The Saccharomyces cerevisiae Piccolo NuA4 Histone Acetyltransferase Complex Requires the Enhancer of Polycomb A Domain and Chromodomain To Acetylate Nucleosomes

Abstract

Chromatin modification complexes are key gene regulatory factors which posttranslationally modify the histone component of chromatin with epigenetic marks. To address what features of chromatin modification complexes are responsible for the specific recognition of nucleosomes compared to naked histones, we have performed a functional dissection of the Esa1-containing Saccharomyces cerevisiae Piccolo NuA4 histone acetyltransferase complex. Our studies define the Piccolo determinants sufficient to assemble its three subunits into a complex as well as Piccolo determinants sufficient to specifically acetylate a chromatin template. We find that the conserved Enhancer of Polycomb A (EPcA) homology region of the Epl1 component and the Nterminal 165 amino acids of the Yng2 component of Piccolo are sufficient with Esa1 to specifically act on nucleosomes. We also find that the Esa1 chromodomain plays a critical role in Piccolo's ability to distinguish between histones and nucleosomes. In particular, specific point mutations in the chromodomain putative hydrophobic cage which strongly hinder growth in yeast greatly reduce histone acetyltransferase activity on nucleosome substrates, independent of histone methylation or other modifications. However, the chromodomain is not required for Piccolo to bind to nucleosomes, suggesting a role for the chromodomain in a catalysis step after nucleosome binding.

Expression and purification of recombinant yeast Ada2/Ada3/Gcn5 and Piccolo NuA4 histone acetyltransferase complexes

“…However, in contrast to SAGA which is apparently directed to the promoter through its Tra1 subunit, the Ada2/Ada3/Gcn5 complex (which may be identical to the HAT-A2 complex [10,11]) may act as a global, untargeted nucleosome acetyltransferase in the cell [12]. We have similarly identified the Piccolo NuA4 complex of Epl1, Yng2 and Esa1 as the catalytic core of the megadalton NuA4 complex [13,14]. Interestingly, Piccolo NuA4 possesses even greater HAT activity on nucleosomes than the whole NuA4 complex [14].…”

“…Furthermore, if the Esa1 subunit is HIS-tagged, it is possible that the Piccolo NuA4 prep will be contaminated with some Esa1 polypeptide that is not associated with the Epl1 and Yng2 subunits of Piccolo NuA4. This could complicate quantitative measurements of the nucleosomal HAT activity of Piccolo NuA4 relative to its naked histone HAT activity since both Esa1 and Piccolo acetylate histones but Piccolo possesses a much higher specific activity on nucleosomes than Esa1 does [13,14]. Therefore, it may be advisable to further purify the Piccolo NuA4 complex by additional chromatography steps.…”

The MYST family of histone acetyltransferases and their intimate links to cancer

“…Within NuA4, the N terminus of EPC1 binds Tip60 and ING3, forming the trimeric piccolo NuA4 (picNuA4), whereas the C terminus bridges it to the rest of the NuA4 complex. Thus, picNuA4 contains HAT activity and, in fact, is the minimal module within NuA4 capable of acetylating chromatin substrates (Boudreault et al, 2003;Doyon et al, 2004;Selleck et al, 2005). Conversely, PHF1 is homologous to the Drosophila polycomblike protein (PCL), which is associated with transcriptional repression and heterochromatin formation.…”

Structure of Arabidopsis thaliana At1g77540 Protein, a Minimal Acetyltransferase from the COG2388 Family,

“…Taken together these comparisons suggest that At1g77540 likely represents a novel adaptation of the acetyltransferase fold tuned for a currently unknown substrate. Testing of the proposed mechanism will depend on identification of the true physiological substrate of At1g77540 and detailed kinetic studies to confirm or refute the presence of the acetylated intermediate and the roles of conserved Cys 76 and His 41 . To date the only other COG2388 family member that has been structurally characterized is a putative N-acetyltransferase from Staphylococcus aureus (PDB # 1r57).…”

“…Other structures shown are: PDB # 1bob in blue, PDB # 1mja in magenta, and other members of the GNAT superfamily (PDB # 1bo4, 1cm0, 1i12, 1j4j, 1qsm, 1qsn, 1s5k, and 1tiq) in light blue. The sidechains of His 41 and Cys 76 are displayed as sticks. The black arrow denotes the approximate location of the loop between helices 2 and 3 of At1g77540, which contains the CoA binding motif.…”