Ji Hyun Kim^1,†, Chae Young Yoon^1,†, Yukyung Jun^2,†, Bo Bae Lee¹, Ji Eun Lee¹, So Dam Ha¹, Hyeonju Woo¹, Ahyoung Choi³, Sanghyuk Lee^2,3, Woojin Jeong¹, Ji Hyung Kim⁴ and TaeSoo Kim^1,*

¹Department of Life Science and the Research Center for Cellular Homeostasis, Ewha Womans University, Seoul 03760, Korea,
²Ewha-JAX Cancer Immunotherapy Research Center, Ewha Womans University, Seoul 03760, Korea,
³Department of Bio-Information Science, Ewha Womans University, Seoul, 03760, Korea and
⁴Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Korea

^*To whom correspondence should be addressed.

^†The authors wish it to be known that, in their opinion, the first three authors should be regarded as Joint First Authors.

In yeast, NuA3 histone acetyltransferase (NuA3 HAT) promotes acetylation of histone H3 lysine 14 (H3K14) and transcription of a subset of genes through interaction between the Yng1 plant homeodomain (PHD) finger and H3K4me3. Although NuA3 HAT has multiple chromatin binding modules with distinct specificities, their interdependence and combinatorial actions in chromatin binding and transcription remain unknown. Modified peptide pulldown assays reveal that the Yng1 N-terminal region is important for the integrity of NuA3 HAT by mediating the interaction between core subunits and two methyl-binding proteins, Yng1 and Pdp3. We further uncover that NuA3 HAT contributes to the regulation of mRNA and lncRNA expression dynamics by antagonizing the histone deacetylases (HDACs) Rpd3S and Rpd3L. The Yng1 N-terminal region, the Nto1 PHD finger and Pdp3 are important for optimal induction of mRNA and lncRNA transcription repressed by the Set2-Rpd3S HDAC pathway, whereas the Yng1 PHD finger–H3K4me3 interaction affects transcriptional repression memory regulated by Rpd3L HDAC. These findings suggest that NuA3 HAT uses distinct chromatin readers to compete with two Rpd3-containing HDACs to optimize mRNA and lncRNA expression dynamics.