Journal of Shanghai Jiao Tong University (Medical Science) >

2022 , Vol. 42 >Issue 4: 455 - 463

DOI: https://doi.org/10.3969/j.issn.1674-8115.2022.04.007

Basic research

Negative-stain electron microscopic study of the nucleosome remodeling and deacetylase complex

  • Yujuan DUAN ,
  • Jing HUANG
Expand
  • Shanghai Institute of Precision Medicine, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200125, China
HUANG Jing, E-mail: huangjing@shsmu.edu.cn

Received date: 2022-01-13

  Accepted date: 2022-03-07

  Online published: 2022-04-28

Supported by

National Natural Science Foundation of China(32022036)

Fold

Abstract

Objective

·To study the structure of human nucleosome remodeling and deacetylase complex (NuRD complex) with negative-stain electron microscopy to obtain the profile information of human NuRD complex.

Methods

·Full length MBD3 (methyl-CpG binding domain protein 3) and GATAD2A (GATA zinc finger domain containing 2A) constructs were cloned into the pMLink vectors with an amino terminal 10×His tag and a carboxyl terminal 3×Flag affinity tag, respectively. Proteins were expressed in human Expi293F cells grown in suspension cultures by using polyethylenimine as the transfection reagent, and were isolated via affinity purifications with Ni-NTA and anti-DYKDDDDK (Flag) G1 affinity resin sequentially. The complex was further purified by Superose 6 Increase 5/150 gel filtration chromatography to improve the homogeneity, identified by Western blotting and liquid chromatography-tandem mass spectrometry (LC-MS/MS), and was then studied by negative-stain electron microscopy and single particle analysis. The existing atomic structural model of the subcomplex (7AO9, 5FXY) in Protein Data Bank (PDB) was automatically docked into the generated structural model by the UCSF Chimera software to predict the localization of multiple protein components in the structural model.

Results

·The Flag-tagged MBD3 and His-tagged GATAD2A proteins could effectively pull down the other endogenous components of the NuRD complex through two-step affinity purifications. The high-purity complex was obtained by gel filtration chromatography and confirmed as the NuRD complex by LC-MS/MS and Western blotting identification. Preliminary study on the three-dimensional (3D) structure of the NuRD complex was carried out with negative-stain electron microscopy and single particle analysis, which revealed that the NuRD complex was in the obvious shape of a long asymmetrical rod. The 3D structural model of the human NuRD complex was finally obtained by further 3D refinement at an overall resolution of 17 ? (1 ?=0.1 nm). The existing atomic structural model (PDB: 7AO9, 5FXY) was automatically docked into the negative staining structural model, and the localizations of MTA1/2/3 (metastasis-associated protein 1/2/3), HDAC1/2 (histone deacetylase 1/2), RBBP4/7 (retinoblastoma-binding protein 4/7) and MBD2/3 (methyl-CpG-binding domain protein 2/3) subunits in the NuRD complex were preliminarily confirmed.

Conclusion

·A low-resolution negative-staining structural model of human NuRD complex is obtained by single particle analysis.

Key words： nucleosome remodeling and deacetylase (NuRD) complex; epigenetic regulation; deacetylation; negative-stain electron microscopy

Cite this article

Yujuan DUAN , Jing HUANG . Negative-stain electron microscopic study of the nucleosome remodeling and deacetylase complex[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022 , 42(4) : 455 -463 . DOI: 10.3969/j.issn.1674-8115.2022.04.007

References

1 CHEN T P, DENT S Y R. Chromatin modifiers and remodellers: regulators of cellular differentiation[J]. Nat Rev Genet, 2014, 15(2): 93-106.
2 ITO K, J BARNES P, M ADCOCK I. Histone acetylation and deacetylation[J]. Methods Mol Med, 2000, 44: 309-319.
3 WANG Z B, ZANG C Z, CUI K R, et al. Genome-wide mapping of HATs and HDACs reveals distinct functions in active and inactive genes[J]. Cell, 2009, 138(5): 1019-1031.
4 MINUCCI S, PELICCI P G. Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer[J]. Nat Rev Cancer, 2006, 6(1): 38-51.
5 ZHANG Y, IRATNI R, ERDJUMENT-BROMAGE H, et al. Histone deacetylases and SAP18, a novel polypeptide, are components of a human Sin3 complex[J]. Cell, 1997, 89(3): 357-364.
6 ZHANG Y, NG H H, ERDJUMENT-BROMAGE H, et al. Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation[J]. Genes Dev, 1999, 13(15): 1924-1935.
7 ZHANG Y, LEROY G, SEELIG H P, et al. The dermatomyositis-specific autoantigen Mi2 is a component of a complex containing histone deacetylase and nucleosome remodeling activities[J]. Cell, 1998, 95(2): 279-289.
8 YOU A, TONG J K, GROZINGER C M, et al. CoREST is an integral component of the CoREST- human histone deacetylase complex[J]. Proc Natl Acad Sci U S A, 2001, 98(4): 1454-1458.
9 BANTSCHEFF M, HOPF C, SAVITSKI M M, et al. Chemoproteomics profiling of HDAC inhibitors reveals selective targeting of HDAC complexes[J]. Nat Biotechnol, 2011, 29(3): 255-265.
10 TORCHY M P, HAMICHE A, KLAHOLZ B P. Structure and function insights into the NuRD chromatin remodeling complex[J]. Cell Mol Life Sci, 2015, 72(13): 2491-2507.
11 FARNUNG L, OCHMANN M, CRAMER P. Nucleosome-CHD4 chromatin remodeler structure maps human disease mutations[J]. eLife, 2020, 9: e56178.
12 LI D Q, KUMAR R. Mi-2/NuRD complex making inroads into DNA-damage response pathway[J]. Cell Cycle, 2010, 9(11): 2071-2079.
13 RAIS Y, ZVIRAN A, GEULA S, et al. Deterministic direct reprogramming of somatic cells to pluripotency[J]. Nature, 2013, 502(7469): 65-70.
14 LUO M, LING T, XIE W B, et al. NuRD blocks reprogramming of mouse somatic cells into pluripotent stem cells[J]. Stem Cells, 2013, 31(7): 1278-1286.
15 MILLARD C J, WATSON P J, CELARDO I, et al. Class Ⅰ HDACs share a common mechanism of regulation by inositol phosphates[J]. Mol Cell, 2013, 51(1): 57-67.
16 MILLARD C J, VARMA N, SALEH A, et al. The structure of the core NuRD repression complex provides insights into its interaction with chromatin[J]. eLife, 2016, 5: e13941.
17 MILLARD C J, FAIRALL L, RAGAN T J, et al. The topology of chromatin-binding domains in the NuRD deacetylase complex[J]. Nucleic Acids Res, 2020, 48(22): 12972-12982.
18 LOW J K K, SILVA A P G, TABAR M S, et al. The nucleosome remodeling and deacetylase complex has an asymmetric, dynamic, and modular architecture[J]. Cell Rep, 2020, 33(9): 108450.
19 TANG G, PENG L W, BALDWIN P R, et al. EMAN2: an extensible image processing suite for electron microscopy[J]. J Struct Biol, 2007, 157(1): 38-46.
20 SCHERES S H W. RELION: implementation of a Bayesian approach to cryo-EM structure determination[J]. J Struct Biol, 2012, 180(3): 519-530.
21 PETTERSEN E F, GODDARD T D, HUANG C C, et al. UCSF Chimera: a visualization system for exploratory research and analysis[J]. J Comput Chem, 2004, 25(13): 1605-1612.
22 SANNA S, ESPOSITO S, MASALA A, et al. HDAC1 inhibition ameliorates TDP-43-induced cell death in vitro and in vivo[J]. Cell Death Dis, 2020, 11(5): 369.
23 DOS SANTOS R L, TOSTI L, RADZISHEUSKAYA A, et al. MBD3/NuRD facilitates induction of pluripotency in a context-dependent manner[J]. Cell Stem Cell, 2014, 15(3): 392.
24 LUO J, SU F, CHEN D, et al. Deacetylation of p53 modulates its effect on cell growth and apoptosis[J]. Nature, 2000, 408(6810): 377-381.
25 ZHANG T Y, WEI G F, MILLARD C J, et al. A variant NuRD complex containing PWWP2A/B excludes MBD2/3 to regulate transcription at active genes[J]. Nat Commun, 2018, 9(1): 3798.
Options
Outlines

/

〈 〉