CHARACTERIZATION OF PROTEIN-PROTEIN INTERACTION DOMAINS WITHIN THE S. CEREVISIAE NUA3 HISTONE ACETYLTRANSFERASE COMPLEX
Embargo until
2019-05-01
Date
2014-12-01
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Johns Hopkins University
Abstract
Eukaryotic DNA is coiled around octamers of core histone proteins (H2A, H2B, H3, and H4), forming nucleosomes- the basic unit of chromatin. Nucleosome organization is coordinated in part by chromatin-associated protein complexes that modulate histone post-translational modifications (PTMs). NuA3, a conserved S. cerevisiae histone acetyltransferase (HAT) complex, engages trimethylation on lysine 4 of histone H3 (H3K4me3) through the plant homeodomain (PHD) finger in the Yng1 subunit. Subsequently, NuA3 acetylates H3K14 through the HAT domain of Sas3, leading to transcription initiation at NuA3-regulated genes. In addition to Yng1, NuA3 contains other potential protein-protein interaction domains, including the proline-tryptophan-tryptophan-proline (PWWP) domain protein, Pdp3, and the extra-terminal (ET) domain protein, Taf14, which have undefined functions.
Here we performed co-immunoprecipitation combined with mass-spectrometry, biochemical binding assays, and genetic analyses to classify the NuA3 HAT complex into two functionally distinct forms: NuA3a and NuA3b. While NuA3a uses the PHD finger of Yng1 to bind H3K4me3 at the 5’-ends of open reading frames (ORFs), NuA3b uses the PWWP domain of the unique subunit, Pdp3, to bind H3K36me3 at gene bodies. We find that deletion of PDP3 decreases NuA3-regulated transcription and results in growth defects when combined with transcription elongation mutants, suggesting NuA3b acts as a positive elongation factor. Therefore, we ascribe a new function to H3K36me3 in yeast- in addition to repressing intergenic transcription, H3K36me3 also positively regulates transcription elongation via HAT complex recruitment into ORFs.
Additionally, we performed mass-spectrometry and biochemical binding assays to further characterize ET domains as mediators of protein-protein interactions. Specifically, the ET domain of Taf14 engages residues 105-125 of Yng1 and this interaction is critical for NuA3-directed HAT activity. Interestingly, the H. sapiens ET domain proteins AF9 and BRD4 also engage residues 105-125 of Yng1. Therefore, this S. cerevisiae Yng1 sequence may contain a conserved ET domain recognition motif and provide a novel mechanism to inhibit pathological ET domain interactions.
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Keywords
epigenetic, transcription initiation, transcription elongation, histone acetyltransferase complex, NuA3, effector protein, Pdp3, Taf14, histone methylation, histone acetylation, proline-tryptophan-tryptophan-proline domain, extra-terminal domain