Recombinant Ashbya gossypii Histone acetyltransferase type B catalytic subunit (HAT1)

What is the molecular function of HAT1 in Ashbya gossypii?

HAT1 in A. gossypii functions as a type B histone acetyltransferase that catalyzes the acetylation of specific lysine residues on histone H4 prior to its incorporation into chromatin. Based on comparative studies with related fungi, A. gossypii HAT1 likely acetylates newly synthesized histone H4 at lysines 5 and 12, creating a conserved pattern that is essential for proper chromatin assembly during DNA replication and repair . As a B-type HAT, it operates primarily in the cytoplasm, though nuclear localization may occur under specific conditions to facilitate histone deposition during DNA damage repair processes.

How is the HAT1 gene structured in the A. gossypii genome?

The A. gossypii HAT1 gene is identified in the Ashbya Genome Database with UniProt accession number Q750F5 . While specific details about the genomic structure are not fully characterized in the provided information, researchers typically analyze this through whole genome sequencing data. The gene likely contains conserved domains characteristic of the GNAT (Gcn5-related N-acetyltransferase) superfamily. To determine the exact structure, researchers should perform genomic mapping and sequence analysis using the fully sequenced A. gossypii genome to identify exon-intron boundaries, regulatory regions, and conserved domains.

What are the recommended methods for creating HAT1 deletion mutants in A. gossypii?

For generating HAT1 deletion mutants in A. gossypii, researchers should employ PCR-based gene targeting techniques that have been optimized for this organism. The procedure involves:

• Designing primers with 45-50 bp homology to regions flanking the HAT1 gene

• Amplifying a selection marker (typically G418 resistance cassette, loxP-kanMX-loxP)

• Transforming A. gossypii spores with the deletion cassette

• Selecting primary heterokaryotic transformants on G418-containing medium

• Isolating homokaryotic clones through sporulation of primary transformants

The correct genomic integration should be confirmed by analytical PCR and DNA sequencing. For marker recycling, transient expression of Cre recombinase can be used to eliminate the loxP-kanMX-loxP marker, allowing its reuse for subsequent genetic modifications . This approach has been successfully used for gene disruption in A. gossypii as demonstrated in studies of other regulatory genes .

How can the HAT1 gene be tagged for protein expression studies?

For HAT1 protein expression studies, researchers can use the molecular tools specifically developed for A. gossypii. A recommended approach involves:

• Using PCR-based gene targeting with modules combining available markers with fluorescent protein or epitope tags

• Designing three series of modules for either C-terminal or N-terminal tagging, with options for promoter exchange

• Including G418 resistance markers (loxP-kanMX-loxP) in the constructs

• Transforming A. gossypii with the tagging cassette and selecting on appropriate media

• Confirming correct integration through PCR and sequencing

For protein localization studies, GFP-tagging is particularly useful, while epitope tags like HA or Myc are valuable for immunoprecipitation and Western blot analysis. The C-terminal tagging approach has been extensively used for visualization of cellular components in A. gossypii, as demonstrated in studies of polarisome components and septins .

How might HAT1 influence hyphal growth and morphogenesis in A. gossypii?

Based on studies of histone acetyltransferases in related fungi, HAT1 likely plays critical roles in hyphal growth and morphogenesis in A. gossypii through its effects on chromatin structure and gene expression. Evidence from Candida albicans indicates that HAT1 deletion causes cells to switch from yeast-like to pseudohyphal growth, suggesting a role in morphological transitions . In A. gossypii, which grows as polarized hyphae, HAT1 may influence:

• Polarized growth mechanisms: HAT1-mediated histone modifications could regulate the expression of genes involved in the establishment and maintenance of polarity, including formins like AgBni1 that are essential for hyphal tip extension .

• Nuclear division and organization: Given that A. gossypii has asynchronously dividing nuclei within a continuous cytoplasm, HAT1 might contribute to the regulation of nuclear division cycles through epigenetic mechanisms .

• Cytoskeletal organization: Proper acetylation of histones may affect the expression of genes encoding components of the actin cytoskeleton, which is crucial for polarized growth in A. gossypii .

Experimental approaches to study these connections would include phenotypic analysis of HAT1 deletion mutants, examining changes in hyphal extension rates, branching patterns, and nuclear distribution.

What is the potential role of HAT1 in DNA damage repair in A. gossypii?

Based on evidence from related fungi, HAT1 likely plays a crucial role in DNA damage repair in A. gossypii. In Candida albicans, HAT1 is required for efficient repair of both exogenous and endogenous DNA damage . Cells lacking HAT1 rapidly accumulate DNA damage and show altered morphology. The mechanism involves:

• Acetylation of newly synthesized histone H4 prior to its incorporation into chromatin during DNA repair processes

• Facilitation of chromatin assembly at sites of DNA damage

• Coordination with other histone chaperones and chromatin remodeling factors

In A. gossypii, which experiences significant DNA replication stress due to its rapid hyphal growth, HAT1 may be particularly important for maintaining genome integrity. Researchers investigating this function should:

• Expose HAT1 deletion mutants to DNA-damaging agents (e.g., hydroxyurea, camptothecin, UV radiation)

• Assess sensitivity through growth and survival assays

• Measure DNA damage accumulation using markers like γH2AX

• Analyze changes in histone H4 acetylation patterns during DNA damage responses

Interestingly, studies in A. gossypii have shown that hydroxyurea and camptothecin treatment leads to enhanced riboflavin production , suggesting potential connections between DNA damage response, histone modifications, and secondary metabolism that warrant further investigation.

How does HAT1 interact with the cellular machinery during cell cycle progression in A. gossypii?

The interaction between HAT1 and the cell cycle machinery in A. gossypii likely involves complex regulatory networks that coordinate nuclear division with hyphal growth. In multinucleated fungi like A. gossypii, nuclei divide asynchronously , presenting a unique context for studying HAT1 function. Potential interactions include:

• Regulation of histone supply during S-phase: HAT1 likely acetylates newly synthesized histones needed for chromatin assembly during DNA replication.

• Coordination with mitotic regulators: HAT1 may influence the activity of cell cycle regulators such as AgSwe1p (a Wee1 homologue) that coordinates mitosis with morphogenesis in A. gossypii .

• Synchronization with growth signals: Given that starvation conditions affect nuclear division in A. gossypii through AgSwe1p-dependent CDK phosphorylation , HAT1-mediated histone modifications might play a role in nutrient-responsive cell cycle regulation.

Experimental approaches to investigate these interactions should include:

• Co-immunoprecipitation of HAT1 with cell cycle proteins

• Analysis of cell cycle progression in HAT1 mutants using nuclear markers

• Investigation of genetic interactions between HAT1 and known cell cycle regulators

How might HAT1 activity influence riboflavin production in A. gossypii?

The connection between HAT1 activity and riboflavin production in A. gossypii represents an intriguing area for research, as histone modifications are known to influence secondary metabolism. Based on existing evidence:

• Histone acetylation states affect riboflavin production: Studies of sirtuins (histone deacetylases) in A. gossypii have shown that altered histone H3 acetylation patterns influence riboflavin biosynthesis . Specifically, the disruption of AgHST3, which has SIRT6-like deacetylase activity, enhances riboflavin production.

• DNA damage response links to riboflavin overproduction: DNA-damaging agents like hydroxyurea and camptothecin enhance riboflavin production in A. gossypii . Since HAT1 is involved in DNA damage repair through histone H4 acetylation , it may provide a mechanistic link between genome integrity and riboflavin metabolism.

• Transcriptional regulation of biosynthetic genes: HAT1-mediated histone modifications could affect the expression of genes involved in the purine pathway, which is connected to riboflavin biosynthesis .

To investigate this relationship, researchers should:

• Generate HAT1 deletion and overexpression strains in A. gossypii

• Measure riboflavin production under various conditions

• Analyze histone H4 acetylation patterns in wild-type and mutant strains

• Assess the expression of riboflavin biosynthetic genes

What is the relationship between HAT1 and other epigenetic regulators in A. gossypii?

The interplay between HAT1 and other epigenetic regulators in A. gossypii likely forms a complex network controlling gene expression and cellular functions. Based on studies in related systems:

• Interaction with histone deacetylases (HDACs): HAT1 activity may be balanced by sirtuin-family HDACs like AgHst1, AgHst2, AgHst3, and AgHst4, which have been shown to influence riboflavin production and stress responses in A. gossypii .

• Coordination with transcription factors: HAT1 may interact with transcription factors like AgBas1, a Myb family member that regulates purine biosynthesis, riboflavin production, and growth phase transitions in A. gossypii .

• Integration with chromatin remodeling complexes: HAT1 likely works in concert with ATP-dependent chromatin remodelers to facilitate proper gene expression during different growth phases.

Research approaches to study these interactions should include:

• Co-immunoprecipitation experiments to identify protein-protein interactions

• Chromatin immunoprecipitation (ChIP) to map genomic binding sites

• Double mutant analyses to identify genetic interactions

• Transcriptome profiling to assess global effects on gene expression

What assays can be used to measure HAT1 enzymatic activity in A. gossypii?

To measure HAT1 enzymatic activity in A. gossypii, researchers can employ several complementary approaches:

For A. gossypii specifically, researchers should:

• Express and purify recombinant HAT1 using the available molecular tools for this organism

• Use the purified enzyme in activity assays with histone H4 substrates

• Compare wild-type activity with site-directed mutants affecting catalytic residues

• Assess activity under various conditions (pH, temperature, salt) to determine optimal parameters

Additionally, in vivo assays can be performed by:

• Creating A. gossypii strains with tagged HAT1

• Immunoprecipitating the enzyme from cell extracts

• Measuring acetyltransferase activity of the immunoprecipitated complex

• Analyzing global histone acetylation patterns in wild-type versus HAT1 mutant strains

What imaging techniques are most effective for studying HAT1 localization in A. gossypii hyphae?

For studying HAT1 localization in the filamentous, multinucleated hyphae of A. gossypii, several advanced imaging techniques can be employed:

• Live-cell confocal microscopy: Using GFP-tagged HAT1, this approach allows for real-time visualization of protein localization within living hyphae. The technique has been successfully used to study polarisome components and growth dynamics in A. gossypii .

• Time-lapse fluorescence microscopy: Particularly valuable for tracking HAT1 dynamics during hyphal growth and in response to environmental changes. This approach has been used to monitor protein localization changes during hyphal development in A. gossypii .

• 3D reconstruction imaging: Given the three-dimensional nature of hyphal growth, z-stack imaging with subsequent 3D reconstruction provides comprehensive spatial information. This technique has been used for analyzing septation events in A. gossypii .

• Super-resolution microscopy (STORM, PALM): These techniques overcome the diffraction limit of conventional microscopy and can provide nanoscale resolution of protein localization, which is valuable for determining precise subcellular compartmentalization.

• Correlative light and electron microscopy (CLEM): Combines fluorescence microscopy with electron microscopy to correlate protein localization with ultrastructural features.

For optimal results in A. gossypii:

• Use integrative gene tagging approaches to create HAT1-GFP fusions at the endogenous locus

• Include nuclear markers (e.g., Histone-RFP) for co-localization studies

• Apply 3D live cell imaging to capture the dynamic nature of multinucleated hyphae

• Consider microfluidic devices to control growth conditions during long-term imaging

How can researchers analyze the global effects of HAT1 deletion on histone acetylation patterns in A. gossypii?

To comprehensively analyze the effects of HAT1 deletion on global histone acetylation patterns in A. gossypii, researchers should employ a multi-faceted approach:

• Chromatin immunoprecipitation followed by sequencing (ChIP-seq):

  • Use antibodies specific for histone H4 acetylation marks (K5, K12)

  • Compare acetylation profiles between wild-type and HAT1 deletion strains

  • Identify genomic regions with altered acetylation patterns

  • Correlate changes with gene expression data

• Quantitative mass spectrometry:

  • Extract and purify histones from wild-type and HAT1 mutant strains

  • Analyze post-translational modifications using LC-MS/MS

  • Quantify differences in acetylation levels at specific residues

  • Identify potential compensatory modifications

• Western blot analysis:

  • Use modification-specific antibodies to detect changes in histone acetylation

  • Perform time-course experiments to track acetylation dynamics

  • Compare acetylation patterns under different growth conditions

  • Analyze nuclear and cytoplasmic fractions separately

• RNA-seq transcriptome analysis:

  • Compare gene expression profiles between wild-type and HAT1 mutant strains

  • Identify pathways affected by altered histone acetylation

  • Correlate expression changes with acetylation pattern differences

  • Analyze under various conditions (e.g., normal growth, DNA damage)

• Genomic footprinting:

  • Use techniques like ATAC-seq to assess chromatin accessibility

  • Compare nucleosome positioning between wild-type and mutant strains

  • Identify regions with altered chromatin structure

This comprehensive approach would provide insights into both the direct targets of HAT1-mediated acetylation and the broader effects on chromatin structure and gene expression in the unique multinucleated hyphal system of A. gossypii.

How does A. gossypii HAT1 compare structurally and functionally to homologs in Saccharomyces cerevisiae?

A comparative analysis of HAT1 between A. gossypii and its close relative Saccharomyces cerevisiae reveals important insights into evolutionary conservation and functional adaptation:

Despite A. gossypii and S. cerevisiae sharing a common ancestor, A. gossypii has evolved a filamentous lifestyle while S. cerevisiae remains unicellular. This divergence likely affects how HAT1 functions in each organism:

• In A. gossypii, HAT1 must operate in a shared cytoplasm with multiple asynchronously dividing nuclei, potentially requiring specialized regulatory mechanisms.

• The rapid polarized growth of A. gossypii hyphae may place unique demands on chromatin assembly pathways, including HAT1-mediated histone acetylation.

• A. gossypii experiences distinctive morphogenetic processes like hyphal branching and tip splitting that may involve HAT1-dependent gene regulation.

Experimental approaches to explore these differences should include cross-species complementation studies to determine functional equivalence, and comparative analysis of protein interaction networks to identify species-specific partners.

What insights can be gained by comparing HAT1 function between A. gossypii and pathogenic filamentous fungi?

Comparing HAT1 function between the non-pathogenic A. gossypii and pathogenic filamentous fungi can provide valuable insights for both basic biology and potential antifungal development:

• Conserved roles in DNA damage repair:

  • In Candida albicans, HAT1 is required for both exogenous and endogenous DNA damage repair

  • HAT1-deficient C. albicans cells accumulate DNA damage and switch to pseudohyphal growth

  • Similar functions in A. gossypii would suggest evolutionary conservation of HAT1's role in genome maintenance

• Morphogenetic regulation:

  • Morphological transitions are crucial for pathogenicity in many fungi

  • HAT1's involvement in cellular morphology in C. albicans suggests it may influence the distinctive hyphal growth patterns in A. gossypii

  • Comparing the molecular mechanisms could reveal how similar epigenetic machinery is adapted for different biological purposes

• Stress response pathways:

  • HAT1-deficient C. albicans shows hypersusceptibility to antifungal drugs like caspofungin

  • Investigating whether A. gossypii HAT1 similarly affects stress resistance could identify conserved stress response mechanisms

• Metabolic regulation:

  • A. gossypii is known for riboflavin overproduction

  • Comparing how HAT1 influences secondary metabolism in A. gossypii versus pathogenic fungi could reveal divergent regulatory networks

• Antifungal targets:

  • HAT1 has been proposed as a potential target for antifungal therapy

  • A. gossypii could serve as a non-pathogenic model for investigating HAT1 inhibitors

  • Comparative analysis could identify selective targeting opportunities

To pursue these comparisons, researchers should:

• Generate equivalent HAT1 mutations in multiple fungal species

• Perform phenotypic analyses under identical conditions

• Use ChIP-seq to compare genomic targets

• Conduct transcriptome analyses to identify shared and species-specific regulated genes

How can CRISPR-Cas9 technology be optimized for HAT1 engineering in A. gossypii?

While CRISPR-Cas9 systems have not been extensively documented for A. gossypii in the provided materials, developing this technology for HAT1 engineering would significantly advance research capabilities. A proposed optimization strategy would include:

• Vector design for A. gossypii:

  • Adapt existing CRISPR-Cas9 systems using promoters functional in A. gossypii

  • Utilize the dual luciferase reporter system described for A. gossypii to test different promoters for Cas9 expression

  • Incorporate selectable markers compatible with A. gossypii (e.g., G418 resistance)

• sgRNA optimization:

  • Design sgRNAs targeting HAT1 locus with minimal off-target effects

  • Test various sgRNA scaffold designs for efficiency in A. gossypii

  • Utilize the native RNA polymerase III promoters (e.g., U6) from A. gossypii for sgRNA expression

• Delivery methods:

  • Adapt transformation protocols used for PCR-based gene targeting in A. gossypii

  • Test both plasmid-based and ribonucleoprotein (RNP) delivery

  • Optimize transformation conditions for spores versus mycelium

• Template design for precise editing:

  • Create repair templates with homology arms appropriate for A. gossypii (typically 45-50 bp)

  • Include selectable markers flanked by loxP sites for marker recycling

  • Design templates for various modifications: knockout, point mutations, domain deletions, and protein tagging

• Validation strategies:

  • PCR-based genotyping for initial screening

  • Sequencing to confirm precise editing

  • Western blotting to verify protein modifications

  • Phenotypic characterization to assess functional impact

• Multiplexing capabilities:

  • Develop systems for simultaneous editing of HAT1 and related genes

  • Create arrays of sgRNAs for multi-locus targeting

  • Design strategies for orthogonal modifications (e.g., knockout of one gene with tagging of another)

This optimized CRISPR-Cas9 system would enable precise engineering of HAT1, including domain-specific mutations, regulatory element modifications, and fusion constructs for advanced functional studies in A. gossypii.

What high-throughput approaches can be used to identify the genome-wide targets of HAT1 in A. gossypii?

Several high-throughput approaches can be employed to comprehensively identify the genome-wide targets of HAT1 in A. gossypii:

• ChIP-seq (Chromatin Immunoprecipitation followed by Sequencing):

  • Generate A. gossypii strains expressing epitope-tagged HAT1 using established gene targeting methods

  • Perform ChIP with antibodies against the epitope tag or directly against HAT1

  • Sequence immunoprecipitated DNA to identify genomic binding sites

  • Compare binding profiles between different growth conditions and developmental stages

• CUT&RUN or CUT&Tag:

  • These newer alternatives to ChIP provide higher signal-to-noise ratio

  • Particularly useful for factors with transient chromatin interactions

  • Can be performed with fewer cells, which may be advantageous for A. gossypii culture conditions

• RNA-seq of HAT1 mutants:

  • Compare transcriptomes of wild-type and HAT1 deletion/mutation strains

  • Identify genes differentially expressed upon HAT1 perturbation

  • Perform time-course experiments during different growth phases

  • Analyze under various stress conditions to identify condition-specific targets

• ATAC-seq (Assay for Transposase-Accessible Chromatin with sequencing):

  • Compare chromatin accessibility patterns between wild-type and HAT1 mutant strains

  • Identify regions with altered accessibility that may represent HAT1 targets

  • Correlate with histone acetylation patterns and transcriptional changes

• Proteomics approaches:

  • Perform immunoprecipitation of HAT1 followed by mass spectrometry (IP-MS)

  • Identify protein interaction partners that may guide HAT1 to specific genomic loci

  • Use cross-linking methods to capture transient interactions

• Histone modification profiling:

  • Use mass spectrometry to quantitatively profile histone modifications in wild-type versus HAT1 mutants

  • Identify specific acetylation marks dependent on HAT1 activity

  • Correlate modifications with gene expression patterns

• Integration of multiple datasets:

  • Combine ChIP-seq, RNA-seq, and ATAC-seq data using computational approaches

  • Identify consistent patterns across datasets to define high-confidence targets

  • Use machine learning to predict additional targets based on sequence and chromatin features