Expression, purification and characterization of the human MTA2-RBBP7 complex.
The repressive Nucleosome Remodeling and histone Deacetylation (NuRD) complex remodels the chromatin structure by coupling ATP-dependent remodeling activity with histone deacetylase function and plays important roles in regulating gene transcription, DNA damage repair and chromatin assembly. The complex is composed of six subunits: Metastasis Associated proteins MTA1/2/3 initially recruit histone chaperones RBBP4/7 followed by the histone deacetylases HDAC1/2 forming a core complex. Further association of the CpG-binding protein MBD2/3, p66α/β and the ATP-dependent helicase CDH3/4 constitutes the NuRD complex. Recent structural studies on truncated human proteins or orthologous have revealed that the stoichiometry of the MTA1-RBBP4 complex is 2:4. This study reports expression and purification of the intact human MTA2-RBBP7 complex using HEK293F cells as expression system. In analogy with findings on the Drosophila NuRD complex, we find that also the human MTA-RBBP can be isolated in vitro. Taken together with previous findings this suggests, that MTA-RBBP is a stable complex, with a central role in the initial assembly of the human NuRD complex. Refined 3D volumes of the complex generated from negative stain electron microscopy (EM) data reveals an elongated architecture that is capable of hinge like motion around the center of the particle.
Biostructural Research, Department of Drug Design and Pharmacology, University of Copenhagen, Jagtvej 162, 2100 Copenhagen, Denmark.
Christoffer
C
Brasen
Biostructural Research, Department of Drug Design and Pharmacology, University of Copenhagen, Jagtvej 162, 2100 Copenhagen, Denmark.
Jerzy
J
Dorosz
Biostructural Research, Department of Drug Design and Pharmacology, University of Copenhagen, Jagtvej 162, 2100 Copenhagen, Denmark.
Anders
A
Wiuf
Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds vej 10C, 8000 Aarhus, Denmark; Interdisciplinary Nanoscience Center, Aarhus University, Gustav Wieds Vej 14, 8000 Aarhus, Denmark.
Thomas
T
Boesen
Biostructural Research, Department of Drug Design and Pharmacology, University of Copenhagen, Jagtvej 162, 2100 Copenhagen, Denmark.
Osman
O
Mirza
Biostructural Research, Department of Drug Design and Pharmacology, University of Copenhagen, Jagtvej 162, 2100 Copenhagen, Denmark. Electronic address: mig@sund.ku.dk.
Michael
M
Gajhede
D000595:Amino Acid Sequence / Q000235:genetics; D042002:Chromatin Assembly and Disassembly / Q000235:genetics*; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D056488:Histone Chaperones / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D056284:Histone Deacetylase 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D056464:Histone Deacetylase 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006655:Histone Deacetylases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D056245:Mi-2 Nucleosome Remodeling and Deacetylase Complex / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D056524:Retinoblastoma-Binding Protein 7 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D056488:Histone Chaperones; C490746:RBBP7 protein, human; D012097:Repressor Proteins; D056524:Retinoblastoma-Binding Protein 7; C120917:MTA2 protein, human; C110550:HDAC1 protein, human; C533423:HDAC2 protein, human; D056284:Histone Deacetylase 1; D056464:Histone Deacetylase 2; D006655:Histone Deacetylases; D056245:Mi-2 Nucleosome Remodeling and Deacetylase Complex
Negative stain electron microscopy; NuRD complex; Transient HEK293F cell expression