Piccolo NuA4-Catalyzed Acetylation of Nucleosomal Histones: Critical
Roles of an Esa1 Tudor/Chromo Barrel Loop and an Epl1 Enhancer of
Polycomb A (EPcA) Basic Region
Jiehuan Huang, Song Tan
Center for Eukaryotic Gene Regulation, Department of Biochemistry & Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA
2). Much has been learned about the identity and activities of
beginning to appreciate the different genetic pathways through
vances, the precise mechanism by which chromatin enzymes
function is largely not understood. In particular, we have a rela-
tively poor understanding of how chromatin enzymes recognize
and interact with their nucleosome substrate.
The discovery that the yeast Gcn5 protein possessed histone
acetyltransferase (HAT) activity and was also a subunit of the
between histone acetylation and gene regulation (3, 4). Biochem-
ical and structural studies of the catalytic domains of HAT en-
zymes equip us with insights into how the catalytic domain pro-
vides the structural environment to allow the acetyl-coenzyme A
cofactor to react with the appropriate histone tail peptide sub-
strates (5–8) and the mechanistic basis for how one histone mod-
ification can enhance or prevent a different modification on the
same histone peptide, also known as cross talk (9, 10). Neverthe-
less, major fundamental issues, including how the histone modi-
unresolved. The catalytic subunit of histone modification en-
zymes often possess little or no activity on their physiologically
tisubunit protein complexes that act on nucleosomes. For exam-
ple, while the Gcn5 subunit possesses very poor HAT activity
when assayed on a nucleosome substrate, the megadalton SAGA
complex is able to acetylate both histone and nucleosome sub-
strates (4). Similarly, the yeast Esa1 HAT fails to acetylate nucleo-
somal histones, but its parent multisubunit NuA4 complex will
acetylate nucleosomes (11). In our previous studies, we identified
he regulation of gene expression in eukaryotic cells involves a
complex orchestration of chromatin enzymes that modify or
NuA4 (Epl1/Yng2/Esa1, also known as the Piccolo NuA4 com-
plex) with activities on nucleosome substrates similar to or even
The ability of chromatin modification enzymes like SAGA or
has important implications for their molecular mechanisms. Al-
though the histone tails are the targets of SAGA- and NuA4-di-
rected acetylation, it is unlikely that the tails are sufficient to en-
gage these enzymes. The fact that the Gcn5 and Esa1 catalytic
subunits require accessory subunits to modify nucleosomal his-
tones suggests that these accessory proteins assist the catalytic
subunit in interacting with the nucleosome and in acting on the
histone tails. Parallels can be drawn for other chromatin modifi-
cation enzymes, including the human histone demethylase LSD1,
which requires CoREST to act on the nucleosome, and the yeast
Set1 and human MLL1 histone methyltransferases, which require
other components of the COMPASS or MLL complex to meth-
ylate nucleosomal histones (13, 15, 16). Some of the key unre-
solved issues include precisely how the surface of the nucleosome
protein-DNA complex is recognized by these chromatin modifi-
cation enzymes and the role of the accessory subunits in nucleo-
somal binding or catalytic activity.
In our previous studies of the Piccolo NuA4 catalytic subcom-
Epl1, Yng2, and Esa1 subunits necessary for the three proteins to
Received 20 August 2012 Returned for modification 24 September 2012
Accepted 23 October 2012
Published ahead of print 29 October 2012
Address correspondence to Song Tan, email@example.com.
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
January 2013 Volume 33 Number 1Molecular and Cellular Biologyp. 159–169mcb.asm.org
on December 22, 2015 by guest
N-terminal to the catalytic HAT domain and a 20-residue region
at the N terminus of the conserved EPcA domain of the Epl1
subunit were necessary for Piccolo NuA4 to acetylate nucleo-
somes. The Tudor domain found in Esa1 is part of the Tudor
domain family, which includes the chromo, PWWP, MBT, and
Tudor domains (18). Unlike the case of the chromodomain,
which contains a peptide binding site often used to bind to meth-
yeast Esa1 and related human MOF Tudor/chromo barrel do-
mains shows that an N-terminal extension to the chromodomain
Thus, the Tudor/chromo barrel domain is unlikely to bind to
chromodomain. The exact function of the Tudor/chromo barrel
domain in Esa1 and MOF is not clear, although RNA binding has
Tudor/chromo barrel domain, no experimental structural infor-
mation is available for the Epl1 EPcA domain.
In this study, we identify key residues within the Esa1 Tudor/
chromo barrel domain and the Epl1 EPcA domains necessary for
loop region that we show is in close proximity to nucleosomal
the Epl1 EPcA domain is close both to the N-terminal tail of his-
tone H2A and to nucleosomal DNA. These results suggest new
insights into how the Piccolo NuA4 enzyme interacts with its
nucleosome substrate and catalyzes acetylation of nucleosomal
MATERIALS AND METHODS
Protein expression and purification. Wild-type Esa1, Yng2(2–18), and
hexahistidine-tagged Epl1(51–380) subunits of Piccolo NuA4 and ap-
propriate mutational variants were expressed using the pST44 polycis-
tronic expression vector in BL21(DE3)pLysS cells as described previ-
ously (17, 23). The coexpressed complexes were purified by Talon
(Clontech) cobalt affinity and SourceQ anion-exchange chromatogra-
phy (24), and the engineered mutations did not adversely affect Pic-
colo NuA4 complex stability.
Recombinant Xenopus core histones and nucleosome core particles
bodies under denaturing conditions. The histone octamer was refolded
from the individual core histones and then purified over a Superdex 200
16/60 size exclusion column. Nucleosome core particles were reconsti-
tuted from the purified histone octamer and recombinant Widom 601
DNA positioning sequence by gradient dialysis before purification by
SourceQ anion-exchange high-performance liquid chromatography
HAT assay. HAT assays were performed at least three times each on
HC1 at pH 8.0, 50 mM KC1, 5% glycerol, 0.1 mM EDTA, 1 mM dithio-
threitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10
mM sodium butyrate using procedures described previously (4, 17). The
HAT activity nucleosome preference was calculated by dividing the HAT
activity on nucleosomes substrates by the HAT activity on free histone
Strep-Tactin pulldown assay. One hundred picomoles of recombi-
nant nucleosome core particles were immobilized on Strep-Tactin resin
via histone H2B tagged with Strep-tag in H50 pulldown buffer (20 mM
HEPES [pH 7.5], 50 mM NaCl, 100 ?g/ml BSA [bovine serum albumin],
1% Triton X-100, and 0.1% sodium deoxycholate). After washing with
H50 pulldown buffer twice, the same buffer or equivalent buffer contain-
ing 70 or 90 mM NaCl was used to wash the resin twice. After the resins
40 ?l SDS-PAGE sample loading buffer and separating on an 18% acryl-
amide SDS-PAGE gel.
Preparation of Piccolo NuA4 containing Bpa and post-32P labeling
assay. Piccolo NuA4 complexes with p-benzoyl-L-phenylalanine (Bpa)
incorporated at specific Esa1 positions were expressed using the pSup-
27), and pST44-yEsa1-Yng2(1-218)-HISNyEpl1(51-380) coexpression
plasmids with the TAG amber codon introduced at appropriate Esa1 po-
sitions by site-directed mutagenesis. Expression of Piccolo NuA4 com-
plexes containing Bpa were performed in 2? tryptone-yeast extract (2 ?
SourceISO hydrophobic chromatography was used to further purify Pic-
colo NuA4 complexes containing Bpa.
Bpa at Esa1 K61 was incubated with 63 pmol recombinant nucleosome
core particles in 40 ?l cross-linking buffer (20 mM HEPES [pH 7.5], 50
mM KCl, 0.1 mM EDTA, and 5% glycerol) for 10 min at room tempera-
ture in the dark. Samples were irradiated with UV light at a distance of 5
After adding 40 ?l of T2400 (20 mM Tris-Cl [pH 8.0] and 2.4 M NaCl)
and 20 ?l of Talon metal affinity resin in 200 ?l T1200 (20 mM Tris-Cl
[pH 8.0] and 1.2 M NaCl), the mixture was mixed with rotation at room
temperature for 20 min before washing twice with T1200 and once with
DNase I buffer (10 mM Tris-HCl [pH 7.6], 2.5 mM MgCl2, and 0.5 mM
DNase I buffer at 37°C for 1 h. After washing with T1200 twice and NEB
buffer 3 (50 mM Tris-HCl [pH 7.9], 100 mM NaCl, and 10 mM MgCl2)
once, the sample was treated with 10 U calf intestinal phosphatase (NEB)
T1200 and once with T4 PNK buffer (70 mM Tris-HCl [pH 7.6] and 10
mM MgCl2) before incubation with 5 U T4 polynucleotide kinase (NEB)
and 0.5 ?l [?-32P]ATP (3,000 Ci/mmol, 10 Ci/ml; MP Biomedical) in 40
?l T4 PNK buffer for 1 h. After this labeling step, the resin was washed 6
times with T4 PNK buffer plus 0.1 mM ATP. The Piccolo NuA4 com-
plexes were eluted from the resin by adding 20 ?l of SDS-PAGE sample
were separated by SDS-PAGE and visualized first by staining the gel with
Coomassie blue and then by phosphorimaging after drying down the gel.
Yeast strains and viability tests. Hemagglutinin (HA)-tagged ESA1
was subcloned into the yeast integrative vector pRS403 HIS marker plas-
mid. Site-directed mutagenesis was performed to create point mutations.
pRS403 plasmids with HA-tagged wild-type or mutant ESA1 were lin-
earized with PstI and transformed into QY118 MATa his3?1 leu2?0
met15?0 ura3?0 esa1?::KanMX pLP795 (ESA1 ARS/CEN URA3) using
standard methods (28). Desired clones were selected on synthetic com-
plete (SC) plates lacking histidine and uracil. Viability tests were per-
formed as follows. Yeast strains integrated with HA-tagged ESA1 con-
structs as well as wild-type ESA1 on a URA3 plasmid were grown
overnight in yeast extract, peptone, adenine, and dextrose (YPAD) me-
dium at 30°C and then diluted to an optical density of 1.5. Tenfold serial
5=-fluoroorotic acid (5-FOA). The plates were grown at 30°C for 2 to 5
days. Yeast strains integrated with HA-tagged esa1-D64A were selected
from the 5-FOA plates.
Chromatin immunoprecipitation and qPCR. One hundred millili-
ters of yeast culture (optical density at 600 nm [OD600] ? 0.8 to 1.0) was
cross-linked with formaldehyde (1% [vol/vol]) for 15 min at room tem-
perature and quenched by adding glycine to 125 mM for 5 min at room
temperature (29). Whole-cell extracts were prepared by zirconia bead
disruption, and chromatin was sheared into fragments averaging 150 to
500 bp in size by using a Bioruptor instrument (Diagenode, Philadelphia
Huang and Tan
mcb.asm.orgMolecular and Cellular Biology
on December 22, 2015 by guest
binding activity of the Esa1 presumed chromodomain. J. Mol. Biol. 378:
22. Akhtar A, Zink D, Becker PB. 2000. Chromodomains are protein-RNA
interaction modules. Nature 407:405–409.
23. Tan S, Kern RC, Selleck W. 2005. The pST44 polycistronic expression
24. Barrios A, Selleck W, Hnatkovich B, Kramer R, Sermwittayawong D,
Tan S. 2007. Expression and purification of recombinant yeast Ada2/
core particle from recombinant histones. Methods Enzymol. 304:3–19.
26. Chin JW, Martin AB, King DS, Wang L, Schultz PG. 2002. Addition of
a photocrosslinking amino acid to the genetic code of Escherichia coli.
Proc. Natl. Acad. Sci. U. S. A. 99:11020–11024.
27. Ryu Y, Schultz PG. 2006. Efficient incorporation of unnatural amino
acids into proteins in Escherichia coli. Nat. Methods 3:263–265.
28. Gietz D, St Jean A, Woods RA, Schiestl RH. 1992. Improved method for
high efficiency transformation of intact yeast cells. Nucleic Acids Res.
29. Sharma VM, Tomar RS, Dempsey AE, Reese JC. 2007. Histone deacety-
lases RPD3 and HOS2 regulate the transcriptional activation of DNA
damage-inducible genes. Mol. Cell. Biol. 27:3199–3210.
30. Gao YG, Su SY, Robinson H, Padmanabhan S, Lim L, McCrary BS,
Edmondson SP, Shriver JW, Wang AH. 1998. The crystal structure of the
hyperthermophile chromosomal protein Sso7d bound to DNA. Nat.
Struct. Biol. 5:782–786.
31. Reference deleted.
32. Cobb CE, Beth AH. 1990. Identification of the eosinyl-5-maleimide re-
action site on the human erythrocyte anion-exchange protein: overlap
with the reaction sites of other chemical probes. Biochemistry 29:8283–
33. Smyth DG, Blumenfeld OO, Konigsberg W. 1964. Reactions of N-
ethylmaleimide with peptides and amino acids. Biochem. J. 91:589–595.
34. Chahal HK, Dai Y, Saini A, Ayala-Castro C, Outten FW. 2009. The
SufBCD Fe-S scaffold complex interacts with SufA for Fe-S cluster trans-
fer. Biochemistry 48:10644–10653.
35. Layer G, Gaddam SA, Ayala-Castro CN, Ollagnier-de Choudens S,
Lascoux D, Fontecave M, Outten FW. 2007. SufE transfers sulfur from
SufS to SufB for iron-sulfur cluster assembly. J. Biol. Chem. 282:13342–
36. Padrick SB, Doolittle LK, Brautigam CA, King DS, Rosen MK. 2011.
Acad. Sci. U. S. A. 108:E472–E479.
37. Conrad T, Cavalli FMG, Holz H, Hallacli E, Kind J, Ilik I, Vaquerizas
JM, Luscombe NM, Akhtar A. 2012. The MOF chromobarrel domain
plex. Dev. Cell 22:610–624.
38. Decker PV, Yu DY, Iizuka M, Qiu Q, Smith MM. 2008. Catalytic-site
mutations in the MYST family histone acetyltransferase Esa1. Genetics
39. Chittuluru JR, Chaban Y, Monnet-Saksouk J, Carrozza MJ, Sapountzi
V, Selleck W, Huang J, Utley RT, Cramet M, Allard S, Cai G, Workman
JL, Fried MG, Tan S, Côté J, Asturias FJ. 2011. Structure and nucleo-
some interaction of the yeast NuA4 and Piccolo-NuA4 histone acetyl-
transferase complexes. Nat. Struct. Mol. Biol. 18:1196–1203.
40. Berndsen CE, Selleck W, McBryant SJ, Hansen JC, Tan S, Denu JM.
2007. Nucleosome recognition by the Piccolo NuA4 histone acetyltrans-
ferase complex. Biochemistry 46:2091–2099.
41. Makde RD, England JR, Yennawar HP, Tan S. 2010. Structure of RCC1
42. White CL, Suto RK, Luger K. 2001. Structure of the yeast nucleosome
particle reveals fundamental changes in internucleosome interactions.
EMBO J. 20:5207–5218.
43. Reference deleted.
Role of Tudor and EPcA Domains in Nucleosome Acetylation
January 2013 Volume 33 Number 1mcb.asm.org 169
on December 22, 2015 by guest