Recombinant Neurospora crassa Histone chaperone rtt-106 (rtt-106)

What is Neurospora crassa RTT106 and what is its primary function?

RTT106 from Neurospora crassa is a 469-amino acid histone chaperone protein that facilitates nucleosome assembly by promoting the deposition of histone complexes onto DNA . As a histone chaperone, RTT106 plays a crucial role in chromatin organization during DNA replication and other cellular processes. The recombinant form (e.g., ABIN1676071) is typically expressed in yeast systems with a His-tag for purification and experimental applications .

The protein functions primarily to bind histone H3-H4 complexes and deposit them onto DNA during nucleosome formation . This activity is particularly important during S phase of the cell cycle when chromatin needs to be reassembled following DNA replication.

How does RTT106 from Neurospora crassa compare structurally to RTT106 from other fungal species?

Neurospora crassa RTT106 shares functional conservation with homologs from other fungal species, though with some structural variations:

While the core functional domains are conserved across species, these length variations may reflect evolutionary adaptations to specific cellular environments . The S. cerevisiae Rtt106 has been characterized to contain two tandem PH (Pleckstrin Homology) domains connected by a disordered loop, which likely represents the conserved structural organization across fungal species .

What experimental evidence demonstrates RTT106's role in histone binding?

RTT106 has been shown to bind specifically to histone H3-H4 complexes through multiple experimental approaches:

• Sequential affinity purification experiments have demonstrated that RTT106 binds preferentially to (H3-H4)2 tetramers rather than H3-H4 dimers .

• Mutational analyses have identified specific amino acid residues involved in histone binding .

• Co-immunoprecipitation assays have confirmed the interaction between RTT106 and histones in vivo .

• The binding affinity of RTT106 for histones is significantly enhanced when histone H3 is acetylated at lysine 56 (H3K56ac) .

These findings collectively establish RTT106 as a specialized histone chaperone with specific binding properties that facilitate its function in nucleosome assembly.

What is the domain organization of RTT106 and how does it relate to function?

Based on structural studies primarily in S. cerevisiae, RTT106 contains:

• An N-terminal domain (residues 1-68 in S. cerevisiae) that facilitates oligomerization

• Two tandem PH (Pleckstrin Homology) domains connected by a disordered loop

• A C-terminal domain that contributes to histone binding

A key structural discovery is that RTT106 forms homodimers through its N-terminal domain, which is critical for its ability to bind (H3-H4)2 tetramers . The full sequence of Neurospora crassa RTT106 (469 amino acids) suggests a similar domain organization with potential variations in the flexible regions .

What specific structural features enable RTT106 to function as a histone chaperone?

RTT106's histone chaperone function depends on several key structural features:

• A conserved basic patch within the N-terminal PH domain that is essential for histone interaction

• A critical loop in the C-terminal domain containing specific residues (such as T265 in S. cerevisiae) that directly participate in histone binding

• N-terminal residues that facilitate oligomerization, which is necessary for binding (H3-H4)2 tetramers

Structural analysis has revealed that these histone-binding regions lie approximately 30Å apart but collectively form a conserved interaction surface that accommodates the histone complexes . Mutational studies have demonstrated that altering the charge characteristics of the basic patch (e.g., S80E and R86A mutations in S. cerevisiae) disrupts RTT106 function, while conservative mutations that maintain charge (S80T, S80A, and R86K) preserve activity .

How does the oligomeric state of RTT106 affect its function?

RTT106 undergoes oligomerization primarily through its N-terminal domain, forming homodimers or possibly higher-order oligomers . This oligomerization is functionally significant for several reasons:

• It creates the appropriate structural configuration to bind (H3-H4)2 tetramers

• Mutations in the N-terminal domain that disrupt oligomerization compromise RTT106's function in transcriptional silencing and genotoxic stress response

• The oligomeric state affects RTT106's ability to mediate nucleosome assembly during DNA replication

Gel filtration chromatography and dynamic light scattering analyses have confirmed RTT106's oligomeric state in vitro, with the full-length protein exhibiting higher apparent molecular mass than expected for a monomer . The N-terminal 66 residues appear particularly important for this oligomerization, as truncated constructs lacking this region migrate at the expected monomeric size .

How does RTT106 interact with histone proteins at the molecular level?

RTT106 interacts with histone proteins through two distinct surfaces that collectively form a conserved binding interface:

• A basic patch within the N-terminal PH domain that contributes to electrostatic interactions with histones

• A specific loop in the C-terminal domain containing crucial residues for histone binding

The interaction is significantly enhanced when histone H3 is acetylated at lysine 56 (H3K56ac), a modification that increases RTT106's binding affinity . Mutational studies have shown that changing the charge characteristics of the basic patch (through mutations like S80E) disrupts histone binding, while conservative mutations that maintain charge (S80T, S80A) preserve this function .

The structure-function relationship has been explored through targeted mutations, revealing that residues in both the N-terminal and C-terminal domains contribute to a unified histone-binding surface despite being spatially separated by approximately 30Å .

What is the stoichiometry of histone binding to RTT106 and why is it significant?

RTT106 binds to (H3-H4)2 tetramers rather than H3-H4 dimers, which has significant implications for nucleosome assembly . This was demonstrated through elegant experiments showing that H3K56R (a mutation that reduces binding to RTT106) could co-purify with RTT106 when wild-type H3 was present, suggesting that RTT106 binds heterotetramers of H3-H4 .

The significance of this stoichiometry includes:

• It suggests that RTT106 deposits complete (H3-H4)2 tetramers onto DNA during nucleosome assembly, rather than sequential deposition of H3-H4 dimers

• It provides insights into how nucleosomes are formed during S phase of the cell cycle

• It distinguishes RTT106's mechanism from other histone chaperones like Asf1, which binds H3-H4 dimers

This tetramer-binding capacity is directly related to RTT106's N-terminal oligomerization, as mutations affecting oligomerization also compromise tetramer binding .

How does RTT106 coordinate with chromatin remodeling complexes?

RTT106 physically interacts with both the SWI/SNF and RSC chromatin remodeling complexes both in vitro and in vivo, as demonstrated through GST pull-down and co-immunoprecipitation assays . This coordination is functionally significant because:

• RTT106 is important for recruiting both SWI/SNF and RSC complexes to HIR-dependent histone genes

• The RTT106-dependent recruitment of SWI/SNF is cell cycle regulated, occurring primarily in late G1 phase just before peak histone gene expression in S phase

• SWI/SNF is required for activation of histone genes in S phase, while RSC is implicated in their repression outside of S phase

These interactions reveal RTT106's broader role in chromatin regulation beyond simple histone deposition, suggesting it functions as a coordinator between histone handling and chromatin remodeling activities during cell cycle progression .

What expression systems are most effective for producing recombinant Neurospora crassa RTT106?

Based on available data, the following expression systems have been used or considered for RTT106 production:

The recombinant Neurospora crassa RTT106 protein available commercially (e.g., ABIN1676071) is expressed in yeast systems with a His-tag for purification, suggesting this is currently the most validated approach .

What analytical methods are most informative for studying RTT106-histone interactions?

Multiple complementary analytical approaches provide comprehensive insights into RTT106-histone interactions:

• Protein-Protein Interaction Assays:

  • GST pull-down assays to detect direct interactions in vitro

  • Co-immunoprecipitation to verify protein associations in vivo

  • Sequential affinity purification to determine binding stoichiometry

• Chromatin Association Studies:

  • Chromatin immunoprecipitation (ChIP) assays to study RTT106's association with specific genomic regions

  • Cell synchronization combined with ChIP to assess cell cycle-dependent chromatin association

• Structural and Biophysical Analyses:

  • Gel filtration chromatography to analyze oligomeric state

  • Dynamic light scattering to assess size and shape of complexes

  • Crystallography for detailed structural information

• Functional Assays:

  • Silencing assays using reporter strains (e.g., HMR-a1::URA3) to assess transcriptional silencing function

  • Genotoxic sensitivity assays (e.g., CPT sensitivity) to evaluate DNA damage response roles

These methodologies collectively provide a comprehensive view of how RTT106 interacts with histones and functions in chromatin-related processes.

How can researchers effectively design mutation studies to analyze RTT106 function?

Based on previous successful studies, an effective approach to RTT106 mutation analysis includes:

• Structure-guided targeting: Focus on conserved residues identified through sequence alignment and structural studies:

  • Basic patch residues in the N-terminal PH domain (e.g., S80, R86 in S. cerevisiae)

  • Critical residues in the C-terminal loop (e.g., T265, T268 in S. cerevisiae)

• Charge-altering vs. conservative mutations:

  • Compare charge-altering mutations (S80E, R86A) with conservative mutations (S80T, S80A, R86K) to distinguish electrostatic effects from structural requirements

  • Include appropriate controls like double-alanine mutations (TT265,268AA) to interpret results correctly

• Functional readouts:

  • Use silencing assays (growth on FOA medium) to assess effects on transcriptional silencing

  • Employ CPT sensitivity assays to evaluate genotoxic stress response

  • Conduct biochemical assays for histone binding to directly measure effects on protein-protein interactions

• Domain deletion studies:

  • Create constructs lacking specific domains (e.g., ΔN-terminal) to assess domain-specific functions

  • Analyze migration patterns in gel filtration to evaluate effects on oligomerization

This comprehensive approach has previously revealed that surprisingly few residues (approximately 10) are critical for RTT106 function, highlighting key surfaces for histone interaction .

What is the role of RTT106 in transcriptional regulation of histone genes?

RTT106 plays a complex role in the transcriptional regulation of histone genes, particularly the HIR-dependent histone gene pairs (HTA1-HTB1, HHT1-HHF1, and HHT2-HHF2) in S. cerevisiae . Key aspects of this regulation include:

• Recruitment of chromatin remodeling complexes:

  • RTT106 mediates the recruitment of SWI/SNF complex to histone genes in late G1 phase, which is required for their activation

  • RTT106 also facilitates RSC complex recruitment, which is implicated in histone gene repression outside of S phase

• Cell cycle-dependent regulation:

  • RTT106 is present at histone gene loci throughout the cell cycle in a HIR- and Asf1-dependent manner

  • The RTT106-dependent recruitment of SWI/SNF is restricted to late G1 phase, just before the peak of histone gene expression in S phase

• Transcriptional repression:

  • RTT106 contributes to transcriptional repression of histone genes outside of S phase, ensuring cell cycle-appropriate expression

This sophisticated regulatory mechanism ensures that histone genes are expressed at the appropriate time during the cell cycle, coordinating histone production with DNA replication requirements.

How do post-translational modifications of histones affect their interaction with RTT106?

Post-translational modifications of histones significantly influence their interaction with RTT106, with H3K56 acetylation being particularly important:

• H3K56 acetylation effects:

  • Acetylation of histone H3 at lysine 56 (H3K56ac) significantly enhances binding to RTT106

  • Mutating lysine 56 of H3 to arginine (H3K56R) substantially reduces binding to RTT106

  • In the presence of wild-type H3, H3K56R can still co-purify with RTT106 when part of a heterotetramer, further supporting the tetramer-binding model

• Cell cycle significance:

  • H3K56ac is prevalent on newly synthesized histones during S phase

  • This modification provides a mechanism for RTT106 to preferentially target newly synthesized histones for deposition during DNA replication

• Structural implications:

  • The enhanced binding of H3K56ac suggests a specific recognition mechanism within RTT106's binding pocket

  • This recognition mechanism likely involves both the N-terminal basic patch and C-terminal loop regions identified as critical for histone binding

The interaction between RTT106 and H3K56ac represents an important link between histone modification status and nucleosome assembly processes during DNA replication.

What are the implications of RTT106's role in genotoxic stress response for DNA damage research?

RTT106's involvement in genotoxic stress response has significant implications for DNA damage research:

• Experimental evidence:

  • Mutations affecting RTT106 function lead to sensitivity to camptothecin (CPT), a genotoxic agent that induces DNA damage

  • The same mutations that affect histone binding also compromise genotoxic stress response, suggesting a direct mechanistic link

• Potential mechanisms:

  • RTT106 may facilitate chromatin reassembly following DNA repair, helping to restore proper chromatin structure

  • By preferentially binding H3K56ac-containing histones, RTT106 might direct newly synthesized histones to sites of DNA damage repair

  • Coordination with chromatin remodeling complexes may help regulate accessibility of DNA repair machinery to damaged sites

• Research applications:

  • RTT106 can serve as a model for understanding how histone chaperones contribute to maintaining genome stability

  • Studying the interplay between RTT106 and DNA damage response pathways may reveal new therapeutic targets for diseases involving genomic instability

  • The conserved nature of RTT106 across fungal species suggests potential relevance of findings to broader eukaryotic systems

This connection between histone chaperone function and DNA damage response highlights the intimate relationship between chromatin dynamics and genome maintenance mechanisms.