Live-cell 3D single-molecule tracking reveals how NuRD modulates enhancer dynamics

Abstract

Enhancer-promoter dynamics are critical for the spatiotemporal control of gene expression, but it remains unclear how these dynamics are controlled by chromatin regulators, such as the nucleosome remodelling and deacetylase (NuRD) complex. Here, we use Hi-C experiments to show that the intact NuRD complex increases CTCF/Cohesin binding and the probability of the interaction of intermediate-range (~1Mb) genomic sequences.To understand how NuRD alters 3D genome structure in this way, we developed an approach to segment and extract key biophysical parameters from trajectories of the NuRD complex determined using live-cell 3D singlemolecule imaging. Unexpectedly, this revealed that the intact NuRD complex decompacts chromatin structure and makes NuRD-bound sequences move faster, thus increasing the overall volume of the nucleus that these sequences explore. Interestingly, we also uncovered a rare fast-diffusing state of chromatin that exhibits directed motion. The intact NuRD complex reduces the amount of time that enhancers/promoters remain in this fastdiffusing state, which we propose would otherwise re-organise enhancer-promoter proximity. Thus, we uncover an intimate connection between a chromatin remodeller and the spatial dynamics of the local region of the genome to which it binds.3D genome organisation is thought to be critical for the spatiotemporal control of gene expression. However, little is known about the multi-scale chromatin dynamics of cis-regulatory elements or how they relate to genome organisation. To probe these dynamics at the single-cell level, one of two complementary methods are typically used: either live-cell imaging 1-4 or single nucleus versions of chromosome conformation capture experiments (such as Hi-C), which reveal the proximity of DNA sequences in different individual fixed cells [5][6][7][8][9][10][11][12][13][14][15][16] . For example, live-cell tracking of enhancers and promoters has revealed fast diffusion (or 'stirring') during transcription 4 , but the mechanisms underlying these changes in enhancer dynamics or how they relate to Hi-C measurements of enhancer-promoter proximity remain unclear.The nucleosome remodelling and deacetylase (NuRD) complex is a highly conserved 1 MDa multisubunit protein complex that we have previously shown clusters in 3D space in the nucleus with active enhancers and promoters 16 . It combines two key enzymatic activities -nucleosome remodelling via its helicasecontaining ATPase (predominantly CHD4 in ES cells) and lysine deacetylation via its HDAC1/2 subunits [16][17][18][19][20][21][22] .These activities are thought to be present in two sub-complexes (Figure 1a). HDAC1/2 associates, along with the histone chaperones RBBP4/7, with the core scaffold proteins MTA1/2/3 (metastasis tumour antigens) to form a stable sub-complex with deacetylase activity 23 . The nucleosome remodeller CHD4 interacts with chromatin by itself and may also form a second sub-complex with GATAD2A/B and DOC1 (CDK2AP1) 23-25 .The methyl-CpG DNA binding domain proteins MBD2/3 ...

Single-molecule imaging of chromatin remodelers reveals role of ATPase in promoting fast kinetics of target search and dissociation from chromatin

“…Chromatin imaged by several distinct methods in living cells displays heterogeneous mobility, which is dependent on its compaction state, subnuclear localization, and ATP-dependent processes (Gasser, 2002; Gu et al, 2018; Marshall et al, 1997; Soutoglou & Misteli, 2007). Remodelers may undergo 1D translocation on DNA (Sirinakis et al, 2011), and alter either local chromatin movement (Basu et al, 2020; Neumann et al, 2012) or higher-order chromatin structure (Lusser, Urwin, & Kadonaga, 2005; Maier, Chioda, Rhodes, & Becker, 2008) in an ATP-dependent fashion. We assessed the diffusive behavior of the chromatin-bound fraction of remodelers relative to the average dynamics of incorporated Halo-H2B histone.…”

Enhancer-promoter interactions and transcription are maintained upon acute loss of CTCF, cohesin, WAPL, and YY1

“…In this model, CTCF, cohesin, and architectural factors contribute to the establishment of E-P interactions, but not to their maintenance. Instead, once established, a molecular memory (e.g., histone modifications 89 , chromatin remodeling [90][91][92] , DNA modification [93][94][95] , lncRNAs 96,97 , etc.) may be sufficient to maintain E-P interactions and gene expression for several hours without the contribution of CTCF, cohesin, and other architectural factors.…”

Single-molecule imaging of chromatin remodelers reveals role of ATPase in promoting fast kinetics of target search and dissociation from chromatin

“…Chromatin imaged by several distinct methods in living cells displays heterogeneous mobility, which is dependent on its compaction state, subnuclear localization, and ATPdependent processes (Gasser, 2002;Gu et al, 2018;Marshall et al, 1997;Soutoglou & Misteli, 2007). Remodelers may undergo 1D translocation on DNA (Sirinakis et al, 2011), and alter either local chromatin movement (Basu et al, 2020;Neumann et al, 2012) or higher-order chromatin structure (Lusser, Urwin, & Kadonaga, 2005;Maier, Chioda, Rhodes, & Becker, 2008) in an ATP-dependent fashion. We assessed the diffusive behavior of the chromatin-bound fraction of remodelers relative to the average dynamics of incorporated Halo-H2B histone.…”