Yeast activators bind specific ATPase domains in chromatin remodelling complexes

Published in Current Genetics, the study details how transcriptional activation domains (TADs) interact with the ATPase subunits of the SWI/SNF, RSC, and INO80 complexes. These interactions are critical for the dynamic organization of nucleosomes along eukaryotic DNA, a process that facilitates access for general transcription factors and initiates gene expression. The work provides a detailed molecular map of these interactions, highlighting distinct functional requirements for different subunits within the complexes.

The study focuses on the ATP-dependent molecular machines known as chromatin remodelling complexes (CRCs). SWI/SNF, RSC, and INO80 are capable of moving positioned nucleosomes in promoter DNA, leading to nucleosome-depleted regions. This activity is strongly supported by transcriptional activators, which interact with various subunits of these complexes. The authors specifically examined the interaction between the activator Ino2, required for the expression of phospholipid biosynthetic genes in yeast, and the subunits of the three major CRCs. By mapping these interactions, the team aimed to understand how activators mediate the access of RSC and INO80 to their target genes, a mechanism that had previously remained unclear.

Using in vitro interaction assays, the researchers demonstrated that SWI/SNF subunits Swi1, Swi2, Snf5, and Snf6 can bind to the activation domains of Ino2. The study identified an activator binding domain (ABD) within the ATPase Swi2, located in the N-terminal region of the protein. This 70-amino-acid domain, spanning residues 238 to 307, was shown to interact not only with Ino2 but also with the activation domains of unrelated activators such as Gal4, Gcn4, Rap1, Aro80, and Swi5. Despite this broad interaction capability, the ABD of Swi2 was found to be functionally dispensable in vivo, suggesting that other subunits of the SWI/SNF complex can compensate for its loss.

In contrast to Swi2, the ABD of the Swi2-related ATPase Sth1, which is the enzymatic core of the RSC complex, proved to be essential for function. Mutational characterization revealed that specific conserved basic and hydrophobic amino acids within the Sth1 ABD are indispensable. The core region of this domain, spanning amino acids 198 to 218, was identified as critical for interaction with transcriptional activators. When these residues were mutated to alanine, the Sth1 protein failed to interact with Ino2 in vitro and could not complement a sth1 null mutation in yeast, indicating that the ABD is vital for the recruitment and function of the RSC complex.

The study also mapped the ABD within the ATPase Ino80, a subunit of the INO80 complex. This domain, spanning amino acids 455 to 620, co-localizes with the HSA domain, which is required for binding actin-related proteins, and the DBINO domain, which interacts with DNA. This multifunctional nature of the Ino80 ABD suggests a complex regulatory mechanism where the same protein region mediates activator binding, actin interaction, and DNA sensing. Deletion of this region in Ino80 resulted in a protein that could not complement an ino80 null mutation, confirming the functional importance of this domain in vivo.

The molecular anatomy of the ATPases Swi2 and Sth1 is highly conserved, and the study found that their ABDs are located at similar positions in the N-terminus of both proteins. This conservation supports the idea of a related molecular organization of functional domains in these ATPases. However, the functional consequences of losing these domains differ significantly between the two complexes. While SWI/SNF can tolerate the loss of the Swi2 ABD due to redundancy among its subunits, RSC cannot compensate for the loss of the Sth1 ABD. This difference highlights the distinct roles and regulatory mechanisms of these chromatin remodelling complexes in yeast.

The researchers used a combination of GST pull-down assays and plasmid shuffling experiments to characterize these interactions and their functional impact. GST pull-down assays allowed for the precise mapping of the minimal domains required for activator binding, while plasmid shuffling enabled the assessment of functional complementation in vivo. By introducing specific missense mutations and deletions into the coding regions of SWI2, STH1, and INO80, the team could determine the essential residues for protein function. These methods provided a robust framework for understanding the structural and functional requirements of the activator binding domains.

The study also examined the interaction of Ino2 with other subunits of the SWI/SNF complex, including Swi1, Snf5, and Snf6. The ABD of Swi1 was mapped to a region distinct from its DNA-binding ARID domain, while Snf5 was found to contain two separate activator binding domains. Snf6, which lacks sequences similar to known functional domains, was also shown to interact with Ino2 activation domains. These findings suggest that multiple subunits of SWI/SNF can be redundantly recruited by activators, providing a layer of regulatory flexibility that may not be present in other complexes.

Limitations of the study include the inability to exclude the possibility that other subunits of RSC and INO80, which were not investigated, may also contain activator binding domains. Additionally, while the study provides strong evidence for the functional importance of the Sth1 ABD, it cannot completely rule out that mutations may also affect the formation of the RSC complex. The interaction of Ino2 with the ATPase Swr1 of the SWR1 complex was also examined, but no interaction was detected with the tested truncated protein, leaving open the question of whether other sequences of Swr1 contain ABDs.

The findings have broader implications for understanding how transcriptional activators regulate gene expression through chromatin remodelling. By identifying the specific domains involved in these interactions, the study provides a foundation for future research into the molecular mechanisms of gene regulation. The distinct functional requirements of the ABDs in different CRCs highlight the complexity of chromatin dynamics and the need for precise regulatory controls. This work contributes to the growing body of knowledge on how eukaryotic cells manage their genomes through the coordinated action of transcriptional activators and chromatin remodelling complexes.

Beyond direct binding assays, the investigators evaluated the functional contribution of Swi2 to gene activation in living yeast cells using a GAL1-lacZ reporter system driven by Gal4 DNA-binding domain hybrids carrying either Ino2 TAD1 or TAD2. In cells lacking SWI2, reporter expression driven by TAD1 plummeted to 13.4 percent of wild-type levels, whereas activation driven by TAD2 decreased only moderately to 72.5 percent. This selective dependence demonstrated that although both activation domains associate with Swi2 in vitro, TAD1 relies far more heavily on the ATPase in vivo. To test whether the newly identified activator binding domain was indispensable for organismal fitness, the authors assayed swi2 deletion strains harboring SWI2 variants lacking residues 240 to 298 or 160 to 298. Both deletion variants fully complemented the growth defects of the null mutant, restoring cell growth without inositol and permitting normal utilization of alternative carbon sources including raffinose, ethanol, and galactose.

To resolve why the Swi2 binding region proved dispensable, the researchers conducted site-directed alanine mutagenesis across nine conserved amino acid clusters within the Swi2 activator binding domain, spanning residues 248 to 293. None of these alanine substitutions impaired in vitro binding to Ino2 TAD1, pointing to functional redundancy among contact residues. By contrast, mutational analysis of the paralogous Sth1 domain uncovered stricter sequence requirements. Focusing on an evolutionary conserved heptapeptide motif from residues 212 to 218 and nearby basic residues, the authors found that double alanine mutations at positions R198A I199A and N212A L213A abolished binding to Ino2 TAD1 in vitro. Although cells expressing either double mutant alone remained viable, combining them into a quadruple mutant or mutating the entire heptapeptide through a septuple alanine substitution completely destroyed the ability of Sth1 to support cell survival in plasmid shuffling assays.

Structural and mapping analyses further clarified the distinct architecture of these coactivator surfaces. In Sth1, the activator binding domain resides within the scaffold II region of the RSC body module, spanning residues 154 to 318, with cryo-electron microscopy models confirming that the core binding sequence between amino acids 198 and 218 is exposed on the outer surface of the complex. In Ino80, the identified binding region spans residues 455 to 620, an unusually large segment that physically overlaps with the conserved HSA domain between positions 462 and 598 and the DBINO region between positions 504 and 601. This trifunctional arrangement allows Ino80 to coordinate activator binding alongside nuclear actin, actin-related proteins Arp4 and Arp8, and extranucleosomal linker DNA. Detailed dissection of additional SWI/SNF components similarly mapped a core activator binding domain in Swi1 to residues 507 to 606, separating it from the adjacent AT-rich interaction domain. For Snf5, binding mapped to two discrete segments, designated ABD1 from residues 130 to 275 and ABD2 from residues 265 to 334, while Ino2 TADs recruited Snf6 via residues 1 to 225.

The authors also evaluated the broader biochemical context governing these interactions. Parallel experiments using protein extracts from yeast transformants and Escherichia coli yielded identical binding patterns, establishing that TAD-ABD associations occur directly and do not require eukaryotic post-translational modifications or auxiliary yeast factors. Reflecting on earlier chromatin immunoprecipitation studies at the INO1 and HO promoters, the authors proposed that activator binding domains serve a dual biological function. Beyond facilitating the initial recruitment of chromatin remodelling machines to promoter regions, activator contacts may actively stimulate enzymatic nucleosome mobilization once gene induction signals arise, allowing tightly packaged promoters to transition into accessible transcription pre-initiation sites.

Subject of Research: Molecular mechanisms of transcriptional activator interaction with yeast chromatin remodelling complexes

Article Title: Transcriptional activation domains interact with ATPase subunits of yeast chromatin remodelling complexes SWI/SNF, RSC and INO80

Article References: Wendegatz, E.-C., Engelhardt, M., & Schüller, H.-J. (2024). Transcriptional activation domains interact with ATPase subunits of yeast chromatin remodelling complexes SWI/SNF, RSC and INO80. Current Genetics, 70(1), Article 15. https://doi.org/10.1007/s00294-024-01300-x

Image Credits: AI Generated

DOI: 10.1007/s00294-024-01300-x

Keywords: chromatin remodelling, yeast genetics, transcriptional activation, ATPase subunits, SWI/SNF, RSC complex, INO80 complex, protein domains, gene regulation, molecular biology, Transcriptional, activation