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Comparative analysis of histone H3 and H4 post-translational modifications of esophageal squamous cell carcinoma with different invasive capabilities Kai Zhanga,b,c,⁎, Liyan Lid,1 , Mengxiao Zhud,1 , Guojuan Wangc , Jianjun Xieb , Yunlong Zhaoc , Enguo Fana , Liyan Xud,⁎⁎, Enmin Lib,⁎⁎ a
Tianjin Key Laboratory of Medical Epigenetics, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China b Department of Biochemistry and Molecular Biology, Key Laboratory of Molecular Biology in High Cancer Incidence Coastal Chaoshan Area of Guangdong Higher Education Institutes, Shantou University Medical College, Shantou 515041, China c State Key Laboratory of Medicinal Chemical Biology, Department of Chemistry, Nankai University, Tianjin 300071, China d Institute of Oncologic Pathology, Shantou University Medical College, Shantou 515041, China
AR TIC LE I N FO
ABS TR ACT
Article history:
Eukaryotic DNA is packaged into a chromatin with the help of four core histones (H2A, H2B, H3,
Received 1 July 2014
and H4). Diverse histone post-translational modifications (PTMs) are hence involved in the
Accepted 4 September 2014
regulation of gene transcription. However, how this regulation does work is still poorly
Available online 16 September 2014
understood and lacks details. Here we used the mass spectrometry-based proteomics approach to perform a comparative analysis of histone marks at a global level in two phenotypes of
Keywords:
esophageal squamous cell carcinoma (ESCC) with different invasiveness. We obtained a
Protein post-translational
comprehensive profiling of histone H3 and H4 PTMs including lysine methylation, acetylation
modifications (PTMs)
and novel butyrylation. The correlation between histone marks and cancer invasive capabilities
Histone
was further characterized and one distinguishable PTM, H4K79me2 was discovered and verified
Mass spectrometry
in this study. Immunohistochemistry analysis suggests that abnormal level of H4K79me2 may be
Esophageal squamous cell carcinoma
related to poor survival of ESCC patients. Our results enrich the dataset of the feature pattern of
Invasive capability
global histone PTMs in ESCC cell lines. Biological significance Core histone proteins, decorated by multiple biological significant protein post-translational modifications (PTMs) such as lysine acetylation and lysine methylation, are considered to regulate gene transcription and be associated with the development of cancer. Recent studies have further shown that global level of histone modifications is the potential hallmark of cancer to predict the clinical outcomes of human cancers. However, the regulation mechanism is
⁎ Correspondence to: K. Zhang, Department of Biochemistry and Molecular Biology, Tianjin Medical University, Tianjin 300070, China. Tel.: + 86 22 24387742. ⁎⁎ Corresponding author. Tel.: + 86 754 88900460. E-mail addresses: [email protected] (K. Zhang), [email protected] (L. Xu). 1 Co-first author.
http://dx.doi.org/10.1016/j.jprot.2014.09.004 1874-3919/© 2014 Elsevier B.V. All rights reserved.
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largely unknown. Here we used the mass spectrometry based proteomics coupled with stable isotope labeling with amino acids in cell culture (SILAC) to characterize the global levels of histone marks in two phenotypes of esophageal squamous cell carcinoma (ESCC) cell lines with different invasive capabilities. To the best of our knowledge, it is the first report about the comparative analysis for histone marks of the different invasive ESCC cell lines. A significantly differential level of histone modification, H4K79me2, was determined and verified. Immunohistochemistry analysis further suggests that abnormal level of H4K79me2 may be related to poor survival of ESCC patients. Our results could contribute to understanding the different expressions of global histone PTMs in different invasive ESCC cell lines. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In eukaryotic chromosomes, genomic DNA is packaged into a compacted chromatin with the help of four core histones (H2A, H2B, H3, and H4). The core histone proteins are characterized by diverse post-translational modifications (PTMs) at their N-terminal tails. The histone modifications play a key role in regulating chromatin structure and transcriptional activity by modulating the packaging of chromatin and recruiting PTM-specific binding proteins [1,2]. Histone PTMs are generally considered to be a major group of important epigenetic marks involved in diverse biological processes such as DNA repair and chromosome condensation. Increasing evidence suggests that dysregulation of histone PTMs has intimately linked with the development and progression of cancer [3]. Furthermore abnormal expressions of specific histone PTMs have been observed in a variety of human cancers including lung [4], prostate [5], kidney [6], breast [7], and stomach [8]. For example, global loss of acetylation and gain of methylation at lysine 9 and lysine 27 in histone H3 have been considered to be associated with the silencing of genes, resulting in the development of cancer [9]. Recent studies have further shown that global level of histone modifications is the potential hallmark of cancer to predict the clinical outcomes of human cancers [10]. Esophageal cancer (EC) is the sixth leading cause of cancer deaths worldwide. In China, esophageal squamous cell carcinoma (ESCC) is the predominant histological subtype and accounts for approximately 90% of all EC [11]. To date, most EC patients with 5-year survival rates remain as low as 10% or even less. Pattern of histone PTMs disruption is largely unknown in ESCC, however it has been reported that the global level of acetylation on H4 was varied with ESCC progression and metastasis [12,13]. Furthermore several histone marks have been demonstrated to be associated with the development of tumors [14]. However, the histone modification has not yet been examined carefully in ESCC cell lines. A complete atlas of histone PTMs should be constructed to understand the relationship between histone code and ESCC properties. To date, mass spectrometry (MS) has become the fundamental tool to characterize histone PTMs [15,16]. At least 11 types of histone PTMs have been reported at over 60 different amino acid residues [1,17]. Besides the known histone PTMs such as lysine methylation and acetylation, several types of new histone PTMs have been discovered at lysine residues such as lysine butyrylation, propionylation [18,19], crotonylation [17] and 2-hydroxyisobutyrylation [20], which suggests even more unknown histone modifications to be discovered [21]. However it is still unclear what role does the novel histone PTM play in the regulation of histone function in a variety of diseases.
Here we used the mass spectrometry based proteomics coupled with stable isotope labeling with amino acids in cell culture (SILAC) to characterize the global levels of histone marks in two phenotypes of ESCC cell lines with different invasive capabilities. Individual specific histone PTMs were carefully determined including lysine methylation, acetylation and novel butyrylation. We measured the abundance of key histone PTMs in two cell lines, and further evaluated the correlation between histone marks and ESCC invasion capabilities. A significantly differential level of histone modification, H4K79me2, was determined and verified in this study. Immunohistochemistry analysis further suggests that abnormal level of H4K79me2 may be related to poor survival of ESCC patients.
2. Materials and methods 2.1. Materials Water and acetonitrile were from Fisher Scientific (Pittsburgh, PA). Trifluoroacetic acid (TFA) was from Sigma-Aldrich (St. Louis, MO). Colloidal Coomassie Blue Staining Kit was from Invitrogen (Carlsbad, CA). Sequencing-grade trypsin was from Promega (Madison, WI). C18 ZipTips were from Millipore (Bedford, MA).
2.2. Cell culture ESCC cell lines (TE3 and KYSE180) were generous gifts from Professor Ming-Zhou Guo, Department of Gastroenterology & Hepatology, Chinese PLA General Hospital. TE3 and KYSE180 cells were cultured with modified RPMI-1640 medium (Thermo, Pittsburgh, PA) supplemented with 10% fetal calf serum at 37 °C with 5% CO2 in atmosphere. The SILAC labeling was carried out as described earlier [22]. In brief, TE3 and KYSE180 cells were maintained in RPMI-1640 medium containing 13C6-lysine and 12 C6-lysine (Thermo, Pittsburgh, PA), respectively, containing 10% fetal calf serum in an atmosphere of 5% CO2 at 37 °C. Two cell lines were grown for more than 6 generations before being harvested in order to achieve more than 99% labeling efficiency.
2.3. Western blotting Total cell lysates were prepared in RIPA buffer, separated by SDS-PAGE and transferred to PVDF membranes (Millipore). The membranes were incubated in blocking buffer and then incubated with histone PTM antibody (histone anti-H3K9me2
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2.4. Cell invasiveness assay Invasiveness assay was performed as described before [23]. Cells (1 × 105) were seeded onto the top chamber of a 24-well matrigel-coated membrane with 8-μm pores (Millipore), and the bottom chamber was filled with culture medium with 10% fetal calf serum. The membranes were fixed and stained by Giemsa reagent, and invasiveness and migration were quantified by counting 10 random fields under a light microscope (400×). The mean value was calculated from data obtained from 3 separate chambers.
400
Invasive cells
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2.5. Histone extraction
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The cells were harvested by centrifugation at 5000 ×g for 5 min and washed once with distilled water. Cells were resuspended in Triton Extraction Buffer (TEB: PBS containing 0.5% Triton X-100 (v/v), 2 mM phenyl-methylsulfonyl fluoride (PMSF), 0.02% (w/v) NaN3) at a cell density of 107 cells ml−1. Lysed cells were put on ice for 10 min with gentle stirring. Cells were removed by centrifugation for 15 min at 2000 ×g. After supernatant removal, the cells were washed with half a volume of TEB and centrifuged as before. The core histones were extracted with 0.4 N H2SO4 on ice overnight. The extract was centrifuged at 4 °C for 10 min at 16,000 ×g. Histone proteins were precipitated using trichloroacetic acid precipitation (TCA). Samples were then washed twice with acetone containing 0.1% HCl (v/v) and twice with pure acetone. The pellet was allowed air drying and was then resolubilized in water.
100 50 0
KYSE180
TE3
Fig. 1 – Invasiveness assay of KYSE180 and TE3 cell lines. Invasiveness assay was used to determine the invasive capability. Invasive cells were fixed and stained, and representative fields were photographed. For quantification, the cells were counted in 10 random fields under a light microscope. Each experiment was performed in triplicate.
2.6. SDS-PAGE separation and in-gel digestion and anti-H4K79me2 antibody were from Millipore, and antiacetyllysine pan antibody and anti-butyryllysine pan antibody were from PTM BioLab, Inc.). Finally, immunoreactive bands were revealed using luminol reagent (Santa Cruz Biotechnology). Photography and quantitative analyses were done using the FluorChemTMIS-8900 (Alpha Innotech).
The core histones were resolved in a 15% SDS-PAGE gel and visualized by colloidal Coomassie blue staining. Gel bands of interest were excised and subjected to in-gel digestion as described previously [24]. Briefly, the gel bands were sliced into small pieces (~1 mm3) and destained with 25 mM ammonium
Bu Bu
H3
Me2
Me2
KTE3
N Kyse180
Ac
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Ac
K9
K14
K18
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Bu
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Bu
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K20
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Ac
Me
R55 Me
Me2
K59
Me
Me2 Me2
Me2
K77 R78 K79 K91 Me
Me2 Me2
C
Me2
Bu
Fig. 2 – Identified post-translational modification sites within core histones H3 and H4 in KYSE180 and TE3 cells. Ac: acetylation; Me: mono-methylation; Me2: di-methylation; Me3: tri-methylation; Buty: butyrylation.
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bicarbonate in ethanol/water (50:50, v/v). The destained gel pieces were washed in an acidic buffer (acetic acid/ethanol/ water, 10:50:40, v/v/v) three times for 1 h each time, and in water two times for 20 min each time. The gel pieces were dehydrated
in acetonitrile and dried in a SpeedVac (ThermoFisher, Waltham, MA). Two hundred nanograms of porcine modified trypsin (Promega, Madison, WI) in 50 mM ammonium bicarbonate was added to the dried gels and incubated overnight at 37 °C. Tryptic
K(Bu)QLATK(Ac)AAR
H3K18 Buty
Retention Time 28.69 min Parent ion intensity 2.14 E4
A M-NH3
y3 317.19
100
y7 772.53
532.37
Relative Abundance
b2 327.33 b1 199.25
y4 487.37 b3 440.36
b(2)* 310.30
y1 175.12
y6 659.41 y5 588.36
b4 511.37
y2 246.27
y8 b7 853.60 900.62
b8 924.62
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m/z y7 772.56
Relative Abundance
100
Retention Time 25.99 min Parent ion intensity 2.55 E6
b2 299.31
M-NH3 518.48 b5 b3 584.33 y5 412.36 b(4)* 466.39 588.38 y4 487.34
y3 b(2)* y2 282.27 317.29 246.26
y1 175.15
y6 659.46 y(7)0 754.49
b7 825.59
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y2 288.37
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y7 964.45 b7 902.46
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b(3)0 296.28
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b10 1231.62
y9 1163.57
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EIAQDFK(Ac)TDLR
671.49
Retention Time 49.87 min Parent ion intensity 4.86 E3
y7
M-H2O
b(5)0
936.47
y5 674.44
b9 1090.51
b7 874.43
539.21
y3 403.29 b4 441.31
b6 704.36 400
y*(8) 1075.64
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y(9)++ 568.39
y2 288.24
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b9 1118.52
b*(8) 986.59
y4 504.27
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y6 849.48
b(7)0 884.31
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y5 702.50
y3 403.24
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Retention Time 54.21 min Parent ion intensity 1.19 E3
y*(5) 685.39
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y8 900.60
EIAQDFK(Bu)TDLR
H3K79 Buty
b2 243.13
K(Ac)QLATK(Ac)AAR
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y6 821.47 b(7)0 856.38 800
b(8)0 957.41 900
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y9 y8 1135.53 1064.56 b10 1203.55 1100
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Fig. 3 – (A) MS/MS of a tryptic peptide ion K(Bu)QLATK(Ac)AAR from histone H3 (up) and MS/MS of a tryptic peptide ion K(Ac) QLATK(Ac)AAR from histone H3 (Bottom). (B) MS/MS of a tryptic peptide ion EIAQDFK(Bu)TDLR from histone H3 (up) and MS/MS of a tryptic peptide ion EIAQDFK(Ac)TDLR from histone H3 (bottom).
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Fig. 4 – (A) The quantitative analysis of lysine methylation sites on histones in KYSE180 and TE3. (B) The quantitative comparison of lysine acetylation sites on histones between KYSE180 and TE3. The relative ratio of abundance of KYSE180 and TE was determined according to the description in Section 2. Mean values and standard deviations derive from 3 technical replicates. peptides were sequentially extracted from the gel pieces with 50% acetonitrile (acetonitrile/water/TFA, 50:45:5, v/v/v) and 75% acetonitrile (acetonitrile/water/TFA, 75:24:1, v/v/v). The peptide extracts were pooled and dried in a SpeedVac. The peptide extracts were desalted using a μ-C18 Ziptip before HPLC/MS/MS analysis.
2.7. Nano-HPLC/mass spectrometric analysis Each tryptic digestion was dissolved in 10 μL of HPLC buffer A (0.1% (v/v) formic acid in water), and 2 μL was injected into a Nano-LC system (Eksigent Technologies, Dublin, CA). Peptides were separated on a homemade capillary HPLC column (100-mm length × 75-μm inner diameter) containing Jupiter C12 resin (4-μm particle size, 90-Å pore diameter, Phenomenex, St. Torrance, CA) with a 120 min HPLC-gradient from 5 to 90% HPLC buffer B (0.1% formic acid in acetonitrile) at a flow rate of 200 nL/min. The HPLC elute was electrosprayed directly into an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Waltham, MA) using a nanospray source. The LTQ-Orbitrap Velos mass spectrometer was operated in a data-dependent mode with resolution R = 60,000 at m/z 400. Full scan MS spectra from m/z 350–2000 were acquired in the
Orbitrap. The twenty most intense ions were sequentially isolated in the linear ion trap and subjected to collisionactivated dissociation (CAD) with a normalized energy of 35% and an isolation width of 4 Da. The exclusion duration for the data-dependant scan was 36 s, the repeat count was 2, and the exclusion window was set at ± 2 Da. AGC settings were 1E6 for full scan Orbitrap analysis, 1E4 for MSn scan in the ion trap and 6E4 for MSn scan in the Orbitrap. Signal threshold for CID acquisition was set at 5000.
2.8. Protein sequencing alignment and bioinformatics analysis Peak lists were first generated by the extract_msn.exe software (v5.0, Thermo Scientific). All MS/MS spectra were searched against the NCBI human protein sequence database (September 20, 2012) using the Mascot search engine (version 2.1.0, Matrix Science, London). Trypsin was specified as digesting enzyme. A maximum of 5 missing cleavages were allowed. Mass tolerances for precursor ions were set at ±0.1 Da for precursor ions and ± 0.5 Da for MS/MS. For label free samples, the specific search parameters included lysine acetylation, lysine mono, di, and tri-methylation, lysine butyrylation and arginine methylation as variable modifications. For SILAC samples, lysine (13C6), lysine
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KYSE180
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A
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H3K9me2
B
an-H3
KYSE180
Pan-buty
TE3
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H3 H4 an-H3 Fig. 5 – Western blotting detection for histone modifications. (A) H3K9me2; (B) H4K79me2; (C) pan lysine-acetylation; and (D) pan lysine-butyrylation.
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Dako, Glostrup, Denmark) and Liquid DAB Substrate Kit (Zymed/Invitrogen, Carlsbad, CA, USA) were used to conduct immunohistochemical staining according to the manufacturer's instructions respectively. Scores were independently assessed by two researchers blinded to clinical data. Each separate tissue core was scored based on both intensity and percentage of positive cells [25]. Briefly, the intensity grade of staining was: 0, negative; 1, weak; 2, moderate; and 3, strong. The percentage of positive cells was: 0, 75%. The final score was calculated by multiplying the intensity grade and the percentage of positive cells, producing a total range from 0 to 12. To assess statistical significance and avoid arbitrary cutoff point selection, the X-tile program was applied to assess H4K79me2 expression score and determine an optimal cutoff value based on outcome. According to the optimal cutoff value, data analysis was performed using SPSS 13.0 software (SPSS, Inc., Chicago, IL). Kaplan–Meier curves were obtained for overall survival analysis using a log-rank test. Each P-value is two-tailed and significance level is 0.05.
3. Results and discussion acetylation (13C6), lysine mono, di, and tri-methylation (13C6), and lysine butyrylation (13C6) were also included as variable modifications. All the identified peptides with Mascot score above 40 and E-value below 0.05 were manually verified according to the rules described previously [24]. Quantification of PTMs was based on the peak areas of precursor ions of peptides labeled by light and heavy stable isotopes. Briefly, for every PTM site, we first validated all the identified peptides containing the targeted PTM type and site, then added up the peak areas of precursor ion of these peptides. The ratio of the light and heavy isotope peak areas was used for comparing the relative abundance of the PTM between the two cell lines. Mean values and standard deviations derive from 3 technical replicates.
2.9. Tissue specimens and immunohistochemical staining A total of 166 ESCC patients underwent curative surgery in the Affiliated Shantou Hospital of Sun Yat-sen University (Shantou, China) between 2000 and 2006. Then the patients were followed up until their deaths. Mean age at surgery was 57.7 (range 31 to 75), and 41 patients were female and 125 were male. Clinicopathological characteristics of the patients were assessed according to the 7th edition of the tumor-node-metastasis (TNM) classification of the International Union against Cancer (UICC). The patients suffered from severe post-operative complications and died of other tumors or other causes were excluded. The study was approved by the ethical committee of the Central Hospital of Shantou City and the ethical committee of the Medical College of Shantou University, and a written informed consent was obtained from each patient to use resected samples for research. All paraffin-embedded samples undergoing tissue microarray (TMA) construction were cut into 4 μm sections. The rabbit polyclonal antibodies to H4K79me2 (1:200, ab2885, Abcam Inc., Cambridge, MA, USA), a Universal LSAB™ + Kit/HRP (K0690,
3.1. Identification and quantitative approach for profiling histone PTMs Accumulated evidence suggests that the dysregulation of histone PTMs might be associated with the development of cancer. However it is still unclear how these epigenetic marks are linked with critical processes of development, such as invasive and metastatic capabilities of tumors. To investigate how histone PTMs correlates with cancer invasive capability, we chose two phenotypes of ESCC cell lines that possess different invasiveness. As shown in Fig. 1, the invasive and metastatic property of TE3 is significantly higher than that of KYSE180 cells. To the best of our knowledge, histone PTMs profiles have not yet been examined carefully in these two cell lines. Our interests focused on the PTMs of histones H3 and H4 because their abnormal expressions have been shown to be involved in the development of tumor [5]. The histone PTMs in each cell lines were mapped, and then compared their abundance at specific PTM sites using a quantitative proteomics technique by coupling SILAC with high-resolution LTQ-Orbitrap mass spectrometry (See Supplementary material 1).
3.2. Characterization of global histone PTMs in two ESCC cell lines MS/MS spectra were searched against the NCBInr human protein sequence database using MASCOT software. After a manual inspection as described previously [24], we identified in total 35 sites that were modified with 5 types of PTMs on core histones on H3 and H4 in TE3 cells, including 11 sites of lysine acetylation, 17 sites of lysine methylation (mono-, di-, and tri-methylation), 4 sites of lysine butyrylation, and 3 sites of arginine methylation. In KYSE180, Likewise, we identified 32 histone PTMs, including 11 sites of lysine acetylation, 15 sites of lysine methylation (mono-, di-, and tri-methylation), 3 sites of lysine butyrylation, and 3 sites of arginine methylation. All the peptides containing PTM marks are summarized in Supplementary material 1 and all the MS/MS
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Fig. 6 – (A) Immunohistochemistry expression pattern of H4K79me2 in ESCC. (B) The X-tile plot showed the chi-square log-rank values created when the cohort was divided into three populations. (C) The optimal cutpoint (0–3 vs. 4–7 vs. 8–12) was demonstrated on a histogram of the entire cohort. (D) Kaplan–Meier survival analysis demonstrated that the overall survival rate was significantly in the H4K79me2 low- and high-expression groups than in the mid-expression group (P = 0.004). “vs.” means versus.
spectra of the modified peptides are presented in Supplementary material 2. To further characterize the histone PTMs in the two cell lines, we analyzed all PTM sites. Fig. 2 demonstrates that the combinatorial histone PTMs are highly similar for both ESCC cell lines. Lysine PTM landscape was shown to be expanded in previous works [21]. In addition to common lysine acetylation and methylation, lysine butyrylation was identified as one of
the novel lysine PTMs in both human and yeast cells [18,19]. Currently the biological function of lysine butyrylation remains largely unknown [26,27]. The presence of pattern of lysine butyrylation in a variety of cancer cells has not drawn much attention yet. Interestingly, the butyrylation was detected in both of two tumor cell lines by western blot (Fig. 5D). Following with MS/MS analysis, four lysine butyrylated sites were identified on H3K18, H3K23, H3K79 and H4K77 in TE3 cell. To
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B
C A
Fig. 7 – The overall structure of human nucleosome core particle and proposed model of H4K79 dimethylation. The structure models are generated with Pymol based on the PDB file (code: 3AFA) published by Tachiwana et al. [35] (A) The cylinders denote the subunits (H2A, H2B, H3 and H4) forming an octameric histone core. H4K79 is located in Loop 2 connecting the two helices of H4. DNA, H2A, H2B, H3.1 and H4 are colored by wheat, yellow, pink, cyan, and green. (B) H4K79 directly contact with the DNA molecule. The potential interactions regarding H4K79 are indicated as blue dotted lines. The H4K79 backbone nitrogen and oxygen forms two hydrogen bonds with two backbone DNA phosphates and one hydrogen bond with R81 in H3 Loop3 respectively [33]. (C) The close-up view of structure around dimethylated H4K79 residue. The side chain Nε–N forms a salt could also interact with a backbone DNA phosphate. The dimethylation was generated using the mutagenesis function in Pymol. And it is observed that two methyl groups interrupt the interaction between the H4K79 side chain and DNA backbone.
further validate the lysine butyrylation, we compared MS/MS spectrum and HPLC retention of the butyrylated peptides with that of its corresponding acetylated counterparts. We observed highly similar fragmentation patterns of pairing fragment ions that contain the two modifications, respectively, as demonstrated in Fig. 3. Obviously butyrylated peptides are more hydrophobic than acetylated ones as indicated by their HPLC retention time. The two lines of evidences both supported the existence of butyrylated peptides in ESCC cells. MS signal intensities suggest that the butyrylation has a pretty low
abundance. For example, the butyrylation on H3K18 is 100 times lower than its corresponding acetylation on the same site.
3.3. Comparative analysis of specific histone PTM sites in two ESCC cell lines To examine the differences between TE3 and KYSE180 cell lines at specific histone PTM sites, we compared the abundance of key histone marks within the two cells. The relative abundance was calculated based on the ratio of the peak areas of the same
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parent ions labeled by light and heavy isotopes in the MS spectra. H3K9me/me2 and H3K27me/me2, which were reported as the repressive transcription marks [28] and considered to be associated with cancer development, if dysregulated [29]. Fig. 4 demonstrates that there is no obvious difference on the level of H3K9me2 between the two cell lines. The result was further validated by western blot assay (Fig. 5A). We also observed the difference of abundance of H3K27me2 in the cell lines. In case of the methylation of H3K27, which has been considered to be an epigenetic mark that mediates tumor suppressor gene silencing [30], it was reported that an elevation of H3K27 methylation was observed in ESCC than in healthy esophageal issue that was from the same individual and was closely associated with the tumor stage [31]. To investigate the relationship between individual modifications and ESCC invasive compatibility, we further compared the global methylation levels identified on H3 and H4. Among these methylated sites, a significant differential abundance was found on H4K79me2, which is 6 times higher in KYSE180 than that in TE3 cell (Fig. 4A). To further verify this finding, H4K79me2 was examined in both cell lines by western blots. As shown in Fig. 5B, H4K79me2 in KYSE180 is indeed more abundant than that in TE3. Although H4K79me2 has been identified in anuran Xenopus laevis [32], its function is still largely unknown. It is also unclear what enzymes regulate the addition or removal of H4K79me2. To check whether H4K79me2 is relevant to the survival of ESCC patients, we examined 166 ESCC specimens by immunohistochemistry analysis. H4K79me2 immunoreactivity was both cytoplasmic and nucleic in ESCC cells. Typical immunostainings are shown in Fig. 6A. According to X-tile plots, the 166 cases were divided into three populations: low-expression (0–3 scores, n = 8), mid-expression (4–7 scores, n = 70) and high-expression (8–12 scores, n = 88) (Fig. 6B and C). Kaplan Meier survival analysis was used to evaluate the association between H4K79me2 expression and the overall survival. The patients with low-expression of H4K79me2 had the lowest 5-year survival rate of 0.0%, compared with 54.4% or 44.0% for H4K79me2 mid- or high-expression (χ2 = 10.892, P =0.004; Fig. 6D). These data suggest that low-expression of H4K79me2 may be correlated with a poor survival of ESCC patients. Our evidence indicates a correlation between H4K79me2 and tumor phenotype in both cell lines and ESCC patients, therefore we assume that H4K79me2 could have a potential effect on ESCC development. However, the complexity of genetic source of cancer makes it difficult to prove this effect and elucidate the mechanism. Here we have just deduced a possible role of H4K79me2 in the global DNA–histone interaction based on a well-solved crystal structure of wild type nucleosome. Fig. 7A illustrates that H4K79 residue localizes in the histone-fold domains in human nucleosome. Structure analysis has uncovered that the unmodified H4 K79 residue may contain three main interactions [33]: (1) a hydrogen bond between the main chain amide nitrogen and a DNA backbone phosphate; (2) a salt bridge between the side chain Nε and a DNA backbone phosphate; and (3) a hydrogen bond with a main chain carbonyl oxygen of the H3 D81 residue (Fig. 7B). Fig. 7C illustrates a close-up view of the structure around dimethylated H4K79 residue. Two methyl groups
might destabilize the salt bridge between the side chain Nε and a DNA backbone phosphate. Although the role of dimethylation on H4K79 is unclear, structural analysis indicates that the H4K79me2 may have a potential effect on the histone–DNA interaction. The histone acetylation (Kac) plays a crucial role in the regulation of gene transcription via the modulation of nucleosomal DNA package. Under hypoacetylated state, nucleosomes are tightly compacted because of the ionic interaction between the protonated amine groups of lysines and the phosphate groups of DNA, thus lead to a transcriptional repression because the transcription factors have restricted access to their target DNA. In contrast, histone Kac results in a relaxed nucleosomal structure thus allows transcription to occur. Histone acetylation and its deacetylation enzyme, HDAC are associated with the development and progress of human cancer [29]. Reduced level of acetylated histones H3 and H4 has been believed to be a potential mark for the diagnosis of cancer at an early stage [31]. Additionally, abnormal expression of the histone marks has been linked to the recurrence-free survival in different ESCC stages [34]. In this study, MS analysis demonstrated that there is no obvious difference between the two cell lines, as shown in Fig. 4B. The result was further confirmed by western blot using anti-acetyllysine pan antibodies (Fig. 5C).
4. Conclusion In this study, we combined quantitative proteomics approach and bioinformatic analysis to characterize the global histone PTMs in two ESCC cell lines. A series of histone PTMs including novel lysine butyrylation were identified. The landscape of histone PTMs in two phenotypes of ESCCs with different invasion capacity was presented. Further quantitative analysis found that abundance of H4K79me2 might be correlated to the invasion capacity of ESCC cell and poor survival of ESCC patients. Structural analysis indicated that the modification may have a potential effect on the histone– DNA interaction. Our results might be valuable for the understanding of the relationship between histone PTMs and cancer invasiveness. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jprot.2014.09.004.
Transparency document Transparency document associated with this article can be found, in the online version.
Acknowledgments This work was supported by the National Basic Research Program of China (Grants 2012cb910601 and 2013cb910903), the National Natural Science Foundation of China with Grants (21275077) and the Tianjin Municipal Science and Technology Commission (no. 14JCYBJC24000).
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REFERENCES [1] Kouzarides T. Chromatin modifications and their function. Cell 2007;128:693–705. [2] Reik W. Stability and flexibility of epigenetic gene regulation in mammalian development. Nature 2007;447:425–32. [3] Esteller M. Cancer epigenomics: DNA methylomes and histone-modification maps. Nat Rev Genet 2007;8:286–98. [4] Barlesi F, Giaccone G, Gallegos-Ruiz MI, Loundou A, Span SW, Lefesvre P, et al. Global histone modifications predict prognosis of resected non small-cell lung cancer. J Clin Oncol 2007;25:4358–64. [5] Seligson DB, Horvath S, Shi T, Yu H, Tze S, Grunstein M, et al. Global histone modification patterns predict risk of prostate cancer recurrence. Nature 2005;435:1262–6. [6] Seligson DB, Horvath S, McBrian MA, Mah V, Yu H, Tze S, et al. Global levels of histone modifications predict prognosis in different cancers. Am J Pathol 2009;174:1619–28. [7] Elsheikh SE, Green AR, Rakha EA, Powe DG, Ahmed RA, Collins HM, et al. Global histone modifications in breast cancer correlate with tumor phenotypes, prognostic factors, and patient outcome. Cancer Res 2009;69:3802–9. [8] Park YS, Jin MY, Kim YJ, Yook JH, Kim BS, Jang SJ. The global histone modification pattern correlates with cancer recurrence and overall survival in gastric adenocarcinoma. Ann Surg Oncol 2008;15:1968–76. [9] Jenuwein T. The epigenetic magic of histone lysine methylation. FEBS J 2006;273:3121–35. [10] Fraga MF, Ballestar E, Villar-Garea A, Boix-Chornet M, Espada J, Schotta G, et al. Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer. Nat Genet 2005;37:391–400. [11] Qi YJ, Chao WX, Chiu JF. An overview of esophageal squamous cell carcinoma proteomics. J Proteomics 2012;75: 3129–37. [12] Toh Y, Yamamoto M, Endo K, Ikeda Y, Baba H, Kohnoe S, et al. Histone H4 acetylation and histone deacetylase 1 expression in esophageal squamous cell carcinoma. Oncol Rep 2003;10: 333–8. [13] Tzao C, Sun GH, Tung HJ, Hsu HS, Hsu WH, Wang YC, et al. Reduced acetylated histone H4 is associated with promoter methylation of the fragile histidine triad gene in resected esophageal squamous cell carcinoma. Ann Thorac Surg 2006; 82:396–401 [discussion]. [14] Tzao C, Tung HJ, Jin JS, Sun GH, Hsu HS, Chen BH, et al. Prognostic significance of global histone modifications in resected squamous cell carcinoma of the esophagus. Mod Pathol 2009;22:252–60. [15] Jensen ON. Interpreting the protein language using proteomics. Nat Rev Mol Cell Biol 2006;7:391–403. [16] Sidoli S, Cheng L, Jensen ON. Proteomics in chromatin biology and epigenetics: elucidation of post-translational modifications of histone proteins by mass spectrometry. J Proteomics 2012;75: 3419–33. [17] Tan MJ, Luo H, Lee S, Jin FL, Yang JS, Montellier E, et al. Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification. Cell 2011;146:1015–27. [18] Zhang K, Chen Y, Zhang Z, Zhao Y. Identification and verification of lysine propionylation and butyrylation in yeast core histones using PTMap software. J Proteome Res 2009;8: 900–6.
189
[19] Chen Y, Sprung R, Tang Y, Ball H, Sangras B, Kim SC, et al. Lysine propionylation and butyrylation are novel post-translational modifications in histones. Mol Cell Proteomics 2007;6:812–9. [20] Dai L, Peng C, Montellier E, Lu Z, Chen Y, Ishii H, et al. Lysine 2-hydroxyisobutyrylation is a widely distributed active histone mark. Nat Chem Biol 2014;10:365–70. [21] Olsen CA. Expansion of the lysine acylation landscape. Angew Chem Int Ed Engl 2012;51:3755–6. [22] Ong SE, Mann M. A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC). Nat Protoc 2006;1: 2650–60. [23] Xie JJ, Xu LY, Xie YM, Zhang HH, Cai WJ, Zhou F, et al. Roles of ezrin in the growth and invasiveness of esophageal squamous carcinoma cells. Int J Cancer 2009;124:2549–58. [24] Chen Y, Kwon SW, Kim SC, Zhao Y. Integrated approach for manual evaluation of peptides identified by searching protein sequence databases with tandem mass spectra. J Proteome Res 2005;4:998–1005. [25] Zhang FR, Tao LH, Shen ZY, Lv Z, Xu LY, Li EM. Fascin expression in human embryonic, fetal, and normal adult tissue. J Histochem Cytochem 2008;56:193–9. [26] Vollmuth F, Geyer M. Interaction of propionylated and butyrylated histone H3 lysine marks with Brd4 bromodomains. Angew Chem Int Ed Engl 2010;49:6768–72. [27] Cheng Z, Tang Y, Chen Y, Kim S, Liu H, Li SS, et al. Molecular characterization of propionyllysines in non-histone proteins. Mol Cell Proteomics 2009;8:45–52. [28] Pan G, Tian S, Nie J, Yang C, Ruotti V, Wei H, et al. Whole-genome analysis of histone H3 lysine 4 and lysine 27 methylation in human embryonic stem cells. Cell Stem Cell 2007;1:299–312. [29] Portela A, Esteller M. Epigenetic modifications and human disease. Nat Biotechnol 2010;28:1057–68. [30] Wei YK, Xia WY, Zhang ZH, Liu JS, Wang HM, Adsay NV, et al. Loss of trimethylation at lysine 27 of histone H3 is a predictor of poor outcome in breast, ovarian, and pancreatic cancers. Mol Carcinog 2008;47:701–6. [31] Chen C, Zhao M, Yin N, He B, Wang B, Yuan Y, et al. Abnormal histone acetylation and methylation levels in esophageal squamous cell carcinomas. Cancer Invest 2011;29:548–56. [32] Shechter D, Nicklay JJ, Chitta RK, Shabanowitz J, Hunt DF, Allis CD. Analysis of histones in Xenopus laevis. I. A distinct index of enriched variants and modifications exists in each cell type and is remodeled during developmental transitions. J Biol Chem 2009;284:1064–74. [33] Iwasaki W, Tachiwana H, Kawaguchi K, Shibata T, Kagawa W, Kurumizaka H. Comprehensive structural analysis of mutant nucleosomes containing lysine to glutamine (KQ) substitutions in the H3 and H4 histone-fold domains. Biochemistry 2011;50:7822–32. [34] I H, Ko E, Kim Y, Cho EY, Han J, Park J, et al. Association of global levels of histone modifications with recurrence-free survival in stage IIB and III esophageal squamous cell carcinomas. Cancer Epidemiol Biomarkers Prev 2010;19: 566–73. [35] Tachiwana H, Kagawa W, Osakabe A, Kawaguchi K, Shiga T, Hayashi-Takanaka Y, et al. Structural basis of instability of the nucleosome containing a testis-specific histone variant, human H3T. Proc Natl Acad Sci 2010;107:10454–9.
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Comparative analysis of histone H3 and H4 post-translational modifications of esophageal squamous cell carcinoma with different invasive capabilities Kai Zhanga,b,c,⁎, Liyan Lid,1 , Mengxiao Zhud,1 , Guojuan Wangc , Jianjun Xieb , Yunlong Zhaoc , Enguo Fana , Liyan Xud,⁎⁎, Enmin Lib,⁎⁎ a
Tianjin Key Laboratory of Medical Epigenetics, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China b Department of Biochemistry and Molecular Biology, Key Laboratory of Molecular Biology in High Cancer Incidence Coastal Chaoshan Area of Guangdong Higher Education Institutes, Shantou University Medical College, Shantou 515041, China c State Key Laboratory of Medicinal Chemical Biology, Department of Chemistry, Nankai University, Tianjin 300071, China d Institute of Oncologic Pathology, Shantou University Medical College, Shantou 515041, China
AR TIC LE I N FO
ABS TR ACT
Article history:
Eukaryotic DNA is packaged into a chromatin with the help of four core histones (H2A, H2B, H3,
Received 1 July 2014
and H4). Diverse histone post-translational modifications (PTMs) are hence involved in the
Accepted 4 September 2014
regulation of gene transcription. However, how this regulation does work is still poorly
Available online 16 September 2014
understood and lacks details. Here we used the mass spectrometry-based proteomics approach to perform a comparative analysis of histone marks at a global level in two phenotypes of
Keywords:
esophageal squamous cell carcinoma (ESCC) with different invasiveness. We obtained a
Protein post-translational
comprehensive profiling of histone H3 and H4 PTMs including lysine methylation, acetylation
modifications (PTMs)
and novel butyrylation. The correlation between histone marks and cancer invasive capabilities
Histone
was further characterized and one distinguishable PTM, H4K79me2 was discovered and verified
Mass spectrometry
in this study. Immunohistochemistry analysis suggests that abnormal level of H4K79me2 may be
Esophageal squamous cell carcinoma
related to poor survival of ESCC patients. Our results enrich the dataset of the feature pattern of
Invasive capability
global histone PTMs in ESCC cell lines. Biological significance Core histone proteins, decorated by multiple biological significant protein post-translational modifications (PTMs) such as lysine acetylation and lysine methylation, are considered to regulate gene transcription and be associated with the development of cancer. Recent studies have further shown that global level of histone modifications is the potential hallmark of cancer to predict the clinical outcomes of human cancers. However, the regulation mechanism is
⁎ Correspondence to: K. Zhang, Department of Biochemistry and Molecular Biology, Tianjin Medical University, Tianjin 300070, China. Tel.: + 86 22 24387742. ⁎⁎ Corresponding author. Tel.: + 86 754 88900460. E-mail addresses: [email protected] (K. Zhang), [email protected] (L. Xu). 1 Co-first author.
http://dx.doi.org/10.1016/j.jprot.2014.09.004 1874-3919/© 2014 Elsevier B.V. All rights reserved.
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largely unknown. Here we used the mass spectrometry based proteomics coupled with stable isotope labeling with amino acids in cell culture (SILAC) to characterize the global levels of histone marks in two phenotypes of esophageal squamous cell carcinoma (ESCC) cell lines with different invasive capabilities. To the best of our knowledge, it is the first report about the comparative analysis for histone marks of the different invasive ESCC cell lines. A significantly differential level of histone modification, H4K79me2, was determined and verified. Immunohistochemistry analysis further suggests that abnormal level of H4K79me2 may be related to poor survival of ESCC patients. Our results could contribute to understanding the different expressions of global histone PTMs in different invasive ESCC cell lines. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In eukaryotic chromosomes, genomic DNA is packaged into a compacted chromatin with the help of four core histones (H2A, H2B, H3, and H4). The core histone proteins are characterized by diverse post-translational modifications (PTMs) at their N-terminal tails. The histone modifications play a key role in regulating chromatin structure and transcriptional activity by modulating the packaging of chromatin and recruiting PTM-specific binding proteins [1,2]. Histone PTMs are generally considered to be a major group of important epigenetic marks involved in diverse biological processes such as DNA repair and chromosome condensation. Increasing evidence suggests that dysregulation of histone PTMs has intimately linked with the development and progression of cancer [3]. Furthermore abnormal expressions of specific histone PTMs have been observed in a variety of human cancers including lung [4], prostate [5], kidney [6], breast [7], and stomach [8]. For example, global loss of acetylation and gain of methylation at lysine 9 and lysine 27 in histone H3 have been considered to be associated with the silencing of genes, resulting in the development of cancer [9]. Recent studies have further shown that global level of histone modifications is the potential hallmark of cancer to predict the clinical outcomes of human cancers [10]. Esophageal cancer (EC) is the sixth leading cause of cancer deaths worldwide. In China, esophageal squamous cell carcinoma (ESCC) is the predominant histological subtype and accounts for approximately 90% of all EC [11]. To date, most EC patients with 5-year survival rates remain as low as 10% or even less. Pattern of histone PTMs disruption is largely unknown in ESCC, however it has been reported that the global level of acetylation on H4 was varied with ESCC progression and metastasis [12,13]. Furthermore several histone marks have been demonstrated to be associated with the development of tumors [14]. However, the histone modification has not yet been examined carefully in ESCC cell lines. A complete atlas of histone PTMs should be constructed to understand the relationship between histone code and ESCC properties. To date, mass spectrometry (MS) has become the fundamental tool to characterize histone PTMs [15,16]. At least 11 types of histone PTMs have been reported at over 60 different amino acid residues [1,17]. Besides the known histone PTMs such as lysine methylation and acetylation, several types of new histone PTMs have been discovered at lysine residues such as lysine butyrylation, propionylation [18,19], crotonylation [17] and 2-hydroxyisobutyrylation [20], which suggests even more unknown histone modifications to be discovered [21]. However it is still unclear what role does the novel histone PTM play in the regulation of histone function in a variety of diseases.
Here we used the mass spectrometry based proteomics coupled with stable isotope labeling with amino acids in cell culture (SILAC) to characterize the global levels of histone marks in two phenotypes of ESCC cell lines with different invasive capabilities. Individual specific histone PTMs were carefully determined including lysine methylation, acetylation and novel butyrylation. We measured the abundance of key histone PTMs in two cell lines, and further evaluated the correlation between histone marks and ESCC invasion capabilities. A significantly differential level of histone modification, H4K79me2, was determined and verified in this study. Immunohistochemistry analysis further suggests that abnormal level of H4K79me2 may be related to poor survival of ESCC patients.
2. Materials and methods 2.1. Materials Water and acetonitrile were from Fisher Scientific (Pittsburgh, PA). Trifluoroacetic acid (TFA) was from Sigma-Aldrich (St. Louis, MO). Colloidal Coomassie Blue Staining Kit was from Invitrogen (Carlsbad, CA). Sequencing-grade trypsin was from Promega (Madison, WI). C18 ZipTips were from Millipore (Bedford, MA).
2.2. Cell culture ESCC cell lines (TE3 and KYSE180) were generous gifts from Professor Ming-Zhou Guo, Department of Gastroenterology & Hepatology, Chinese PLA General Hospital. TE3 and KYSE180 cells were cultured with modified RPMI-1640 medium (Thermo, Pittsburgh, PA) supplemented with 10% fetal calf serum at 37 °C with 5% CO2 in atmosphere. The SILAC labeling was carried out as described earlier [22]. In brief, TE3 and KYSE180 cells were maintained in RPMI-1640 medium containing 13C6-lysine and 12 C6-lysine (Thermo, Pittsburgh, PA), respectively, containing 10% fetal calf serum in an atmosphere of 5% CO2 at 37 °C. Two cell lines were grown for more than 6 generations before being harvested in order to achieve more than 99% labeling efficiency.
2.3. Western blotting Total cell lysates were prepared in RIPA buffer, separated by SDS-PAGE and transferred to PVDF membranes (Millipore). The membranes were incubated in blocking buffer and then incubated with histone PTM antibody (histone anti-H3K9me2
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2.4. Cell invasiveness assay Invasiveness assay was performed as described before [23]. Cells (1 × 105) were seeded onto the top chamber of a 24-well matrigel-coated membrane with 8-μm pores (Millipore), and the bottom chamber was filled with culture medium with 10% fetal calf serum. The membranes were fixed and stained by Giemsa reagent, and invasiveness and migration were quantified by counting 10 random fields under a light microscope (400×). The mean value was calculated from data obtained from 3 separate chambers.
400
Invasive cells
350 300 250 200
2.5. Histone extraction
150
The cells were harvested by centrifugation at 5000 ×g for 5 min and washed once with distilled water. Cells were resuspended in Triton Extraction Buffer (TEB: PBS containing 0.5% Triton X-100 (v/v), 2 mM phenyl-methylsulfonyl fluoride (PMSF), 0.02% (w/v) NaN3) at a cell density of 107 cells ml−1. Lysed cells were put on ice for 10 min with gentle stirring. Cells were removed by centrifugation for 15 min at 2000 ×g. After supernatant removal, the cells were washed with half a volume of TEB and centrifuged as before. The core histones were extracted with 0.4 N H2SO4 on ice overnight. The extract was centrifuged at 4 °C for 10 min at 16,000 ×g. Histone proteins were precipitated using trichloroacetic acid precipitation (TCA). Samples were then washed twice with acetone containing 0.1% HCl (v/v) and twice with pure acetone. The pellet was allowed air drying and was then resolubilized in water.
100 50 0
KYSE180
TE3
Fig. 1 – Invasiveness assay of KYSE180 and TE3 cell lines. Invasiveness assay was used to determine the invasive capability. Invasive cells were fixed and stained, and representative fields were photographed. For quantification, the cells were counted in 10 random fields under a light microscope. Each experiment was performed in triplicate.
2.6. SDS-PAGE separation and in-gel digestion and anti-H4K79me2 antibody were from Millipore, and antiacetyllysine pan antibody and anti-butyryllysine pan antibody were from PTM BioLab, Inc.). Finally, immunoreactive bands were revealed using luminol reagent (Santa Cruz Biotechnology). Photography and quantitative analyses were done using the FluorChemTMIS-8900 (Alpha Innotech).
The core histones were resolved in a 15% SDS-PAGE gel and visualized by colloidal Coomassie blue staining. Gel bands of interest were excised and subjected to in-gel digestion as described previously [24]. Briefly, the gel bands were sliced into small pieces (~1 mm3) and destained with 25 mM ammonium
Bu Bu
H3
Me2
Me2
KTE3
N Kyse180
Ac
Ac
Ac
K9
K14
K18
Ac
Ac
Me2
Ac
Bu
Me2
Me2
Me
Me2
Ac
Ac
Me
K23
Me2
K27
Ac
Me
K36K37
Ac
Me
Me2
Me
Me2
Bu
Me2
Me
Me2
Me
Ac
Ac
K56
K79
Ac
Ac
Me2
C
Me Me2
Bu
H4 KTE3
N Kyse180
Bu Me
Me
K20
K31
R35
Me2
Me
Me
Ac
Ac
Ac
Ac
Me2
K5
K8
K12
K16
Ac
Ac
Ac
Ac
Me
R55 Me
Me2
K59
Me
Me2 Me2
Me2
K77 R78 K79 K91 Me
Me2 Me2
C
Me2
Bu
Fig. 2 – Identified post-translational modification sites within core histones H3 and H4 in KYSE180 and TE3 cells. Ac: acetylation; Me: mono-methylation; Me2: di-methylation; Me3: tri-methylation; Buty: butyrylation.
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bicarbonate in ethanol/water (50:50, v/v). The destained gel pieces were washed in an acidic buffer (acetic acid/ethanol/ water, 10:50:40, v/v/v) three times for 1 h each time, and in water two times for 20 min each time. The gel pieces were dehydrated
in acetonitrile and dried in a SpeedVac (ThermoFisher, Waltham, MA). Two hundred nanograms of porcine modified trypsin (Promega, Madison, WI) in 50 mM ammonium bicarbonate was added to the dried gels and incubated overnight at 37 °C. Tryptic
K(Bu)QLATK(Ac)AAR
H3K18 Buty
Retention Time 28.69 min Parent ion intensity 2.14 E4
A M-NH3
y3 317.19
100
y7 772.53
532.37
Relative Abundance
b2 327.33 b1 199.25
y4 487.37 b3 440.36
b(2)* 310.30
y1 175.12
y6 659.41 y5 588.36
b4 511.37
y2 246.27
y8 b7 853.60 900.62
b8 924.62
0 150
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500
550
600
650
700
750
800
850
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950
1000
m/z y7 772.56
Relative Abundance
100
Retention Time 25.99 min Parent ion intensity 2.55 E6
b2 299.31
M-NH3 518.48 b5 b3 584.33 y5 412.36 b(4)* 466.39 588.38 y4 487.34
y3 b(2)* y2 282.27 317.29 246.26
y1 175.15
y6 659.46 y(7)0 754.49
b7 825.59
0 150
200
250
300
350
400
450
600 550 m/z
500
650
700
504.27
Relative Abundance
y2 288.37
0
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y(9)++ 582.44
y4
Relative Abundance
y7 964.45 b7 902.46
b4 442.31 400
500
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700
m/z
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900
b(3)0 296.28
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1000
b10 1231.62
y9 1163.57
y8 1092.49
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b(10)0 1213.44 1200
1300
EIAQDFK(Ac)TDLR
671.49
Retention Time 49.87 min Parent ion intensity 4.86 E3
y7
M-H2O
b(5)0
936.47
y5 674.44
b9 1090.51
b7 874.43
539.21
y3 403.29 b4 441.31
b6 704.36 400
y*(8) 1075.64
1000
y(9)++ 568.39
y2 288.24
850
b9 1118.52
b*(8) 986.59
y4 504.27
200
y6 849.48
b(7)0 884.31
100
0
y5 702.50
y3 403.24
300
800
Retention Time 54.21 min Parent ion intensity 1.19 E3
y*(5) 685.39
B 100
750
y8 900.60
EIAQDFK(Bu)TDLR
H3K79 Buty
b2 243.13
K(Ac)QLATK(Ac)AAR
500
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m/z
y6 821.47 b(7)0 856.38 800
b(8)0 957.41 900
1000
y9 y8 1135.53 1064.56 b10 1203.55 1100
1200
1300
Fig. 3 – (A) MS/MS of a tryptic peptide ion K(Bu)QLATK(Ac)AAR from histone H3 (up) and MS/MS of a tryptic peptide ion K(Ac) QLATK(Ac)AAR from histone H3 (Bottom). (B) MS/MS of a tryptic peptide ion EIAQDFK(Bu)TDLR from histone H3 (up) and MS/MS of a tryptic peptide ion EIAQDFK(Ac)TDLR from histone H3 (bottom).
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Fig. 4 – (A) The quantitative analysis of lysine methylation sites on histones in KYSE180 and TE3. (B) The quantitative comparison of lysine acetylation sites on histones between KYSE180 and TE3. The relative ratio of abundance of KYSE180 and TE was determined according to the description in Section 2. Mean values and standard deviations derive from 3 technical replicates. peptides were sequentially extracted from the gel pieces with 50% acetonitrile (acetonitrile/water/TFA, 50:45:5, v/v/v) and 75% acetonitrile (acetonitrile/water/TFA, 75:24:1, v/v/v). The peptide extracts were pooled and dried in a SpeedVac. The peptide extracts were desalted using a μ-C18 Ziptip before HPLC/MS/MS analysis.
2.7. Nano-HPLC/mass spectrometric analysis Each tryptic digestion was dissolved in 10 μL of HPLC buffer A (0.1% (v/v) formic acid in water), and 2 μL was injected into a Nano-LC system (Eksigent Technologies, Dublin, CA). Peptides were separated on a homemade capillary HPLC column (100-mm length × 75-μm inner diameter) containing Jupiter C12 resin (4-μm particle size, 90-Å pore diameter, Phenomenex, St. Torrance, CA) with a 120 min HPLC-gradient from 5 to 90% HPLC buffer B (0.1% formic acid in acetonitrile) at a flow rate of 200 nL/min. The HPLC elute was electrosprayed directly into an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Waltham, MA) using a nanospray source. The LTQ-Orbitrap Velos mass spectrometer was operated in a data-dependent mode with resolution R = 60,000 at m/z 400. Full scan MS spectra from m/z 350–2000 were acquired in the
Orbitrap. The twenty most intense ions were sequentially isolated in the linear ion trap and subjected to collisionactivated dissociation (CAD) with a normalized energy of 35% and an isolation width of 4 Da. The exclusion duration for the data-dependant scan was 36 s, the repeat count was 2, and the exclusion window was set at ± 2 Da. AGC settings were 1E6 for full scan Orbitrap analysis, 1E4 for MSn scan in the ion trap and 6E4 for MSn scan in the Orbitrap. Signal threshold for CID acquisition was set at 5000.
2.8. Protein sequencing alignment and bioinformatics analysis Peak lists were first generated by the extract_msn.exe software (v5.0, Thermo Scientific). All MS/MS spectra were searched against the NCBI human protein sequence database (September 20, 2012) using the Mascot search engine (version 2.1.0, Matrix Science, London). Trypsin was specified as digesting enzyme. A maximum of 5 missing cleavages were allowed. Mass tolerances for precursor ions were set at ±0.1 Da for precursor ions and ± 0.5 Da for MS/MS. For label free samples, the specific search parameters included lysine acetylation, lysine mono, di, and tri-methylation, lysine butyrylation and arginine methylation as variable modifications. For SILAC samples, lysine (13C6), lysine
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H3 H4 an-H3 Fig. 5 – Western blotting detection for histone modifications. (A) H3K9me2; (B) H4K79me2; (C) pan lysine-acetylation; and (D) pan lysine-butyrylation.
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Dako, Glostrup, Denmark) and Liquid DAB Substrate Kit (Zymed/Invitrogen, Carlsbad, CA, USA) were used to conduct immunohistochemical staining according to the manufacturer's instructions respectively. Scores were independently assessed by two researchers blinded to clinical data. Each separate tissue core was scored based on both intensity and percentage of positive cells [25]. Briefly, the intensity grade of staining was: 0, negative; 1, weak; 2, moderate; and 3, strong. The percentage of positive cells was: 0, 75%. The final score was calculated by multiplying the intensity grade and the percentage of positive cells, producing a total range from 0 to 12. To assess statistical significance and avoid arbitrary cutoff point selection, the X-tile program was applied to assess H4K79me2 expression score and determine an optimal cutoff value based on outcome. According to the optimal cutoff value, data analysis was performed using SPSS 13.0 software (SPSS, Inc., Chicago, IL). Kaplan–Meier curves were obtained for overall survival analysis using a log-rank test. Each P-value is two-tailed and significance level is 0.05.
3. Results and discussion acetylation (13C6), lysine mono, di, and tri-methylation (13C6), and lysine butyrylation (13C6) were also included as variable modifications. All the identified peptides with Mascot score above 40 and E-value below 0.05 were manually verified according to the rules described previously [24]. Quantification of PTMs was based on the peak areas of precursor ions of peptides labeled by light and heavy stable isotopes. Briefly, for every PTM site, we first validated all the identified peptides containing the targeted PTM type and site, then added up the peak areas of precursor ion of these peptides. The ratio of the light and heavy isotope peak areas was used for comparing the relative abundance of the PTM between the two cell lines. Mean values and standard deviations derive from 3 technical replicates.
2.9. Tissue specimens and immunohistochemical staining A total of 166 ESCC patients underwent curative surgery in the Affiliated Shantou Hospital of Sun Yat-sen University (Shantou, China) between 2000 and 2006. Then the patients were followed up until their deaths. Mean age at surgery was 57.7 (range 31 to 75), and 41 patients were female and 125 were male. Clinicopathological characteristics of the patients were assessed according to the 7th edition of the tumor-node-metastasis (TNM) classification of the International Union against Cancer (UICC). The patients suffered from severe post-operative complications and died of other tumors or other causes were excluded. The study was approved by the ethical committee of the Central Hospital of Shantou City and the ethical committee of the Medical College of Shantou University, and a written informed consent was obtained from each patient to use resected samples for research. All paraffin-embedded samples undergoing tissue microarray (TMA) construction were cut into 4 μm sections. The rabbit polyclonal antibodies to H4K79me2 (1:200, ab2885, Abcam Inc., Cambridge, MA, USA), a Universal LSAB™ + Kit/HRP (K0690,
3.1. Identification and quantitative approach for profiling histone PTMs Accumulated evidence suggests that the dysregulation of histone PTMs might be associated with the development of cancer. However it is still unclear how these epigenetic marks are linked with critical processes of development, such as invasive and metastatic capabilities of tumors. To investigate how histone PTMs correlates with cancer invasive capability, we chose two phenotypes of ESCC cell lines that possess different invasiveness. As shown in Fig. 1, the invasive and metastatic property of TE3 is significantly higher than that of KYSE180 cells. To the best of our knowledge, histone PTMs profiles have not yet been examined carefully in these two cell lines. Our interests focused on the PTMs of histones H3 and H4 because their abnormal expressions have been shown to be involved in the development of tumor [5]. The histone PTMs in each cell lines were mapped, and then compared their abundance at specific PTM sites using a quantitative proteomics technique by coupling SILAC with high-resolution LTQ-Orbitrap mass spectrometry (See Supplementary material 1).
3.2. Characterization of global histone PTMs in two ESCC cell lines MS/MS spectra were searched against the NCBInr human protein sequence database using MASCOT software. After a manual inspection as described previously [24], we identified in total 35 sites that were modified with 5 types of PTMs on core histones on H3 and H4 in TE3 cells, including 11 sites of lysine acetylation, 17 sites of lysine methylation (mono-, di-, and tri-methylation), 4 sites of lysine butyrylation, and 3 sites of arginine methylation. In KYSE180, Likewise, we identified 32 histone PTMs, including 11 sites of lysine acetylation, 15 sites of lysine methylation (mono-, di-, and tri-methylation), 3 sites of lysine butyrylation, and 3 sites of arginine methylation. All the peptides containing PTM marks are summarized in Supplementary material 1 and all the MS/MS
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Fig. 6 – (A) Immunohistochemistry expression pattern of H4K79me2 in ESCC. (B) The X-tile plot showed the chi-square log-rank values created when the cohort was divided into three populations. (C) The optimal cutpoint (0–3 vs. 4–7 vs. 8–12) was demonstrated on a histogram of the entire cohort. (D) Kaplan–Meier survival analysis demonstrated that the overall survival rate was significantly in the H4K79me2 low- and high-expression groups than in the mid-expression group (P = 0.004). “vs.” means versus.
spectra of the modified peptides are presented in Supplementary material 2. To further characterize the histone PTMs in the two cell lines, we analyzed all PTM sites. Fig. 2 demonstrates that the combinatorial histone PTMs are highly similar for both ESCC cell lines. Lysine PTM landscape was shown to be expanded in previous works [21]. In addition to common lysine acetylation and methylation, lysine butyrylation was identified as one of
the novel lysine PTMs in both human and yeast cells [18,19]. Currently the biological function of lysine butyrylation remains largely unknown [26,27]. The presence of pattern of lysine butyrylation in a variety of cancer cells has not drawn much attention yet. Interestingly, the butyrylation was detected in both of two tumor cell lines by western blot (Fig. 5D). Following with MS/MS analysis, four lysine butyrylated sites were identified on H3K18, H3K23, H3K79 and H4K77 in TE3 cell. To
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B
C A
Fig. 7 – The overall structure of human nucleosome core particle and proposed model of H4K79 dimethylation. The structure models are generated with Pymol based on the PDB file (code: 3AFA) published by Tachiwana et al. [35] (A) The cylinders denote the subunits (H2A, H2B, H3 and H4) forming an octameric histone core. H4K79 is located in Loop 2 connecting the two helices of H4. DNA, H2A, H2B, H3.1 and H4 are colored by wheat, yellow, pink, cyan, and green. (B) H4K79 directly contact with the DNA molecule. The potential interactions regarding H4K79 are indicated as blue dotted lines. The H4K79 backbone nitrogen and oxygen forms two hydrogen bonds with two backbone DNA phosphates and one hydrogen bond with R81 in H3 Loop3 respectively [33]. (C) The close-up view of structure around dimethylated H4K79 residue. The side chain Nε–N forms a salt could also interact with a backbone DNA phosphate. The dimethylation was generated using the mutagenesis function in Pymol. And it is observed that two methyl groups interrupt the interaction between the H4K79 side chain and DNA backbone.
further validate the lysine butyrylation, we compared MS/MS spectrum and HPLC retention of the butyrylated peptides with that of its corresponding acetylated counterparts. We observed highly similar fragmentation patterns of pairing fragment ions that contain the two modifications, respectively, as demonstrated in Fig. 3. Obviously butyrylated peptides are more hydrophobic than acetylated ones as indicated by their HPLC retention time. The two lines of evidences both supported the existence of butyrylated peptides in ESCC cells. MS signal intensities suggest that the butyrylation has a pretty low
abundance. For example, the butyrylation on H3K18 is 100 times lower than its corresponding acetylation on the same site.
3.3. Comparative analysis of specific histone PTM sites in two ESCC cell lines To examine the differences between TE3 and KYSE180 cell lines at specific histone PTM sites, we compared the abundance of key histone marks within the two cells. The relative abundance was calculated based on the ratio of the peak areas of the same
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parent ions labeled by light and heavy isotopes in the MS spectra. H3K9me/me2 and H3K27me/me2, which were reported as the repressive transcription marks [28] and considered to be associated with cancer development, if dysregulated [29]. Fig. 4 demonstrates that there is no obvious difference on the level of H3K9me2 between the two cell lines. The result was further validated by western blot assay (Fig. 5A). We also observed the difference of abundance of H3K27me2 in the cell lines. In case of the methylation of H3K27, which has been considered to be an epigenetic mark that mediates tumor suppressor gene silencing [30], it was reported that an elevation of H3K27 methylation was observed in ESCC than in healthy esophageal issue that was from the same individual and was closely associated with the tumor stage [31]. To investigate the relationship between individual modifications and ESCC invasive compatibility, we further compared the global methylation levels identified on H3 and H4. Among these methylated sites, a significant differential abundance was found on H4K79me2, which is 6 times higher in KYSE180 than that in TE3 cell (Fig. 4A). To further verify this finding, H4K79me2 was examined in both cell lines by western blots. As shown in Fig. 5B, H4K79me2 in KYSE180 is indeed more abundant than that in TE3. Although H4K79me2 has been identified in anuran Xenopus laevis [32], its function is still largely unknown. It is also unclear what enzymes regulate the addition or removal of H4K79me2. To check whether H4K79me2 is relevant to the survival of ESCC patients, we examined 166 ESCC specimens by immunohistochemistry analysis. H4K79me2 immunoreactivity was both cytoplasmic and nucleic in ESCC cells. Typical immunostainings are shown in Fig. 6A. According to X-tile plots, the 166 cases were divided into three populations: low-expression (0–3 scores, n = 8), mid-expression (4–7 scores, n = 70) and high-expression (8–12 scores, n = 88) (Fig. 6B and C). Kaplan Meier survival analysis was used to evaluate the association between H4K79me2 expression and the overall survival. The patients with low-expression of H4K79me2 had the lowest 5-year survival rate of 0.0%, compared with 54.4% or 44.0% for H4K79me2 mid- or high-expression (χ2 = 10.892, P =0.004; Fig. 6D). These data suggest that low-expression of H4K79me2 may be correlated with a poor survival of ESCC patients. Our evidence indicates a correlation between H4K79me2 and tumor phenotype in both cell lines and ESCC patients, therefore we assume that H4K79me2 could have a potential effect on ESCC development. However, the complexity of genetic source of cancer makes it difficult to prove this effect and elucidate the mechanism. Here we have just deduced a possible role of H4K79me2 in the global DNA–histone interaction based on a well-solved crystal structure of wild type nucleosome. Fig. 7A illustrates that H4K79 residue localizes in the histone-fold domains in human nucleosome. Structure analysis has uncovered that the unmodified H4 K79 residue may contain three main interactions [33]: (1) a hydrogen bond between the main chain amide nitrogen and a DNA backbone phosphate; (2) a salt bridge between the side chain Nε and a DNA backbone phosphate; and (3) a hydrogen bond with a main chain carbonyl oxygen of the H3 D81 residue (Fig. 7B). Fig. 7C illustrates a close-up view of the structure around dimethylated H4K79 residue. Two methyl groups
might destabilize the salt bridge between the side chain Nε and a DNA backbone phosphate. Although the role of dimethylation on H4K79 is unclear, structural analysis indicates that the H4K79me2 may have a potential effect on the histone–DNA interaction. The histone acetylation (Kac) plays a crucial role in the regulation of gene transcription via the modulation of nucleosomal DNA package. Under hypoacetylated state, nucleosomes are tightly compacted because of the ionic interaction between the protonated amine groups of lysines and the phosphate groups of DNA, thus lead to a transcriptional repression because the transcription factors have restricted access to their target DNA. In contrast, histone Kac results in a relaxed nucleosomal structure thus allows transcription to occur. Histone acetylation and its deacetylation enzyme, HDAC are associated with the development and progress of human cancer [29]. Reduced level of acetylated histones H3 and H4 has been believed to be a potential mark for the diagnosis of cancer at an early stage [31]. Additionally, abnormal expression of the histone marks has been linked to the recurrence-free survival in different ESCC stages [34]. In this study, MS analysis demonstrated that there is no obvious difference between the two cell lines, as shown in Fig. 4B. The result was further confirmed by western blot using anti-acetyllysine pan antibodies (Fig. 5C).
4. Conclusion In this study, we combined quantitative proteomics approach and bioinformatic analysis to characterize the global histone PTMs in two ESCC cell lines. A series of histone PTMs including novel lysine butyrylation were identified. The landscape of histone PTMs in two phenotypes of ESCCs with different invasion capacity was presented. Further quantitative analysis found that abundance of H4K79me2 might be correlated to the invasion capacity of ESCC cell and poor survival of ESCC patients. Structural analysis indicated that the modification may have a potential effect on the histone– DNA interaction. Our results might be valuable for the understanding of the relationship between histone PTMs and cancer invasiveness. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jprot.2014.09.004.
Transparency document Transparency document associated with this article can be found, in the online version.
Acknowledgments This work was supported by the National Basic Research Program of China (Grants 2012cb910601 and 2013cb910903), the National Natural Science Foundation of China with Grants (21275077) and the Tianjin Municipal Science and Technology Commission (no. 14JCYBJC24000).
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REFERENCES [1] Kouzarides T. Chromatin modifications and their function. Cell 2007;128:693–705. [2] Reik W. Stability and flexibility of epigenetic gene regulation in mammalian development. Nature 2007;447:425–32. [3] Esteller M. Cancer epigenomics: DNA methylomes and histone-modification maps. Nat Rev Genet 2007;8:286–98. [4] Barlesi F, Giaccone G, Gallegos-Ruiz MI, Loundou A, Span SW, Lefesvre P, et al. Global histone modifications predict prognosis of resected non small-cell lung cancer. J Clin Oncol 2007;25:4358–64. [5] Seligson DB, Horvath S, Shi T, Yu H, Tze S, Grunstein M, et al. Global histone modification patterns predict risk of prostate cancer recurrence. Nature 2005;435:1262–6. [6] Seligson DB, Horvath S, McBrian MA, Mah V, Yu H, Tze S, et al. Global levels of histone modifications predict prognosis in different cancers. Am J Pathol 2009;174:1619–28. [7] Elsheikh SE, Green AR, Rakha EA, Powe DG, Ahmed RA, Collins HM, et al. Global histone modifications in breast cancer correlate with tumor phenotypes, prognostic factors, and patient outcome. Cancer Res 2009;69:3802–9. [8] Park YS, Jin MY, Kim YJ, Yook JH, Kim BS, Jang SJ. The global histone modification pattern correlates with cancer recurrence and overall survival in gastric adenocarcinoma. Ann Surg Oncol 2008;15:1968–76. [9] Jenuwein T. The epigenetic magic of histone lysine methylation. FEBS J 2006;273:3121–35. [10] Fraga MF, Ballestar E, Villar-Garea A, Boix-Chornet M, Espada J, Schotta G, et al. Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer. Nat Genet 2005;37:391–400. [11] Qi YJ, Chao WX, Chiu JF. An overview of esophageal squamous cell carcinoma proteomics. J Proteomics 2012;75: 3129–37. [12] Toh Y, Yamamoto M, Endo K, Ikeda Y, Baba H, Kohnoe S, et al. Histone H4 acetylation and histone deacetylase 1 expression in esophageal squamous cell carcinoma. Oncol Rep 2003;10: 333–8. [13] Tzao C, Sun GH, Tung HJ, Hsu HS, Hsu WH, Wang YC, et al. Reduced acetylated histone H4 is associated with promoter methylation of the fragile histidine triad gene in resected esophageal squamous cell carcinoma. Ann Thorac Surg 2006; 82:396–401 [discussion]. [14] Tzao C, Tung HJ, Jin JS, Sun GH, Hsu HS, Chen BH, et al. Prognostic significance of global histone modifications in resected squamous cell carcinoma of the esophagus. Mod Pathol 2009;22:252–60. [15] Jensen ON. Interpreting the protein language using proteomics. Nat Rev Mol Cell Biol 2006;7:391–403. [16] Sidoli S, Cheng L, Jensen ON. Proteomics in chromatin biology and epigenetics: elucidation of post-translational modifications of histone proteins by mass spectrometry. J Proteomics 2012;75: 3419–33. [17] Tan MJ, Luo H, Lee S, Jin FL, Yang JS, Montellier E, et al. Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification. Cell 2011;146:1015–27. [18] Zhang K, Chen Y, Zhang Z, Zhao Y. Identification and verification of lysine propionylation and butyrylation in yeast core histones using PTMap software. J Proteome Res 2009;8: 900–6.
189
[19] Chen Y, Sprung R, Tang Y, Ball H, Sangras B, Kim SC, et al. Lysine propionylation and butyrylation are novel post-translational modifications in histones. Mol Cell Proteomics 2007;6:812–9. [20] Dai L, Peng C, Montellier E, Lu Z, Chen Y, Ishii H, et al. Lysine 2-hydroxyisobutyrylation is a widely distributed active histone mark. Nat Chem Biol 2014;10:365–70. [21] Olsen CA. Expansion of the lysine acylation landscape. Angew Chem Int Ed Engl 2012;51:3755–6. [22] Ong SE, Mann M. A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC). Nat Protoc 2006;1: 2650–60. [23] Xie JJ, Xu LY, Xie YM, Zhang HH, Cai WJ, Zhou F, et al. Roles of ezrin in the growth and invasiveness of esophageal squamous carcinoma cells. Int J Cancer 2009;124:2549–58. [24] Chen Y, Kwon SW, Kim SC, Zhao Y. Integrated approach for manual evaluation of peptides identified by searching protein sequence databases with tandem mass spectra. J Proteome Res 2005;4:998–1005. [25] Zhang FR, Tao LH, Shen ZY, Lv Z, Xu LY, Li EM. Fascin expression in human embryonic, fetal, and normal adult tissue. J Histochem Cytochem 2008;56:193–9. [26] Vollmuth F, Geyer M. Interaction of propionylated and butyrylated histone H3 lysine marks with Brd4 bromodomains. Angew Chem Int Ed Engl 2010;49:6768–72. [27] Cheng Z, Tang Y, Chen Y, Kim S, Liu H, Li SS, et al. Molecular characterization of propionyllysines in non-histone proteins. Mol Cell Proteomics 2009;8:45–52. [28] Pan G, Tian S, Nie J, Yang C, Ruotti V, Wei H, et al. Whole-genome analysis of histone H3 lysine 4 and lysine 27 methylation in human embryonic stem cells. Cell Stem Cell 2007;1:299–312. [29] Portela A, Esteller M. Epigenetic modifications and human disease. Nat Biotechnol 2010;28:1057–68. [30] Wei YK, Xia WY, Zhang ZH, Liu JS, Wang HM, Adsay NV, et al. Loss of trimethylation at lysine 27 of histone H3 is a predictor of poor outcome in breast, ovarian, and pancreatic cancers. Mol Carcinog 2008;47:701–6. [31] Chen C, Zhao M, Yin N, He B, Wang B, Yuan Y, et al. Abnormal histone acetylation and methylation levels in esophageal squamous cell carcinomas. Cancer Invest 2011;29:548–56. [32] Shechter D, Nicklay JJ, Chitta RK, Shabanowitz J, Hunt DF, Allis CD. Analysis of histones in Xenopus laevis. I. A distinct index of enriched variants and modifications exists in each cell type and is remodeled during developmental transitions. J Biol Chem 2009;284:1064–74. [33] Iwasaki W, Tachiwana H, Kawaguchi K, Shibata T, Kagawa W, Kurumizaka H. Comprehensive structural analysis of mutant nucleosomes containing lysine to glutamine (KQ) substitutions in the H3 and H4 histone-fold domains. Biochemistry 2011;50:7822–32. [34] I H, Ko E, Kim Y, Cho EY, Han J, Park J, et al. Association of global levels of histone modifications with recurrence-free survival in stage IIB and III esophageal squamous cell carcinomas. Cancer Epidemiol Biomarkers Prev 2010;19: 566–73. [35] Tachiwana H, Kagawa W, Osakabe A, Kawaguchi K, Shiga T, Hayashi-Takanaka Y, et al. Structural basis of instability of the nucleosome containing a testis-specific histone variant, human H3T. Proc Natl Acad Sci 2010;107:10454–9.
