Recombinant Neurospora crassa Histone acetyltransferase esa-1 (esa-1), partial

What is ESA-1 and what is its function in Neurospora crassa?

ESA-1 (Essential SAS2-related Acetyltransferase) is a histone acetyltransferase (HAT) in Neurospora crassa that functions as the catalytic subunit of the NuA4 complex. It belongs to the MYST family of acetyltransferases, which includes members implicated in transcriptional regulation, silencing, and various cellular processes across species. In N. crassa, ESA-1 is encoded by the gene NCU05218 and is essential for survival, similar to its yeast ortholog .

ESA-1 catalyzes the transfer of acetyl groups to specific lysine residues on histone proteins, particularly H4, H3, and H2A, thereby modifying chromatin structure and potentially influencing gene expression . Recent research has demonstrated that ESA-1 plays a crucial role in the regulation of the circadian clock in N. crassa, as downregulation of esa-1 severely impairs circadian rhythmicity .

What is the substrate specificity of recombinant N. crassa ESA-1?

Microsequence analyses have revealed that ESA-1 acetylates specific lysine residues in these histones. In H4, ESA-1 targets lysines 5, 8, 12, and 16. In H2A, it acetylates lysines 5, 9, 13, and 15 . This pattern of site utilization differs from that of other HATs like Gcn5p, confirming that ESA-1 has a distinct functional profile in chromatin modification .

How is ESA-1 expression regulated in Neurospora crassa?

The expression of ESA-1 in N. crassa appears to be tightly regulated, consistent with its essential role in cellular function. While specific mechanisms controlling ESA-1 expression are not comprehensively detailed in the available literature, research indicates that ESA-1 is dynamically expressed and regulated .

The regulation of ESA-1 activity likely involves both transcriptional and post-translational mechanisms. Like other components of the NuA4 complex, ESA-1 expression may be responsive to cellular conditions and developmental stages. Its expression pattern appears to be particularly important for maintaining proper circadian rhythmicity, suggesting potential temporal regulation .

What experimental systems are used to study ESA-1 function in Neurospora crassa?

Several experimental approaches have been employed to study ESA-1 function in N. crassa:

• Knockout/knockdown systems: Since ESA-1 is essential in N. crassa, researchers have utilized inducible RNA interference (RNAi) systems to study its function. Specifically, hairpin RNA constructs targeting esa-1 driven by the quinic acid (QA)-inducible qa-2 promoter have been transformed into N. crassa strains to create regulatable knockdown systems .

• Dominant-negative mutations: Researchers have created dominant-negative mutants of esa-1 (esa-1^E395Q) to study the effects of reduced ESA-1 activity on cellular processes, particularly circadian rhythms .

• Luciferase reporter assays: To monitor circadian rhythms in ESA-1-modified strains, researchers have utilized luciferase reporters driven by the frq promoter or C-box .

• Biochemical assays: In vitro histone acetyltransferase activity assays using recombinant ESA-1 and various histone substrates have been employed to characterize its enzymatic properties and substrate preferences .

How does ESA-1 contribute to circadian clock regulation in Neurospora crassa?

ESA-1 plays a crucial role in maintaining normal circadian rhythmicity in N. crassa. Experimental evidence demonstrates that downregulation of esa-1 using an inducible RNAi system severely impairs circadian clock function . When a hairpin RNA construct targeting esa-1 is induced with quinic acid (QA), luciferase signals driven by the frq C-box show significantly reduced robustness compared to uninduced controls .

Furthermore, expression of a dominant-negative mutant of esa-1 (esa-1^E395Q) under the control of the QA-inducible promoter results in a period extension of approximately 2 hours compared to uninduced controls . This suggests that ESA-1-mediated histone acetylation likely influences the transcriptional dynamics of clock-controlled genes.

The mechanism by which ESA-1 regulates the circadian clock may involve acetylation of histones at promoters of clock genes, potentially facilitating the binding of transcription factors or the release of RNA polymerase II for transcriptional elongation. This is consistent with observations in mammals, where the orthologous Tip60 complex acetylates BMAL1, a core clock component, to promote circadian transcription .

What are the key methodological considerations when expressing and purifying recombinant N. crassa ESA-1?

Expressing and purifying functional recombinant N. crassa ESA-1 requires careful consideration of several factors:

How does the substrate specificity of N. crassa ESA-1 compare to orthologs in other species?

The substrate specificity of N. crassa ESA-1 shows similarities and differences compared to its orthologs in other species:

N. crassa ESA-1 shows substrate specificity that is most similar to that of human Tip60, particularly in its ability to acetylate histone H4 . This is consistent with their evolutionary relationship within the MYST family of histone acetyltransferases.

Unlike some other HATs like Gcn5p, which preferentially acetylates H3 at Lys-14, ESA-1 shows a broader acetylation pattern across multiple histones . This suggests that ESA-1 may have evolved to regulate chromatin structure through more widespread acetylation of nucleosomes rather than through highly targeted modification of specific residues.

What are the strategies to study ESA-1 function given its essential nature in Neurospora?

Studying ESA-1 function in N. crassa presents challenges due to its essential nature, as homokaryotic deletion mutants cannot be obtained . Researchers have developed several strategies to overcome this limitation:

• Conditional knockdown systems: The use of inducible RNAi constructs targeting esa-1 allows for controlled reduction of ESA-1 levels. The qa-2 promoter system, inducible by quinic acid, has been effectively employed for this purpose . This approach permits the study of ESA-1 function without completely eliminating the protein, which would be lethal.

• Dominant-negative mutations: Introduction of catalytically inactive versions of ESA-1, such as the esa-1^E395Q mutant, provides another approach to studying ESA-1 function. When expressed, these mutants can interfere with endogenous ESA-1 activity, creating a hypomorphic phenotype rather than a complete loss of function .

• Heterokaryotic mutants: Although homokaryotic deletion of esa-1 is lethal, it may be possible to maintain heterokaryotic mutants where some nuclei contain the deletion while others retain the wild-type gene. These heterokaryons could potentially be used to study partial loss of ESA-1 function.

• Temperature-sensitive alleles: Development of temperature-sensitive esa-1 mutants would allow for conditional inactivation of the protein by temperature shifts, providing temporal control over ESA-1 function.

• Biochemical complex analysis: Studying the composition and function of the NuA4 complex as a whole can provide insights into ESA-1 function. Affinity purification of tagged ESA-1 or other NuA4 components followed by mass spectrometry can identify interaction partners and potentially reveal functional domains .

How do mutations in ESA-1 affect its enzymatic activity and cellular functions?

Mutations in ESA-1 can significantly impact its enzymatic activity and subsequent cellular functions. The E395Q mutation in ESA-1 has been particularly well-studied and functions as a dominant-negative form of the enzyme . When expressed in N. crassa, this mutant causes a period extension of approximately 2 hours in the circadian rhythm .

The E395Q mutation likely affects the catalytic center of ESA-1, reducing its ability to acetylate histones without completely eliminating its capacity to interact with other components of the NuA4 complex or bind to chromatin. This results in a partially functional protein that can interfere with normal ESA-1 function, possibly by competing for binding sites or complex formation.

Other potential mutations in ESA-1 might affect:

• Substrate recognition: Mutations in regions involved in histone binding could alter substrate specificity or affinity, leading to changes in the pattern of histone acetylation.

• Complex formation: ESA-1 functions as part of the NuA4 complex, and mutations affecting protein-protein interactions could disrupt complex assembly or stability.

• Chromatin targeting: Mutations might alter the recruitment of ESA-1/NuA4 to specific genomic loci, affecting gene-specific regulation.

• Enzymatic activity: Beyond the E395Q mutation, other alterations in the catalytic domain could result in partial or complete loss of HAT activity, or potentially even altered specificity.

What are the optimal conditions for in vitro histone acetyltransferase assays with recombinant ESA-1?

Optimal conditions for in vitro histone acetyltransferase assays with recombinant ESA-1 include:

• Buffer composition: Typically, HAT assays are performed in buffers containing:

  • Tris-HCl (50 mM, pH 8.0) or HEPES (50 mM, pH 7.5-8.0)

  • NaCl (50-100 mM)

  • EDTA (1 mM)

  • DTT or β-mercaptoethanol (1-5 mM) to maintain reducing conditions

  • Glycerol (5-10%) for protein stability

  • Protease inhibitors to prevent degradation

• Reaction conditions: Based on published protocols:

  • Temperature: 30°C is commonly used for ESA-1 assays

  • Incubation time: 20-30 minutes is typically sufficient for detectable activity

  • Enzyme concentration: ~150 ng of purified recombinant ESA-1 per reaction

  • Substrate amount: 5 μg of each histone per reaction

  • Acetyl-CoA: 5 μCi of [³H]acetyl-CoA for radiometric assays

• Substrate preparation: For optimal results, histones should be purified and, if possible, deacetylated prior to the assay to avoid background acetylation. Tetrahymena histones are often preferred for sequence analysis as their H4 has an unblocked amino terminus .

• Detection methods:

  • Radiometric assays using [³H]acetyl-CoA followed by SDS-PAGE and fluorography

  • Microsequencing of acetylated histones to determine specific lysine residues modified

  • Western blotting with acetyl-lysine-specific antibodies

  • Mass spectrometry for comprehensive site identification

• Controls: Proper controls should include reactions without enzyme, without histone substrate, and with known HATs (e.g., Gcn5p, Hat1p) for comparison .

How can researchers generate conditional ESA-1 mutants for functional studies?

Researchers can employ several strategies to generate conditional ESA-1 mutants for functional studies:

• Inducible RNAi system: The qa-2 promoter-driven hairpin RNA approach has been successfully used to create conditionally silenced esa-1 strains . This system:

  • Utilizes a hairpin construct specific to esa-1 sequence

  • Is controlled by the quinic acid (QA)-inducible qa-2 promoter

  • Can be targeted to specific loci (e.g., csr) for stable integration

  • Allows for controlled knockdown by adding QA to the growth medium

• Dominant-negative mutants: The esa-1^E395Q mutation creates a dominant-negative effect when expressed . Researchers can:

  • Place this mutant under an inducible promoter (e.g., qa-2)

  • Integrate it at a neutral locus (e.g., his-3)

  • Induce expression with QA to create a conditional loss-of-function phenotype

  • Monitor effects using appropriate reporter systems (e.g., luciferase)

• Degron systems: Alternatively, researchers could employ degron-based approaches:

  • Fusion of auxin-inducible degron (AID) to ESA-1

  • Expression of the TIR1 F-box protein

  • Addition of auxin to trigger rapid degradation of the fusion protein

• Temperature-sensitive alleles: Through random or directed mutagenesis, temperature-sensitive alleles of esa-1 could be generated:

  • Screen for mutations that allow growth at permissive temperature (e.g., 25°C)

  • But cause loss of function at restrictive temperature (e.g., 37°C)

  • These provide temporal control over ESA-1 activity

• Tetracycline-regulated systems: Alternative inducible systems such as the Tet-ON/OFF systems could be adapted for N. crassa to regulate ESA-1 expression.

What approaches can be used to study the genome-wide occupancy and activity of ESA-1?

Several approaches can be employed to study the genome-wide occupancy and activity of ESA-1:

• Chromatin Immunoprecipitation sequencing (ChIP-seq):

  • Requires specific antibodies against ESA-1 or epitope tags (e.g., V5, as used in previous studies )

  • Can reveal genomic binding sites of ESA-1 across the N. crassa genome

  • Can be conducted under different conditions or time points to study dynamic binding

• CUT&RUN or CUT&Tag:

  • More sensitive alternatives to ChIP-seq

  • Utilize targeted nuclease activity to release protein-bound DNA fragments

  • Particularly useful if ESA-1 antibodies have limited efficiency in ChIP

• Histone acetylation profiling:

  • ChIP-seq using antibodies against specific acetylated lysine residues (e.g., H4K5ac, H4K8ac, H4K12ac, H4K16ac)

  • Compare acetylation patterns in wild-type and ESA-1-depleted conditions

  • Identify genomic regions dependent on ESA-1 for proper histone acetylation

• RNA-seq combined with ESA-1 manipulation:

  • Transcriptome analysis after ESA-1 knockdown or dominant-negative expression

  • Identifies genes whose expression depends on ESA-1 activity

  • Can be performed at different time points to study temporal dynamics

• Nascent RNA sequencing:

  • Techniques like NET-seq or PRO-seq to measure active transcription

  • Can reveal immediate effects of ESA-1 on transcriptional elongation

  • Particularly relevant given ESA-1's potential role in transcription

• ChIP-mass spectrometry:

  • Combines chromatin immunoprecipitation with mass spectrometry

  • Identifies proteins associated with ESA-1 at chromatin

  • Helps understand the composition of ESA-1/NuA4 complexes at specific genomic loci

• Circadian time-course experiments:

  • Any of the above techniques performed across a circadian time course

  • Reveals temporal dynamics of ESA-1 occupancy and activity

  • Particularly relevant given ESA-1's role in circadian regulation

How does ESA-1 integrate with other chromatin modifiers in Neurospora crassa?

Understanding the integration of ESA-1 with other chromatin modifiers presents a significant research challenge. ESA-1, as the catalytic subunit of the NuA4 complex, likely functions within a broader network of chromatin-modifying enzymes to regulate gene expression and other chromatin-dependent processes.

Potential interactions and functional relationships to investigate include:

• Coordination with other HATs: N. crassa contains multiple HATs including Gcn5p homologs. Research should address how these different HATs coordinate or complement each other's functions. For instance, while ESA-1 preferentially acetylates H4 and H2A, Gcn5p shows preference for H3 . These distinct specificities suggest potential division of labor in regulating different aspects of chromatin structure.

• Interplay with histone deacetylases (HDACs): The dynamic balance between acetylation and deacetylation is crucial for proper gene regulation. Investigating how ESA-1 activity is counterbalanced by specific HDACs would provide insights into the dynamic regulation of histone acetylation.

• Relationship with DNA methylation machinery: In N. crassa, DNA methylation is primarily associated with relics of RIP (Repeat-Induced Point mutation) . Research could explore whether ESA-1-mediated histone acetylation influences DNA methylation patterns or vice versa, particularly in regions affected by RIP.

• Interactions with chromatin remodeling complexes: Histone acetylation often facilitates the action of ATP-dependent chromatin remodelers. Understanding how ESA-1 activity coordinates with chromatin remodeling would provide insights into higher-order chromatin dynamics.

• Relationship with the circadian clock machinery: Given ESA-1's role in circadian rhythm regulation , investigating its specific interactions with clock components could reveal mechanisms of transcriptional regulation in the circadian system.

What explains the discrepancies between in vitro and in vivo observations of ESA-1 activity?

Several factors may contribute to discrepancies between in vitro and in vivo observations of ESA-1 activity:

How can researchers address the technical challenges of studying ESA-1's role in the circadian clock?

Studying ESA-1's role in the circadian clock presents several technical challenges that researchers can address through specialized approaches:

• Temporal resolution limitations: Traditional methods may lack the temporal resolution needed to study dynamic changes in ESA-1 activity throughout the circadian cycle. Researchers can address this by:

  • Implementing automated sampling systems for time-course experiments

  • Using real-time reporters (such as luciferase assays already employed )

  • Developing methods for rapid fixation of samples at specific circadian times

  • Employing nascent RNA sequencing to capture immediate transcriptional effects

• Circadian desynchronization: Cell populations can become desynchronized over time, obscuring rhythmic patterns. Solutions include:

  • Temperature entrainment protocols to resynchronize cells

  • Single-cell analysis techniques where feasible

  • Mathematical deconvolution methods to extract rhythmic components from population data

• Distinguishing direct from indirect effects: When ESA-1 activity is manipulated, distinguishing direct clock effects from secondary consequences is challenging. Approaches include:

  • Acute induction of ESA-1 knockdown or dominant-negative expression

  • Phase response curve analyses to determine when the clock is sensitive to ESA-1 perturbation

  • ChIP-seq of ESA-1 and core clock proteins to identify co-regulated loci

  • Targeted ESA-1 recruitment to specific clock gene loci using CRISPR/dCas9 fusion systems

• Quantifying subtle period effects: Period changes in response to ESA-1 manipulation (e.g., the ~2-hour extension observed with esa-1^E395Q expression ) require precise measurement. Researchers can:

  • Employ longer time-course experiments to improve period estimation accuracy

  • Use multiple analytical methods for period determination

  • Develop more sensitive reporters for circadian rhythmicity

  • Implement statistical approaches designed for oscillatory data

• Integration with metabolic state: The circadian clock is influenced by metabolic conditions, which may confound ESA-1 studies. Researchers should:

  • Control for metabolic variables in experimental design

  • Monitor key metabolites alongside circadian measurements

  • Test whether ESA-1 activity responds to metabolic cues that affect the clock

What novel approaches could reveal the mechanism of ESA-1 in circadian regulation?

Several innovative approaches could help elucidate the precise mechanisms by which ESA-1 regulates the circadian clock in N. crassa:

• Circadian ChIP-seq time courses: Performing ChIP-seq for ESA-1 and histone acetylation marks across a full circadian cycle would reveal the temporal dynamics of ESA-1 recruitment and activity at clock-controlled genes. This could be combined with nascent transcription analysis to correlate histone acetylation with transcriptional activity.

• Proximity labeling approaches: Techniques such as BioID, TurboID, or APEX2 fused to ESA-1 could identify proteins that interact with ESA-1 in a circadian time-dependent manner, potentially revealing clock-specific interaction partners.

• Single-molecule imaging: Development of systems to visualize ESA-1 localization and activity in living N. crassa cells throughout the circadian cycle could provide unprecedented insights into its temporal and spatial regulation.

• Synthetic biology approaches: Engineering synthetic clock circuits with modified ESA-1 binding sites could help determine the minimum requirements for ESA-1-mediated clock regulation and the effects of altered ESA-1 recruitment patterns.

• Targeted protein degradation: Development of systems for rapid, inducible degradation of ESA-1 (e.g., auxin-inducible degron systems) would allow for precise temporal control over ESA-1 levels, enabling investigation of phase-dependent effects on the clock.

• Comparative analysis across fungal species: Examining the role of ESA-1 orthologs in circadian regulation across multiple fungal species could reveal evolutionarily conserved mechanisms and species-specific adaptations.

• Integration of multi-omics data: Combining ChIP-seq, RNA-seq, proteomics, and metabolomics data in a circadian time-course framework would provide a systems-level understanding of how ESA-1 influences the broader circadian network.

How might structural studies of ESA-1 inform the development of specific inhibitors or activators?

Structural studies of ESA-1 could significantly advance our understanding of its function and facilitate the development of specific modulators:

• High-resolution structural determination: X-ray crystallography or cryo-electron microscopy (cryo-EM) of ESA-1, both alone and in complex with the NuA4 components, would reveal:

  • The detailed architecture of the catalytic domain

  • Substrate binding pockets and specificity determinants

  • Potential allosteric regulatory sites

  • Interfaces with other NuA4 components

  • Structural changes associated with activation/inhibition

• Structure-guided inhibitor design: Identification of unique structural features in the ESA-1 catalytic domain could enable the design of specific inhibitors that:

  • Target the active site with high specificity for ESA-1 over other HATs

  • Exploit allosteric sites unique to ESA-1

  • Disrupt specific protein-protein interactions within the NuA4 complex

  • Feature fungal-specific targeting to create tools with minimal cross-reactivity to mammalian orthologs

• Dynamic structural studies: Techniques such as hydrogen-deuterium exchange mass spectrometry (HDX-MS) or nuclear magnetic resonance (NMR) spectroscopy could reveal:

  • Conformational changes during catalysis

  • Dynamics of substrate recognition

  • Allosteric networks within the protein

  • Effects of potential modulators on protein dynamics

• Computational approaches: Molecular dynamics simulations and virtual screening could:

  • Identify potential binding pockets not evident in static structures

  • Screen virtual libraries for compounds with high binding probability

  • Predict effects of mutations on protein stability and function

  • Model interactions with novel synthetic substrates or inhibitors

• Fragment-based drug discovery: This approach could identify small molecules that bind to different sites on ESA-1, which could then be linked or optimized to create potent and specific modulators.

What potential applications might emerge from a deeper understanding of ESA-1 function?

A comprehensive understanding of ESA-1 function could lead to several innovative applications:

• Chronobiological tools: Given ESA-1's role in circadian rhythm regulation , specific modulators of its activity could serve as valuable tools for chronobiology research. These could allow precise manipulation of circadian timing in experimental systems, potentially helping to:

  • Dissect molecular mechanisms underlying circadian rhythms

  • Develop methods to reset or entrain biological clocks

  • Investigate circadian dysregulation in disease models

• Biotechnological applications: Understanding how ESA-1 regulates gene expression could inform the development of:

  • Synthetic biology tools for temporal control of gene expression

  • Improved protein production systems in fungal hosts

  • Methods to enhance or suppress specific metabolic pathways in fungi

• Agricultural applications: Knowledge of ESA-1 function could contribute to:

  • Development of novel fungicides targeting ESA-1 or the NuA4 complex

  • Creation of fungal strains with modified circadian properties for optimal production of valuable compounds

  • Strategies to control fungal pathogens by disrupting their temporal regulation

• Comparative epigenetics: Understanding the distinctive features of fungal ESA-1 compared to orthologs in other organisms could provide insights into the evolution of epigenetic regulation and potentially reveal:

  • Conserved mechanisms of chromatin regulation across eukaryotes

  • Lineage-specific adaptations in histone acetylation systems

  • Novel regulatory mechanisms that could be applied in other systems

• Disease model insights: As the human ortholog of ESA-1, Tip60, has been implicated in various diseases including cancer and neurodegenerative disorders , insights from the fungal system could potentially inform understanding of human disease mechanisms and therapeutic approaches.