results

Lentiviral induced knockdown andexpression ofH3.3G34V

To characterize the epigenetic eﬀects of H3.3G34V

mutant protein in pediatric glioma, we compared global

methylation and Histone H3 enrichment patterns in

isogenic cell lines. We used doxycycline-induced lenti

viral delivery of genetic constructs designed to silence

(shRNA) or express (cDNA) mutant and wild-type

H3F3A in H3.3G34V mutant glioma cells (KNS42),

and H3 wild type astrocytes (NHA). Using ﬂuorescent

imaging, we measured lentiviral RFP expression after

DOX-induction to conﬁrm vector expression (Fig. 1a).

Western blot analysis of whole-cell extracts conﬁrmed

successful knockdown of H3.3G34V mutant protein in

glioma cells treated with doxycycline, compared to those

not treated with doxycycline and those transduced with

H3F3A for wild-type H3.3 expression, with no signiﬁcant

reduction in H3K36 trimethylation across conditions

(Fig.1b). In turn, we observed DOX-induced expression

of H3.3G34V mutant protein in cDNA transduced astro

cytes, with no mutant protein detected in untreated cells,

or in cells transduced with H3F3A (Fig. 1c). While we

achieved signiﬁcant overexpression of H3.3G34V mutant

protein in astrocytes compared to control, the maxi

mum level of mutant protein we could express in astro-

cytes is only 28.9% of the level observed in H3.3G34V

mutant glioma KNS42 (Additional ﬁle 1: Figure S1A,

B). Flow cytometry was used to select the top 30% cells

with successful H3.3G34V knockdown and knock-in, for

subsequent ChIP-Seq (Fig.1d). We did not observe sig

niﬁcant diﬀerence in cell viability (Additional ﬁle1: Fig-

ure S1C) or proliferation (Additional ﬁle1: Figure S1D, E)

between KNS42 with and without doxycycline-induced

H3.3G34 knockdown, nor between NHA overexpressed

with H3.3G34V and H3.3 control. On ChIP-Seq analysis,

we observed decreased H3.3G34V enrichment at H3F3A

in KNS42 glioma cells after DOX-induced transduc

tion of H3F3A shRNA, relative to KNS42 cells that were

transduced without DOX induction, or not transduced

(Fig. 1e). We also conﬁrmed enrichment of H3.3G34V

mutant protein at H3F3A in DOX-induced astrocytes,

with no mutant protein in untreated NHAs or those

transduced with wild-type H3F3A cDNA (Fig.1e). Out

side of H3F3A, metagene plots of H3.3G34V enrichment

within the gene body for the top 1000 most H3.3G34V-

enriched genes demonstrated reduction, but not com

plete elimination, of H3.3G34V deposition in KNS42

with DOX-induced knockdown compared to KNS42

cells that were transduced but without DOX induction.

Higher H3.3G34V enrichment is also observed in NHAs

transduced for H3.3G34V expression, compared to

NHAs with only wild-type H3.3 expression (Fig.1f).

H3.3G34V Co‑enriches withWild‑type H3.3

Overall, we observed greater H3.3 enrichment in cells

expressing H3.3G34V mutant protein. Speciﬁcally, we

identiﬁed 270,656 wild-type H3.3 peaks in glioma cells

after H3.3G34V knockdown (6628 promoter, 144,735

gene body), and signiﬁcantly fewer H3.3 peaks in KNS42

glioma cells with intact H3.3G34V expression (164,785

total, 4071 promoter, 87,770 gene body, Fig. 2a). e

same trend was observed in astrocytes induced to

express H3.3G34V (77,018 total peaks, 2019 promoter,

40,530 gene body) compared to controls (15,9235 total

peaks, 3952 promoter, 84,746 in gene body, Fig.2a). At

genes previously reported to have greater WT H3.3

enrichment in H3,3G34V mutant tumors compared to

WT, manipulation of H3.3G34V mutant protein expres

sion had no eﬀect on WT H3.3 enrichment (Fig.2b). To

further determine the eﬀect of H3.3G34V expression on

genomic enrichment of WT and mutant H3.3, we identi

ﬁed the top 10,000 loci most-enriched for H3.3G34V in

transduced KNS42 cells without DOX induction (nega

tive control), and compared H3.3G34V and WT H3.3

enrichment at these loci across experimental conditions.

Fig. 1 Genetic Modiﬁcation of Histone H3.3 expression in Pediatric Glioma Cells and Astrocytes. a Expression of RFP reporter was conﬁrmed in

> 95% of Doxycycline‑induced cells. b H3.3G34V expression was signiﬁcantly reduced in lentiviral‑transduced KNS42 cells treated with Doxycycline,

compared to no Doxycycline and control conditions. **p < 0.01, ****p < 0.0001. c H3.3G34V protein was expressed in lentiviral‑transduced NHAs

treated with doxycycline, with no H3.3G34V expression observed in controls. d Flow cytometry was used to select the top 30% cells with H3.3G34V

knockdown and knock‑in for subsequent ChIP‑Seq analysis. e Genome browser view of H3.3G34V enrichment at the H3F3A locus across cell lines

studied. Tracks highlighted in red are those conditions without H3.3G34V expression. f Metagene proﬁle of H3.3G34V enrichment in the gene body

across the top 1000 most H3.3G34V‑enriched genes in KNS42. Reduction of H3.3G34V enrichment is observed following doxycycline‑induced

knockdown (light blue versus navy blue lines). Greater H3.3G34V enrichment is also observed in NHA transduced for H3.3G34V expression (green

line) compared to control transduced for H3.3WT expression (orange line)

(See ﬁgure on next page.)

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Huangetal. acta neuropathol commun (2020) 8:219

b c

Page 6 of 13

Huangetal. acta neuropathol commun (2020) 8:219

Fig. 2 H3.3G34V Co‑enriches With Wild‑type H3.3. a Relative proportion of H3.3WT enrichment across gene elements with H3.3G34V expression

or knock‑down. b H3.3 enrichment at SOX2 was greatest KNS42 cell s expressing H3.3G34V, compared to NHAs lacking H3.3G34V expression (red

highlight) or after H3.3G34V transduction. c, d Co‑enrichment of H3.3G34V and H3.3WT was observed across experimental conditions at the top

10,000 loci most‑enriched for C) H3.3G34V, and D) H3.3WT in transduced KNS42 cells without DOX induction. e When the top 10,000 loci most

enriched for H3.3G34V, H3.3 wild‑type, H3K36me3 and H3K27me3 in transduced KNS42 cells without DOX induction are compared, greatest overlap

is observed between H3.3G34V and wild‑type H3.3 enrichments patterns, with 3041 common loci between these two groups

Page 7 of 13

Huangetal. acta neuropathol commun (2020) 8:219

We detected co-enrichment of H3.3G34V and WT H3.3

in all glioma cell treatment conditions at these 10,000 loci

most enriched for H3.3G34V (Fig.2c), as well as at the

10,000 loci most enriched for WT H3.3 (Fig.2d). When

these loci are mapped to the nearest gene, H3.3G34V and

WT H3.3 enriched loci have the most overlap, with 3041

shared loci between groups (Fig. 2e). Taken together,

these data suggest that glioma H3.3G34V expression is

associated with increased WT H3.3 enrichment through

out the genome, that WT H3.3 and H3.3G34V strongly

co-enrich, and that loci most enriched by these proteins

do not signiﬁcantly change with H3.3G34V knockdown.

H3.3G34V does notaect global H3K36me3 enrichment

levels

Next, we set to determine the eﬀects of H3.3G34V expres-

sion on patterns of genomic H3K36me3 enrichment.

Consistent with previous studies [17], H3.3G34V knock

down in glioma cells did not result in signiﬁcant change in

the level of global H3K36me3 enrichment across all gene

elements evaluated, with a total of 354,857 K36me3 peaks

after H3.3G34V knockdown (7187 promoter, 202,284

gene body), compared to 254,315 H3K36me3 peaks cells

in KNS42 cells with intact H3.3G34V expression (5193

promoter, 145,856 gene body, Fig.3a). Similarly, we did

not observe a signiﬁcant diﬀerence in the number of

H3K36me3 peaks in astrocytes after H3.3G34V knock-in

compared to WT cells (Fig.3a).

In H3.3G34V mutant KNS42 cells, we observed no

diﬀerence in H3K36me3 enrichment with or without

DOX-induced H3.3G34V knockdown, at neither the

1000 most H3K36me3 enriched loci, nor at the 1000

most H3.3G34V enriched loci (Fig. 3c, top left and

top right panels). Additionally, we not observe any dif

ference in H3K36me3 enrichment in NHAs, with or

without induced H3.3G34V expression. It is possible

that H3.3G34V knockdown in KNS42 did not occur

at loci with highest H3.3G34V enrichment, nor at

loci with highest H3K36me3 enrichment. To test this

hypothesis, we examined the loci with highest diﬀer

ence in H3.3G34V and H3K36me3 enrichment, with

and without DOX-induced H3.3G34V knockdown. At

the 1000 loci with the greatest diﬀerence in H3.3G34V

enrichment between no DOX and DOX-induced

H3.3G34V knockdown conditions, we also saw no dif

ference in H3K36me3 enrichment (Fig.3c, bottom left

panel). Interestingly, at the 1000 loci with the greatest

diﬀerence in H3K36me3 enrichment with versus with

out H3.3G34V knockdown, we observed much greater

H3K36me3 enrichment with H3.3G34V knockdown

compared to no DOX control (Fig. 3c, bottom right

panel). For example, we saw reduced enrichment with

H3K36me3 at neuroligin-2, NLGN2 with H3.3G34V

knockdown, compared to transduced KNS42 with no

DOX control and KNS42 non-transduced cell lines

(Fig.3b). ese data are consistent with previous obser

vations that H3.3G34V mutation leads to local reduc-

tions in H3K36me3 enrichment. Additionally, our data

indicate that the loci with diﬀerences in H3K36me3

enrichment after H3.3G34V knockdown are neither

those with greatest H3.3G34V enrichment, nor those

with the greatest H3K36me3 enrichment.

In contrast, induced expression of H3.3G34V was not

suﬃcient to change genomic H3K36me3 enrichment

patterns. Evaluation of the top 1000 loci with the most

signiﬁcantly diﬀerent H3K36me3 enrichment between

no DOX and DOX-induced knockdown of H3.3G34V

in KNS42 showed that, compared to no DOX control,

KNS42 cells with DOX-induced H3.3G34V knock

down have greater overall H3K36me3 enrichment at

698 loci (Fig.3d). It has been previously suggested that

H3.3G34V expression leads to diﬀerential distribu

tion of H3K36me3 on approximately 150 genes [19].

Taken together, our data suggest that DOX-induced

H3.3G34V knockdown does not aﬀect the most

H3.3G34V or H3K36me3 enriched loci, but does have

speciﬁc genomic eﬀects on a limited subset of biologi

cally relevant genes.

Fig. 3 G34V mutation does not aﬀect global H3K36me3. Increased H3K36me3 is observed at loci with G34V knockdown. a Relative proportion of

H3K36me3 enrichment across gene elements with H3.3G34V expression or knock‑down. b H3K36me3 enrichment at the NLGN2 gene is higher in

KNS42 after G34V knockdown, compared to KNS42 no DOX control and non‑transduced KNS42. However, transduced H3.3G34V into NHAs had

no eﬀect on H3K36me3 enrichment patterns. c Metagene proﬁle of H3K36me3 enrichment. No signiﬁcant diﬀerence in H3K36me3 enrichment

was observed at the 1000 most H3K36me3 enriched loci (top left), nor at the 1000 most H3.3G34V enriched loci (top right) with or without

DOX‑induced H3.3G34V knockdown. No diﬀerence H3K36me3 enrichment was also observed at the 1000 loci with the greatest diﬀerence in

H3.3G34V enrichment between KNS42 no DOX and DOX‑induced H3.3G34V knockdown (bottom left). In contrast, greater H3K36me3 enrichment

was observed in 1000 loci with the greatest diﬀerence in H3K36me3 enrichment with H3.3G34V knockdown, compared to no DOX control (bottom

right). d Heatmap proﬁles of K36me3 peaks at loci with the top 1000 most diﬀerence in enrichment before and after knockdown. Signals from (left)

KNS42 with DOX‑induced G34V knockdown, (middle) KNS42 without G34V knockdown (no DOX control), and (left) the diﬀerence between them (Δ,

left minus middle). Positive values indicate that KNS42 with DOX‑induced G34V knockdown has higher K36me3 enrichment compared to no DOX

control

(See ﬁgure on next page.)

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Huangetal. acta neuropathol commun (2020) 8:219

Page 9 of 13

Huangetal. acta neuropathol commun (2020) 8:219

H3.3G34V isassociated withhigher H3K27me3 enrichment

Several studies have shown that H3.3G34V directly

impacts the modiﬁcation state of adjacent K27 and K36

residues on the mutant H3.3 protein [2]. In line with this,

we observed greater H3K27me3 enrichment in KNS42

cells with H3.3G34V (740,497 peaks total, 11,516 pro

moter, 332,437 gene body), compared to KNS42 cells after

H3.3G34V knockdown (457,844 peaks total, 8270 pro

moter, 214,405 gene body) (Fig.4a). e greater enrich-

ment of H3.3K27me3 in non-transduced KNS42 and

transduced KNS42 with no DOX control compared to

KNS42 cells with DOX-induced H3.3G34V knockdown

was observed at active enhancers (Fig. 4b). In turn, at

HOXA13 we saw lower enrichment of H3K27me3 in

KNS42 cells with DOX-induced H3.3G34V knockdown,

compared to transduced KNS42 with no DOX control and

non-transduced KNS42, indicating that the H3.3G34V

mutation is associated with higher H3K27me3 enrich

ment (Fig.4c). Indeed, at the top 1000 most H3.3G34V-

enriched loci in KNS42 without DOX, we observed that

KNS42 cells without DOX-induced H3.3G34V knock

down harbored higher HK27me3 enrichment relative

to other cell lines studied (Fig.4d, top panel). is same

ﬁnding was observed at loci with greatest diﬀerential

H3.3G34V enrichment between DOX-induced H3.3G34V

knockdown cells and no-DOX controls (Fig. 4d, bot

tom panel). Heatmap proﬁles of the top 1000 most

H3K27me3 enriched loci in KNS42 showed that DOX-

induced knockdown of H3.3G34V results in lower relative

H3K27me3 enrichment, compared to no DOX controls.

H3.3G34V enrichment implicates distinct molecular

pathways

As H3.3G34V mutation is associated with distinct

changes in H3K36me3 and H3K27me3 enrichment pat

terns, we sought to determine functional pathways of

gene expression implicated by diﬀerential enrichment

patterns of mutant and wild-type H3.3. Functional path

ways analysis revealed cellular assembly and organiza-

tion (p = 1.12 × 10

−3

), cellular function and maintenance

(p = 1.18 × 10

−3

), and cell signaling (p = 8.81 × 10

−5

) as

the top molecular functions associated with the gene set

co-enriched in H3.3G34V and H3K36me3. Cell death

and survival (p = 1.12 × 10

−2

), cellular development

(p = 1.25 × 10

−4

), and cellular function and maintenance

(p = 1.25 × 10

−4

), are the top molecular and cellular func-

tions implicated by the gene set co-enriched in H3.3G34V

and K27me3. As expected, the top disease associated

with both these gene sets was cancer (p = 2.11 × 10

−10

GO and KEGG analyses were also performed to iden

tify functional annotations of genes with the most diﬀer-

ential H3.3G34V, H3.3WT, and H3K36me3 enrichment

between KNS42 with intact H3.3G34V expression and

H3.3G34V knockdown. Cellular metabolism and onco

genesis were consistently the top two enriched pathways

in cells expressing H3.3G34V (Additional ﬁle 2: Figure

S2), with metabolic pathways implicated as the top cellu

lar function of this gene set (−log(p value) = 12.92–21.15,

Additional ﬁle3 Figure S3). Cellular neuron projection

morphogenesis and neuron diﬀerentiation were also

highly enriched in this gene set, consistent with previous

studies showing genes enriched in H3K36me3 contribute

to in neuronal diﬀerentiation and cell proliferation [19].

Discussion

Somatic missense mutations that alter histone H3.3 struc-

ture and function are uniquely common in pediatric

high-grade glioma. Tumors harboring the H3.3G34V/R

mutation are clinically and biologically distinct from wild

type tumors, with poorer progression free and overall

survival, and unique genomic, proteomic, methylomics

and epigenetic proﬁles relative to wild-type tumors [5,

9]. However, the mechanisms by which these mutations

lead to tumorigenesis are still not completely understood.

While prior studies have attempted to compare the eﬀects

of H3.3G34V invitro, these studies did not employ iso

genic cell lines. Here, we genetically modiﬁed pediatric

glioma cells and normal astrocytes using a DOX-induci

ble construct in order to more accurately determine the

eﬀects of H3.3G34V expression on the glioma epigenetic

landscape that may contribute to tumorigenesis.

Prior studies have shown that unlike the K27 and K36

amino acid residues on the histone H3 N-terminal tail,

the H3G34 residue is not post-translationally modiﬁed.

(See ﬁgure on next page.)

Fig. 4 H3.3G34V is associated with higher H3K27me3 enrichment. a Relative proportion of H3K27me3 enrichment across gene elements

with H3.3G34V expression or knock‑down in KNS42 cells. b Metagene proﬁle of H3K27me3 enrichment at active enhancers (H3K27ac peaks

2.5 kb away from TSS). Higher H3K27me3 was observed in transduced KNS42 without DOX and non‑transduced KNS42 compared to KNS42

with G34V knockdown. c At HOXA13, H3K27me3 enrichment is lower in KNS42 with G34V knockdown, compared to KNS42 no dox control and

non‑transduced KNS42. Blue tracks are cell lines that harbor, or overexpressed, H3.3G34V; red tracks are cell lines that do not harbor, or knockdown,

H3.3G34V. d Metagene proﬁle of H3K27me3 enrichment. H3K27me3 enrichment in the gene body across the top 1000 (top) most G34V‑enriched

genes in KNS42 TRIP no dox control. (bottom) most diﬀ genes in G34V enrichment before and after KD in KNS42 TIRP ± DOX. e Heatmap proﬁles of

K27me3 peaks at the top 1000 most K27me3 enriched loci. Signals from (left) KNS42 with DOX‑induced G34V knockdown, (middle) KNS42 without

G34V knockdown (no DOX control), and (left) the diﬀerence between them (Δ, left minus middle). Negative values in the third column indicate that

KNS42 with DOX‑induced G34V knockdown has lower K27me3 enrichment compared to no DOX control

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Huangetal. acta neuropathol commun (2020) 8:219

However, H3G34 does lie in close proximity to H3K36,

which undergoes methylation during transcriptional

elongation. As a result, H3.3G34V/R mutations may

alter accessibility of H3K36 to lysine methyltrans

ferases, thereby aﬀecting H3K36 methylation and hence

gene expression. For example, a recent study reported

d e