The N-terminal Region of Chromodomain Helicase DNA-binding Protein 4 (CHD4) Is Essential for Activity and Contains a High Mobility Group (HMG) Box-like-domain That Can Bind Poly(ADP-ribose)

Abstract

Chromodomain Helicase DNA-binding protein 4 (CHD4) is a chromatin-remodeling enzyme that has been reported to regulate DNA-damage responses through its N-terminal region in a poly(ADP-ribose) polymerase-dependent manner. We have identified and determined the structure of a stable domain (CHD4-N) in this N-terminal region. The-fold consists of a four-␣-helix bundle with structural similarity to the high mobility group box, a domain that is well known as a DNA binding module. We show that the CHD4-N domain binds with higher affinity to poly(ADP-ribose) than to DNA. We also show that the N-terminal region of CHD4, although not CHD4-N alone, is essential for full nucleosome remodeling activity and is important for localizing CHD4 to sites of DNA damage. Overall, these data build on our understanding of how CHD4-NuRD acts to regulate gene expression and participates in the DNA-damage response.The nucleosome remodeling and deacetylase (NuRD) 3 complex is a conserved transcription co-regulatory complex that is found in all complex animals. It is unique in that it contains both nucleosome remodeling and deacetylase activity (1-6); other co-regulator complexes such as Sin3 (7), Rpd3S (8), and PRC2 (9) only carry a single enzymatic activity. The NuRD complex has been shown to have essential roles in both activation and repression of gene transcription and has links to stem cell renewal and differentiation (10), cell cycle control (11), cancer (for review see Ref. 12), and the DNA-damage response, including DNA double strand break (DSB) repair (13). DSBs are highly cytotoxic; if repaired erroneously or left unrepaired, they can lead to cancer, neurodegeneration, and immunodeficiency (14).One of the defining components of the NuRD complex is chromodomain helicase DNA-binding protein 4 (CHD4). CHD4 mediates the chromatin remodeling activity of the NuRD complex via a SNF2-type DNA translocase domain (15), and evidence has accumulated that CHD4 is important for an effective DNA-damage response and for the maintenance of genomic integrity. For example, the expression level of CHD4 has been shown to increase upon UV irradiation (16). CHD4 has been also reported to be a target of the DNA-damage response apical kinases and to be recruited to DSB sites as part of the NuRD complex (20). This recruitment occurs in a poly(ADP-ribose) polymerase (PARP)-dependent manner. It has been shown that a 750-residue N-terminal fragment of CHD4 (residues 1-758) was sufficient for its recruitment and that this region binds poly(ADP-ribose) (PAR) polymers with the same apparent affinity as full-length CHD4 (20), suggesting that the N-terminal portion of CHD4 contains one or more PAR binding motifs. Similarly, the Drosophila homologue of CHD4 (dMi-2) has been shown to be recruited to active heat shock genes in a PARP-dependent manner (21). Proteomic studies have also indicated that CHD4 and other proteins of the CHD family can undergo poly-ADP-ribosylation; however, the biological effects of this modification are still unknown (22,23).Poly(ADP-ri...

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

“…The cut sites avoided crossing known domains or predicted secondary structures. CHD4N contains the recently reported HMG-box-like domain [35], CHD4M contains the PHD, chromo- and helicase domains and CHD4C1C2 contains two predicted helical domains with unknown folds or function. Figure 5b shows the western blots for which positive interactions were observed, while the rest of the pulldowns performed are shown in Figures 4b and 6.…”

“…In the case of immunoprecipitations and pulldowns, 'Protein 1' is the bait, and 'Protein 2' the prey. 35 GATAD2B HeLa cells 1 [40] for other interactions between pairs of subunits, including CHD4 and RBBP7 ( Fig. 3E).…”

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

“…When full-length BRD3 was co-expressed in HEK293 cells with the N-terminal, middle or C-terminal sections of CHD4 (Figure 2A), only the Nterminal third (residues 1-355) was able to robustly bind BRD3 ( Figure 2B). This portion of the protein contains an HMG-box-like domain (48) and is otherwise predicted to be disordered (by programs such as DISOPRED3 (49)). We next made a series of bacterially expressed, GST-tagged CHD4 fragments focused on the Nterminal third of the protein (Figure 2A) and used these as baits to pull down HA-tagged BRD3-L expressed in mammalian cells ( Figure 2C).…”

Nucleosome-CHD4 chromatin remodeller structure explains human disease mutations

“…However, the resolution of these studies was limited, such that atomic details were not resolved. The human CHD family member CHD4 (Woodage et al, 1997) shows nucleosome spacing activity (Silva et al, 2016). CHD4 is also known as Mi-2 in Drosophila melanogaster (Kehle et al, 1998) and together with CHD3 forms subfamily II, which differs in domain architecture from subfamily I. CHD3 and CHD4 contain two N-terminal plant homeodomain zinc fingers (Schindler et al, 1993) (PHD fingers 1 and 2), a DNA-interacting double chromodomain, and the ATPase motor.…”

“…CHD4 is also known as Mi-2 in Drosophila melanogaster (Kehle et al, 1998) and together with CHD3 forms subfamily II, which differs in domain architecture from subfamily I. CHD3 and CHD4 contain two N-terminal plant homeodomain zinc fingers (Schindler et al, 1993) (PHD fingers 1 and 2), a DNA-interacting double chromodomain, and the ATPase motor. CHD4 contains an additional high mobility group (HMG) box-like domain in its N-terminal region (Silva et al, 2016) and two additional domains of unknown function located in the C-terminal region. CHD4 is implicated in the repression of lineage-specific genes during differentiation (Liang et al, 2017) and is required for the establishment and maintenance of more compacted chromatin structures (Bornelöv et al, 2018).…”