CHD4 Human

What is CHD4 and what is its primary role in human chromatin regulation?

CHD4 (Chromodomain Helicase DNA Binding Protein 4) is an ATP-dependent chromatin remodeling enzyme that plays crucial roles in gene regulation during development, differentiation, and disease processes. It functions primarily as a catalytic subunit of multi-protein complexes involved in gene repression, most notably the Nucleosome Remodeling Deacetylase (NuRD) complex and the ChAHP complex. CHD4 utilizes ATP hydrolysis to alter histone-DNA contacts within nucleosomes, facilitating chromatin reorganization that ultimately influences gene expression patterns. Unlike some other chromatin remodelers associated with gene activation, CHD4 primarily functions in transcriptional repression contexts, supporting the establishment and maintenance of compacted chromatin structures .

CHD4 has been implicated in several essential cellular processes including repression of lineage-specific genes during differentiation, cell cycle progression, DNA damage response, and heterochromatin formation and maintenance. Its role in establishing more compacted chromatin structures is consistent with its function in gene silencing and maintenance of repressed genomic regions. This activity contrasts with some other chromatin remodelers that function primarily in gene activation by promoting more accessible chromatin states .

What is the domain architecture of human CHD4 and how does it relate to function?

Human CHD4 contains multiple functional domains organized in a specific architecture that enables its diverse interactions with chromatin and other proteins. At the N-terminus, CHD4 contains a high mobility group (HMG) box-like domain involved in DNA interactions. This is followed by two plant homeodomain (PHD) zinc finger domains that recognize specific histone modifications. The protein also contains a DNA-interacting double chromodomain followed by the central ATPase motor, which catalyzes ATP hydrolysis to provide energy for chromatin remodeling. Additional domains of unknown function are located in the C-terminal region .

This domain organization places CHD4 (along with CHD3 and CHD5) in CHD subfamily II, which differs from subfamily I members (including Chd1) in their domain architecture. The presence of PHD fingers is particularly significant as they allow CHD4 to recognize specific histone modification patterns, providing a mechanism for targeted recruitment to particular chromatin regions. The ATPase motor is composed of two RecA-like lobes that together form the catalytic core responsible for ATP binding and hydrolysis, which drives the mechanical work of chromatin remodeling .

What protein complexes contain CHD4 and how do they function in gene regulation?

CHD4 functions primarily as a component of multi-protein complexes rather than as an isolated protein. The most well-characterized CHD4-containing complex is the Nucleosome Remodeling Deacetylase (NuRD) complex, which uniquely combines chromatin remodeling and histone deacetylase activities. The NuRD complex includes CHD4 as the remodeling enzyme, histone deacetylases HDAC1/2, metastasis-associated proteins (MTA1/2/3), methyl-CpG-binding domain proteins (MBD2/3), histone binding proteins (RBBP4/7), and transcriptional repressors (GATAD2A/B) .

Additionally, CHD4 is part of the heterotrimeric ChAHP complex, which includes CHD4, ADNP (activity-dependent neuroprotective protein), and HP1 (heterochromatin protein 1). This complex is also involved in gene repression pathways. The NuRD complex participates in gene silencing (though it can also contribute to gene activation in some contexts), cell cycle progression, DNA damage response, heterochromatin establishment, and cellular differentiation .

Through these complex associations, CHD4 coordinates chromatin remodeling with other epigenetic modifications, particularly histone deacetylation, creating a cooperative system for establishing and maintaining repressive chromatin environments. This integrated approach to chromatin modification allows for sophisticated control of gene expression during development and in response to cellular signals .

How does CHD4 interact with nucleosomes at the molecular level?

The molecular interaction between CHD4 and nucleosomes has been revealed through high-resolution cryo-electron microscopy (cryo-EM) structures at 3.1Å resolution. CHD4 engages with nucleosomal DNA specifically at the superhelical location (SHL) +2, where it binds and distorts the DNA structure in the presence of ATP or ATP analogues like AMP-PNP. This interaction supports the "twist defect" model of chromatin remodeling, wherein ATP-dependent distortion of DNA propagates along the nucleosome surface to reposition DNA relative to the histone octamer .

How does the ATPase mechanism of CHD4 drive chromatin remodeling?

The ATPase mechanism of CHD4 involves a sophisticated sequence of molecular events that convert chemical energy from ATP hydrolysis into mechanical force for chromatin remodeling. Based on high-resolution structural studies, we now understand that the ATPase motor of CHD4 contains two RecA-like lobes that engage nucleosomal DNA at SHL +2. Upon binding the ATP analogue AMP-PNP, CHD4 induces a significant conformational change in the DNA at this location, creating what is known as a "twist defect" in the nucleosomal DNA .

This twist defect represents a local distortion in the DNA that alters its normal path around the histone octamer. Through cycles of ATP binding, hydrolysis, and product release, this distortion propagates along the DNA, effectively breaking and reforming histone-DNA contacts in a directional manner. The binding of ATP and subsequent hydrolysis induce conformational changes in the ATPase motor itself, which are translated into movement of the engaged DNA segment. The presence of a coordinated magnesium ion in the active site, clearly visible in the 3.1Å resolution structure, plays a critical role in ATP hydrolysis .

Importantly, different nucleotide states (apo, ADP-bound, or ATP/AMP-PNP-bound) correspond to different conformations of the ATPase motor and different DNA distortion patterns, representing successive steps in the remodeling process. Unlike some other remodelers, CHD4's mechanism preserves nucleosome integrity rather than promoting DNA unwrapping, which aligns with its biological role in maintaining repressive chromatin states .

What is the significance of the "twist defect" model in chromatin remodeling by CHD4?

The "twist defect" model provides a mechanistic framework for understanding how CHD4 and other chromatin remodelers physically alter nucleosome positioning. According to this model, when CHD4's ATPase motor binds at SHL +2, it induces a localized twist distortion in the nucleosomal DNA. This creates a structural strain that must be accommodated and eventually propagated along the DNA wrapped around the histone octamer .

High-resolution cryo-EM structures at 3.1Å clearly reveal specific DNA distortions at SHL +2 in the presence of the ATP analogue AMP-PNP. These distortions differ from those observed in apo and ADP-bound states, providing structural evidence for the twist defect model. Through successive cycles of ATP binding and hydrolysis, this twist defect propagates along the DNA, effectively "pumping" DNA around the nucleosome in small increments of 1-2 base pairs per ATP hydrolysis event .

How do mutations in CHD4 contribute to human diseases?

Mutations in CHD4 have been implicated in several human diseases, with different genetic alterations affecting distinct aspects of protein function and leading to context-specific pathologies:

• Cancer: CHD4 mutations have a high incidence in certain carcinomas and in thyroid and ovarian cancers. The high-resolution structure of CHD4 suggests that these mutations can disrupt DNA binding, impede ATP hydrolysis, or uncouple ATP hydrolysis from DNA translocation, potentially leading to dysregulated gene expression contributing to oncogenesis .

• Intellectual Disability Syndromes: Specific mutations in CHD4 are associated with Sifrim-Hitz-Weiss syndrome, an intellectual disability disorder. The structural mapping of these mutations provides insights into how they may impair critical interfaces in the ATPase motor or disrupt interactions with DNA or protein partners .

• Congenital Heart Defects: A specific missense mutation in CHD4 (M202I in humans, M195I in mice) causes ventricular non-compaction, a form of congenital heart disease. Studies in a humanized mouse model demonstrate that this single mutation leads to failure of heart compaction, retention of cardiomyocytes in an immature proliferating state, increased extracellular matrix proteins, and reduced expression of the ECM protease ADAMTS1 .

What is particularly remarkable is how a single missense mutation in CHD4, a broadly expressed chromatin remodeler, can lead to tissue-specific phenotypes such as cardiac defects. This suggests that CHD4 has context-dependent roles that are particularly sensitive to specific mutations in different tissues and developmental stages .

What methodologies are used to study CHD4-nucleosome interactions at the structural level?

Studying CHD4-nucleosome interactions at the structural level requires sophisticated methodologies spanning various resolution scales:

• Cryo-Electron Microscopy (Cryo-EM): This technique has been pivotal in revealing the structure of CHD4-nucleosome complexes at high resolution (3.1Å). Sample preparation involves purifying recombinant CHD4 and reconstituted nucleosomes, forming the complex, and flash-freezing on EM grids. Data collection on instruments like the Titan Krios microscope equipped with a K2 direct electron detector allows visualization of molecular details including the orientation of protein domains, DNA path, and even the ATP analogue AMP-PNP and coordinated magnesium ion in the active site .

• Nucleosome Reconstitution: For structural studies, precisely defined nucleosomes are assembled using recombinant histones and DNA fragments containing positioning sequences like the Widom 601 sequence. Additional DNA extensions (e.g., 4 and 30 bp of extranucleosomal DNA) can be included to mimic the native chromatin context .

• Protein Expression and Purification: Full-length human CHD4 can be expressed using baculovirus systems in insect cells (Hi5 cells) and purified using affinity tags (such as 6xHis-MBP tags) with TEV protease cleavage sites. Size exclusion chromatography is then used to isolate the properly formed nucleosome-CHD4 complex .

• Computational Structure Analysis: The reconstructed density maps are interpreted through model building, where crystal structures of components (such as the nucleosome and CHD4 domains) are placed into the density and refined. Additional structural elements are built de novo and the entire structure undergoes real-space refinement to ensure good stereochemistry .

These approaches collectively provide unprecedented insights into how CHD4 engages with nucleosomes and the conformational changes associated with its remodeling activity, which has been essential for understanding both the mechanistic basis of CHD4 function and the potential impacts of disease-associated mutations .

How does CHD4 differ functionally from other CHD family members?

Despite sharing the core chromodomain-helicase-DNA binding architecture, CHD4 exhibits several key functional differences from other CHD family members, particularly from subfamily I members like Chd1:

• Domain Architecture: CHD4 (as part of subfamily II) contains two N-terminal PHD zinc finger domains and an HMG box-like domain that are absent in Chd1 (subfamily I). These additional domains enable distinct chromatin interactions and protein partnerships that influence targeting and function .

• Nucleosome Engagement: A crucial functional difference revealed by structural studies is that CHD4 does not induce unwrapping of terminal DNA during nucleosome remodeling, unlike Chd1. This difference directly correlates with their opposing roles in gene regulation, with CHD4 primarily involved in gene repression and Chd1 in gene activation .

• Complex Membership: CHD4 functions primarily as a component of large multi-protein complexes (NuRD, ChAHP) that integrate multiple chromatin-modifying activities. The NuRD complex uniquely combines CHD4's remodeling activity with histone deacetylase activity from HDAC1/2, creating a coordinated system for establishing repressive chromatin states .

• Biological Function: CHD4 is primarily associated with gene repression, heterochromatin formation, and maintenance of compact chromatin structures. It plays essential roles in lineage-specific gene repression during differentiation, cell cycle progression, and DNA damage response. In contrast, Chd1 is involved in gene activation, nucleosome assembly during transcription, and maintaining open chromatin .

These functional differences highlight how variations in domain architecture and protein interactions among chromatin remodelers can lead to distinct and sometimes opposing roles in gene regulation and chromatin organization .

What considerations are important when designing experiments to study CHD4 function?

Designing robust experiments to investigate CHD4 function requires careful consideration of multiple factors that address the complex nature of chromatin remodeling enzymes:

• Protein Expression Systems: For biochemical and structural studies, full-length human CHD4 requires expression in eukaryotic systems, typically using baculovirus-infected insect cells. The search results describe successful expression in Hi5 cells grown in ESF-921 media, with viral infection protocols optimized for this large, multi-domain protein .

• Purification Strategy: Effective purification typically employs affinity tags (such as the 6xHis-MBP tag described) with specific protease cleavage sites (TEV protease). Buffer composition is critical, with attention to salt concentration, pH, and additives that maintain protein stability and activity. Size exclusion chromatography provides a final purification step to isolate properly folded, homogeneous protein .

• Nucleosome Substrate Design: For in vitro studies, nucleosome design is crucial. The Widom 601 sequence provides a strongly positioning sequence for consistent nucleosome assembly. Researchers should consider including extra DNA (as described with 4bp and 30bp extensions) to mimic the nucleosomal context more accurately. The choice of symmetric versus asymmetric nucleosome constructs will depend on the specific questions being addressed .

• Complex Assembly: When studying CHD4 within the context of larger complexes like NuRD, researchers must decide whether to co-express components or reconstitute complexes from purified subunits. The approach chosen will influence the homogeneity and activity of the resulting complexes .

• Mutation Design: For studying disease-relevant mutations, precise design is essential. The humanized mouse model of CHD4 M195I (equivalent to human M202I) provides an excellent example of translating human disease mutations into experimental systems. CRISPR/Cas9 technology enables the creation of precise mutations that faithfully recapitulate human disease conditions .

• Functional Assays: Multiple complementary assays should be employed, including ATPase activity assays, nucleosome sliding assays, binding studies, and in vivo functional tests. The choice of readout (biochemical, cellular, or organismal) should align with the specific aspect of CHD4 function being investigated .

By addressing these considerations systematically, researchers can design experiments that yield meaningful insights into CHD4's role in chromatin remodeling across different biological contexts .

What approaches are effective for studying disease-associated CHD4 mutations?

Studying disease-associated CHD4 mutations requires an integrated approach spanning multiple levels of biological organization:

• Structural Mapping: High-resolution structures of CHD4-nucleosome complexes (3.1Å) allow precise mapping of disease-associated mutations. This mapping provides initial insights into potential mechanisms of disruption, such as interference with DNA binding, ATP hydrolysis, or protein-protein interactions. The structural context of mutations can guide hypotheses about their functional consequences .

• In Vitro Biochemical Assays: Recombinant proteins carrying disease mutations can be characterized through multiple assays:

  • ATPase activity assays to assess catalytic function

  • Nucleosome binding and remodeling assays to evaluate substrate interactions

  • Protein interaction studies to identify disrupted complex formation
    These approaches can determine whether mutations cause loss of function, altered activity, or novel properties .

• Cellular Models: CRISPR/Cas9 gene editing allows the introduction of patient-specific mutations into cellular models. RNA-seq, ChIP-seq, and ATAC-seq can then characterize changes in transcriptional profiles, chromatin binding, and chromatin accessibility. Cell type selection should reflect the disease context - for cardiac phenotypes, cardiomyocyte models are particularly relevant .

• Animal Models: The humanized mouse model of CHD4 M195I demonstrates an effective approach for studying CHD4 mutations in vivo. This model recapitulates key aspects of human congenital heart defects, including failure of ventricular compaction and retention of cardiomyocytes in an immature proliferating state. Phenotypic analysis should focus on the specific tissues affected in human patients .

• Molecular Mechanism Investigations: Beyond characterizing phenotypes, investigating the molecular mechanisms underlying disease manifestations is essential. In the CHD4 M195I mouse model, researchers identified downstream effects including altered extracellular matrix composition and reduced expression of the ECM protease ADAMTS1, connecting the chromatin remodeling defect to specific developmental abnormalities .

This multi-level approach allows researchers to connect structural alterations to molecular dysfunctions and ultimately to disease phenotypes, providing a comprehensive understanding of how CHD4 mutations contribute to human disorders .

What techniques are optimal for analyzing CHD4's genomic distribution and activity?

Analyzing CHD4's genomic distribution and activity requires sophisticated techniques that span from genome-wide profiling to locus-specific functional analysis:

• Chromatin Immunoprecipitation Approaches: ChIP-seq remains the gold standard for mapping genome-wide binding patterns of CHD4. This can be enhanced with newer techniques like CUT&RUN or CUT&Tag, which offer improved signal-to-noise ratios and require less starting material. For CHD4 specifically, optimization of fixation conditions and sonication parameters is crucial due to its association with compacted chromatin regions .

• Multi-omics Integration: CHD4 function is best understood through integrated approaches that combine:

  • ChIP-seq to map CHD4 binding

  • RNA-seq to assess transcriptional consequences

  • ATAC-seq to evaluate chromatin accessibility changes

  • Histone modification ChIP-seq to correlate with chromatin states
    This integration helps distinguish direct from indirect effects and reveals the relationship between CHD4 binding and functional outcomes .

• Co-occupancy Analysis: Since CHD4 functions within multi-protein complexes, analyzing co-occupancy with other NuRD components (HDACs, MBD proteins) or ChAHP complex members provides insights into context-specific activities. Sequential ChIP (Re-ChIP) can determine whether these factors simultaneously occupy the same genomic regions .

• Genome Editing Approaches: CRISPR/Cas9-based approaches can be used to:

  • Disrupt CHD4 binding at specific loci

  • Introduce tagged versions for improved detection

  • Create domain deletions to assess domain-specific functions
    The mouse model with the M195I mutation demonstrates how precise genome editing can provide insights into disease mechanisms .

• Nascent Transcription Assays: Techniques like PRO-seq or TT-seq can capture immediate transcriptional changes following CHD4 perturbation, distinguishing direct regulatory effects from secondary consequences. This is particularly important given CHD4's role in gene repression .

• Chromatin Conformation Analysis: HiChIP or PLAC-seq can reveal how CHD4 influences three-dimensional chromatin organization, potentially connecting its remodeling activity to higher-order chromatin structure .

By combining these approaches, researchers can develop a comprehensive understanding of how CHD4 is distributed across the genome, which loci it regulates, and how its activity contributes to chromatin organization and gene expression patterns .

How can researchers effectively study CHD4 within multi-protein complexes?

Studying CHD4 within its native multi-protein complexes presents unique challenges that require specialized approaches:

• Complex Purification Strategies: To isolate intact CHD4-containing complexes:

  • Tandem affinity purification using tagged CHD4 or other complex components

  • Size exclusion chromatography to separate different complex assemblies

  • Density gradient ultracentrifugation for high-purity separation
    Buffer conditions must be carefully optimized to maintain complex integrity throughout purification .

• Compositional Analysis: Determining the exact composition of CHD4-containing complexes is essential:

  • Mass spectrometry for unbiased identification of all components

  • Western blotting to confirm the presence of known subunits

  • Native gel electrophoresis to assess complex integrity and homogeneity
    These approaches can distinguish between NuRD and ChAHP complexes and identify potential novel associations .

• Structural Characterization: Understanding the architecture of CHD4-containing complexes:

  • Cryo-EM can resolve structures of intact complexes

  • Crosslinking mass spectrometry can map protein-protein interfaces

  • Integrative structural modeling can combine data from multiple sources
    The 3.1Å structure of CHD4-nucleosome provides a foundation for building more complete complex models .

• Functional Reconstitution: Bottom-up approaches to understand complex assembly and function:

  • Recombinant expression of individual components

  • Stepwise assembly of subcomplexes

  • Functional assays comparing partial and complete complexes
    This approach can identify the minimal functional units and the contribution of each component .

• In Vivo Complex Analysis: Techniques to study native complexes in cellular contexts:

  • Proximity labeling (BioID, APEX) to identify context-specific interactions

  • Live-cell imaging of fluorescently tagged components to assess dynamics

  • Rapid immunoprecipitation for time-resolved interaction studies
    These approaches capture the dynamic nature of complex assembly and function .

• Differential Complex Analysis: Methods to distinguish different CHD4-containing complexes:

  • Complex-specific antibodies targeting unique components

  • Density gradient separation of different complexes

  • Functional assays that distinguish complex-specific activities
    This is particularly important given CHD4's participation in both NuRD and ChAHP complexes .

These complementary approaches provide a comprehensive understanding of how CHD4 functions within its native complexes, which is essential for interpreting its diverse roles in gene regulation and chromatin organization .

How should researchers interpret contradictory findings regarding CHD4 function?

The scientific literature contains seemingly contradictory findings regarding CHD4 function, requiring careful interpretation strategies:

• Context Dependency Recognition: Many apparent contradictions regarding CHD4 function stem from biological context differences. For example, while CHD4 primarily functions in gene repression, some studies report roles in gene activation. These seemingly contradictory findings can often be reconciled by recognizing that CHD4's function depends on:

  • Cell type (stem cells vs. differentiated cells)

  • Developmental stage (embryonic vs. adult tissues)

  • Genomic context (promoters vs. enhancers)

  • Complex composition (variations in NuRD subunits)
    The tissue-specific effects of the CHD4 M202I mutation exemplify this context dependency .

• Methodological Considerations: Experimental approaches significantly influence results:

  • Acute vs. chronic perturbation (transient knockdown vs. genetic knockout)

  • Global vs. locus-specific analysis (genome-wide profiling vs. reporter assays)

  • In vitro vs. in vivo studies (purified components vs. cellular contexts)
    Researchers should explicitly address these differences when comparing studies .

• Multifunctional Nature Appreciation: CHD4 performs multiple roles that may seem contradictory:

  • It can both establish and maintain chromatin states

  • It functions in both global chromatin organization and locus-specific regulation

  • It participates in both developmental processes and adult homeostasis
    These diverse functions require nuanced interpretation rather than forcing data into simplistic models .

• Integrated Data Analysis: Rather than focusing on individual studies, researchers should:

  • Conduct systematic literature reviews with explicit attention to experimental conditions

  • Perform meta-analyses where appropriate

  • Integrate multiple data types to develop comprehensive models
    This approach can reveal patterns across seemingly conflicting studies .

• Direct Experimental Comparison: When faced with contradictory findings, researchers should consider:

  • Side-by-side testing under identical conditions

  • Introducing controlled variables to identify sources of discrepancy

  • Using multiple complementary assays to measure the same phenomenon
    This approach can identify the specific factors driving apparent contradictions .

By applying these strategies, researchers can develop more nuanced models of CHD4 function that accommodate apparently conflicting data and better reflect the complexity of chromatin regulation across different biological contexts .

What approaches help integrate structural data with functional genomic findings?

Integrating structural data with functional genomic findings requires methodologies that bridge molecular-level mechanisms with genome-wide patterns:

• Structure-Function Mapping: High-resolution structural data of CHD4-nucleosome complexes (3.1Å) provides atomic details that can be connected to genomic functions:

  • Map functional domains identified in structures to genomic binding patterns

  • Correlate DNA distortion mechanisms observed at SHL +2 with nucleosome positioning changes

  • Link ATP binding and hydrolysis states to temporal aspects of chromatin remodeling
    This mapping connects the biophysical mechanism with genomic consequences .

• Mutation Analysis Pipeline: Disease mutations provide valuable connection points:

  • Structurally map mutations (as done for Sifrim-Hitz-Weiss syndrome mutations)

  • Predict functional consequences based on structural context

  • Test predictions through genomic approaches (ChIP-seq, ATAC-seq)

  • Correlate altered binding or remodeling with expression changes
    The CHD4 M202I/M195I mutation demonstrates how structural insights can inform understanding of genomic consequences .

• Multi-scale Modeling: Computational approaches can bridge scales:

  • Molecular dynamics simulations based on cryo-EM structures

  • Integration of structural details into models of nucleosome arrays

  • Prediction of genomic regions susceptible to CHD4 remodeling based on structural constraints
    These models connect atomic-level interactions to chromosome-scale organization .

• Domain-specific Genomics: Structure-guided design of domain mutations:

  • Create variants that specifically disrupt DNA binding, ATP hydrolysis, or protein interactions

  • Assess genome-wide consequences through ChIP-seq, ATAC-seq, and RNA-seq

  • Correlate domain-specific functions with genomic outcomes
    This approach dissects the contribution of individual structural elements to genomic function .

• Mechanistic Validation: Test structural mechanisms through genomic approaches:

  • Design nucleosomes with modifications predicted to alter CHD4 binding

  • Assess genomic regions with varying DNA sequences at SHL +2

  • Correlate ATP hydrolysis rates with remodeling efficiency at different loci
    These approaches validate structural insights in genomic contexts .

By implementing these integrative approaches, researchers can develop comprehensive models that connect the atomic details of CHD4-nucleosome interactions to genome-wide chromatin organization and gene regulation patterns .

What are the current frontiers in CHD4 research?

Research on the human CHD4 chromatin remodeler continues to advance rapidly, with several exciting frontiers emerging at the intersection of structural biology, disease genetics, and functional genomics. The high-resolution cryo-EM structure of the CHD4-nucleosome complex at 3.1Å has transformed our understanding of the molecular mechanisms underlying chromatin remodeling, particularly supporting the "twist defect" model. This structural foundation now enables precise mapping of disease-associated mutations and investigation of their effects on CHD4 function .

The connection between CHD4 mutations and specific human diseases represents another critical frontier. The discovery that a single missense mutation (M202I in humans, M195I in mice) can cause ventricular non-compaction highlights the challenge of understanding how mutations in a broadly expressed chromatin remodeler can lead to tissue-specific phenotypes. This paradox points to context-dependent roles of CHD4 that require further investigation to fully understand the pathogenesis of CHD4-associated disorders .

The integration of structural insights with genomic functions remains an important frontier. While we now have detailed knowledge of how CHD4 engages individual nucleosomes, connecting this to genome-wide chromatin organization and gene regulation requires further research. Similarly, understanding how CHD4 functions within different complexes (NuRD versus ChAHP) and how complex composition varies across cellular contexts represents an ongoing challenge .