Recombinant Neurospora crassa Histone H3-like centromeric protein cse-4 (cse-4)

What is Neurospora crassa CSE-4 and why is it important for research?

CSE-4 is the Neurospora crassa version of the centromeric histone H3 variant, which in humans is called CENP-A and in yeast is called Cse4. This specialized histone replaces conventional H3 at centromeres and serves as a foundation for kinetochore assembly, which is essential for proper chromosome segregation during cell division. CSE-4 is crucial for research because it provides insights into fundamental cellular processes of chromosome segregation and centromere specification across eukaryotes. In Neurospora crassa, CSE-4 has been identified through chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq), showing that it localizes to specific 150-300 kbp regions on each chromosome .

How does CSE-4 differ structurally from canonical histone H3?

While both CSE-4 and canonical histone H3 share a histone fold domain, CSE-4 contains unique structural features that distinguish it from H3. The N-terminal domain of CSE-4 shows significant divergence from conventional H3 and contains motifs that mediate specific protein-protein interactions with other centromere and kinetochore components. For example, in the related yeast system, the Cse4 N-terminus binds to the Ame1/Okp1 complex through the Okp1 core domain, establishing one of the specific recognition points for centromeric nucleosomes . These structural differences are essential for the specialized function of CSE-4 in centromere specification and kinetochore assembly.

How is CSE-4 localized to centromeres in Neurospora crassa?

The localization of CSE-4 to centromeres in Neurospora crassa appears to depend on heterochromatin components. ChIP-seq experiments have revealed that CSE-4 colocalizes extensively with CEN-C, CEN-T, and histone H3K9 trimethylation (H3K9me3) at centromeric regions . Unlike some other organisms, Neurospora centromeres do not show enrichment of H3K4me2. Importantly, mutations in the dim-5 gene, which encodes an H3K9 methyltransferase responsible for nearly all H3K9me3 in Neurospora, result in altered distribution of CSE-4. Similarly, the absence of HP1, the chromodomain protein that binds to H3K9me3, also disrupts proper CSE-4 localization . This suggests that in Neurospora crassa, heterochromatin plays a critical role in establishing and maintaining CSE-4 at centromeres.

What techniques are commonly used to study CSE-4 in Neurospora crassa?

Several methodological approaches are employed to study CSE-4 in Neurospora crassa:

• ChIP-seq: Chromatin immunoprecipitation followed by high-throughput sequencing has been used to identify the genomic locations of CSE-4, revealing its enrichment at 150-300 kbp regions on each chromosome .

• Fluorescence microscopy: GFP-tagged CSE-4 allows visualization of its localization patterns in live cells and assessment of how mutations in other genes affect its distribution .

• Genetic manipulation: Creation of mutant strains with deletions or modifications of CSE-4 or interacting proteins helps elucidate functional relationships. Studies in related systems have shown how specific mutations (e.g., cse4-R143A in yeast) affect centromere stability and kinetochore function .

• Protein interaction studies: Techniques like co-immunoprecipitation are used to identify proteins that physically interact with CSE-4, helping to map the centromere-kinetochore interaction network.

How do post-translational modifications of CSE-4 regulate centromere function?

Post-translational modifications of CSE-4/Cse4 play crucial roles in regulating centromere function, though most detailed studies have been conducted in yeast rather than Neurospora specifically. In Saccharomyces cerevisiae, methylation at arginine 143 (R143me) and lysine 131 (K131me) of Cse4 affects centromere stability and kinetochore function . These modification sites are located in the core region of the centromeric nucleosome, near the entry/exit sites of DNA.

The functional significance of these modifications was demonstrated when mutation of Cse4-R143 (cse4-R143A) exacerbated kinetochore defects in strains with mutations in outer kinetochore components (spc25-1) and the MIND complex (dsn1-7) . Suppressor mutations of the spc25-1 cse4-R143A growth defect highlighted residues in Spc24, Ndc80, and Spc25 that localize to the tetramerization domain of the NDC80 complex and the Spc24-Spc25 stalk, suggesting these mutations enhance interactions among NDC80 complex components to stabilize the complex .

Additionally, the Set2 histone methyltransferase appears to inhibit kinetochore function in spc25-1 cse4-R143A cells, possibly by methylating Cse4-K131 . While these specific modifications have been characterized in yeast, similar regulation likely exists in Neurospora crassa CSE-4, and investigating these modifications represents an important avenue for future research.

How does the COMA complex interact with CSE-4 and what are the functional implications?

The COMA complex (consisting of Ctf19, Okp1, Mcm21, and Ame1 proteins) plays a critical role in centromere function by interacting with CSE-4/Cse4. Studies using chemical crosslinking and mass spectrometry (XLMS) combined with biochemical reconstitution have characterized how the COMA complex contributes to establishing a selective Cse4-nucleosome core particle (NCP) binding environment .

Specifically, the Cse4 N-terminus binds to Ame1/Okp1 through the Okp1 core domain. This interaction, along with Mif2 binding to other Cse4 motifs, establishes dual recognition of the Cse4-NCP, creating specificity for centromeric nucleosomes . Beyond CSE-4 recognition, the COMA complex also interacts with the chromosome passenger complex (CPC), specifically through Sli15/Ipl1 (Aurora B kinase in humans) binding to the Ctf19 C-terminus .

This COMA-CPC interaction is functionally important, as demonstrated by synthetic lethality between the sli15ΔN mutant and deletions of Ctf19 or Mcm21. This lethality can be rescued by fusing Sli15ΔN to the COMA complex, indicating that physical proximity of these components is essential for proper chromosome segregation . These findings illustrate how the COMA complex serves as a hub that both recognizes centromeric nucleosomes and positions key regulatory proteins like Aurora B kinase at the kinetochore.

What genetic approaches can be used to study CSE-4 function in Neurospora crassa?

Several sophisticated genetic approaches can be employed to study CSE-4 function in Neurospora crassa:

• Anchor-away techniques: This approach allows conditional depletion of proteins from specific cellular compartments. For example, Ctf19 anchor-away strains have been used to assess minichromosome loss frequency when COMA complex components are depleted .

• Site-directed mutagenesis: Creating specific mutations in CSE-4 can reveal functional domains. Based on studies in yeast, mutations analogous to cse4-R143A could be introduced in Neurospora CSE-4 to examine effects on centromere function .

• Fusion proteins and rescue experiments: Fusion constructs can test functional relationships between proteins. For instance, fusing Sli15ΔN to COMA complex components rescued synthetic lethality in yeast , and similar approaches could be applied in Neurospora.

• Synthetic genetic array analysis: Systematically combining CSE-4 mutations with mutations in other genes can reveal genetic interactions and functional relationships within the centromere-kinetochore network.

• Regulatable promoters: Replacing the native CSE-4 promoter with inducible promoters allows controlled expression to study dosage effects and requirements for CSE-4 loading.

What is the optimal protocol for ChIP-seq analysis of CSE-4 in Neurospora crassa?

For optimal ChIP-seq analysis of CSE-4 in Neurospora crassa, researchers should follow this methodological approach:

• Sample preparation: Culture Neurospora crassa under appropriate conditions, ensuring synchronized growth if cell cycle-specific analysis is desired. Crosslink proteins to DNA using 1% formaldehyde for approximately 10 minutes.

• Chromatin extraction and fragmentation: Lyse cells and isolate chromatin, then fragment to approximately 200-500 bp using sonication or enzymatic digestion. Neurospora cell walls require special consideration, so optimization of lysis conditions is essential.

• Immunoprecipitation: Use either antibodies specific to native CSE-4 or, more commonly, antibodies against epitope tags (e.g., FLAG, Myc, or GFP) on recombinant CSE-4. Include appropriate controls such as input chromatin and non-specific antibody immunoprecipitation.

• DNA purification and library preparation: Purify immunoprecipitated DNA, prepare sequencing libraries, and perform high-throughput sequencing. Paired-end sequencing is recommended for better mapping to repetitive regions that often surround centromeres.

• Data analysis: Map sequencing reads to the Neurospora crassa genome, identify enriched regions, and compare to known genomic features and other chromatin marks like H3K9me3 . Analysis should account for the 150-300 kbp size of centromeric regions identified in previous studies.

This approach has successfully identified centromeric regions in Neurospora crassa, revealing colocalization of CSE-4 with other centromeric proteins and specific chromatin modifications .

How can recombinant CSE-4 be expressed and purified for in vitro studies?

For successful expression and purification of recombinant Neurospora crassa CSE-4:

• Expression system selection: The E. coli BL21(DE3) strain is commonly used for histone protein expression. For better folding and solubility, consider using specialized strains like Rosetta or Arctic Express. Alternative expression systems include insect cells (using baculovirus) for post-translational modifications.

• Construct design:

  • Include an N-terminal affinity tag (His6 or GST) with a precision protease cleavage site

  • Codon-optimize the CSE-4 sequence for the expression host

  • Consider expressing just the histone fold domain if solubility issues arise

• Expression conditions:

  • Induce at lower temperatures (16-20°C) overnight to enhance proper folding

  • Use 0.2-0.5 mM IPTG for induction in E. coli

  • Include additives like 5-10% glycerol and 1-5 mM β-mercaptoethanol in growth media

• Purification protocol:

  • Perform initial affinity purification under denaturing conditions (8M urea)

  • Gradually remove denaturant through dialysis or on-column refolding

  • Follow with ion exchange chromatography (SP Sepharose)

  • Conclude with size exclusion chromatography

  • Maintain reducing conditions throughout purification

• Quality control:

  • Verify protein identity by mass spectrometry

  • Assess purity by SDS-PAGE (>95% purity)

  • Test functionality through nucleosome assembly assays

This approach allows production of recombinant CSE-4 suitable for biochemical studies, including nucleosome reconstitution, protein interaction studies, and structural analysis.

What approaches can be used to study interactions between CSE-4 and other centromere/kinetochore proteins?

Several complementary approaches can be used to characterize interactions between CSE-4 and other centromere/kinetochore proteins:

• Chemical crosslinking coupled with mass spectrometry (XLMS): This method has been successfully used to characterize CTF19c/CCAN subunit connectivity and protein interfaces that establish selective Cse4-NCP binding . It provides spatial information about protein proximity within complexes.

• Yeast two-hybrid assays: While traditionally used in yeast, this approach can be adapted for Neurospora proteins to screen for direct binary interactions between CSE-4 and other proteins.

• Co-immunoprecipitation (Co-IP): Using antibodies against CSE-4 or epitope-tagged versions, researchers can immunoprecipitate CSE-4 and identify associated proteins by mass spectrometry or Western blotting.

• Biochemical reconstitution: In vitro assembly of centromeric nucleosomes with recombinant CSE-4 and histone partners, followed by binding assays with purified centromere/kinetochore proteins, can reveal direct interactions and binding affinities.

• Biolayer interferometry or surface plasmon resonance: These techniques provide quantitative binding data including association/dissociation rates and equilibrium constants for CSE-4 interactions.

• Fluorescence microscopy with FRET/FLIM: Fluorescence resonance energy transfer or fluorescence lifetime imaging microscopy can detect protein-protein interactions in living cells with spatial resolution.

• Genetic approaches: Synthetic genetic interactions, suppressor screens, and rescue experiments can reveal functional relationships, as demonstrated by the synthetic lethality between sli15ΔN and deletions of Ctf19 or Mcm21, which was rescued by fusion constructs .

What are the key unresolved questions about CSE-4 function in Neurospora crassa?

Several critical questions remain unresolved regarding CSE-4 function in Neurospora crassa:

• Post-translational modification landscape: While specific modifications like R143me and K131me have been characterized in yeast Cse4 , the complete set of post-translational modifications on Neurospora CSE-4 and their functional significance remains to be determined.

• Centromere specification mechanism: The precise mechanism by which CSE-4 is initially deposited at centromeres in Neurospora is unclear. While heterochromatin components like DIM-5 and HP1 are required for proper CSE-4 distribution , the sequence of events in de novo centromere establishment is unknown.

• Cell cycle regulation: How CSE-4 deposition and maintenance are regulated throughout the cell cycle in Neurospora remains largely unexplored, including the timing of new CSE-4 incorporation and potential recycling of existing CSE-4.

• Structural details: The atomic-level structure of Neurospora CSE-4 nucleosomes and how they differ from canonical nucleosomes has not been determined, leaving questions about how structural differences contribute to centromere identity.

• Evolutionary conservation and divergence: While CSE-4 is functionally conserved across eukaryotes, Neurospora shows some distinct features in centromere organization, including the role of heterochromatin. Understanding these evolutionary differences would provide insight into centromere evolution.

How might CSE-4 research in Neurospora contribute to understanding centromere biology across species?

Neurospora crassa offers unique advantages for comparative centromere biology studies for several reasons:

• Distinctive heterochromatin dependence: Unlike some other model organisms, Neurospora centromeres show a clear dependence on heterochromatin components (DIM-5 and HP1) for proper CSE-4 localization . This provides insights into alternative mechanisms of centromere specification and maintenance across eukaryotes.

• Regional centromere organization: Neurospora possesses regional centromeres (150-300 kbp) rather than point centromeres like Saccharomyces cerevisiae or the large, satellite-rich centromeres of mammals. This intermediate centromere type helps elucidate the spectrum of centromere architectures.

• Evolutionarily informative position: As a filamentous fungus, Neurospora occupies an important evolutionary position, allowing comparisons with both yeasts and more complex eukaryotes to trace the evolution of centromere specification mechanisms.

• Well-characterized epigenetic mechanisms: Neurospora is a model organism for understanding epigenetic processes including DNA methylation and histone modifications, enabling integrated studies of how these mechanisms contribute to centromere function.

• Genetic tractability: The sophisticated genetic tools available in Neurospora facilitate functional studies that are challenging in other organisms with regional centromeres.

By comparing CSE-4 biology in Neurospora with CENP-A/Cse4 in other organisms, researchers can develop more comprehensive models of centromere specification that account for both conserved and divergent mechanisms across eukaryotes.

What methodological advances would accelerate research on CSE-4 in Neurospora crassa?

Several methodological advances would significantly enhance research capabilities for studying CSE-4 in Neurospora crassa:

• Improved genome editing techniques: While CRISPR-Cas9 has been adapted for Neurospora, further refinement of this technology specifically for centromeric regions would enable more precise manipulation of CSE-4 and associated factors.

• Live-cell super-resolution microscopy: Enhanced imaging techniques with higher spatial and temporal resolution would allow better visualization of CSE-4 dynamics throughout the cell cycle and its interactions with other kinetochore components.

• Single-molecule approaches: Techniques like single-molecule tracking or force spectroscopy applied to CSE-4 would provide insights into its dynamic behavior and physical properties within the centromeric chromatin environment.

• Centromere-specific proteomics: Developing methods to isolate intact centromere complexes from Neurospora for comprehensive proteomic analysis would reveal the complete protein composition of these structures.

• In vitro centromere assembly systems: Establishing cell-free systems that recapitulate centromere assembly with Neurospora components would allow mechanistic studies of CSE-4 deposition and centromere formation.

• Chromosome conformation capture techniques: Applying Hi-C or related methods specifically to centromeric regions would reveal three-dimensional organization of Neurospora centromeres and how CSE-4 influences chromatin architecture.

• Inducible centromere systems: Developing synthetic or conditional centromeres in Neurospora would enable controlled studies of de novo centromere establishment and the role of CSE-4 in this process.

How does Neurospora CSE-4 compare to its homologs in other model organisms?

Comparative analysis reveals both conservation and divergence in CSE-4/CENP-A properties across model organisms:

Neurospora CSE-4 shares the fundamental role of centromere specification with its homologs but exhibits unique features in its regulation and genomic context. Unlike human CENP-A or Drosophila CID, Neurospora CSE-4 does not show enrichment of H3K4me2 at centromeric cores . Instead, it shows extensive colocalization with H3K9me3, a heterochromatic mark. The dependence on DIM-5 (H3K9 methyltransferase) and HP1 for proper CSE-4 localization represents a distinctive feature of Neurospora centromere organization .

The comparative analysis suggests that while the presence of a specialized H3 variant at centromeres is conserved across eukaryotes, the mechanisms for its deposition and maintenance have diversified throughout evolution.