人源SUV39H1-HP1α复合物识别内源核小体的冷冻电镜结构分析

doi:10.3969/j.issn.1674-8115.2026.06.005

摘要/Abstract

摘要：

目的·利用冷冻电镜（cryo-electron microscopy，cryo-EM）技术，解析人源甲基转移酶SUV39H1与异染色质结合蛋白1α（heterochromatin protein 1α，HP1α）形成的复合物SUV39H1-HP1α结合内源核小体（endogenous nucleosome core particle，NCP^endo）时SUV39H1-HP1α-NCP^endo复合物的三维结构。方法·将人源SUV39H1基因在其N端添加6×His-3×Flag标签后，克隆至pMLink载体中；同时，将人源HP1α基因在N端添加2×HA标签后，也克隆至pMLink载体。采用聚乙烯亚胺瞬时转染法，于悬浮培养的哺乳动物细胞Expi293F中实现SUV39H1与HP1α复合物的共表达。依次利用anti-DYKDDDDK 亲和树脂进行亲和层析，以及通过甘油密度梯度离心（glycerol gradient ultracentrifugation，Grafix）结合化学交联，对复合物进行纯化。进一步借助cryo-EM数据采集与单颗粒重构技术，获取SUV39H1-HP1α-NCP^endo复合物的三维电镜密度图；并运用UCSF Chimera软件，将AlphaFold2预测模型拟合至该电镜密度图。结果·经由亲和层析与甘油密度梯度离心，成功获取了高纯度的SUV39H1-HP1α与内源核小体结合形成的稳定复合物SUV39H1-HP1α-NCP^endo。利用cryo-EM单颗粒重构技术，初步获得了SUV39H1-HP1α-NCP^endo复合物分辨率3.6 Å（1 Å=10^-10 m）的电镜密度图。在该密度图中，除能清晰分辨出核小体核心颗粒外，还在其外侧观察到一段不连续的附加密度。基于该附加密度的体积大小和空间分布特征，对其进行初步结构拟合后，推测该附加密度可能对应于SUV39H1蛋白中的SET结构域。结论·借助cryo-EM单颗粒重构技术，初步获得了人源SUV39H1与HP1α复合物识别并结合内源核小体的电镜密度图。尽管外侧附着密度不连续，暂时无法进行可靠的原子级建模，但基于密度体积与空间分布特征，推测其主要对应SUV39H1的SET结构域。此外，SUV39H1的染色质结构域（chromo domain，CD）及HP1α未被清晰捕捉，提示二者在该复合物中可能呈现构象动态性。

关键词: 甲基转移酶SUV39H1, 异染色质结合蛋白1α, 核小体, 表观遗传调控, 冷冻电镜

Abstract:

Objective ·To analyze the three-dimensional structure of the human histone methyltransferase SUV39H1 in complex with heterochromatin protein 1α bound to the endogenous nucleosome core particle (NCP^endo) through cryo-electron microscopy (cryo-EM). Methods ·The human SUV39H1 gene was cloned into the pMLink vector with an N-terminal 6×His-3×Flag tag, and the human HP1α gene was cloned into the pMLink vector with an N-terminal 2×HA tag. The SUV39H1-HP1α complex was co-expressed in Expi293F mammalian suspension cells through transient transfection using polyethylenimine. The expressed complex was sequentially purified by affinity chromatography with anti-DYKDDDDK resin and glycerol gradient ultracentrifugation combined with chemical cross-linking. Subsequently, cryo-EM data collection and single-particle reconstruction were performed to obtain the three-dimensional electron density map of the SUV39H1-HP1α-NCP^endo complex. AlphaFold2-predicted models were docked into the EM density using UCSF Chimera. Results ·The SUV39H1-HP1α-NCP^endo complex was successfully obtained with high purity by affinity chromatography and glycerol density gradient ultracentrifugation. Cryo-EM single-particle reconstruction yielded a preliminary density map of the SUV39H1-HP1α-NCP^endo complex at a resolution of approximately 3.6 Å (1 Å=10^-10 m). In addition to the nucleosome core particle, an extra discontinuous peripheral density was observed. Based on its size and spatial distribution, preliminary structural fitting suggested that this density may correspond to the SET domain of SUV39H1. Conclusion ·The density map of the SUV39H1-HP1α-NCP^endo complex was obtained by cryo-EM and single-particle reconstruction. Although the peripheral density is discontinuous and does not support reliable atomic modeling, its size and spatial features are consistent with the SET domain (including pre-SET, SET, and post-SET sub-modules) of SUV39H1. In addition, the chromo domain (CD) of SUV39H1 and HP1α were not been clearly resolved, suggesting that these regions may exhibit conformational dynamics within this complex.

Key words: SUV39H1 methyltransferase, heterochromatin protein 1α (HP1α), nucleosome, epigenetic regulation, cryo-electron microscopy

中图分类号:

Q518.2

胡琼, 黄晶. 人源SUV39H1-HP1α复合物识别内源核小体的冷冻电镜结构分析[J]. 上海交通大学学报（医学版）, 2026, 46(6): 732-739.

Hu Qiong, Huang Jing. Structural insights into the recognition of endogenous nucleosomes by the human SUV39H1- HP1α complex[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2026, 46(6): 732-739.

图/表 7

表1 PCR引物序列（5′→3′）

Tab 1 Primer sequences for PCR （5′→3′）

Gene	Forward primer sequence	Reverse primer sequence
SUV39H1	CGCGGATCCATGGCGGAAATTTAAAAGGCTGCAGCG	CCGCTCGAGCTAGAAGAGGTATTTGCGGCAGGACTCAGTCC
HP1α	CGCGGATCCATGGGAAAGAAAACCAAGCGGACAGC	CCGCTCGAGTTAGCTCTTTGCTGTTTCTTTCTCTTTGTTTTCC

图1 SUV39H1-HP1α-NCPendo 复合物表达与纯化Note： A. Domain organization of human SUV39H1-HP1α-NCPendo. B. SDS-PAGE analysis of the SUV39H1-HP1α-NCPendo complex after anti-DYKDDDDK G1 affinity purification. C. Western blotting analysis of histone modifications on nucleosomes co-purified with the SUV39H1-HP1α-NCPendo complex. CD-chromodomain; SET-suppressor of variegation, enhancer of zeste, and trithorax domain; CSD-chromoshadow domain. The asterisk (*) denotes HSP70 (70 kDa).

Fig 1 Purification of the SUV39H1-HP1α-NCPendo complex

表2 SUV39H1-HP1α-NCPendo 复合物的LC-MS/MS鉴定结果

Tab 2 LC-MS/MS identification results of the purified SUV39H1-HP1α-NCPendo complex

Protein	Gene name	PSM	Unique peptide
Histone-lysine N-methyltransferase SUV39H1	SUV39H1	118	33
Chromobox protein homolog 5	HP1α	115	15
Histone H4	H4C9	107	18
Histone H2B type 1-K	H2BC12	62	5
Histone H2B type 1-J	H2BC11	59	1
Histone H2B type 3-B	H2BC26	55	2
Histone H2A type 1-B/E	H2AC8	33	2
Histone H2A type 1	H2AC17	33	1
Histone H3.1	H3C12	28	5

图2 SUV39H1-HP1α-NCPendo 复合物cryo-EM数据收集与分析Note： A. A representative cryo-EM micrograph of the SUV39H1-HP1α-NCPendo complex. B. The fourier transform pattern corresponding to the cryo-EM image shown in panel (A). C. Representative two-dimensional (2D) class averages obtained from electron microscopy particles of the SUV39H1-HP1α-NCPendo complex. D. Local resolution assessment of the SUV39H1-HP1α-NCPendo complex. E. The FSC curve of the SUV39H1-HP1α-NCPendo electron density map. The global resolution was estimated to be 3.6 Å based on the gold-standard FSC=0.143 criterion. F. Angular distribution of the reconstructed particles of the SUV39H1-HP1α-NCPendo complex.

Fig 2 cryo-EM data collection and analysis of the SUV39H1-HP1α-NCPendo complex

表3 cryo-EM数据收集与处理

Tab 3 cryo-EM data collection and processing

Data collection and processing	SUV39H1-HP1α-NCP^endo complex
Magnification	81 000
Voltage /kV	300
Electron exposure/(e^-·Å^-2)	50.0
Defocus range/μm	-0.8‒-2.5
Pixel size /Å	1.1
Symmetry imposed	C1
Initial particle images/n	5 295 046
Final particle images /n	2 167 181
Map resolution/Å (FSC threshold 0.143)	3.6
Map resolution range/Å	2.5‒7.5

图3 SUV39H1-HP1α-NCPendo 复合物的cryo-EM结构分析Note： A. Single-particle reconstruction of SUV39H1-HP1α-NCPendo complex shown in three orthogonal views. B. Docking of the AlphaFold2-predicted SET domain of SUV39H1 into the cryo-EM density map.

Fig 3 cryo-EM structural analysis of the SUV39H1-HP1α-NCPendo complex

图4 SUV39H1SET 与HP1α在核小体表面的空间占位叠合分析Note： A. Density map of the SUV39H1SET-nucleosome complex. B. Density map of the HP1α dimer-nucleosome complex (EMD-42774). C. Superposition of the SUV39H1SET-NCP complex with the HP1α dimer-NCP complex (EMD-42774), revealing steric incompatibility on the nucleosome surface.

Fig 4 Spatial occupancy analysis of SUV39H1SET and HP1α on the nucleosome surface

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