非经典型多梳抑制复合物1.6的电镜结构分析

doi:10.3969/j.issn.1674-8115.2024.09.008

摘要/Abstract

摘要：

目的·通过负染色及透射电子显微镜（电镜）技术分析非经典型多梳抑制复合物1.6（polycomb repressive complex 1.6，PRC1.6）的蛋白结构，获得人源PRC1.6七元复合物的三维轮廓信息。方法·将PRC1.6复合物中7个组分基因RNF2、PCGF6、RYBP、L3MBTL2、CBX3、E2F6和TFDP1分别克隆至N端带有6×His-3×Flag标签的pMLink载体中，利用聚乙烯亚胺瞬时转染的方式在悬浮培养的哺乳动物细胞Expi293F中表达PRC1.6七元复合物，依次使用anti-DYKDDDDK标签亲合树脂、分子筛Superdex 200 Increase 10/300 GL（凝胶过滤层析）和甘油密度梯度离心分离纯化PRC1.6复合物；通过液相色谱-串联质谱法（liquid chromatography-tandem mass spectrometry，LC-MS/MS）确认PRC1.6七元复合物组分；利用泛素化活性实验和电泳迁移率变动分析（electrophoretic mobility shift assay，EMSA）对复合物的体外泛素化活性以及与核小体结合亲和力进行验证；使用乙酸双氧铀进行样品制备并通过透射电镜观察蛋白样品，随后通过单颗粒重构技术对PRC1.6复合物的三维结构信息进行分析；利用UCSF Chimera软件将蛋白质结构数据库（Protein Data Bank，PDB）中的目的蛋白相关原子结构模型与重构获得的PRC1.6电子密度图进行拟合，预测PRC1.6七元复合物中各亚基的定位。结果·通过真核表达、亲和层析、凝胶过滤层析和甘油密度梯度离心，以及LC-MS/MS验证，获得了纯度较高、均一性较好的PRC1.6七元复合物，且泛素化活性实验和EMSA发现该复合物在体外具有泛素化活性和核小体结合亲和力。利用负染色、透射电镜和单颗粒重构技术初步解析了分辨率约为15.2 ?（1 ?=10^-10 m）的PRC1.6七元复合物的三维结构，将已有RNF2、PCGF6、RYBP、L3MBTL2、CBX3和DP1部分氨基酸序列的原子模型以及E2F6的AlphaFold2预测结构与重构获得的电子密度图进行匹配，初步确认了7个亚基在PRC1.6复合物三维结构中的定位情况。结论·使用负染色和透射电镜，以及单颗粒重构技术搭建了人源PRC1.6七元复合物的三维结构模型。

关键词: 多梳抑制复合物1, 表观遗传调控, 组蛋白泛素化, 负染色, 透射电子显微镜, 蛋白质三维结构

Abstract:

Objective ·To analyse the structure of non-canonical polycomb repressive complex 1.6 (PRC1.6) by negative staining and transmission electron microscopy (TEM), and obtain the three-dimensional (3D) profile information of human PRC1.6 heptameric complex. Methods ·Seven PRC1.6 components, RNF2, PCGF6, RYBP, L3MBTL2, CBX3, E2F6, and TFDP1, were cloned into the pMLink vector with a 6×His-3×Flag tag at the N-terminus, respectively. The proteins were expressed in Expi293F cells grown in suspension cultures by using transfection with polyethylenimine. The tagged proteins were isolated via affinity purification with anti-DYKDDDDK G1 affinity resin, followed by gel filtration chromatography with Superdex 200 Increase 10/300 GL and glycerol density gradient centrifugation. The components of the PRC1.6 heptameric complex were confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The in vitro ubiquitination activity and nucleosome-binding affinity of the purified heptameric complex were verified by the ubiquitination activity assay and the electrophoretic mobility shift assay (EMSA). The protein samples were stained by uranyl acetate and observed by TEM. The 3D information of the PRC1.6 complex was studied by single particle analysis. To predict the localization of the seven components within the structure model of PRC1.6 complex, the structure models of proteins in Protein Data Bank (PDB) were docked into the electron density map of PRC1.6 complex by using UCSF Chimera software. Results ·The PRC1.6 complex with high purity and good homogeneity was obtained by eukaryotic expression, affinity purification, gel filtration chromatography and glycerol density gradient centrifugation, and confirmed as the heptameric complex by LC-MS/MS. The purified proteins showed ubiquitination activity and nucleosome-binding affinity in vitro. The 3D structure of the PRC1.6 heptameric complex with a resolution of 15.2 ? (1 ?=10^-10 m) was preliminarily resolved by negative staining, TEM, and single particle analysis. The available structure models of RNF2, PCGF6, RYBP, L3MBTL2, CBX3, and DP1 proteins, as well as the predicted E2F6 structure by AlphaFold2, were docked into the reconstructed density map of PRC1.6 complex. The position of each component in the complex was preliminarily confirmed. Conclusion ·The 3D structural model of the human PRC1.6 heptameric complex is obtained by negative staining, TEM, and single particle analysis.

Key words: polycomb repressive complex 1 (PRC1), epigenetic regulation, histone ubiquitination, negative staining, transmission electron microscopy (TEM), three-dimensional protein structure

中图分类号:

蔡单, 黄晶. 非经典型多梳抑制复合物1.6的电镜结构分析[J]. 上海交通大学学报（医学版）, 2024, 44(9): 1136-1145.

CAI Dan, HUANG Jing. Electron microscopic study of the non-canonical polycomb repressive complex 1.6[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(9): 1136-1145.

图/表 7

图1 PRC1.6七元复合物成分与蛋白结构域组成示意图Note： A. The schematic diagram of the PRC1.6 heptameric complex depicts its ability to recognize and modify nucleosomes. B. Domain organization of human PRC1.6 heptameric complex. RING—really interesting new gene; RAWUL—ring finger and WD40-associated ubiquitin-like; ZnF—zinc finger; MBT—malignant brain tumor; CD—chromodomain; CSD—chromodomain-shadow; DBD—DNA binding domain; LZ—leucine zipper; MB—marked-box; CC—coiled coil; Ub-ubiquitin.

Fig 1 Schematic diagram of components and domain organization of the PRC1.6 heptameric complex

表1 PCR引物序列

Tab 1 Primer sequences for PCR

Gene	Forward sequence (5′→3′)	Reverse sequence (5′→3′)
RNF2	CTGTTCCAGGGCCCCGGATCCATGTCTCAGGCTG	CTAGCAAGCTTCTCGAGTCATTTGTGCTCCTTTGTAGG
PCGF6	GTTCCAGGGCCCCGGATCCATGGAGGGGGTCGCGGTGGTG	CTAGCAAGCTTCTCGAGTCAAGTTATCTTCAGAGGAGAAAC
E2F6	CTGTTCCAGGGCCCCGGATCCATGAGTCAGCAGCGGCCG	GCAAGCTTCTCGAGTCAGTTGCTTACTTCAAGCAATTC
L3MBTL2	GTTCCAGGGCCCCGGATCCATGGAGAAGCCCCGGAGTATTG	CTAGCAAGCTTCTCGAGTCAGTCGTCTGTTTCCTGCTTG
CBX3	CAGGGCCCCGGATCCATGGCCTCCAACAAAACTACATTG	CTAGCAAGCTTCTCGAGTTATTGAGCTTCATCTTCTGGAC
TFDP1	GTTCCAGGGCCCCGGATCCATGGCAAAAGATGCCGGTCTAATTG	GCAAGCTTCTCGAGTCAGTCGTCCTCGTCATTCTCGTTG
RYBP	CTGTTCCAGGGCCCCGGATCCATGACCATGGGCGACAAGAAG	CTAGCAAGCTTCTCGAGTCAGAAAGATTCATCATTGACTGC

图2 人源PRC1.6七元复合物蛋白纯化Note： A. SDS-PAGE analysis of the PRC1.6 heptameric complex after anti-DYKDDDDK G1 affinity purification. B. Gel filtration chromatography of the PRC1.6 heptameric complex with Superdex 200 Increase 10/300 GL. C. SDS-PAGE of gel filtration chromatography in B. D. SDS-PAGE of protein fractions after glycerol density gradient centrifugation. * denotes TRIM28 (Mr 110 000), ** denotes HSP70 (Mr 70 000), and *** denotes HRV-3C enzyme (Mr 66 400). AU—absorbance unit.

Fig 2 Purification of the human PRC1.6 heptameric complex

表2 LC-MS/MS分析纯化得到的PRC1.6七元复合物

Tab 2 Analysis of the purified PRC1.6 heptameric complex by LC-MS/MS

Subunit	Coverage/%	PSM	Unique peptide
RNF2	86	218	23
PCGF6	68	169	29
E2F6	89	145	33
L3MBTL2	64	133	46
CBX3	78	115	15
DP1	67	113	21
RYBP	78	104	19

图3 PRC1.6七元复合物的泛素化活性实验和EMSANote： A. Ubiquitination assay of the PRC1.6 complex. The upper two figures are Western blotting analysis of ubiquitination assay, and the lower figure is SDS-PAGE of ubiquitination assay. B. EMSA of the PRC1.6 complex. The upper figure on the left is Native-PAGE analysis of EMSA, which was stained by Coomassie brilliant blue in the lower figure. The right figure is SDS-PAGE analysis of EMSA input. * denotes TRIM28 and ** denotes HSP70. Ub—ubiquitin; H2Aub—H2A with one ubiquitin; H2Aub2—H2A with two ubiquitins.

Fig 3 Ubiquitination assay and EMSA of the PRC1.6 heptameric complex

图4 通过负染色及透射电子显微镜技术分析PRC1.6七元复合物Note： A. A representative negative staining electron micrograph image of PRC1.6 complexes (×57 000). Scale bar=200 nm. B. Representative two-dimensional (2D) class averages obtained from negative staining particles of PRC1.6 complexes. C. Angular distribution of particle projections of the PRC1.6 complex reconstruction. D. Gold-standard Fourier shell correlation (FSC) curve of the final reconstructed electron micrograph map of the PRC1.6 complex.

Fig 4 Analysis of the PRC1.6 heptameric complex by negative staining and transmission electron microscopy

图5 PRC1.6七元复合物电子密度图和各亚基定位Note： A. 3D reconstruction of PRC1.6 heptameric complex shown in three orthogonal views. B. Docking of the structure models (RN2, PCGF6, RYBP, L3MBTL2, CBX3, and DP1) and the predicted structure model (E2F6) into the electron density map of the PRC1.6 heptameric complex in three orthogonal views.

Fig 5 Electron density map of the PRC1.6 heptameric complex and localization of each subunit

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