Biochemical Characterization of the TINTIN Module of the NuA4 Complex Reveals Allosteric Regulation of Nucleosome Interaction

ABSTRACT

Trimer Independent of NuA4 involved in Transcription Interactions with Nucleosomes (TINTIN) is an integral module of the essential yeast lysine acetyltransferase complex NuA4 that plays key roles in transcription regulation and DNA repair. Composed of Eaf3, Eaf5, and Eaf7, TINTIN mediates targeting of NuA4 to chromatin through the chromodomain-containing subunit Eaf3 that is shared with the Rpd3S histone deacetylase complex. How Eaf3 mediates chromatin interaction in the context of TINTIN and how is it different from what has been observed in Rpd3S is unclear. Here, we reconstituted recombinant TINTIN and its subassemblies and characterized their biochemical and structural properties. Our coimmunoprecipitation, AlphaFold2 modeling, and hydrogen deuterium exchange mass spectrometry (HDX-MS) analyses revealed that the Eaf3 MRG domain contacts Eaf7 and this binding induces conformational changes throughout Eaf3. Nucleosome-binding assays showed that Eaf3 and TINTIN interact non-specifically with the DNA on nucleosomes. Furthermore, integration into TINTIN enhances the affinity of Eaf3 toward nucleosomes and this improvement is a result of allosteric activation of the Eaf3 chromodomain. Negative stain electron microscopy (EM) analysis revealed that TINTIN binds to the edge of nucleosomes with increased specificity in the presence of H3K36me3. Collectively, our work provides insights into the dynamics of TINTIN and the mechanism by which its interactions with chromatin are regulated.

KEYWORDS:

• chromatin
• transcription
• NuA4
• lysine acetyltransferase
• Eaf3
• TINTIN
• hydrogen deuterium exchange mass spectrometry
• electron microscopy
• single-particle analysis

Declaration of Interests

The authors declare no conflict of interest.

SUPPLEMENTAL MATERIAL

Supplemental material is available online only.

ACKNOWLEDGMENTS

This work was supported a Discovery Grant from the Natural Science and Engineering Research Council of Canada (RGPIN-2018-03951), a Foundation Grant from the Canadian Institutes of Health Research (FDN-143228) to C.K.Y., a NSERC Discovery Grant (NSERC-2020-04241, J.E.B.), and a Michael Smith Foundation for Health Research Scholar award (17686, J.E.B.). This research project was supported in part by the UBC High Resolution Macromolecular Cryo-Electron Microscopy Facility (HRMEM).

U.D. conceived the project, designed all experiments, carried out all biochemical and structural studies, analyzed the biochemical and structural data, and wrote the manuscript. E.C. assisted in completing protein purification and protein-DNA binding studies and analyzed the results. S.-E.N. conducted the immunoprecipitation experiment. K.D.F., B.E.M., and J.E.B. designed and carried out the hydrogen deuterium exchange mass spectrometry experiments and analyzed the data. C.K.Y. supervised the project and revised the manuscript.

We declare that we have no competing interests.