HP1α is highly expressed in glioma cells and facilitates cell proliferation and survival

Accepted Manuscript HP1α is highly expressed in glioma cells and facilitates cell proliferation and survival Xianliang Lai, Zhifeng Deng, Hua Guo, Xingen Zhu, Wei Tu PII:

S0006-291X(17)31188-9

DOI:

10.1016/j.bbrc.2017.06.056

Reference:

YBBRC 37962

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 29 May 2017 Accepted Date: 12 June 2017

Please cite this article as: X. Lai, Z. Deng, H. Guo, X. Zhu, W. Tu, HP1α is highly expressed in glioma cells and facilitates cell proliferation and survival, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.06.056. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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HP1α Is Highly Expressed in Glioma Cells and Facilitates Cell Proliferation

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Xianliang Lai1, Zhifeng Deng1, Hua Guo1, Xingen Zhu1, Wei Tu1,a

Department of Neurosurgery, the Second Affiliated Hospital of Nanchang University, Nanchang

Corresponding author: Wei Tu.

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E-mail address: [email protected]

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330006, China

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and Survival

LAI et al: HP1α promotes glioma cell proliferation and survival.

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Abstract Epigenetic alteration plays critical roles in gliomagenesis by regulating gene expression through modifications of Histones and DNA. Trimethylation of H3K9, an essential repressed

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transcription mark, and one of its methyltransferase, SUV39H1, are implicated in glioma pathogenesis and progression. We find that the protein level of HP1α, a reader of H3K9me3 is elevated in cultured glioma cell lines and glioma tissues. H3K9me3 is also upregulated. Depletion of HP1α and SUV39H1 weakens glioma cell proliferation capacity and results in

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apoptosis of cells. Furthermore, we find that HP1α and H3K9me3 are enriched in the FAS and PUMA promoters, which suggests that upregulated HP1α and H3K9me3 contribute to cell survival by suppressing apoptotic activators. These data suggests that up-regulated HP1α and

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H3K9me3 in glioma cells are functionally associated with glioma pathogenesis and progression

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and may serve as novel biomarkers for diagnostic and therapeutic targeting of brain tumors.

Keywords: HP1α; H3K9 methylation; glioma; transcriptional repression

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Introduction Human gliomas are highly invasive and malignant brain tumors [1]. Despite the progress in glioma diagnosis and therapy achieved with decades of study, survival of glioma patients still

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needs improvement [2; 3; 4; 5; 6]. Genetic mutation and epigenetic aberrance have been proposed to explain the underlying mechanisms of glioma pathogenesis [7]. Isocitrate dehydrogenase (IDH1) and isocitrate dehydrogenase (IDH2) mutations are suggested to be a survival predictor of glioma patients [8;

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9; 10; 11]. Heat shock protein B11 is related to prognosis in patients with high-grade glioma [12]. Recently, DNA and Histone modification alteration was also identified to be involved in glioma tumorigenesis and studied for searching potential clinical treatment [3; 13; 14; 15]. Effect

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of O6-methylguanine (MGMT) promoter methylation regulation in glioma cells highlights the roles of DNA methylation in glioma pathogenesis and treatment [10; 11; 16]. Histone modification, especially H3 methylation, is largely and widely paid attention [17; 18; 19; 20]. The previous study suggested the roles of H3K27 methylation in glioma tumorigenesis and progression [17; 18; 21]. Mutation and somatic alterations of Histone H3 define the prognosis improvement and even potential therapy of gliomas by altering H3 modification [17; 18; 19; 20].

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It is also worth being mentioned that the potential significance of linker Histone variant H1.0 in gliomas was also proposed [22].

Suppressor of variegation 3-9 homology 1 (SUV39H1) is a Histone methyltransferase that specifically trimethylates ‘Lys-9’ of histone H3 using monomethylated or dimethylated H3

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‘Lys-9’ as substrate [23]. Trimethylates ‘Lys-9’ of Histone H3 represent a specific tag for epigenetic transcriptional repression by recruiting heterochromatin protein 1 (HP1) family

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(including CBX1, CBX3, and CBX5), which specifically recognize and bind to Histone H3 tail with H3K9me3 [24]. SUV39H1 and HP1 bind to each other to form a complex and mainly function in heterochromatin regions by playing a central role in the establishment of constitutive heterochromatin at pericentric and telomere regions [25; 26; 27; 28]. SUV39H1 and HP1 are also demonstrated to be important for regulating euchromatin gene expression by interaction with retinoblastoma 1 (Rb1) [29]. Recent studies have linked the suppression function of SUV39H1 and H3K9me3 to cancer. Upregulation of SUV39H1 and high level of H3K9me3 in glioma cells and patients’ glioma tissues suggested the roles of Histone methylation in brain tumorigenesis

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and progression [22; 30]. HP1 proteins are the main readers of H3K9me3, but there is little information about the potential roles of HP1 in gliomas tomorigenesis or progression [24; 28]. We find that HP1α is upregulated in glioma cells and glioma tumor tissues. HP1α and SUV39H1 are required for glioma cell proliferation and protect cells from apoptosis. HP1α and

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H3K9me3 are enriched in the FAS and PUMA promoters and reduce the protein expression, which suggests that up-regulated HP1α and H3K9me3 prevent apoptosis by suppressing apoptotic activators in glioma cells. Therefore, our data indicates that high levels of HP1α, H3K9me3 are potential biomarkers of gliomas, and enrichs the roles of epigenetic regulation of

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Histone modifications in tumorigenesis.

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Materials and Methods

Antibodies

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Anti-Actin (mabcam8224, abcam) 1:500 WB (western blotting); anti-SUV39H1 (ab38637, abcam) 1:500 WB, 1:50 IP (immunoprecipitation); anti-HP1α (730019, ThermoFisher) 1:500 WB; anti-H3K9me3 (61013, Active motif) 1:1000 WB; anti-G9a (07-551, Millipore) 1:500 WB; anti-BrdU (ab8152, abcam) 1:50 IF (immunofluorescence).

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The secondary antibodies used for immunofluorescence were Alexa fluor 568-conjugated goat anti-mouse IgG (Alexa fluor series, Molecular probes) 1:200. The secondary antibodies

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used for WB were HRP-conjugated goat anti-mouse/rabbit IgG (GE healthcare) 1:5000.

Cell cultures

Human brain astrocytoma glioma cell line 1321N1 (86030402, SIGMA), human glioblastoma cell lines DBTRG-05MG (93061119, SIGMA) and ANGM-CSS (08040401, SIGMA), human Caucasian glioblastoma-derived cell line T98G (92090213, SIGMA), and human astrocytomaderived cell line GOS3 (ACC-408, DSMZ) were cultured in RPMI 1640 medium (Gibco)

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containing 10% FBS (Gibco) and 0.1% Fungizone antimycotic (Gibco). Normal human astrocyte (NHA) cell strain (Clonetics Corporation) was maintained in MEM supplemented with L-

under 5% CO2.

Tissue samples

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glutamine and 10% FBS (Gibco). Cells were plated in 15 cm plastic cell culture dishes at 37°C

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Archival human normal brain tissues and glioma tissues samples were from Department of Neurosurgery, the Second Affiliated Hospital of Nanchang University with approval from the institutional review boards. Histological grading of tumors was performed by following the latest principles of World Health Organization Classification [31]. All samples were deidentified before analysis.

Reverse transcription (RT) PCR and quantitative PCR analysis Tissue was broken by douncing and the total RNA was extracted using RNeasy Mini Kit (Qiagen) by following the manufacturer’s instructions. OneTaq RT-PCR Kit (NEB) was used to 5

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produce cDNA library according to the manufacturer’s instructions. The produced cDNA was used as template for amplification and quantification with specific primer sets for HP1α and Actin using HotStarTaq Plus Master Mix Kit (Qiagen). The conditions of the PCR were as follows: the first activation for 5 min at 95°C, denaturation at 95°C for 30 s, annealing at 58°C

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for 30 s, extension at 72°C for 1 min, and final extension for 5 min at 72°C. The cycle number was 38. PCR-produced fragments were analyzed by running 2% agarose gel and then measured using Image J (National Institutes of Health). The primer sequences are below:

HP1α reverse primer: 5’-GTAGATATTCCACTTGT-3’.

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HP1α forward primer: 5’-GGAAAGAAAACCAAGCGGACA-3’.

Actin forward primer: 5’-GATGATGATATCGCCGCGCTC-3’.

Western blotting

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Actin reverse primer: 5’-TGGGGCGCCCCACGATGGAG-3’.

Western blotting was performed as previous description [32]. Briefly, SDS-PAGE was performed to separate the proteins that were further transferred to a PVDF membrane (Millipore). The PVDF membrane was sequentially incubated with primary antibodies and

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secondary HRP-conjugated antibodies.

Prognostic significance analysis

GeneChip (Affymetrix Human Genome U133 Plus 2.0) mRNA expression profiling data was

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downloaded from two published datasets, dataset 1 (GSE16011, n = 270) and dataset 2 (GSE13041-GPL570, n = 27) [33; 34]. The clinical information of all cases (patient age at diagnosis, tumor grade, surgery, and survival time) was available. Affymetrix GeneChip probes

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Mapping was performed using customchip definition files based on the NCBI Entrez Gene v.11 (http://brainarray.mbni.med.umich.edu/Brainarray). Probesets were summarized by median intensity. To separate samples into low expression and high expression groups, recursive partitioning analysis was performed. The two groups were compared by the Kaplan-Meier method and the significance was defined by the log-rank test.

BrdU incorporation assay Cells were washed with PBS, followed by fixation and permeabilization in methanol for 7-10

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min at –20°C. Fixed cells were incubated with primary antibodies in PBS containing 3% bovine serum, followed by incubating with secondary antibodies and 1 µg/ml DAPI (Wako pure chemical industries) [35]. A microscope (NIKON) equipped with a 100×/1.40 NA objective lens

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was used to observe the cells.

Chromatin immunoprecipitation assays

H3K9me3 and HP1α chromatin immunoprecipitations (ChIPs) were performed using 1 µg antiH3K9me3 antibody (61013, Active motif) and 2 µg anti-HP1α antibody (05-689, Millipore)

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respectively. A mouse IgG isotype was used as a control for antibody specificity. Cells (on plates) and tissues (cut up) were washed with PBS, fixed with 1% formaldehyde for 20 min, quenched

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with 0.125 M glycine, and then lysed in sonication buffer (50 mM Hepes, pH 7.9, 140 mM NaCl, 1 mM EDTA, 1 mM PMSF, 0.1% SDS, 0.1% NaDeoxycholate, and 1% Triton X-100). Crosslinking

was

reversed

by

incubating

at

65°C

overnight.

After

chromatin

immunoprecipitation, DNA was purified and analysed in duplicate using a Sybr GreenER mix (Invitrogen) and quantified on a RT-qPCR machine (Applied biosystems). The primer sets were

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described previously [36]. RT-qPCR signal of ChIP DNA was normalized to its input DNA.

MTT assay

Cells were collected and plated into 96-well plates, and then left overnight. The next day, cells were incubated with 1 mg/ml MTT (Sigma) for 3 h in cell culture incubator. The MTT formazan

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nm as background.

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product was solubilized in isopropanol and the absorbance was measured at 570 nm by using 690

Apoptosis assay

Cells were plated in 12-well plates at a density of approximately 50,000 cells per well. Cells were trypsinized, washed once with PBS, and harvested by centrifugation. Annexin V/propidium iodide kit (ThermoFisher) was used to double stain cells. Cell apoptosis was measured by using a fluorescence-activated

cell

sorter

(FACSCalibur,

Ecton-Dickinson).

positive/propidium iodide-negative cells were numbered as apoptotic cells.

Quantitative and Statistical analysis 7

Annexin

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The fluorescence signals and western blotting bands intensity were measured with Image J software (National Institutes of Health). Statistical analyses were performed by using SPSS (SPSS Inc.) and GraphPad Prism 5 software (GraphPad Software Inc.). The statistical

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significance calculation was based on the unpaired two-tailed student’s t-test.

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Results HP1α, H3K9me3, and SUV39H1 are upregulated in glioma cell lines Oncogenic role of SUV39H1, a critical H3K9me3 methyltransferase [23; 26], has been proposed

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in several cancer cell types [37; 38], especially in gliomas [30]. Consistantly, H3K9me3 exhibited up-regulated level in some cancer cell lines [39]. However, it is unclear that how H3K9me3 is connected to the tumorigenesis. In order to address the issue, we focused on investigating the roles of H3K9me3 in gliomas. First, we detected the levels of H3K9me3 and its

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reader HP1α protein in normal human astrocyte (NHA) cell line, human brain astrocytoma glioma cell line 1321N1, human astrocytoma-derived cell line GOS3, and human glioblastoma

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cells (T98G, DBTRG-05MG, and ANGM-CSS) by western blotting assays (Fig. 1A). HP1α protein levels were elevated by 110% (in GOS3), 100% (in DBTRG-05MG), 160% (in T98G), 115% (in ANGM-CSS), and 114% (in 1321N1) compared to NHA cells (Fig. 1A,B). H3K9me3 was up-regulated by 98% (in GOS3), 90% (in DBTRG-05MG), 95% (in T98G), 70% (in ANGM-CSS), and 99% (in 1321N1) compared to NHA cells (Fig. 1A,C). Then we checked the levels of HP1α and H3K9me3 in three normal brain tissues and fifteen glioma tissues of different

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grades (2, 3, and 4). HP1α protein levels were elevated by 120% (in grade 2), 190% (in grade 3), and 230% (in grade 4) compared to normal brain (Fig. 1D,E). H3K9me3 was increased by 180% (in grade 2), 190% (in grade 3), and 210% (in grade 4) compared to NHA cells (Fig. 1D,F). Consistant with the elevation of HP1α protein levels in the glioma tissues, the mRNA levels of

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HP1α also elevated (by ~120%) in all the three grades of glioma tissues compared to normal brain (Fig. 1G,H). Thus, these data indicates that HP1α and H3K9me3 are up-regulated in

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glioma cell lines and tumor tissues.

To examine whether expression of HP1α is clinically correlated with glioma patients

survival, we analyzed the mRNA level of HP1α in 270 cases (dataset 1) and 27 cases (dataset 2), respectively. High expression of HP1α was associated with pool survival [a median survival of 11 months in the high-expressing cases vs. 24 months in the low-expressing cases for dataset 1 (P = 0.00003, log rank) and 9 months in the high-expressing cases vs. 25 months in the lowexpressing cases for dataset 2 (P = 0.027, log rank)] (Fig. 1I,J).

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Depletion of HP1α and SUV39H1 reduces survival of glioma cells In order to test whether HP1α and H3K9me3 are connected to glioma progression, we performed

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RNA interference assays. GOS3, 1321N1 and T98G cells were transiently transfected with negative control-siRNA, HP1α-siRNA, or SUV39H1-siRNA, and analyzed by western blotting (Fig. 2A-F). HP1α expressions were significantly reduced in HP1α siRNA 1 or siRNA 2 treated cells compared to negative control cells (Fig. 2A-C). SUV39H1 expression was also much less in

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SUV39H1 siRNA transfected cells than in negative control cells (Fig. 2D-F), but another H3K9 methylation enzyme G9a was not significantly changed (Fig. 2A-F). SUV39H1 protein level was

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not significantly affected by HP1α knockdown (Fig. 2A), and vice versa (Fig. 2D). To test whether high expression levels of HP1α are required for glioma cell survival, we analyzed cell apoptosis by flow cytometry. HP1α depleted cells showed a highly increased apoptotic cell percentage. HP1α siRNA 1 induced 23% increase of apoptotic cell percentage and HP1α siRNA 2 induced 21% in GOS3 cells (Fig. 2G). SUV39H1 depleted cells also revealed an increased percentage of apoptotic cells (by 18% in GOS3 cells) (Fig. 2G). Similar results were

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obtained in 1321N1 cells (Fig. 2H) and (Fig. 2I). These data suggests that HP1α and SUV39H1 contribute to glioma cells survival by inhibiting cell apoptosis.

HP1α and H3K9me3 regulate the expression of FAS and PUMA

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To further investigate the mechanisms by which HP1α and SUV39H1 promote glioma cell survival, we performed HP1α and H3K9me3 chromatin immunoprecipitation assays (ChIP).

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It showed that HP1α and H3K9me3 had higher enrichment at the FAS and PUMA promoters instead of Actin promoter in GOS3 cells than in NHA cells (Fig. 3A-C). With depletion of HP1α, enrichment of HP1α at the FAS and PUMA promoters were significantly weakened (Fig. 3A-C). H3K9me3 at the FAS and PUMA promoters was also slightly affected by HP1α depletion (Fig. 3A-C). FAS and PUMA are essential activators of apoptosis and suppress tumor progression [40; 41]. Consistant with the ChIP results, FAS and PUMA protein levels were much lower in GOS3 cells than NHA cells (Fig. 3D). With HP1α depletion, FAS and PUMA protein levels were significantly elevated (Fig. 3E).

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By performing HP1α and H3K9me3 ChIP assays using glioma tissues, we observed that HP1α and H3K9me3 were accumulated at FAS and PUMA promoters in three grades (grade 2, grade 3, and grade 4) of glioma tissues (Fig. 3F-H). FAS and PUMA proteins were much less in the glioma tissues than in normal brain (Fig. 3I).

apoptosis by suppressing expression of FAS and PUMA.

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Therefore, these results indicate that up-regulated H3K9me3 and HP1α prevent cell

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Depletion of HP1α and SUV39H1 weakens proliferation ability of glioma cells

Further, we examined glioma cells proliferation by MTT assays after HP1α and SUV39H1 depletion. GOS3 and 1321N1 cells proliferation in SUV39H1 depleted cells was decreased

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compared to negative control cells (Fig. 4A-B). Especially at the 48 h time point, the proliferation was significantly reduced (P<0.05) (Fig. 4A-B). HP1α depletion also induced a remarkable decrease of cell proliferation in both GOS3 and 1321N1 cells (P<0.01) (Fig. 4A-B). To further determine whether HP1α and SUV39H1 were necessary for the growth of GOS3 and 1321N1 cells, we measured S phase entry by determining the rate of BrdU incorporation of cells treated with siRNAs. The rates of BrdU incorporation were remarkably reduced with HP1α

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suppression by 20% (siRNA 1) and 16% (siRNA 2) (Fig. 4C-D). The percentage of BrdU positive cells was also decreased (by 12%) with SUV39H1 suppression (Fig. 4C-D). The similar percentage decrease of BrdU cells were also observed in 1321N1 cells (Fig. 4E). Therefore, these data suggests the suppressive effects of HP1α and SUV39H1 depletion in the proliferation

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capacity of glioma cells.

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Discussion In our study, we find the high protein levels of HP1α, and high level of trimethylation in Histone H3 Lysine 9 in glioma cell lines and glioma tumor tissues. Depletion of HP1α and SUV39H1 attenuates glioma cell proliferation and increases cell apoptosis. HP1α and H3K9me3 are

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aberrantly enriched at the promoters of two apoptosis activators FAS and PUMA in both glioma cells and tissues. Our data indicates HP1α and H3K9me3 are functionally associated with glioma cell viability and proliferation capability. Therefore, HP1α and H3K9me3 may be biomarkers of gliomas, and study of epigenetic regulation in glioma cells potentially promotes novel and more

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efficient therapy strategy development.

Brain tumors are characterized by alterations in genetic and epigenetic mechanisms [7].

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The importance of Histone modifications have begun being revealed in glioma progression [22; 30; 39]. Understanding the molecular basis of roles of epigenetic factors in tumorigenesis and cancer cell survival or death potentially facilitates novel therapeutic progression. Unlike genetic alteration, epigenetic changes are more reversible [25; 29]. Histone lysine methylation is a dynamic process and the maintenance of epigenetic marks on Histones highly relies on the related modification enzymes [23; 26], and its effect to the cells is mainly achieved

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by recruiting its readers [26]. SUV39H1 and H3K9me3 are up-regulated in some glioma cells lines, and the roles of H3K9me3 in glial cell differentiation and in affecting high-grade astrocytomas patients’ survival have been implicated [30]. HP1α, an essential reader of H3K9me3, has been reported to be required for

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tumorigenicity and aggressiveness of lung carcinoma in vivo, and high expression level of HP1α protein negatively correlates with patient survival [42]. We found that HP1α was up-regulated in

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glioma cells and tissues. High level of HP1α and H3K9me3 in glioma cells could function cooperatively in inducing aberrant chromatin domain silencing and promoting cell survival and proliferation. Elevated SUV39H1 expression level could contribute to establishment and maintenance of a high level of H3K9me3 [30], which is recognized by HP1 proteins and acts as a binding site for HP1 proteins [26]. H3K9me3 in the glioma cells may be accumulated in the transcriptionally active chromatin region, which further results in aberrant enrichment of HP1α proteins in these H3K9me3 sites and abnormal silent chromatin domains formation, and represses some specific genes that are supposed to be active in normal cells. We have found that HP1α and H3K9me3 were accumulated in the FAS and PUMA promoters. Therefore, HP1α and 12

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H3K9me3 possibly prevent cell apoptosis by targeting apoptotic activators, including FAS and PUMA [40; 41]. There may be cooperation between high levels of HP1α and SUV39H1 in glioma cells as well. By interacting with SUV39H1, highly expressed HP1α may promote the recruitment of

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SUV39H1 to the sites to reinforce the silencing and contribute to the maintenance of high-level H3K9me3 in glioma cells. However, knockdown of HP1α or SUV39H1 did not significantly decrease the protein level of each other (Fig. 2 A and D). Therefore, induction of proliferation and apoptosis change after HP1α knockdown is most likely independent of SUV39H1 and vice

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versa.

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Acknowledgement This work was supported by the Jiangxi Provincial Science and Technology Department funding

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(20161BBH80075).

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Conflict of Interest

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We declare that we have no conflict of interest.

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Figure Legends Figure 1. The expression of HP1α protein is increased in glioma cells and tissues. (A) Western blots showing levels of HP1α protein and Histone H3K9me3 in NHA, GOS3, DBTRG05MGT98G, ANGM-CSS, and 1321N1 cells. Actin protein levels were used as control. (B-C)

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Quantification of HP1α protein level (B) and H3K9me3 level (C) in NHA, GOS3, DBTRG05MGT98G, ANGM-CSS, and 1321N1 cells. The signals were normalized to Actin, and the signal from NHA cells was normalized to 1.0. (D) Western blots showing levels of HP1α protein and Histone H3K9me3 in Grade 2, Grade 3, and Grade 4 glioma tissues, and normal brain. Actin

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protein levels were used as control. (E-F) Quantification of HP1α protein level (E) and H3K9me3 level (F) in glioma tissues and normal brain. The signals were normalized to Actin,

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and the signal from NHA cells was normalized to 1.0. (G) Quantitative PCR showing levels of HP1α mRNA in glioma tissues and normal brain. Actin mRNA level was used as control. (H) Quantification of HP1α mRNA level in glioma tissues and normal brain. The signals were normalized to Actin, and the signal from NHA cells was normalized to 1.0. Each experiment was repeated three times. Data are mean ± s.e.m.. ***P<0.001; **P<0.01. (I-J) The role of HP1α in gliomas was suggested by analyzing gene expression profiling data from two independent data

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sets of the brain cancer, dataset 1 (I) and dataset 2 (J). Kaplan-Meier curves of survival for 270 patients and 27 patients are presented. Samples were separated into high (red) and low (green) HP1α expression groups by recursive partitioning method.

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Figure 2. HP1α prevents apoptosis of GOS3, 1321N1 and T98G cells. (A-C) Western blots showing knockdown of HP1α in GOS3 (A), 1321N1 (B), and T98G (C) cells by siRNA

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transfection for 48 h. NC, negative control. (D-F) Western blots showing knockdown of SUV39H1 in GOS3 (D), 1321N1 (E), and T98G (F) cells by siRNA transfection for 48 h. NC, negative control. (G-I) Quantification of apoptotic cells after indicated siRNA treatment in GOS3 (G), 1321N1 (H), T98G (I) cells. Annecin V/propidium iodide double staining was performed to label the apoptotic cells. The ratio of apoptotic cells was calculated. NC, negative control. Each experiment was repeated three times. Data are mean ± s.e.m.. **P<0.01; ***P<0.001.

Figure 3. HP1α and H3K9me3 repress expression of FAS and PUMA. (A-C) NHA, GOS3 wild type cells, and HP1α siRNA-transfected GOS3 cells were used for chromatin 19

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immunoprecipitation (ChIP) with non-immune mouse IgG, anti-HP1α and anti-H3K9me3 antibodies. ChIPs were quantified by RT-qPCR, normalized relatively to input DNA, and the % recovery for ChIP is plotted on the y-axis. Actin (A), FAS (B) and PUMA (C) promoters were analyzed. (D) Western blots showing PUMA and FAS protein levels in NHA and GOS3 cells.

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Actin protein levels were used as control. (E) Western blots showing PUMA and FAS protein levels in GOS3 cells after HP1α depletion. NC, negative control. Actin protein levels were used as control. (F-H) glioma tissues and normal brain were used for chromatin immunoprecipitation (ChIP) with non-immune mouse IgG, anti-HP1α and anti-H3K9me3 antibodies. ChIPs were

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quantified by RT-qPCR, normalized relatively to input DNA, and the % recovery for ChIP is plotted on the y-axis. Actin (F), FAS (G) and PUMA (H) promoters were analyzed. (I) Western

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blots showing PUMA and FAS protein levels in glioma tissues and normal brain. Actin protein levels were used as control.

Figure 4. HP1α facilitates GOS3 and 1321N1 cell proliferation. (A, B) MTT proliferation assays of GOS3 (A) and 1321N1 (B) cells transfected with negative-siRNA, SUV39H1-siRNA, HP1α-siRNA 1, and HP1α-siRNA 2 for 24 h or 48 h. Each experiment was repeated three times.

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Data are mean ± s.e.m.. *P<0.05; **P<0.01; ***P<0.001. (C) BrdU incorporation assays of GOS3 cells. Cells were transfected with negative-siRNA, SUV39H1-siRNA, HP1α-siRNA 1, and HP1α-siRNA 2 for 48 h, followed by immunofluorescence of BrdU (red). DNA was stained with DAPI. Scale bar, 10 um. (D) Quantification of percentage of BrdU positive cells from (C).

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Each experiment was repeated three times. Data are mean ± s.e.m.. *P<0.05; **P<0.01. (E) Quantification of percentage of BrdU positive 1321N1 cells transfected with negative-siRNA,

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SUV39H1-siRNA, HP1α-siRNA 1, and HP1α-siRNA 2 for 48 h. Each experiment was repeated three times. Data are mean ± s.e.m.. *P<0.05; **P<0.01.

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HP1α and H3K9me3 are upregulated in gliomas Depletion of HP1α results in apoptosis. HP1α and H3K9me3 suppress expression of FAS and PUMA.