The Nucleosome Remodeling and Deacetylase Complex NuRD Is Built from Preformed Catalytically Active Sub-modules

Abstract

The nucleosome remodeling deacetylase (NuRD) complex is a highly conserved regulator of chromatin structure and transcription. Structural studies have shed light on this and other chromatin modifying machines, but much less is known about how they assemble and whether stable and functional sub-modules exist that retain enzymatic activity. Purification of the endogenous Drosophila NuRD complex shows that it consists of a stable core of subunits, while others, in particular the chromatin remodeler CHD4, associate transiently. To dissect the assembly and activity of NuRD, we systematically produced all possible combinations of different components using the MultiBac system, and determined their activity and biophysical properties. We carried out single-molecule imaging of CHD4 in live mouse embryonic stem cells, in the presence and absence of one of core components (MBD3), to show how the core deacetylase and chromatin-remodeling sub-modules associate in vivo. Our experiments suggest a pathway for the assembly of NuRD via preformed and active sub-modules. These retain enzymatic activity and are present in both the nucleus and the cytosol, an outcome with important implications for understanding NuRD function.

Refinement of the subunit interaction network within the nucleosome remodelling and deacetylase (NuRD) complex

“…A similar suggestion has been made based on work on Drosophila NuRD [41]. Further, the observation that immunoprecipitation of GATAD2Af from mammalian cells pulls down CHD4 but no other NuRD components (Figure 5e) suggests that the connection between CHD4 and the rest of the NuRD complex is predominantly through the GATAD2Af region, not via multiple interactions with other subunits.…”

“…Together, these structural data suggest that a HDAC-MTA-RBBP subcomplex containing full-length MTA has a 2 : 2 : 4 stoichiometry. This stoichiometry was also observed recently for the homologous Drosophila melanogaster complex, using multiangle laser light scattering (MALLS) and label-free iBAQ quantitative MS [41]. Two papers from the Vermeulen laboratory also used iBAQ MS to estimate the subunit stoichiometry of the mammalian NuRD complex [39,40].…”

“…The same paper also observes a 2:2 for HDAC:MBD, suggesting two MBD-GATAD2 units might exist in a single NuRD complex. In contrast, however, MALLS and iBAQ data on D. melanogaster NuRD argue for a 2:1 HDAC:MBD ratio and a sub-stoichiometric amount of GATAD2 [41]. A sub-stoichiometric GATAD2 is unlikely; we have previously shown, using blue-native-PAGE (BN-PAGE) and two-dimensional BN-SDS-PAGE that GATAD2 is associated with the NuRD complex in all its forms (including the CHD-less NuDe complex) [32].…”

The BRD3 ET domain recognizes a short peptide motif through a mechanism that is conserved across chromatin remodelers and transcriptional regulators

“…The current study, together with published data (33,57), indicates that different partners display a range of affinities for the BRD3 and -4 ET domains. In addition, there are residues in the α2-α3 loop of the ET domain (namely Arg607-Asn610 in BRD3) that exhibit large chemical shift changes upon peptide binding but are not conserved between BET family members ( Figure S2A), and these might conceivably influence binding affinity and/or specificity.…”

The Nucleosome Remodelling and Deacetylation complex suppresses transcriptional noise during lineage commitment

“…Nucleosome Remodelling and Deacetylation is comprised of two enzymatically and biochemically distinct subcomplexes: a chromatin remodelling and a deacetylase subcomplex. The chromatin remodelling subcomplex contains a nucleosome remodelling ATPase protein (Chd3/4/5) along with one of the zinc finger proteins Gatad2a/b and the Doc1/Cdk2ap1 protein, while the deacetylase subcomplex contains class I histone deacetylase proteins Hdac1/2, the histone chaperones Rbbp4/7, the Metastasis Tumour Antigen family of proteins, Mta1, Mta2 and Mta3 and, in pluripotent cells, the zinc finger proteins Sall1/4 (Lauberth & Rauchman, ; Allen et al , ; Kloet et al , ; Bode et al , ; Low et al , ; Miller et al , ; Spruijt et al , ; Zhang et al , ). These two subcomplexes are bridged by Mbd2/3, creating intact NuRD (Fig A).…”