Recombinant Danio rerio Chromodomain-helicase-DNA-binding protein 1-like (chd1l), partial

What is CHD1L and what are its key structural domains?

CHD1L (Chromodomain helicase/ATPase DNA binding protein 1-like gene), also known as ALC1 (amplified in liver cancer 1) in humans, is a member of the SNF2-like subfamily of the SNF2 family of proteins . The full-length CHD1L protein contains four conserved domains:

• SNF2_N domain: Involved in various cellular processes including DNA repair, chromatin unwinding, and transcription regulation

• Helicase superfamily domain (HELICc): Contains the DEAD-like helicase superfamily ATP-binding region that participates in ATP-dependent DNA or RNA unwinding

• Selenoprotein S (SelS) region: Found in many cell types and tissues

• Macro domain: Recognizes poly ADP-ribose (PAR) which is crucial for its chromatin remodeling function

Unlike CHD1, CHD1L contains a macro domain that recognizes PAR rather than a chromo domain that recognizes methylated histone tails . This structural organization enables CHD1L to utilize energy from ATP hydrolysis to remodel chromatin, particularly at sites of DNA damage.

How does CHD1L function in DNA repair mechanisms?

CHD1L plays a critical role in DNA repair through its PAR-dependent chromatin remodeling activity. The process follows these key steps:

• Single-stranded DNA breaks (SSB) activate PARP1/2, which produces PAR at damage sites

• CHD1L is recruited to these sites through its macro domain binding to PAR

• Once recruited, CHD1L uses its ATPase activity to catalyze nucleosome sliding, leading to chromatin relaxation at damage sites

• This chromatin relaxation facilitates access for DNA repair factors involved in Base Excision Repair (BER) and Nucleotide Excision Repair (NER)

CHD1L specifically mediates the efficient handover between PARP1/2, DNA glycosylases, and APEX1 downstream of lesion excision . Studies have shown that loss of CHD1L confers sensitivity to methyl-methanesulfonate, PARP inhibitors, and formyl-dU, and is synthetic lethal with homologous recombination deficiency (HRD) . This indicates CHD1L's essential role in maintaining genomic integrity through efficient DNA repair mechanisms.

What phenotypes are associated with chd1l manipulation in zebrafish models?

Zebrafish models have revealed dose-dependent phenotypic effects of chd1l manipulation:

• Overexpression effects: Overexpression of CHD1L in zebrafish embryos leads to macrocephaly (enlarged head) and increased larval body length

• Deletion effects: Conversely, chd1l deletion results in microcephaly (smaller head) and decreased body length

These mirrored phenotypes have been consistently observed across zebrafish and mouse embryo models, confirming the conserved developmental function of CHD1L across vertebrates . The bidirectional, dose-dependent phenotypic effects highlight CHD1L's critical role in regulating head size and body growth during development, which correlates with the neuroanatomical and growth abnormalities observed in human 1q21.1 copy number variant carriers.

What experimental conditions should be considered when working with recombinant Danio rerio CHD1L?

When working with recombinant Danio rerio CHD1L protein:

• Storage considerations: Store working aliquots at 4°C and avoid repeated freeze-thaw cycles to maintain protein integrity and activity

• ATP requirements: Ensure ATP availability in functional assays as CHD1L is an ATP-dependent chromatin remodeler

• PAR dependency: Consider that CHD1L's helicase activity is strongly stimulated upon poly(ADP-ribose) binding, which may necessitate the inclusion of PARP1 and NAD+ in activity assays

• Buffer composition: Use buffers that maintain protein stability while allowing for optimal enzymatic activity, typically including components that stabilize protein structure without interfering with the ATPase domain

• Partial protein considerations: When working with partial recombinant protein, verify which domains are included and consider how this might affect functional assays compared to the full-length protein

How can one design experiments to investigate CHD1L's role in neuronal differentiation?

Designing experiments to investigate CHD1L's role in neuronal differentiation requires a multifaceted approach:

• CRISPR-Cas9 gene editing:

  • Design guide RNAs targeting chd1l exons, similar to the approach for human iPSCs using guides like 5'-TCATACTGAGGGCCGAGCCGAGG-3'

  • Create knockout, knockdown, and overexpression models in zebrafish using established protocols

  • Validate edits using PCR screening and Western blot analysis

• Neuronal differentiation assays:

  • Employ neural progenitor cell (NPC) differentiation protocols using wild-type and chd1l-modified cell lines

  • Monitor expression of neuronal markers by qPCR and immunostaining across differentiation timepoints

  • Assess morphological changes, neurite outgrowth, and synaptogenesis using high-content imaging

• Transcriptomic analysis:

  • Perform RNA-seq at different stages of neuronal differentiation comparing control and chd1l-modified samples

  • Analyze differentially expressed genes (DEGs) using packages such as DESeq2, adjusting for batch effects

  • Focus on genes involved in neuronal differentiation and synaptogenesis

  • Use STRING analysis (string-db.org) and DECIPHER database (decipher.sanger.ac.uk/ddd/ddgenes) for gene annotation and pathway analysis

• Chromatin accessibility studies:

  • Implement ATAC-seq to assess chromatin accessibility changes

  • Analyze binding sites for pioneer transcription factors like SOX2 and OTX2 that interact with CHD1L

  • Use tools like IGV v2.17.4 to generate sashimi plots for visualizing splicing events and chromatin states

• In vivo developmental assessment:

  • Monitor telencephalon development in chd1l-modified zebrafish using lineage tracing and live imaging

  • Quantify head size and brain region measurements across developmental stages

  • Compare neurobehavioral outcomes in control versus chd1l-modified zebrafish

What methods can be used to analyze CHD1L's chromatin remodeling activity in experimental settings?

To analyze CHD1L's chromatin remodeling activity, researchers can employ these methodological approaches:

• Nucleosome sliding assays:

  • Reconstitute positioned nucleosomes using purified histones and DNA containing specific positioning sequences

  • Incubate with recombinant CHD1L in the presence of ATP

  • Analyze nucleosome repositioning using native PAGE or restriction enzyme accessibility assays

  • Include controls with ATP-binding deficient CHD1L mutants

• PARP1-dependent remodeling assays:

  • Set up in vitro systems containing CHD1L, PARP1, NAD+, and positioned nucleosomes

  • Induce DNA damage using UV or chemical agents to activate PARP1

  • Monitor chromatin relaxation and nucleosome repositioning in response to PAR synthesis

  • Measure ATPase activity in parallel to correlate with remodeling efficiency

• Real-time fluorescence-based assays:

  • Employ FRET-labeled nucleosomes to monitor conformational changes during remodeling

  • Track remodeling kinetics in the presence and absence of PAR

  • Analyze the effect of PAR chain length and branching on CHD1L activation

• Chromatin immunoprecipitation sequencing (ChIP-seq):

  • Map CHD1L binding sites genome-wide in control and DNA damage conditions

  • Correlate CHD1L occupancy with chromatin accessibility changes and repair factor recruitment

  • Analyze the relationship between CHD1L binding and transcriptional outcomes

• Super-resolution microscopy:

  • Visualize CHD1L recruitment to DNA damage sites in live cells

  • Track the kinetics of chromatin relaxation following damage using fluorescently labeled histones

  • Correlate CHD1L activity with repair factor assembly at damage sites

How does CHD1L interact with transcriptional networks during zebrafish neurodevelopment?

CHD1L's interaction with transcriptional networks during zebrafish neurodevelopment can be characterized through these experimental approaches:

• Integrated omics analysis:

  • Combine RNA-seq, ATAC-seq, and ChIP-seq data from chd1l-modified and control zebrafish embryos at key developmental stages

  • Identify transcription factors whose binding sites show altered accessibility in chd1l mutants

  • Use tools like LISA.cistrome for transcription regulator prediction based on differential gene expression patterns

• Transcription factor binding studies:

  • CHD1L facilitates chromatin accessibility for pioneer transcription factors including SOX2 and OTX2 during forebrain regionalization

  • Simultaneously, it compacts chromatin through interaction with the repressor NuRD complex

  • This dual activity creates a precise balance of activation and repression required for proper telencephalon development

• Gene Ontology enrichment analysis:

  • Apply PANTHER tool (geneontology.org) to identify biological processes enriched among CHD1L-regulated genes

  • Focus on neurodevelopmental pathways, particularly those involved in telencephalon development and neuronal differentiation

  • Visualize enrichment results using R package ggplot2 for clearer interpretation

• Key target genes:

  Gene Category	Role in Neurodevelopment	CHD1L Regulatory Effect
  Autism-associated genes	Synaptogenesis, neuronal connectivity	Direct regulation of chromatin accessibility
  Telencephalon development genes	Forebrain regionalization	Enhanced accessibility for SOX2/OTX2 binding
  Neuronal differentiation markers	Cell fate determination	Balanced activation/repression through NuRD interaction

• Temporal dynamics:

  • Monitor stage-specific effects of chd1l manipulation through developmental time series experiments

  • Track critical windows when CHD1L activity is essential for normal neuronal differentiation

  • Correlate molecular changes with the emergence of structural phenotypes like head size alterations

What is the relationship between CHD1L and poly(ADP-ribose) in the context of DNA repair mechanisms?

The relationship between CHD1L and poly(ADP-ribose) (PAR) in DNA repair involves several key molecular interactions:

• Activation mechanism:

  • CHD1L contains an auto-inhibitory mechanism wherein its macrodomain interacts with its bilobate ATPase module

  • PAR binding to the macrodomain triggers a conformational change that relieves this auto-inhibition

  • This activation enables CHD1L's ATP-dependent chromatin remodeling activity

• Recruitment dynamics:

  • DNA damage (particularly single-strand breaks) activates PARP1/2, which synthesizes PAR chains at damage sites

  • CHD1L is recruited to these sites through specific binding of its macrodomain to PAR

  • The temporal dynamics of this recruitment are rapid, making CHD1L one of the early responders to DNA damage

• Functional consequences:

  • Once activated, CHD1L promotes chromatin relaxation at damage sites, creating accessibility for repair factors

  • CHD1L-dependent nucleosome remodeling facilitates the handover between PARP1/2, DNA glycosylases, and APEX1

  • This process is particularly important in Base Excision Repair (BER) and Nucleotide Excision Repair (NER)

• Experimental verification:

  • The interaction can be verified through PAR-binding assays using recombinant CHD1L macrodomain

  • Mutations in the macrodomain that disrupt PAR binding (similar to those identified in CAKUT patients) demonstrate the importance of this interaction

  • The three variants identified in CAKUT patients (Gly700Arg, Ile765Met, and Ile827Val) all showed decreased interaction with PARP1 compared to wild-type CHD1L

How can researchers effectively design and implement CHD1L knockout or knockdown studies in zebrafish?

Effective design and implementation of CHD1L knockout or knockdown studies in zebrafish requires careful consideration of these methodological aspects:

• CRISPR-Cas9 knockout strategy:

  • Design multiple guide RNAs targeting early exons of the chd1l gene

  • Test guide RNA efficiency using in vitro cleavage assays

  • Inject Cas9 protein with validated guide RNAs into one-cell-stage zebrafish embryos

  • Screen F0 embryos for mutations using PCR and sequencing

  • Establish stable knockout lines through selective breeding of F0 founders

• Morpholino-based knockdown approach:

  • Design splice-blocking or translation-blocking morpholinos specific to zebrafish chd1l

  • Determine optimal morpholino concentration through dose-response testing

  • Include appropriate controls (standard control morpholino and rescue experiments)

  • Validate knockdown efficiency through RT-PCR (for splice-blocking) or Western blot (for translation-blocking)

• Phenotypic analysis pipeline:

  • Head size measurement: Use standardized imaging and analysis protocols to quantify head circumference at defined developmental stages

  • Body length assessment: Implement consistent methodology for measuring larval body length

  • Brain development: Apply neuroanatomical staining and imaging to assess specific brain regions, particularly the telencephalon

  • Behavioral testing: Conduct standardized assays for locomotor activity, response to stimuli, and learning behaviors

• Molecular characterization:

  • Transcriptome analysis: Perform RNA-seq on whole embryos or isolated brain tissue from control and chd1l mutants

  • Chromatin accessibility: Implement ATAC-seq to identify regions with altered chromatin structure

  • Target gene validation: Use in situ hybridization and immunohistochemistry to confirm changes in expression of key neuronal and developmental genes

• Rescue experiments:

  • Generate mRNA encoding wild-type human or zebrafish CHD1L

  • Co-inject with morpholinos or introduce into knockout backgrounds

  • Test domain-specific mutants (e.g., macro domain or ATPase domain mutants) to dissect functional requirements

  • Compare rescue efficiency with different CHD1L dosages to establish dose-dependency

How can CHD1L studies in zebrafish inform our understanding of human 1q21.1 copy number variation disorders?

Zebrafish CHD1L studies provide valuable insights into human 1q21.1 copy number variation (CNV) disorders through translational research approaches:

• Phenotypic correlation:

  • The macrocephaly observed in CHD1L-overexpressing zebrafish mirrors the increased head circumference seen in humans with 1q21.1 duplications

  • Conversely, chd1l deletion in zebrafish causes microcephaly, similar to humans with 1q21.1 deletions

  • These mirrored phenotypes establish CHD1L as a critical dosage-sensitive gene within the 1q21.1 region

• Transcriptional network analysis:

  • Transcriptomic studies in CHD1L-modified zebrafish and human cell models reveal disruption of genes involved in neuronal differentiation and synaptogenesis

  • Many affected genes overlap with known autism-associated genes, providing a molecular link between 1q21.1 CNVs and neurodevelopmental disorders

  • Gene Ontology analysis of these datasets highlights pathways relevant to the clinical manifestations of 1q21.1 CNV syndromes

• Developmental mechanisms:

  • Zebrafish studies demonstrate that CHD1L regulates telencephalon development by modulating chromatin accessibility for key transcription factors like SOX2 and OTX2

  • This mechanism explains how CHD1L dosage affects brain size and potentially contributes to cognitive phenotypes in humans with 1q21.1 CNVs

  • The NuRD complex interaction suggests a balance between activation and repression that, when disrupted, leads to abnormal brain development

• Therapeutic implications:

  • Understanding the downstream effects of CHD1L dosage imbalance identifies potential therapeutic targets

  • Zebrafish models allow for high-throughput screening of compounds that might mitigate the effects of CHD1L dosage abnormalities

  • Chromatin-modifying drugs could potentially normalize gene expression patterns disrupted by CHD1L imbalance

What are the methodological challenges in studying the interactions between CHD1L and PARP1 during DNA repair?

Studying CHD1L-PARP1 interactions during DNA repair presents several methodological challenges that require sophisticated experimental approaches:

• Temporal resolution challenges:

  • The interaction occurs rapidly after DNA damage, requiring high temporal resolution techniques

  • Solutions include:

    • Synchronized damage induction using micro-irradiation coupled with live-cell imaging

    • Time-course sampling with rapid fixation to capture transient interactions

    • Development of biosensors that report CHD1L-PARP1 interactions in real-time

• Spatial organization visualization:

  • Understanding the nanoscale organization of CHD1L and PARP1 at damage sites is challenging

  • Approaches to address this:

    • Super-resolution microscopy techniques like STORM or PALM

    • Proximity ligation assays to detect protein-protein interactions with spatial context

    • Correlative light and electron microscopy to combine functional and structural information

• Biochemical reconstitution complexities:

  • Recapitulating the correct stoichiometry and activity of components in vitro is difficult

  • Strategies include:

    • Stepwise assembly of defined nucleosome substrates with purified proteins

    • Microfluidic approaches to control reaction conditions precisely

    • Use of nuclear extracts with defined genetic backgrounds (e.g., PARP1-null) to study dependency relationships

• PAR chain heterogeneity:

  • PAR chains vary in length and branching, potentially affecting CHD1L recruitment and activation

  • Methods to address this:

    • Mass spectrometry to characterize PAR chain composition

    • Synthetic PAR chains with defined structures to test specificity

    • PAR-binding domain mutations to probe structural requirements for interaction

• Genetic redundancy:

  • Functional overlap between CHD1L and other chromatin remodelers may mask phenotypes

  • Approaches to overcome this:

    • Combined depletion of multiple remodelers

    • Acute protein degradation systems like auxin-inducible degrons

    • Domain-specific mutations that affect specific functions while preserving others

How does CHD1L influence gene expression patterns during zebrafish embryogenesis?

CHD1L's influence on gene expression patterns during zebrafish embryogenesis encompasses multiple regulatory mechanisms:

• Stage-specific transcriptional regulation:

  • CHD1L modulates chromatin accessibility at developmental gene promoters and enhancers

  • This creates permissive or restrictive chromatin states for stage-specific transcription factors

  • Experimental approaches should include:

    • Time-series RNA-seq and ATAC-seq throughout embryogenesis in wild-type and chd1l-modified embryos

    • In situ hybridization to spatially map expression changes of key developmental genes

    • Single-cell transcriptomics to identify cell type-specific effects

• Developmental signaling pathway integration:

  • CHD1L likely coordinates responses to morphogenetic signals that drive embryonic patterning

  • Key pathways to investigate include:

    • Wnt signaling: Critical for anterior-posterior axis formation and telencephalon development

    • Sonic hedgehog (Shh): Important for ventral forebrain specification

    • Retinoic acid signaling: Essential for hindbrain patterning

  • Experiments should examine how CHD1L occupancy changes in response to pathway activation/inhibition

• Epigenetic landscape shaping:

  • CHD1L participates in establishing developmental epigenetic patterns through:

    • Facilitating pioneer transcription factor binding (SOX2, OTX2)

    • Interacting with the NuRD complex to modulate repressive chromatin

    • Potentially influencing DNA methylation patterns at regulatory elements

  • CUT&RUN or CUT&Tag approaches can map CHD1L occupancy alongside histone modifications

• Lineage-specific effects:

  Developmental Lineage	CHD1L Function	Experimental Approach
  Neural ectoderm	Promotes telencephalon development	Neural-specific chd1l knockout
  Neural crest	Influences migration and differentiation	Lineage tracing in chd1l mutants
  Mesoderm	Affects somitogenesis and growth	Time-lapse imaging of somite formation
  Endoderm	Potential role in organ development	Organ-specific marker analysis

• Differential sensitivity periods:

  • CHD1L function may be particularly critical during specific developmental windows

  • Conditional knockout or chemical inhibition approaches at different stages can identify these sensitive periods

  • Correlating molecular changes with morphological outcomes will reveal the developmental consequences of CHD1L dysregulation